THE CALCIUM CHANNEL CACNA1C GENE: MULTIPLE …np206cw1776...the calcium channel cacna1c gene:...
Transcript of THE CALCIUM CHANNEL CACNA1C GENE: MULTIPLE …np206cw1776...the calcium channel cacna1c gene:...
THE CALCIUM CHANNEL CACNA1C GENE: MULTIPLE PROTEINS, DIVERSE FUNCTIONS
A DISSERTATION
SUBMITTED TO THE DEPARTMENT OF CHEMICAL AND SYSTEMS BIOLOGY
AND THE COMMITTEE ON GRADUATE STUDIES
OF STANFORD UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
Natalia Gomez-Ospina
May 2010
http://creativecommons.org/licenses/by-nc/3.0/us/
This dissertation is online at: http://purl.stanford.edu/np206cw1776
© 2010 by Natalia Gomez-Ospina. All Rights Reserved.
Re-distributed by Stanford University under license with the author.
This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 United States License.
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I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Richard Dolmetsch, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Thomas Clandinin
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Gerald Crabtree
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Tobias Meyer
Approved for the Stanford University Committee on Graduate Studies.
Patricia J. Gumport, Vice Provost Graduate Education
This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.
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ABSTRACT
Voltage-gated calcium channels are an important route of calcium entry into cells and
are essential for converting electrical activity into biochemical events. In neurons
these channels are vital for synaptic vesicle release and have been implicated in almost
every activity-dependent process including survival, dendritic arborization, synaptic
plasticity, and gene expression. One of the ways in which these channels regulate
cellular behavior is by regulating gene expression but the mechanisms that link
calcium channels to the transcription machinery are not well understood. In this thesis
I show that a C-terminal fragment of CaV1.2, an L-type voltage-gated calcium
channel, translocates to the nucleus and regulates transcription. I show that this
calcium channel associated transcription regulator (CCAT), binds to a nuclear protein,
associates with an endogenous promoter, and regulates the expression of a variety of
endogenous genes that are important for the function of neurons and muscle cells. The
nuclear localization of CCAT is regulated by changes in intracellular calcium on a
time scale of minutes, suggesting that CCAT integrates information about the
electrical activity of the cell. Together these findings reveal an entirely unexpected
function for a well-characterized calcium channel.
This works also addresses the question of how CCAT is generated. I show that CCAT
is not released from proteolysis of full-length Cav1.2 channel but is generated from an
mRNA that is transcribed from the 3’ end of the Cav1.2 gene (CACNA1C). Consistent
with this, I find that CCAT expression is independent of full-length channel protein.
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Furthermore, Exon 46 of the CACNA1C gene contains a promoter whose
transcriptional activity is required for the expression of CCAT. Activity at this
promoter, and consequently CCAT expression, is regulated spatially and temporally in
the brain having highest expression during embryonic stages and in regions of the
brain rich in inhibitory neurons. Analysis of 5’ transcriptional starts from CACNA1C
and Cap Analysis of Gene Expression (CAGE) tags from genome-wide studies show
at least two mRNAs one of which encodes CCAT in vivo and a second transcript that
is predicted to encode a membrane bound CCAT containing a voltage sensor. These
findings reveal an unexpected mechanism by which CCAT is generated in neurons
and provide a unique example by which two proteins with distinct biologic functions
can be derived from a single gene. Such transcriptional phenomena may be at play in
many other genes throughout the genome and has far reaching implications for
prediction of gene products and interpretation of phenotypes in gene mutations and
knockout studies.
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ACKNOWLEDGEMENTS
Over the past (not so few but enjoyable) years several people helped me reach
the completion of this work. First and foremost, I must thank my advisor Ricardo. He
has consistently supported this project and my learning through intellectual advice,
financial support, and fostering a nourishing work environment that developed
independence, creativity, and camaraderie. Through a series of improbable
coincidences I became his first graduate student and never looked back. In every
situation, Ricardo is always personable, always available, and always has the utmost
confidence in his students.
My foundation as a scientist also rests on the tutelage of my previous mentors--
Dr Andrew Staehelin and Dr Tomas Giddings who first gave me the opportunity to
work in a lab. Dr Staehelin allowed me to join his when I spoke broken English, when
I was new to biology, and when I had never used a word processor. Later he would tell
me how much he had watched my “growth” and thereby let me know that he truly
understood the distance I had traveled. Tom, who was one of the kindest persons I
have ever met and who opened opportunities for me by entrusting me with difficult
projects for several accomplished scientists.
I also would like to acknowledge my colleagues in the Dolmetsch lab with
whom I am proud to have spent these formative years working side-by-side—Jocelyn,
Eric, Jake, Matthieu, Fuminori, Chan, Agatha and Georgia will be lifelong friends.
I owe a warm thank you to my family, in particular my mother Cielo. She followed
me to California and supported me in all those small ways that make the world go
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around at a smooth pace without creaking or faltering. Only with her help did I have
the luxury of dedicating so much time to my research. I have yet to explain the
importance of calcium channels or CCAT to her but it does not matter.
Thanks to Anil, who can make anything fun and believes in me more that I do. In a
world of unexpected things, difficult choices and constant compromise he gives me
the certainty that at least one thing is right and always better than I could predict or
imagine.
Lastly, none of this would have been possible without the support of
Stanford’s Medical Scientist Training Program.
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TABLE OF CONTENTS
List of Tables……………....…………………………………………………………..x List of Figures…………………………………………………………………………xi Chapter 1: Activity-dependent L-type channel regulation of gene expression Abstract….……………………………………………………………………..2 L-type calcium channels.………………………………………………………2 L-type channels and c-fos: The beginning.………….…………………………4 L-type channels and CREB.……………………………………………………5 What mechanisms link LTCs to CREB? .……………………...………………8
A. Biophysical Properties………………………….…………….……9 B. Localization…………………………………….…………………11 C. L-type calcium channels and nuclear calcium.…………….…..…12 D. Local calcium signaling………………………..…………………14
Isoform Specific Considerations……………...………………………………18 Chapter 2: The C-terminus of the L-type voltage-gated calcium channel Cav1.2 encodes a novel transcription factor
Summary……………………………………………………………………...20 Introduction……………………………………………………………...……20 Results
CCAT is found in the nucleus of neurons in the brain………………..22 The concentration of CCAT is regulated by intracellular calcium.…..26 Nuclear CCAT is regulated developmentally………………………...28 CCAT binds to a nuclear protein……………………………………..29 CCAT activates transcription………………………………..………..29 CCAT regulates transcription of endogenous genes…….……………32 CCAT bind and regulates the promoter of Cx31.1………….………..33 Endogenous Cav1.2 and CCAT regulate transcription of Cx31.1……36 CCAT expression promoted neurite growth………………….………38 Discussion…………………………………………………………..………...39 Experimental Procedures……………………………………………………..45 Future Experiments………………………………………………….………..56 Figures………………………………………………………………………...62 Supplementary Figures……………………………………………………….69 Tables…………………………………………………………………………73 Figure legends………………………………………………………………...78
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Chapter 3: An independent promoter in the CACNA1C channel gene generates the transcription factor CCAT
Summary……………………………………………………………………...88 Introduction……………………………………………………………...……89 Results
CCAT is not generated by Proteolytic Cleavage of Exogenously Expressed Channels…………………………………………………..92 Cav1.2 Channel Protein is not necessary for CCAT Expression In Vivo……………………………………………………………………………95 CCAT is translated from a Separate Transcript from the cDNA………………………………………………………………....97 An Exonic Promoter Drives CCAT Expression…………………………………………………………….98 CCAT is Translated from a Separate Transcript In Vivo whose Expression is Spatiotemporally Regulated in the Brain……………..101 CACNA1C has Multiple TSS Predicting Multiple Proteins…………104
Discussion…………………………………………………………..…….....108 Experimental Procedures…………………………………………………....117 Future Experiments………………………………………………….………130 Figures……………………………………………………………………….133
Supplementary Figures……………………………………………………...137 Tables………………………………………………………………………..141 Figure legends……………………………………………………………….142 List of References………………………………………………………..………….150
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List of Tables Chapter 2 Table 1 Genes Significantly Up-regulated by CCAT versus CCAT∆TA…………..………...73 Table 2 CCAT versus GFP Up-regulated genes…………………..…………………………..74 Table 3 CCAT versus GFP Down-regulated genes…………………………...…………...75-76 Table 4 Genes Regulated by CCAT in All Experiments………………...……………………77 Chapter 3 Table 1 Summary of transcription start sites and nearby CAGE tags………………………..141
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List of Figures Chapter 1 Figure 1 Schematic Representation of VGCC Showing the Topology of the Pore-forming α1C Subunit, and β, α2δ Accessory Subunits……………………………..………………..3 Figure 2 Schematic Representation of Signaling pathways from LTCs to CREB……………17 Chapter 2 Figure 1 The C Terminus of Cav1.2 Is Found in the Nucleus of Neurons…………...…….…..62 Figure 2 Ectopically Expressed CCAT Localizes to the Nucleus of Neurons via a Nuclear Retention Domain…………………………………………………………………….63 Figure 3 The Nuclear Localization of CCAT Is Regulated by Intracellular Calcium and by Developmental Processes in the Brain………………………………………………..64 Figure 4 The C Terminus of Cav1.2 Binds to Nuclear Proteins and Activates Transcription…65 Figure 5 CCAT Regulates Endogenous Genes………………………………………………...66 Figure 6 Endogenous CCAT Regulates Transcription Driven by the Cx31.1 Promoter………67 Figure 7 CCAT Regulates Neurite Growth in Primary Neurons……………...………….……68 Figure S1 CCAT’s Nuclear Localization and TA Domain are Conserved Among Cav1.2 Channels in Vertebrates……………………………………………...……………….69 Figure S2 CCAT Derived from Cav1.2-YFP channels is Regulated by Depolarization…….…..70 Figure S3 CCAT Regulates Expression of Endogenous Genes: Summary of Microarray Data...71
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Figure S4 sh-RNA Knockdown of Endogenous Cav1.2 in Neurons Decreases CREB Activation Induced by K+ ………………………………………………………...………………72 Chapter 3 Figure 1 CCAT is Not Generated by Proteolytic Cleavage of Exogenously Expressed or Endogenous Cav1.2 Channels…………………………………………………….…133 Figure 2 CCAT is Translated from a Separate Transcript Driven by an Exonic Promoter.…134 Figure 3 CCAT is Translated from a Separate Transcript whose Expression is Cell-type and Developmentally Regulated In Vivo……………………………………………...…135 Figure 4 CACNA1C has Multiple Transcriptional Start Sites Predicting Multiple Proteins Including CCAT……………………………………………………………………..136 Figure S1 CCAT staining in Cav1.2 knockout embryos……………………………………….137 Figure S2 Multiple Sequence alignment of Cav1.2 C-termini from multiple species………….138 Figure S3 Developmental CCAT staining……………………………………………………...139 Figure S4 Leuzine Zipper mutations and Multiple Sequence alignment from Cav1 channels...140
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Chapter 1:
Activity-dependent L-type channel regulation of gene expression
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Abstract
Calcium-regulated transcription plays a key role in converting electrical
activity at the membrane into long-lasting structural and biochemical changes in
excitable cells. Although several calcium influx pathways contribute to the
intracellular calcium rise that follows membrane depolarization in neurons, a
preponderance of data suggest that calcium entry through voltage-gated L-type
calcium channels and NMDA receptors is particularly important in activating gene
expression. In this chapter, we review seminal work implicating L-type channels in
the induction of gene expression in response to neuronal activity and discuss some of
the mechanisms that explain the dependence of activity-induced transcription on
LTCs. We will focus our discussion on studies that explore the biophysical, structural,
and cell biological features of LTCs that allow them to activate CREB-dependent
transcription.
L-type Calcium Channels
Voltage-gated calcium channels (VGCC) are an important route of calcium
entry into neurons and are essential for converting electrical activity into biochemical
events in excitable cells (Catterall et al., 2005). All VGCCs have a common ability to
carry calcium in response to depolarization of the membrane but they differ in their
subcellular localization, biophysical properties and in their ability to regulate specific
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biochemical processes. VGCCs are classified into L, N, P/Q, R and T types based on
their pharmacological and biophysical properties and are composed of four protein
subunits: a pore forming α1 subunit, and β, α2δ and γ subunits that modulate gating
and trafficking (Tsien and Tsien, 1990) (Figure 1). Neuronal L-type channels contain
one of three α1 subunits: Cav1.2, Cav1.3 or Cav1.4. Cav1.2 and Cav1.3 form the
predominant LTCs in the brain and have been implicated in a wide variety of neuronal
functions including promoting survival, increasing dendritic arborization and
regulating synaptic plasticity (Galli et al., 1995; Moosmang et al., 2005; Redmond et
al., 2002).
Figure 1: Schematic representation of VGCC showing the topology of the pore forming α1C subunit,
and β, α2δ accessory subunits
LTCs have a number of features that set them apart from other types of
VGCCs. They exhibit high sensitivity to dihydropyridines (DHP), are activated by
strong depolarization and have slow activation and inactivation kinetics1 (Tsien and
1 Some LTCs including Cav1.3 can be low voltage-activated and have fast kinetics of activation Lipscombe, D., Helton, T.D., and Xu, W. (2004). L-type calcium channels: the low down. J Neurophysiol 92, 2633-2641, Xu, W., and Lipscombe, D. (2001).
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Tsien, 1990). LTCs are localized in the cell body, dendrites and postsynaptic
membranes of adult neurons, making them ideally poised to control the signal
transduction pathways that are activated post-synaptically (Hell et al., 1993;
Westenbroek et al., 1990). Finally, LTCs have been shown to be particularly effective
at activating gene expression in response to electrical activity. A key question,
however, is what features of LTCs allow them to activate the signaling pathways that
lead to the nucleus.
L-type calcium channels and c-fos: The beginning
Morgan and Curran first reported this peculiar link between LTCs and the
nucleus more than two decades ago. They discovered that depolarizing concentrations
of potassium provoked an influx of calcium ions via VGCCs that led to the
transcription of the immediate-early gene c-fos (Morgan and Curran, 1986). DHPs
and calmodulin (CaM) inhibition were found to block this effect suggesting a role for
LTC activity and the ubiquitous calcium sensor, CaM, in the expression of c-fos in
response to neuronal activity. Contemporaneous studies implicated LTCs downstream
of nicotinic receptors in the ensuing induction of c-fos and actin expression in the
same cells (Greenberg et al., 1986). In another influential study, Murphy and
colleagues showed that blocking and activating LTCs respectively eliminated and
increased basal c-fos expression in spontaneously active neuronal cultures (Murphy et
Neuronal Ca(V)1.3alpha(1) L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. J Neurosci 21, 5944-5951.
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al., 1991). This implied that LTCs play a role in the induction of c-fos expression in
response to endogenous electrical activity. To examine the possibility that this
observed LTC specificity was related to a greater ability of LTCs to elevate
intracellular calcium during neuronal activity, the authors measured the relative
contributions of LTCs and other ligand-gated glutamate receptors to the synaptically
evoked calcium rise. They found that LTCs contributed less than 20% of the
synaptically-induced calcium elevation, significantly less than NMDA or kainate
receptors, suggesting that the route of calcium entry rather than the absolute amplitude
of the calcium rise was important for the activation of c-fos. This implied that specific
mechanisms other than bulk calcium elevations must exist that link these channels to
the nucleus. Thenceforth, much effort has been underway to uncover such
mechanisms.
L-type channels and CREB
Dissection of the c-fos promoter by a number of groups identified two main
calcium-regulated response elements, the calcium response element (CRE) and the
serum response element (SRE) (Miranti et al., 1995; Sheng et al., 1988). The CRE
binds to the transcription factor CREB and the SRE binds to serum response factor
(SRF) both of which are activated by calcium influx in neurons. CREB has emerged as
a major regulator of calcium signaling in the brain and has been implicated in neuronal
development, survival and plasticity (Lonze and Ginty, 2002). Early studies by Sheng
and Greenberg first demonstrated that calcium influx through LTCs is particularly
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effective at activating CREB-dependent transcription (Sheng et al., 1990). Blockers of
LTCs potently block the activation of CREB reporter genes, and calcium influx
through LTCs in developing cortical neurons is substantially more effective at
activating CREB than equivalent calcium elevations through NMDA receptors,
suggesting that LTCs are specifically linked to CREB activity (Bading et al., 1993).
The most compelling illustration of the central role of LTCs in activating CREB is a
study of LTC knockout mice. Eliminating Cav1.2 specifically in the hippocampus and
cortex of mice using CRE recombinase-mediated recombination resulted in a loss of
CREB phosphorylation in response to electrical activity, a reduction in an LTC-
dependent form of long term potentiation, and in learning deficits (Moosmang et al.,
2005). This result demonstrates the importance of LTCs in activating CREB and in
regulating neuronal plasticity of neurons in vivo.
While the mechanisms that link calcium influx through LTCs to the activation
of CREB are not completely understood, a great deal is known about how intracellular
calcium elevations can activate CREB-dependent transcription. Activation of CREB-
dependent transcription is a multi-step process that involves both the recruitment of
CREB to CRE elements, the phosphorylation of CREB and the recruitment of other
co-activators. Recent studies suggest that CREB does not constitutively occupy CRE
sites and that activation of CREB involves its recruitment to CREs via a nitric oxide-
dependent cascade (Riccio et al., 2006). At the same time as CREB is recruited to
CRE elements, it is also phosphorylated at Ser133 which enhances its transactivation
potential. This phosphorylation event is strongly calcium-dependent and is absolutely
required for the activation of CREB-dependent transcription (Gonzalez and
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Montminy, 1989). Phosphorylation of Ser133 allows CREB to recruit the CREB
binding protein (CBP), which acts as a transcriptional co-activator by means of its
intrinsic histone acetyl transferase activity and by promoting binding to basal
transcriptional machinery (Chrivia et al., 1993; Kwok et al., 1994). CBP is also
subject to regulation by calcium via two calcium-inducible transactivation domains
(Hu et al., 1999) and via calcium induced phosphorylation by CamKIV (Impey et al.,
2002). LTCs promote CamKIV mediated phosphorylation of CBP suggesting that
LTC-specific recruitment of co-activators can help explain the need for LTC activity
in the transcriptional activation of CREB (Hardingham and Bading, 1999). Other
CREB co-activators such as the Transducers of Regulated CREB activity (TORCs)
translocate to the nucleus is response to intracellular calcium elevations (Conkright et
al., 2003; Impey et al., 2002). In addition to phosphorylation at Ser133, CREB is also
phosphorylated at several other serines including Ser142 and Ser143 although how
these phosphorylation events regulate transcription has not been elucidated yet
(Kornhauser et al., 2002). Thus CREB is subject to calcium-dependent regulation at
many different points during its activation.
Calcium influx through LTCs simultaneously activates several signaling
pathways culminating in Ser133 phosphorylation. Two of these signaling systems, the
calcium Calmodulin (CaM) activated kinases CaMKIV and CamKI and the mitogen
activated kinases (MAPK), seem to be particularly important for linking CREB to
calcium influx through LTCs. CamKIV, downstream of the calcium-calmodulin
dependent kinase pathway, and RSK2, downstream of the canonical Ras/MAPK
pathway, are thought to be the major players in phosphorylating CREB in response to
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depolarization in neurons (Lonze and Ginty, 2002). CaMKIV is activated by the
concerted action of Calcium-bound calmodulin and CaMKK phosphorylation
(Tokumitsu et al., 1994). The canonical MAP kinase cascade includes Ras, Raf, MEK,
ERK, and the nuclear kinases RSK1, RSK2 and MSK1, which phosphorylate CREB
on Ser133. Phosphorylation of CREB and CREB-dependent transcription are defective
in mice lacking CaMKIV (Ho et al., 2000), or MAP kinase MSK1 (Arthur et al., 2004;
Wiggin et al., 2002) and in cells whose CaMKI levels have been reduced using
siRNAs (Wayman et al., 2006), showing that activation of these signaling molecules is
important for CREB-mediated transcription. Furthermore, the importance of the LTC
induced activation of the Ras/MAPK pathway is highlighted by the markedly reduced
MAPK activation observed in the hippocampus and cortex specific L-type channel
knockouts (Moosmang et al., 2005). Activation of LTCs therefore leads to the
activation of several signaling cascades that result in phosphorylation of CREB.
Precisely what role each of these kinases plays in regulating the activation of
CREB is still a subject of controversy. It has been proposed that the kinetics of
activation of each of these kinases results in specific CREB phosphorylation profiles.
The CaM kinases, for instance, are activated rapidly and transiently in response to
calcium influx, whereas the MAP kinase cascade is activated more slowly and is more
sustained. CaMK activation therefore leads to rapid, transient CREB phosphorylation,
whereas activation of MAP kinase allows CREB to remain phosphorylated for a
prolonged period of time (Wu et al., 2001).
What mechanisms link LTCs to CREB?
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Despite a wealth of information on the biochemical signaling pathways that
regulate CREB phosphorylation in response to depolarization, the mechanisms that
specifically link calcium influx through LTCs to the activation of CREB are not
completely understood. It is likely that multiple features of LTCs contribute to their
ability to activate CREB. At least three features of LTCs seem to be important for
their ability to activate transcription: their biophysical properties, their localization in
the dendrites and cell bodies of neurons, and their association with signaling proteins
that activate nuclear signaling cascades.
A. Biophysical Properties
Two distinct biophysical properties make LTCs particularly well suited to
activate CREB: their high voltage of activation and their slow activation/inactivation
kinetics (Tsien and Tsien, 1990; Xu and Lipscombe, 2001). In other words, LTCs
open relatively slowly and thus require sustained bursts of action potentials or
continuous depolarization for maximal activity (Deisseroth et al., 1996; Nakazawa and
Murphy, 1999)2. Consistent with this, depolarization using concentrated potassium or
strong electrical stimulation3 has been observed to trigger a far more sustained CREB
phosphorylation response than bath stimulation of NMDA receptors4 (Bito et al.,
2 Interestingly LTCs and the pathways leading to CREB activation can be recruited by somatic action potentials if these are delivered as tetha burts, suggesting you may not need signaling evoked from synaptic NMDA receptors. 3 Prolongued vs Transient: 180s vs. 18s 5Hz electrical stimulation or 90mM K+ 3min vs. 1min bath depolarization 4 Synaptic stimulation of NMDA receptors can also trigger sustained phosphorylation (Hardingham, 2002).
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1996; Sala et al., 2000). Conceivable, their selective recruitment during enhanced
activity can explain their privileged pathway to CREB. In order to investigate whether
selective activation of LTCs takes place during more complex or physiologically
relevant patterns of neuronal activity, Liu and colleagues looked at how different
VGCCs respond to waveforms that mimic synaptic stimuli in the form of gamma and
theta frequency stimulation (Liu et al., 2003). They found that these type of stimuli
lead to the inactivation of non-L-type VGCCs leading to a calcium current mostly of
L-type suggesting that these channels by virtue of their activation/inactivation kinetics
are selectively recruited and conduct most of the calcium during strong neuronal
activity. Hence the observed importance of these channels in transcriptional induction
during electrical activity.
Though many stimuli can cause CREB phosphorylation, not all can lead to
transcriptional activation. This is in part because the stimulus must cause sustained
phosphorylation that persists for at least 30 minutes. To maintain this sustained
phosphorylation, calcium levels must be elevated for prolonged periods of time. In
contrast to other types of VGCCs, LTCs inactivate slowly and incompletely and so
they contribute a disproportionate amount of the calcium current under conditions of
tonic electrical stimulation (Liu et al., 2003). Consistent with this mutations that slow
voltage dependent inactivation of channels, such as in timothy syndrome, lead a faster
more sustained phosphorylation of CREB ((Splawski et al., 2004) and unpublished
data). A sustained calcium rise would best engage signaling molecules in the locality
of the channel that would normally deactivate quickly after a drop in calcium
concentration. In principle, a prolonged calcium rise could also lead to the selective
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inactivation of a negative pathway that prevents long-lasting CREB phosphorylation.
A reported example of a molecule involved in such negative feedback mechanism is
the calcium regulated phosphatase, calcineurin. Inhibition of calcineurin with FK506
leads to prolonged CREB phosphorylation and transcription even under conditions
where phosphorylation would be transient and insufficient for transcriptional
activation (Bito et al., 1996; Liu and Graybiel, 1996). Whether LTC activation
promotes calcineurin inactivation and calcineurin ultimately leads to the
dephosphorylation of CREB has not been elucidated.
B. Localization
The biophysical properties of LTCs, however, do not account entirely for the
ability of these channels to activate CREB-dependent transcription. In developing
cortical and hippocampal neurons, sustained calcium elevations mediated by NMDA
receptors or generated by the addition of calcium ionophores are significantly less
effective at activating CREB-dependent transcription than calcium influx through
LTCs (Bading et al., 1993). This suggests that there are additional features of LTCs
that link them to the signaling pathways that activate transcription. Another feature of
LTCs that may be involved in their ability to activate transcription is their subcellular
localization.
Early immunohistochemical studies described LTCs as concentrated at the
soma and basal dendrites of neurons (Ahlijanian et al., 1990; Hell et al., 1996; Hell et
al., 1993; Westenbroek et al., 1990). Other VGCCs such as Cav2.1 (P/Q) and Cav2.2
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(N) are thought to be presynaptic and primarily involved in synaptic vesicle release.
Intuitively, one could posit that their proximity to the nucleus and their strategic
position to summate and respond to depolarizing activity that reaches the soma
explains at least in part why LTCs can more effectively convey signals to the nucleus.
Somatic localization of the channels could imply that LTCs could be more effective at
elevating somatic calcium following depolarization or alternatively that they activate
relevant signaling molecules which are closer to the nucleus and poised for activation
by calcium influx through these channels. However, in several systems it has been
clearly demonstrated that P/Q and N-type channels are also abundant in the soma
where they contribute significantly and in fact more to the somatic calcium rise than
the L-types (Deisseroth et al., 1998; Dolmetsch et al., 2001; West et al., 2001). In
addition, subsequent immunolabeling studies revealed that LTCs also localize to the
synapse and co-localize with synaptic markers and their synaptic localization may
enhance their ability to signal to CREB (Zhang et al., 2006). Consequently, somatic
localization of these channels does not entirely explain the observed L-type channel
specificity.
C. L-type calcium channels and nuclear calcium
Cytoplasmic calcium transients are normally accompanied by large nuclear
calcium rises. Under some conditions, elevations in nuclear calcium have been shown
to be required for CREB activation. Nuclear microinjection of the non-diffusible
calcium chelator, BAPTA-dextran, blocks expression mediated by the CRE element in
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response to depolarization suggesting that nuclear calcium is necessary for CREB-
dependent transcription (Chawla et al., 1998; Hardingham and Bading, 1998). Both
CaMKK and CaMKIV are localized in the nucleus, and therefore a nuclear calcium
elevation would lead to activation of these signaling proteins and phosphorylation of
CREB. However, it is unclear how BAPTA-dextran works in these experiments given
that BAPTA’s chelating activity is exhausted within seconds (given the large amounts
of calcium that enters the nucleus) and the fact the stimulation is in the order of
several minutes. Furthermore, loading cells with the calcium buffer EGTA, which
prevents nuclear calcium elevation, has no effect on activity–induced CREB
phosphorylation (Deisseroth et al., 1996), suggesting that if nuclear calcium plays a
role, it does so in other stages of CREB activation beyond Ser133 phosphorylation.
Another line of evidence for the role of nuclear calcium is the observation that isolated
nuclei can support CREB phosphorylation (). However, it is unknown whether this
phosphorylation would be sustained and whether isolated nuclei would support
transcriptional activation of CREB. In general, the activation of CREB is response to
LTC activation cannot be solely explained on the basis of nuclear calcium. First, other
calcium channels elevate nuclear calcium as or even more effectively than LTCs
(Deisseroth et al., 1998; Dolmetsch et al., 2001). Secondly, activation of the serum
response factor SRF, which also happens downstream of LTC activity is entirely
independent of nuclear calcium (Deisseroth et al., 1996; Hardingham et al., 1997).
Together, the data suggest the nuclear calcium is not sufficient to lead to CREB-
dependent transcription but suggest a role for other nuclear, calcium regulated players.
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D. Local calcium signaling
Studies by Deisseroth and colleagues showed that loading neurons with EGTA,
a slow calcium buffer that prevents calcium elevation in the cell body and nucleus but
allows calcium elevations close to the membrane, does not inhibit CREB
phosphorylation in response to depolarization (Deisseroth et al., 1996). On the other
hand loading neurons with BAPTA, a fast calcium buffer that chelates calcium close
to the mouth of the channels, blocks CREB phosphorylation. This suggested a role for
calcium and calcium sensor molecules near the mouth of the channels in triggering the
signaling pathways that lead to the nucleus. Furthermore, mutations of LTCs that do
not alter their ability to carry calcium or to elevate nuclear calcium prevent LTCs from
inducing CREB phosphorylation and CREB-dependent transcription (Dolmetsch et al.,
2001). To investigate the features of LTCs that specifically couple them to the
activation of CREB, a functional knock-in technique was developed where DHP
resistant recombinant channels could be introduced into neurons and thus their
behavior could be distinguished from their endogenous/DHP-sensitive counterpart.
Using this functional knock-in approach it was found that point mutations that disrupt
Calmodulin binding to the LTC prevent LTC activation of CREB. These mutations
did not affect the ability of the LTC to activate the CaMK signaling pathway but
prevented activation of MAP kinase, suggesting that local calcium elevations around
LTCs activate MAP kinase signaling that is necessary for CREB-dependent
transcription. Together, these findings clearly demonstrated that the coupling of LTCs
to signaling pathways that activate gene expression goes beyond the calcium conduit
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properties of the channel. They have led to the idea that global calcium elevations
(including elevations of nuclear calcium) are required for activation of the CaMK
signaling cascade, but that full activation of CREB-dependent transcription requires
the activation of MAP kinase by signaling molecules close to the mouth of LTCs.
CaM binding to the channels cannot constitute the molecular basis of
specificity since other VGCCs contain IQ motifs and are regulated by calmodulin5.
By means of the functional knock-in approach, other structural domains in the channel
have been found to impinge on the channels ability to signal to CREB. PDZ motifs in
the structures of Cav1.2 and Cav1.3 proteins have also been shown to be necessary for
signaling CREB. Inhibition of the interaction of Cav1.2’s PDZ motif with its
endogenous binding proteins attenuated CREB phosphorylation and CRE-dependent
transcription following depolarization (Weick et al., 2003). In the case of Cav1.3 an
association with post-synaptic density protein shank is necessary for CREB
phosphorylation in response to Cav1.3 activation (Zhang et al., 2006; Zhang et al.,
2005). Taken together these data further provides support for the idea that calcium
responses at the mouth of calcium channels are centrally important for transcriptional
regulation by LTCs.
The identity of the molecule that transmits the signal from the nucleus is not
known. Two candidates have been proposed, CaM itself and elements of the
Ras/MAPK signaling cascade. Because CREB activation depends on CaM-binding
proteins such as CaMKK and CaMKIV, and CaM is found enriched in the vicinity of
5 In addition, calmodulin regulates the channels activation and inactivation kinetics, and thus perturbation of calmodulin binding may have effects on the dynamics of the local calcium concentration.
16
the channels (Mori et al., 2004), calmodulin has been proposed as the herald of LTC
activation to the nucleus. Consistently with this hypothesis CaM translocates to the
nucleus of neurons in response to increases in intracellular calcium and this
translocation seems to be, under conditions of synaptic stimulation, L-type dependent
and NMDA receptor dependent (Deisseroth et al., 1998). However, CaM is also found
in high levels in the nucleus of resting neurons so CaM is unlikely to be the only
signal that conveys information from LTCs to the nucleus.
As discusses earlier, LTCs also activate the MAPK pathway and this signaling
cascade seems to be important for the prolonged phosphorylation of CREB that is
required for CREB-dependent transcription. The importance of the LTC induced
activation of the Ras/MAPK pathway is highlighted by markedly reduced MAPK
activation in response to strong LTC stimuli in the hippocampus and cortex of specific
L-type channel knockouts (Moosmang et al., 2005). Surprisingly, nothing is known
about how calcium influx through LTCs lead to sustained ERK activation. In
mammalian cells calcium can trigger Ras activity via Pyk2 a calcium regulated
tyrosine kinase/scaffolding protein (Lev et al., 1995), via calcium-sensitive K-Ras
(Villalonga et al., 2002) or calcium-regulated Ras-guanine nucleotide exchange factors
(GEFs) including RAS-GRF (Farnsworth et al., 1995), RAS-GRP (Ebinu et al., 1998),
CAPRI (Lockyer et al., 2001), and RASL (Liu et al., 2005). However, which and how
any of these proteins are preferentially activated by LTCs remains to be discovered.
Despite more than 20 years of study, the signaling molecules that connect
LTCs to the activation of CREB-dependent transcription have not been defined. It is
likely that there is a complex of proteins around LTCs that senses calcium and
17
converts calcium elevations into activation of the MAP kinase signaling cascade (Fig.
2). This complex includes calmodulin, which binds directly to the LTC. Calmodulin
has multiple effects on LTCs, and mediates both calcium-dependent inactivation and
calcium-dependent potentiation of the channel in addition to connecting the channels
to activation of CREB. Calmodulin therefore alters the conformation of LTCs in
response to local calcium elevations, and this conformational change might activate
signaling proteins bound to the channel leading to CREB-dependent transcription.
Understanding the molecular mechanisms that connect calmodulin to the activation of
CREB-dependent transcription is a critical question in channel signaling.
Figure 2: Schematic representation of signaling pathways from LTCs to CREB
18
ISOFORM SPECIFIC CONSIDERATIONS
In neurons, Cav1.3 is co-expressed with Cav1.2 where they share
somatodendritic and postsynaptic localization (Hell et al., 1993). Among VGCCs
Cav1.2 and Cav1.3 channels share the highest sequence similarity and have both been
implicated in mediating signaling from the membrane to the nucleus (Zhang et al.,
2006). Some important differences do exist which impact how we view the
contributions of these channels to transcriptional regulation.
Although the traditional view of L-type channels is that they are high-voltage
activated, have slow activation kinetics and are highly sensitive to DHP inhibition,
there is increasing evidence that channels composed by the Cav1.3 subunit have in fact
fast activation kinetics and low activation thresholds (Xu and Lipscombe, 2001).
Consistent with the biophysical properties of clones Cav1.3 channels it has been
reported that Cav1.3 preferentially mediates CREB phosphorylation at low (20mM
KCL 30sec and 5Hz 30sec) but not high levels of stimulation (Zhang et al., 2006).
Therefore it is possible that under low levels of stimulation, perhaps spontaneous
activity Cav1.3 may carry out most of the signaling to the nucleus.
19
Chapter 2:
The c-terminus of the l-type voltage-gated calcium channel Cav1.2 encodes a
transcription factor
20
SUMMARY
Voltage-gated calcium channels play a central role in regulating the electrical and
biochemical properties of neurons and muscle cells. One of the ways in which
calcium channels regulate long-lasting neuronal properties is by activating signaling
pathways that control gene expression, but the mechanisms that link calcium channels
to the nucleus are not well understood. We report that a C-terminal fragment of
CaV1.2, an L-type voltage-gated calcium channel (LTC), translocates to the nucleus
and regulates transcription. We show that this calcium channel associated
transcription regulator (CCAT), binds to a nuclear protein, associates with an
endogenous promoter, and regulates the expression of a wide variety of endogenous
genes important for neuronal signaling and excitability. The nuclear localization of
CCAT is regulated both developmentally and by changes in intracellular calcium,
suggesting that CCAT integrates information about the developmental history and
electrical activity of the cell. These findings provide the first evidence that voltage-
gated calcium channels can directly activate transcription, and suggest a novel
mechanism linking voltage-gated channels to the function and differentiation of
excitable cells.
INTRODUCTION
Changes in intracellular calcium regulate many cellular events including
synaptic transmission, cell division, survival, and differentiation. Voltage-gated
21
calcium channels are an important route of calcium entry and are essential for
converting electrical activity into biochemical events in excitable cells (Catterall et al.,
2005). Among the ten different types of neuronal voltage gated calcium channels, L-
type channels (LTC), encoded by the Cav1.2 and Cav1.3 pore forming subunits are
particularly effective at inducing changes in gene expression that underlie plasticity
and adaptive neuronal responses (Bading et al., 1993). Calcium influx through LTCs
activates transcription factors such as CREB, MEF, and NFAT (Graef et al., 1999;
Mao et al., 1999; Sheng et al., 1990) that lead to the expression of genes such as c-fos
and BDNF (Morgan and Curran, 1986; Murphy et al., 1991; Zafra et al., 1990). Two
mechanisms link LTCs, particularly CaV1.2, to the activation of transcription factors
such as CREB. Calcium entering through the channels can diffuse to the nucleus and
activate nuclear calcium-dependent enzymes, such as CaMKIV, that regulate the
activity of transcription factors and co-regulators (Hardingham et al., 2001). In
addition, calcium entering cells through LTCs can activate calcium-dependent
signaling proteins around the mouth of the channel which propagate the signal to the
nucleus (Deisseroth et al., 1998; Dolmetsch et al., 2001).
In this study we have identified a new mechanism by which calcium channels
control gene expression. We report that neurons produce a C-terminal fragment of
CaV1.2 that can regulate transcription and which we call the calcium channel
associated transcriptional regulator or CCAT. CCAT is located in the nucleus of
many inhibitory neurons in the developing and adult brain, and its production and
nuclear localization are regulated developmentally. In addition, calcium influx
through LTCs and NMDA receptors causes CCAT export from the nucleus. In the
22
nucleus, CCAT interacts with the transcriptional regulator p54(nrb)/NonO and can
activate transcription of both reporter and endogenous genes. Using microarrays and
real-time PCR, we show that CCAT affects the transcription of a many neuronal genes
including a gap junction, an NMDA receptor subunit, and the sodium calcium
exchanger. CCAT binds to the enhancer of the Connexin 31.1 gene (Cx31.1) and
directly regulates both the expression of a Cx 31.1 reporter gene and the expression of
the endogenous gene. Finally, we show that CCAT expression can cause an increase
in neurite extension in primary neurons. This is the first example of a calcium channel
having a dual function as an ion pore and a transcription factor.
RESULTS
CCAT Is Found in the Nucleus of Neurons in the Brain
Experiments in neurons and cardiac myocytes have suggested that the C-
terminus of CaV1.2 is proteolytically cleaved, yielding a truncated channel and a
cytoplasmic C-terminal fragment (De Jongh et al., 1994; Gerhardstein et al., 2000).
To investigate the function of the C-terminal fragment we developed an antibody to a
fourteen-amino acid peptide in the C-terminus of CaV1.2 (a.a. 2106-2120) and used it
to probe HEK 293T cells expressing CaV1.2. The C-terminal antibody (anti-CCAT)
recognizes both the intact channel and a short cleavage product that corresponds to the
C-terminal fragment. In contrast, an antibody recognizing an epitope in the II-III
23
cytoplasmic loop of CaV1.2 (anti-II-III loop) detects full-length and C-terminally
truncated channels only (Figure 1A).
To determine where CCAT is localized in cells in the brain, we purified
nuclear, cytoplasmic and membrane fractions of postnatal day 7 (P7) rat brain cortex
and used western blotting to probe them with the anti-CCAT antibody (Figure 1B).
Surprisingly, we found that the nuclear extracts contained high levels of CCAT
suggesting that the C-terminus of CaV1.2 is localized in the nucleus of cells in the
brain. In contrast the N–terminal portion of the channel was localized in the membrane
and cytoplasmic fractions as expected for an ion channel. To provide further
evidence that CCAT is indeed nuclear in neurons or glial cells, we examined its
localization by immunostaining primary cortical cultures. The anti-CCAT antibody
stained the cell body and dendrites of neurons weakly (Figure 1D), suggesting that the
anti-CCAT antibody recognizes some intact CaV1.2 channels. Importantly, however,
a significant number of neurons (10 ± 5%) exhibited very strong nuclear CCAT
staining (Figure 1C). In contrast, the II-III loop antibody stained the cell bodies and
dendrites of neurons but was excluded from the nucleus, suggesting that the full-length
channel is not nuclear (Figure 1E).
To investigate which types of neurons have nuclear CCAT, we co-stained
neurons with anti-CCAT and with antibodies that stain precursor cells (nestin), glial
cells (GFAP), excitatory neurons (NR2A) or inhibitory neurons (GAD65) in the
cortex. We found that cells that have strong nuclear CCAT also expressed glutamic
acid decarboxylase (GAD65), suggesting that CCAT is strongly nuclear in inhibitory
neurons that produce GABA (Figure 1F). To determine if CCAT is also in the nucleus
24
of neurons in vivo, we used the anti-CCAT antibody to stain P30 rat brain sections. A
subset of cells in the thalamus (data not shown), inferior colliculus (Figure 1G),
inferior olivary nucleus (Figure 1H), and in the olfactory bulb (Figure 1I) displayed
prominent nuclear CCAT staining. In the cortex and the hippocampus, CCAT was
nuclear in a small number of neurons, consistent with its localization in a subset of
GAD65 positive neurons in cortical cultures (data not shown). Taken together, these
experiments indicate that CCAT is localized in the nucleus of inhibitory neurons, in
culture and in restricted regions of the brain in vivo.
To provide further evidence that CCAT can translocate to the nucleus, we
fused yellow fluorescent protein (YFP) to the C-terminus of full-length CaV1.2
(CaV1.2-YFP). We observed cytoplasmic and nuclear fluorescence when CaV1.2-YFP
was expressed in neurons (Figure 2A), cardiac myocytes (data not shown), or
Neuro2A glioblastoma cells (Figure S2). In contrast, in neurons expressing CaV1.2
tagged at its N-terminus with YFP, the channel was localized in the membrane and in
the endoplasmic reticulum (Figure 2B). We did not observe nuclear fluorescence in
HEK 293T cells expressing CaV1.2-YFP, consistent with previous reports that in HEK
293T cells the C-terminus of CaV1.2 remains associated with the plasma membrane
following cleavage (Gao et al., 2001; Gerhardstein et al., 2000; Hulme et al., 2005).
However, a fusion of YFP and the last 503 amino acids of CaV1.2 was nuclear and
formed distinct nuclear punctae in neurons, myocytes and HEK 293T cells (c503
Figure 2C, E). Interestingly, this punctate pattern did not seem to be the result of
overexpression, as it was also observed in some neurons by confocal imaging of
endogenous CCAT staining (Figure 2D) and it was enhanced by incubation in low
25
calcium media (Figure 3B). These experiments provide further evidence that CCAT is
indeed nuclear, and suggest that formation of punctae by endogenous CCAT is
modulated by signaling events in the cell.
Nuclear CCAT does not contain a canonical nuclear localization sequence
suggesting that it enters the nucleus via an alternative pathway, perhaps as has been
described for Stat1 protein where nuclear import is mediated by direct interaction with
nucleoporins (Marg et al., 2004). To identify the regions of CCAT that are necessary
for its nuclear localization, we made truncations of the 503-YFP protein and
introduced them in HEK 293T cells. Deletion of the carboxyl end of CCAT and of
amino acids 1642-1814 of CaV1.2 (c330) had little effect on the protein’s localization.
In contrast, deletion of amino acids 1814-1864 (c280) decreased nuclear retention and
abolished punctae formation (Figure 2E and F). Comparison of the CaV1.2 sequence
from other vertebrates indicates that this nuclear retention domain is conserved
evolutionarily (Figure S1A) suggesting that it plays an important role in the function
of CaV1.2 and CCAT proteins.
Endogenous CCAT is predicted to be a 75 kD protein; therefore, nuclear
translocation of CCAT is likely to involve an active process rather than passive
diffusion across nuclear pores. To estimate the rate of CCAT import into the nucleus,
we used fluorescence recovery after photobleaching (FRAP) and time-lapse
microscopy of Neuro2A cells expressing CaV1.2-YFP. After photobleaching of
nuclear CCAT, nuclear fluorescence recovered over the course of 300 seconds with a
single exponential time course (t=48 +/-16 sec; n=11), while cytoplasmic fluorescence
declined over the same time period (Figure 2G-H). In control cells expressing YFP
26
alone, we observed an almost instantaneous recovery of nuclear fluorescence after
photobleaching concomitant with a decrease in cytoplasmic fluorescence, consistent
with the observation that YFP diffuses rapidly through nuclear pores. The slow rate of
recovery of CCAT-YFP nuclear fluorescence suggests that this protein is actively
imported into the nucleus at a rate similar to that of NFAT, another transcription factor
that translocates to the nucleus (Shibasaki et al., 1996). Measurements of CCAT
export by bleaching cytoplasmic fluorescence indicate that CCAT returns to the
cytoplasm with a time course of approximately 400 seconds (t=62 +/-21 sec; n=5)
(data not shown). These results are consistent with the idea that CCAT is
constitutively transported into the nucleus, and that CCAT shuttles between the
cytoplasm and the nucleus of unstimulated cells.
The Concentration of Nuclear CCAT Is Regulated by Intracellular Calcium
To determine whether the nuclear localization of CCAT is regulated by
changes in intracellular calcium, we assessed the distribution of CCAT by
immunocytochemistry in cortical neurons following treatment with agents that affect
intracellular calcium levels. Decreasing free extracellular calcium using 2.5mM
EGTA caused a robust increase in nuclear CCAT fluorescence (Figure 3A, C), and
caused CCAT to aggregate into punctae in the nucleus of many neurons (Figure 3B).
Conversely, treatment with 65mM KCl, which mimics tonic electrical activity by
increasing the activity of VGCCs, and treatment with 100µM glutamate caused a
significant decrease in the nuclear fluorescence (Figure 3A and 3C). The decrease in
27
nuclear CCAT could be reliably detected after five minutes and reached a maximum
after 30 minutes of stimulation with either depolarization or glutamate, although
depolarization had a more pronounced effect at earlier time points (Figure 3D). The
nuclear fluorescence of Neuro2A cells expressing the CaV1.2-YFP also declined with
tonic depolarization, providing further evidence that electrical activity leads to a net
decrease of CCAT from the nucleus (Figure S2A-B). The decrease in nuclear CCAT
triggered by depolarization was blocked by removing extracellular calcium or by
treating cells with the CaV1.2 blocker nimodipine. Application of NMDA receptor
blocker MK-801 partially blocked the activity-induced decrease in nuclear CCAT but
treatment with the AMPA receptor inhibitor NBQX had no effect (Figure 3E),
suggesting that NMDA but not AMPA receptors can also influence the export of
CCAT from the nucleus of cortical neurons.
The decrease in nuclear CCAT observed in response to high intracellular
calcium could be due to a net export from the nucleus or to selective degradation of
CCAT in the nucleus. To determine if CCAT is degraded following a rise in
intracellular calcium, we measured total CCAT immunoreactivity before and after
depolarization. We found that depolarization had no effect on the total CCAT staining
in neurons, or on the levels of CCAT-YFP expressed in Neuro2A cells (Figure 3F).
Furthermore, addition of the proteosome inhibitor lactacystin failed to block the
depolarization-induced decrease in nuclear CCAT (Figure 3G). The lack of a decrease
in total CCAT levels in depolarized neurons and Neuro2A cells and the lack of effect
of lactacystin on CCAT nuclear localization argue that the decrease in CCAT
following depolarization is not due to protein degradation.
28
Nuclear CCAT Is Regulated Developmentally
The levels of nuclear CCAT vary considerably among neurons in the
developing brain (Figure 1G-I). Since neurons in the central nervous system
differentiate at different rates, we considered whether the levels of CCAT in the
nucleus could be regulated developmentally. To investigate this possibility, we
assessed the levels of nuclear and total CCAT found in brains taken from embryonic
day eighteen (E18), postnatal day one (P1), three-week old (P21), and adult rats. The
levels of CCAT immunoreactivity in the nuclear fractions increased substantially with
age (Figure 3H; middle panel), whereas the amount of CCAT-containing channel at
the membrane appeared to decrease (Figure 3H; upper panel). This is consistent with
increasing cleavage of CaV1.2 during development. The total levels of CaV1.2, as
determined by immunoreactivity of the CaV1.2 internal loop antibody, were also
regulated developmentally. CaV1.2 levels were low at E18 and increased through P8
before declining in P21 and adult brains (Figure 3I; upper panel). Interestingly, early
in development a long and a short form of CaV1.2 could be detected whereas only the
short form of the channel and a new, 150 kD band were observed in both p21 and
adult brains, suggesting that there is increasing cleavage and possibly different
cleavage events in older brains. Together, these results indicate that the levels of
CCAT in the nucleus, the cleavage of CaV1.2, and the levels of CaV1.2 are regulated
independently to yield a complex pattern of channel and transcription factor
expression.
29
CCAT Binds to a Nuclear Protein
To get an indication of CCAT’s function, we looked for proteins that interact
with CCAT in the nucleus. We expressed CCAT or a mutant form lacking the nuclear
localization domain in Neuro2A cells, immunoprecipitated them via epitope tags, and
identified interacting proteins by mass spectrometry. One of the proteins that co-
immunoprecipitated with full length CCAT was p54(nrb)/NonO, a nuclear protein that
plays a role in regulating transcription downstream of the neuronal Wiscott Aldrich
Protein (Wu et al., 2006), the retinoic acid receptor, and the thyroid hormone receptor
(Mathur et al., 2001). We verified the interaction of p54 (nrb)/NonO with CCAT by
co-immunoprecipitation followed by Western blotting against endogenous p54
(nrb)/NonO (Figure 4A). These results indicate that CCAT is associated with a
nuclear protein that participates in transcriptional regulation and regulates mRNA
splicing, and suggest a role for the C-terminus of CaV1.2 in the nucleus.
CCAT Activates Transcription
Based on its nuclear localization and its binding to p54 (nrb)/NonO, we
hypothesized that CCAT might regulate transcription. To investigate whether CCAT
can activate transcription when recruited to a promoter by a heterologous DNA
binding domain, we made a C-terminal fusion of the intact channel and the Gal4 DNA
30
binding domain from yeast (CaV1.2-Gal4, Figure 4B). The Gal4 DNA binding
domain recognizes the UAS DNA sequence but requires a transcriptional activation
domain to activate transcription. We introduced CaV1.2-Gal4 into neuro2A cells
along with a UAS luciferase reporter gene and measured luciferase expression. We
found that CaV1.2-Gal4 activated transcription approximately 80 times better than
Gal4 alone or than the channel lacking the Gal4 DNA binding domain (Figure 4C).
These results suggest that the C-terminus of CaV1.2 is produced as a soluble protein in
cells, that it translocates to the nucleus, and that it activates transcription when
recruited to a heterologous gene.
To identify the domains of CaV1.2 that are required for transcriptional
activation, we made a family of proteins containing fragments of the C-terminus of
CaV1.2 fused to Gal4 and tested them in primary neurons for their ability to activate
the expression of a UAS luciferase reporter gene. A fragment containing 503 amino
acids of the CaV1.2 C-terminus fused to Gal4 activated transcription almost as well as
a CREB-Gal4 fusion protein, and about 130 times better than the Gal4-DNA binding
domain alone (Figure 4D). Deleting 170 amino acids from the N-terminus of this C-
terminal CaV1.2 fragment (c330-Gal4) reduced but did not completely abolish the
ability of the C-terminus to activate transcription. In contrast deletion of a second
domain consisting of the most C-terminal 133 amino acids (c503∆133-Gal4)
completely eliminated the ability of CCAT to activate transcription (Figure 4D).
Deletion of these final 133 amino acids in the full length CaV1.2-Gal4 also produced a
channel unable to activate transcription (Figure 4C; bar 4) suggesting that this domain
is required for transcriptional regulation by the intact channel. These experiments
31
suggest that CCAT has two domains that are necessary to activate transcription: an N-
terminal domain that modulates transcriptional activation, and a C-terminal domain
that is essential for transcription (red and blue boxes in Figure 4B). Significantly, both
transactivation domains are highly conserved in vertebrates (Figure S1B and D), and
the N-terminal transactivation domain has 42% similarity and 27% identity to a
conserved transactivation domain of the transcription factor GATA4, suggesting that it
has a bona fide role in transcriptional regulation (Figure S1C).
Because recruiting proteins to DNA via Gal-4 DNA binding domains can
produce ectopic transcriptional regulators, we also fused various other calcium
channel C-terminal domains to Gal4 and expressed these with the UAS reporter gene.
We found that the C-termini of CaV1.3 and CaV2.1 when fused to Gal4 had no effect
on transcription, suggesting that CaV1.2’s C-terminal domain is specific in its ability
to activate transcription in neurons (Figure 4E).
In earlier experiments we observed that the amount of CCAT in the nucleus
decreased in response to tonic electrical activity. To determine whether this activity-
induced decrease in nuclear CCAT has functional relevance, we depolarized cells
expressing CaV1.2-Gal4 and measured activation of the UAS luciferase reporter
(Figure 4F). Prolonged depolarization led to a 30% decline in transcription from the
reporter gene, and removing extracellular calcium blocked this effect. These results
provide evidence that the nuclear localization of CCAT is important for its activation
of transcription and are consistent with the observation that nuclear CCAT
concentration is regulated by electrical activity.
32
CCAT Regulates Transcription of Endogenous Genes
To determine whether CCAT regulates transcription of endogenous genes, we
used oligonucleotide microarrays to identify mRNAs that are transcriptionally
regulated by CCAT over-expression. We built two plasmids that encode either full
length CCAT or a CCAT∆TA that lacks the N-terminal transcriptional activation
domain. Both plasmids also contain a GFP gene driven by a separate promoter that
was used to identify transfected cells. We introduced these plasmids into Neuro2A
cells and used fluorescence activated cell sorting (FACS) to select transfected cells.
We then compared the mRNA expression profile of cells expressing full-length CCAT
to cells expressing either CCAT∆TA or GFP alone, using Agilent mouse whole
genome arrays. In three independent experiments, we found 23 mRNAs that were up-
regulated more than two fold (p<0.005) in cells expressing CCAT relative to cells
expressing CCAT∆TA, and 22 genes that were down-regulated more than two fold by
CCAT relative to CCAT∆TA (Table S1). Because we subsequently discovered the
CCAT∆TA still activates transcription albeit at a much lower level than full length
CCAT (see Figure 4D), we also compared mRNA expression profiles of cells
expressing CCAT and GFP to cells expressing GFP alone. In three additional
experiments, we found 66 mRNAs up-regulated more than 1.8 fold (p<0.005) in cells
expressing CCAT relative to those expressing GFP (Figure S1 and Table S2). The
genes that were up-regulated by CCAT include the genes for the gap junction protein
Connexin 31.1 (Cx31.1), the axon guidance factor Netrin4, the regulator of G protein
signaling RGS5, the tight junction protein claudin19 and a broad array of other genes
33
(Figure 5A). Approximately 206 genes were repressed more than 0.55 fold (p<0.005)
by CCAT, including the sodium calcium exchanger, the cation channel TRPV4, the
potassium channel Kcnn3, and the transcription factor GATA6 (Figure 5A, Figure S3
and Table S3; Raw data available at http://ncbi.nlm.nih.gov/geo; account:
Dolmetsch_rev; password: reviewer; series #: GSE4180). Combining the results from
all six of our micro-array experiments (CCAT vs. CCAT∆TA and CCAT vs. GFP)
revealed that 16 mRNAs were significantly up-regulated (Table S4) and 31 genes
were significantly down-regulated by CCAT. These results suggest that CCAT can
both increase and decrease the expression of a wide set of genes that regulate neuronal
differentiation, connectivity, and function.
To verify the results of the microarray experiments, we measured changes in
mRNA expression due to CCAT expression using RT-PCR (Figure 5B). CCAT
changed the expression of all seven mRNAs tested, in accordance with the results
from the array experiments. As normalizing controls we used β-actin and GAPDH,
which showed no detectable change in response to overexpression of CCAT. These
data provide independent evidence that CCAT regulates expression of endogenous
genes, some of which are important for the function of excitable cells.
CCAT Binds and Regulates the Promoter of Cx31.1
The microarray and RT-PCR experiments suggested that connexin 31.1
(Cx31.1) was strongly regulated by CCAT in cells. To study the regulation of Cx31.1
by CCAT in more detail, we constructed a reporter gene consisting of the 2 Kb
34
promoter/enhancer region of Cx31.1 in front of the firefly luciferase coding sequence.
We introduced this Cx31.1 luciferase reporter gene into neurons along with either the
full length CCAT or CCAT∆TA, a version of CCAT lacking the C-terminal
transcriptional activation domain. Full length CCAT increased the expression of the
Cx31.1 reporter by 3.4 ± 0.4 fold (n=12) relative to a control vector or to CCAT∆TA
(Figure 5C) providing additional evidence that CCAT regulates the expression of
Cx31.1.
CCAT could affect the transcription of Cx31.1 either by regulating the
transcriptional machinery in the nucleus directly or by modifying signaling proteins in
the cytoplasm of cells that lead to changes in transcription. To determine if CCAT
acts in the nucleus, we fused CCAT to the ligand binding domain of a modified
estrogen receptor (ER) that binds 4-hydroxytamoxifen (4OHT) but not endogenous
estrogen (Littlewood et al., 1995). When expressed in Neuro2A cells, ER-CCAT is
largely excluded from the nucleus but brief treatment with 4OHT causes ER-CCAT to
move into the nucleus (Figure 5D). Treatment of cells expressing ER-CCAT with
4OHT caused a fifty-fold increase in the transcription of Cx31.1 relative to untreated
cells (Figure 5E). 4OHT had no effect on cells expressing ER alone, and caused a
much smaller effect in cells expressing ER-CCAT∆TA. These results provide
compelling evidence that CCAT regulates the transcription of Cx31.1 when it is in the
nucleus of cells.
To identify regions of the Cx31.1 promoter that are important for its regulation
by CCAT, we made a series of deletions of the Cx31.1 promoter and placed them
upstream of the firefly luciferase gene (Figure 5F). We introduced this library of
35
deletion mutants of the Cx31.1 promoter into Neuro2A cells along with full length
CCAT and measured luciferase activity in these cells. CCAT regulation of the Cx31.1
promoter was critically dependent on 148 base pairs at the 3’ end of the Cx31.1
promoter. Deletion of this domain eliminated the ability of CCAT to activate
transcription of the Cx31.1 reporter gene, and this domain alone was sufficient to
confer CCAT regulation on to a reporter gene (Figure 5F). Together, this data
suggests that CCAT regulates the expression of Cx31.1 in a sequence-specific manner,
and that the CCAT recognition element lies in the final 148 base pairs of the Cx31.1
promoter sequence.
In the nucleus, CCAT could affect transcription directly by binding to a
complex of proteins on the promoter of genes, or indirectly by binding to other
proteins in the transcriptional activation pathway. We used chromatin
immunoprecipitation (ChIP) to determine whether CCAT binds to the promoter of Cx
31.1 directly. We introduced an epitope-tagged CCAT into cells, crosslinked the
protein to the DNA and immunoprecipitated CCAT from these cells, and used PCR to
determine if any region in the promoter of the Cx 31.1 gene was co-
immunoprecipitated by CCAT. We found that CCAT could reproducibly
immunoprecipitate a fragment of the endogenous Cx31.1 promoter approximately 1
Kb upstream of the transcriptional start site but not other regions, suggesting that the
CCAT is bound close to this region of the Cx 31.1 promoter (Figure 5G). These
results suggest that CCAT regulates transcription by binding, either directly or through
protein-protein interactions, to the promoter of Cx31.1, providing further evidence that
CCAT is a transcriptional regulator.
36
Endogenous CaV1.2 and CCAT Regulate Transcription of Cx31.1
We have provided evidence that exogenous expression of CaV1.2 leads to the
production of CCAT, which in turn affects transcription. To determine whether
endogenous CaV1.2 regulates transcription by generating CCAT, we asked whether
reducing the levels of endogenous CCAT in the nucleus by depolarization had an
effect on expression of the Cx31.1 reporter gene or of the endogenous Cx 31.1 gene.
Depolarization of cortical neurons reduced activation of the Cx31.1 reporter gene by
2.12 ± 0.12 fold (Figure 6A) and caused a 2.4 fold decrease in the expression of the
Cx31.1 mRNA levels as measured by RT-PCR (Figure 6B). The effects of
depolarization on Cx31.1 mRNA levels were also apparent in Neuro2As and in
cultured thalamic neurons suggesting that CCAT regulates the expression of Cx 31.1
in multiple cell types (Figure 6B). These results support the conclusion that CCAT-
dependent transcription of the Cx31.1 gene requires nuclear localization of CCAT.
Because CCAT is derived from CaV1.2, we also asked whether Cx31.1
expression depends on the expression of endogenous CaV1.2. We designed several
short hairpin RNAs (shRNAs) and asked whether introducing these shRNAs into
neurons reduced the expression of Cx31.1. Two shRNAs targeting the rat CaV1.2
(RCav1.2 sh6410 and RCaV1.2 sh6500) reduced the expression of rat CaV1.2
expressed in Neuro2A cells, whereas an shRNA targeting the mouse CaV1.2 sequence
had no effect on the expression of the rat channel (Figure 6C; lanes 1-3). The shRNAs
37
targeting the rat CaV1.2 also reduced CaV1.2-dependent signaling to CREB in rat
cortical neurons, suggesting that these shRNAs reduce the expression of endogenous
CaV1.2 and prevent activation of CREB-dependent transcription (Bading et al., 1993;
Dolmetsch et al., 2001; Murphy et al., 1991) (Figure S4A). We next introduced the
shRNAs targeting the rat CaV1.2 into cortical neurons and measured the activation of
the Cx31.1 reporter. Both rat shRNAs decreased the expression of Cx31.1 by
approximately six-fold, indicating that CaV1.2 regulates the expression of Cx31.1 in
neurons. (Figure 6D). CaV1.2 knockdown had no effect on Renilla luciferase
expression from the control vector, suggesting that the decrease in Cx31.1 reporter
activity was not due to decreased viability. To assess whether the effect of the
shRNAs targeting CaV1.2 on the transcription of Cx31.1 was the result of the loss of
calcium influx through the channel, we tested whether L-type calcium channel
blockers affected Cx31.1 transcription. Twenty-four hour (24h) treatment of neurons
with 10µM nimodipine had no effect on the expression of Cx31.1 in the presence or
absence of CCAT, suggesting that Cx31.1 is not regulated by calcium influx through
CaV1.2 in unstimulated cells (Figure S4B). To determine if the inhibitory effects of
CaV1.2 shRNAs on the Cx31.1 promoter are due to reduction of CCAT, we
constructed a version of CCAT that is insensitive to the rat CaV1.2 shRNA (CCAT*;
Figure 6E) and expressed it in cells along with the shRNA targeting rat CaV1.2.
Expression of CCAT* rescued the effect of knocking down the endogenous CaV1.2 on
the expression of the Cx 31.1 gene (Figure 6F, n=6). In contrast, CCATDTA* that
lacked the transcriptional activation domain did not rescue the effects of the CaV1.2
shRNA on Cx 31.1 expression. This suggests that CCAT alone can restore expression
38
of Cx 31.1 in cells in which CaV1.2 has been reduced by an shRNA, and that this
effect depends on the transcriptional activation domain of CCAT. We also made a
version of CaV1.2 that is insensitive to the rat CaV1.2 shRNA (CaV1.2*) (Figure 6G)
and asked whether this channel can rescue Cx31.1 expression in cells lacking
endogenous CaV1.2 (Figure 6H). Expression of CaV1.2* in neurons partially rescued
the effect of the CaV1.2 shRNA on Cx31.1 expression while a form of CaV1.2* that
lacks the C-terminal transcriptional activation domain did not restore the effects of
CaV1.2 knockdown on Cx31.1 expression. Together these results support the
conclusion that endogenous CaV1.2 modulates transcription of the Cx31.1 gene, and
that this transcriptional regulation depends on the production of CCAT from the C-
terminus of CaV1.2.
CCAT Expression Promotes Neurite Growth
Our microarray and RT PCR experiments suggested that CCAT regulates the
transcription of a number of genes important in neuronal function and excitability. To
explore the cell biological functions of CCAT, we measured the effect of expressing
CCAT on the morphology and survival of cerebellar granule neurons. We selected
these cells because they are a largely homogenous population of neurons that have low
basal levels of CCAT and that have well-characterized survival and dendritic
arborization patterns. Expression of CCAT or CCAT∆TA did not significantly affect
granule cell survival, but it did cause a dramatic change in the length of neurites
(Figure 7A and B). Full-length CCAT doubled the average length of neurites to 10
39
µm (Figure 7C; bottom panel and 7D) whereas the CCAT∆TA decreased the average
length of neurites to approximately 2.7 um (Figure 7C; top panel and 7D). There was
also a small but statistically significant effect of CCAT on the number of neurites,
suggesting that under some circumstances CCAT could affect the growth and
formation of new dendrites (Figure 7E). Interestingly, expressing CCAT in other cell
types such as Neuro2As also caused a change in the morphology of the cells, causing
an increase in the production of filopodial extensions (data not shown). This data
suggests that CCAT-dependent transcription can lead to rearrangement of the
cytoskeleton and may contribute to changes in the connectivity of neurons during
development.
DISCUSSION
Neurons and myocytes generate characteristic patterns of electrical activity and
intracellular calcium that are essential for cell function. The reliability of the calcium
signal requires a delicate balance of proteins that import and export calcium from the
cytoplasm – proteins whose individual expression is regulated independently in
response to cellular function. The expression of voltage gated calcium channels is
closely coordinated with the expression of other ion channels, pumps and signaling
proteins that regulate membrane excitability and calcium homeostasis. In this paper
we describe a novel mechanism by which cells coordinate the expression of voltage
gated calcium channels with the expression of other molecules. LTCs generate a
transcription factor that integrates information both about the number of calcium
40
channels and the electrical activity of a cell. CCAT is generated from the L-type
channel, and its nuclear localization is negatively regulated by the electrical activity of
the cell, it is therefore in a privileged position to integrate information about the
number of channels with information about the calcium history of a cell.
Several laboratories have reported that LTCs are cleaved at their C-terminus,
and the site of cleavage of Cav1.1, the homologous LTC in skeletal muscle, was
recently identified (Hulme et al., 2005). The cleaved channel carries more calcium,
so channel cleavage could have profound effects on the electrical properties of a
neuron by changing the properties of the LTC. The proteolytically processed C-
terminal domain is also thought to bind to truncated channels, where it exerts an
inhibitory effect on channel function (Hulme et al., 2006b). This hypothesis does not
preclude the idea that the C-terminus of CaV1.2 also acts as a transcription factor. By
analogy with the potassium channel-binding protein KChip/DREAM, which is also a
calcium-sensitive transcriptional repressor, we propose that CCAT both regulates
transcription and reduces calcium influx through CaV1.2 (An et al., 2000; Carrion et
al., 1999). This hypothesis is appealing in light of the observation that CCAT is
exported from the nucleus by elevations in intracellular calcium, suggesting that under
conditions of tonically elevated calcium, CCAT would both alter the transcription of
specific genes and inhibit the activity of CaV1.2. Thus CCAT may be an important
part of a negative feedback pathway regulating both gene expression and calcium
influx in the neurons.
In addition to CaV1.2, it has also been reported that CaV1.3 (Hell et al., 1993),
CaV2.1 (Kubodera et al., 2003), and CaV2.2 (Westenbroek et al., 1992) are cleaved in
41
neurons. In the case of CaV2.1, the cleavage product is also approximately 75 kD and
has been localized to the nucleus of Purkinje neurons in the cerebellum (Kordasiewicz
et al., 2006). This suggests that C-terminal cleavage is a general feature of CaV
channels and that other members of this family may also be transcriptional regulators.
In our studies we did not find that the C-terminal domains of CaV1.3 or CaV2.1
activated transcription in cortical neurons, but it is possible that the C-terminal
domains of other channels may act in other types of neurons or may be transcriptional
repressors or regulators of chromatin structure. This would be consistent with our
finding that in addition to activating transcription CCAT also represses the
transcription of many genes.
Despite more than a decade of experiments, the stimuli and mechanisms that
lead to cleavage of CaV1.2 remain enigmatic. It has been reported that cleavage of
CaV1.2 is triggered by NMDA stimulation in hippocampal slices (Hell et al., 1996),
and CaV1.2 cleavage has also been reported to occur in response to sex hormone
stimulation of uterine muscle (Helguera et al., 2002). We did not observe any obvious
increase in CCAT following stimulation of neurons in culture with NMDA or
potassium chloride, however it is possible that CaV1.2 cleavage only occurs in the
context of hippocampal slices. In cortical neurons, cerebellar granule cells, cardiac
myocytes, Neuro2A cells and PC12 cells exogenous CaV1.2 appears to be cleaved
constitutively to yield nuclear and cytoplasmic CCAT. While the production of
CCAT did not appear to be regulated, its nuclear localization and its transcriptional
effects on the Cx31.1 gene were strongly regulated by changes in cytoplasmic
calcium. Therefore, we favor the idea that CCAT is produced in proportion to the
42
number of CaV1.2 channels in cells and that cytoplasmic calcium levels regulate its
nuclear localization and transcriptional activity. In addition to being regulated by
calcium, nuclear CCAT levels were also regulated in a cell-specific manner and its
appearance in brain nuclear fractions increased substantially over the course of
postnatal development. In cultured neurons, CCAT levels were highest in GABAergic
inhibitory neurons, while in brain slices CCAT staining was particularly strong in the
inferior colliculus, inferior olive and thalamus. These data suggest that CCAT may
play an important role in the development of neurons and in regulation of neuronal
properties in specific cell types.
Our studies have identified many interesting genes regulated by CCAT, and
these genes offer clues to understanding CCAT’s physiologic function. CCAT
regulates the expression of several gap junction proteins, a glutamate receptor, several
potassium channels, a sodium-calcium exchanger and of signaling proteins such as
RGS5, Formin and Nitric Oxide Synthase. One of the main targets of CCAT in the
nucleus is the gap junction protein Cx31.1. Cx31.1 is expressed in the retina
(Guldenagel et al., 2000), in developing embryos (Davies et al., 1996), and in
GABAergic striatal output neurons of the thalamus (Venance et al., 2004). Our array
and RT PCR studies suggest that Cx31.1 is also well expressed in neuroblastoma cells
and in thalamic neurons. Transcription of the Cx31.1 gene correlates well with the
amount of endogenous CCAT in the nucleus and depolarization, which reduces the
amount of nuclear CCAT, also decreases the amount of Cx 31.1 transcript suggesting
that these two are correlated. Finally CCAT binds to the promoter of Cx 31.1
providing compelling evidence that CCAT is a regulator of Cx 31.1 expression in
43
neurons. Connexins play a key role in forming electrical connections between
developing neurons and form conduits for signaling molecules that can regulate a
developing tissue. The expression of Cx 31.1 during development in response to
changes in CCAT could thus play an important role in regulating the electrical
coupling of neurons and the overall excitability of the brain.
We have found that CCAT expression in neurons increases dendritic length.
This effect is blocked by CCAT lacking a transcriptional activation domain. There are
many possible mechanisms for this effect of CCAT on neuronal morphology. The
observation that CCAT up-regulates Cx31.1, formin, claudin 19, procolagen type XI
and an α-catenin-like protein suggests that it might promote the formation of adhesion
complexes or junctional contacts between neurons and the extracellular matrix.
Alternatively, since CCAT increases the production of Netrin4 and of two chemokines
that regulate axonal and dendritic growth, it could lead to increases in neurite length
via these mechanisms (Adler and Rogers, 2005; Barallobre et al., 2005). Finally, by
down-regulating a potassium channel and a sodium calcium exchanger, CCAT could
increase the excitability of neurons and thus regulate their morphology indirectly.
Understanding how CCAT modulates dendritic length might help uncover the
mechanisms by which L-type calcium channels regulate neuronal morphology.
We provide strong evidence that CaV1.2 encodes a transcription factor that can
regulate expression of a variety of genes that are important for the function of neurons
and muscle cells. This finding reveals an entirely unsuspected function for a well-
characterized calcium channel that plays an essential role in electrical tissues. This
44
new function of CaV1.2 will be a rich area for future study in ion channel physiology
and neurobiology.
45
EXPERIMENTAL PROCEDURES
Materials
Nimodipine, MK-801, NBQX and 4OHT were purchased from Sigma. Lactacystin
was from Calbiochem and L-glutamate was from Tocris Bioscience.
Anti-CCAT was used 1:1000 for western blots. Anti-CaV1.2 II-III loop (1:1000) was
purchased from Chemicon or BD Biosciences, anti-CREB (1:1000) was from Upstate
Biotechnologies, anti-p54nrb/NonO (1:1000) and anti-DsRed (1:400) from BD
Biosciences, anti-b-actin (1:2000) and anti-GAPDH (1:2000) were from Ambion, anti-
gal4 (1:500) and anti-GST (1:500) were from Santa Cruz Biotechnology. Anti-flag M2
(1:1000) was purchased from Sigma.
Cell culture and transfection
HEK 293T cells, Neuro2A and PC12 cells were cultured in Dulbecco’s Minimal
Essential Media (DMEM) containing 10% fetal bovine serum (FBS; 15% for PC12s),
penicillin, streptomycin (P/S) and L-glutamine (LQ). Cortical neurons were
dissociated from E17-19 Sprague Dawley rats as described (Xia et al., 1996) and
maintained for 6 to14 days in culture in Basal Medium Eagle with 5% FBS, P/S, LQ
and 1% glucose or in Neurobasal medium containing B27 supplement (Invitrogen).
Cardiac myocytes were cultured from P0-P1 rats using a neonatal myocyte isolation
kit (Cellutron Life Technology) and maintained in DMEM with 10% FBS, P/S, LQ
and 0.1mM BRDU for 3 to 4 days. Cerebellar granule cells were cultured from P5
46
Sprague Dawley rats and grown as described elsewhere (Dudek et al., 1997). For
description of thalamic neuron cultures see Supplemental experimental procedures.
HEK 293T (24h) cells, cortical and granule neurons (72h) were transfected using a
standard calcium phosphate method at a concentration of 2 µg of DNA/106 cells.
Neuro2As (24h), cortical neurons (96h), cardiac myocytes (24h) and PC12s (24h)
were transfected using lipofectamine 2000 according to manufacturer’s instructions.
For luciferase reporter gene experiments and see Supplemental experimental
procedures.
Thalamic Neuron cultures
Thalamic neurons were dissected from E17-19 Sprague Dawley rats in ice-cold Hank's
Balanced Salt Solution without Ca++ and Mg++ (HBSS, Gibco). Thalami were
enzymatically digested using trypsin (Worthington, 10mg/ml), DNase (Sigma, 200
U/ml) in HBSS at room temperature for 5 min. Thalami were washed 3x in Basal
Medium Eagle with 5% FBS, P/S, LQ and 1% glucose and gently triturated in the
same media. Neurons were plated at 25,000/cm2.
Plasmid construction
Construction of the dihydropyridine resistant (DHP- CaV1.2) in the pcDNA4/HisMax
vector has been previously described (Dolmetsch et al., 2001). CaV1.2-YFP and
CaV1.2-Gal4 fusion proteins were constructed by the insertion of the YFP and Gal4
DBD coding sequences into the AfeI/ NotI sites in DHP- CaV1.2. The plasmid
encoding the N-terminal tagged YFP-CaV1.2 was generated using Gateway
47
technology (Invitrogen) by first cloning the CaV1.2 coding sequence from the DHP-
CaV1.2 plasmid into the TOPO sites of the pCR8 entry vector and subsequently
transferring the CaV1.2 coding sequence into a destination vector called pDEST-
pGWYFP that contains a CMV promoter and an N-terminal YFP in frame with the
ATTR acceptor sequences. CaV1.2-flag was made by PCR amplification of CaV1.2
coding sequence with the tag in the 3’ primer and topo cloning into pcDNA4/HisMax
vector (Invitrogen). The CCAT YFP fusion proteins were constructed by inserting
PCR amplified portions of the CaV1.2 C-terminal tail into the HindIII and Kpn1 sites
of pcDNA3.0-YFP, a C-terminal YFP fusion vector. Plasmids encoding the following
amino acids of the CaV1.2 (accession # AAA18905) fused to YFP were generated:
1642-2143, 1642-2011, 1814-2143, 1864-2143, 1841-2101, 1814-2051, 1814-2000,
accession number AAA18605. N-terminal Gal4 DBD fusion proteins encompassing
the following amino acids of CaV1.2 1642-2143, 1642-2011, 1814-2143, 1864-2143
were generated by PCR cloning into BamHI/HindIII sites of PFA-CMV vector
(Stratagene). For microarray experiments and granule cell morphology assays the
sequence encoding amino acids 1642-2143 and 1642-2011 was cloned into the
HindIII/Kpn1 site of the PA1 expression vector, which was a kind gift from Dr.
Michael Lin. CCAT-ER fusions were made by inserting PCR amplified sequences
1642-2143 and 1642-2011 into BglII site of pCS4-myc. The ER sequence was
amplified from pBlu-ER-KS vector (kind gift from Dr. Ann Brunet) and inserted as a
C-terminal fusion into BamHI and EcorI sites.
Cx31.1-luciferase reporter was constructed by PCR amplification of a 2Kb promoter
segment (chr4 (-): 126860469-126862515) from mouse tail DNA using the primers:
48
Cx31.1 2Kb fwd: 5’-AGAGGAGCCCCAGGTAACACAG-3’ and Cx31.1-2kb rev:
5’-AGCCCAGGCGTGTCCTGTTGG-3’. The promoter was first cloned into
pCR8/GW/TOPO and later subcloned into XhoI, HindIII sites of the pGL3-Basic
vector (Promega).
Rat CaV1.3 and Human CaV2.1 were kind gifts from Dr. Diane Lipscombe and Dr.
Richard Tsien respectively. Gal4 fusions were made by PCR cloning into PFA-CMV
of C-terminal domains including amino acids 1669-2203 for CaV1.3 (NP_058994)
and 1975-2505 for CaV2.1 (AAB64179).
For shRNA knockdown experiments, the following 21mer oligonucleotides sequences
were selected using Dharmacon and Invitrogen’s design tools at
www.dharmacon.com/sidesign and rnaidesigner.invitrogen.com/rnaiexpress:
RCav1.2 6410 GGGACAGTTTGCTCAAGATCC, RCav1.2 6500
CGCCGCAGACAACATCCTC and MCav1.2 6203
GCTCAAGATCCCAAGTTTATC; Accession numbers, RATRBCII and AY728090,
for rat and mouse respectively.
Short hairpin Oligonucleotides were designed and inserted into RNAi-Ready pSIREN-
DNR-DsRed-Express vector (Clontech) by ligation into the BamHI and EcoRI sites.
RNAi resistant CCAT and CaV1.2 were made by introducing four silent mutations in
CaV1.2-flag and CCAT-pcDNA3.0 vectors using site-directed mutagenesis
(Stratagene). The resulting sequence was CGCAGCCGATAATATCCTC.
The CRE-luciferase reporter has been previously described (Dolmetsch et al., 2001).
Antibody generation
49
Peptides of the following sequence DPGQDRAVVPEDES were synthesized
(Covance) coupled to KLH (Pierce), injected into rabbits and affinity purified as
previously described (Datta et al., 1997).
Subcellular fractionation and Western blotting
The brain was rapidly removed and homogenized in 320 mM sucrose and 20 mM
HEPES homogenization buffer, pH 7.2, containing 1 mM EDTA, 1 mM dithiothreitol,
Complete protease inhibitors (Roche Applied Science), and calpain inhibitors (A.G.
Scientific). The homogenate was centrifuged for 10 min at 1000g to obtain the nuclear
fraction. The supernatant was then centrifuged for 30 min at 100,000g at 4 °C to
obtain the cytoplasmic and membrane fractions. The nuclear pellet was extracted
using the Dignam method (Dignam et al., 1983). For membrane-bound channel
visualization, proteins were extracted as described previously (Haase et al., 2000).
Western blotting was conducted using standard protocols. Antibodies and dilutions are
included in Supplemental experimental procedures. Protein concentration was
measured by the BCA method (Pierce).
Immunofluorescence
6-day old cortical cultures cells were fixed in 4% paraformaldehyde/2% sucrose,
permeabilized, and blocked with 3% BSA in PBS. Neurons were stained with either
rabbit anti-CCAT or rabbit anti-Cav1.2 II-III loop (each diluted 1:100) and anti-
GAD65 followed by 1:500 dilutions of Alexa 594-conjugated anti-mouse and Alexa
488-conjugated anti-rabbit antibodies (Molecular Probes). Nuclei were stained using
50
Hoechst 33258 (Molecular Probes). Neuro2A cells expressing CCAT-ER fusions were
stained with mouse anti-myc tag (Upstate). P30 rat brain sections were a gift from Dr.
Ben Barres. Sections were blocked and permeabilized 30 min using 10% goat serum,
0.25% triton X-100 in PBS. Primary and secondary antibody incubations were done as
described above. Slides were visualized by conventional epifluorescence microcopy
using a cooled CCD camera (Hamamatsu) coupled to an inverted Nikon Eclipse
E2000-U microscope. Confocal images were obtained using the Volocity grid
confocal microscope (Improvision Inc).
Quantitative Image analysis
Images were analyzed using OpenLab 4.0.4 software (Improvision, Inc). For
measurements of nuclear and cytoplasmic fluorescence, nuclear and whole cell regions
of interest (ROI) were generated by density slicing the Hoechst and anti-CCAT
images respectively and cytoplasmic ROIs were obtained by subtraction. Fluorescence
measurements were analyzed using Igor Pro software (Wavemetrics).
Fluorescence Recovery after Photobleaching (FRAP)
FRAP experiments were conducted at 37° C using a Zeiss Axiovert 200M inverted
microscope coupled to a Coolsnap cooled CCD camera controlled by Slidebook
software. Bleaching was achieved with a 100 ms long 488 nm laser pulse. Images
were captured every 400 ms.
51
Luciferase assays
Channels were transfected in a ratio of 1:1:1 CaV1.2, b1b and a2d subunits. For
luciferase assays, channels were transfected in a ratio of 2:1:1:0.5 CaV1.2, b1b, Firefly
luciferase reporter, and Renilla luciferase reporter and C-term constructs were
transfected at a ratio of 2:1:0.5, C-term, Firefly luciferase reporter and Renilla
luciferase reporter.
For shRNA experiments neurons and PC12s were transfected at a ratio of 1:1:0.5
shRNA vector, channel or c-term construct, Firefly luciferase reporter for 2.5-4 days.
PC12s were arrested and differentiated by switching them to 0.5% media with
50ng/ml NGF.
Most luciferase assays were performed 24 hours after transfection using the Dual-Glo
luciferase assay kit from Promega. For shRNA experiments, assays were performed 72
hours post-transfection. A Veritas 96 well luminometer (Turner biosystems) was used
to measure light emission. CREB-Gal4, constitutively active PKA, and PFA-CMV
constructs were obtained as part of the Path-Detect Trans-Reporting system from
Strategene. Data sets were analyzed using Igor Pro and Prism4 software. Two-paired
t-tests were performed between relevant conditions.
Immunoprecipitation and mass spectrometry
HEK 293T cells were transfected with c503-Gal4 or c280-Gal4. 24 hours after
transfection, immunoprecipitations were carried out using the ProFound Co-
Immunoprecipitation Kit (Pierce) and mouse or rabbit anti-Gal4 antibodies (Santa
Cruz Biotechnology). SDS-PAGE gels were silver stained using the SilverQuest
52
system from Invitrogen. Individual bands were analyzed by Stanford Mass
Spectrometry facility using LC-MS/MS as previously described (Shevchenko et al.,
1996).
RNA isolation and oligonucleotide microarrays
Neuro2A cells were transfected using the CCAT-PA1, CCAT∆TA-PA1 or PA1
control vectors. Twenty-four (24) hours after transfection, cells were trypsinized and
resuspended in fresh media without phenol red and GFP positive cells were sorted
using FACS. RNA was isolated from 2 x 106 cells using an RNAeasy kit from Qiagen.
RNA was hybridized to Agilent whole mouse oligo microarrrays by Mogene, Inc
(Saint Louis). Expression data was analyzed using GeneSpring GX 7.3 software
(Agilent).
Real-Time PCR
First strand synthesis was conducted using the first-strand cDNA synthesis kit from
Invitrogen. 500 ng of cDNA was used as a template for RT PCRs performed using an
Mx3000P Real-Time System (Stratagene), and the reactions were carried out using
Quantitec SYBR green PCR master mix (Qiagen). For a list of primers see
Supplemental experimental procedures.
Cycling parameters were 95°C for 10 min, followed by 45 cycles of 95°C for 30 s,
55°C for 1 min, 72°C for 30 s. Fluorescence intensities were analyzed using the
53
manufacturer’s software, and relative amounts were obtained using the 2–∆∆Ct method
(Livak and Schmittgen, 2001).
The following primers were used:
Mouse Cx31.1 (NM_010291) (F)5’- TTCTGATGCTTGCTGAACCCC -3’
(R)5’- GGAGTCCCTCAAAAACACTCC -3’;
Rat Cx31.1 A: (F) 5’- TTTTGATGCTTGCTGAACCCC-3’
(R)5’- GGAGCCCCTCAAAGACGCTCC-3’
Rat Cx3.1 B: (F)5’- CTGAGTGTGCACCAGCGAAGAGACC-3’
(R)5’-CGAGGGCGATCAGGTAACAGAGGTG-3’
Fmn (NM_010230) (F)5’-GGTCACCCCCAGCTATGTGTTT-3’
(R)5’-GCGGTGGGATTGATTTTCTTTG-3’;
Ppef2 (NM_011148) (F)5’-ATGCATTGCCAGGGTAGTCGG-3’
(R)5’-CAGGCACCTAACCCATGTCTG-3’;
Rgs5 (BC005656) (F)5’-TCACTTGCTCCCCCTTCCTC-3’
(R)5’-TCCTGGAATGTCTGGCAAGC-3’;
SCL8A1 (AF115505) (F)5’-CCTCGGTGCCAGACACATTTG-3’
(R)5’-GCCGGGGGACACTTTGAACT-3’;
Artn (NM_009711) (F)5’-TAGGTGGCAGTCAGCCTGGT-3’
(R)5’-GGGGTCGCAGGGTTCTTTC-3’;
NOS1 (NM_008712) (F)5’-CTCCCACCCTGCACCATCTT-3’
(R)5’-ATTCCTGAAGCCCCTTGCTG-3’;
b-actin (F)5’-CTTTGCAGCTCCTTCGTTGCC-3’
(R)5’-CGATGGAGGGGAATACAGCC-3’.
54
ChIP
ChIP was carried out as described in the Upstate Biotechnology protocol. Briefly 107
cells were transfected with CCAT-GST or CCAT-Gal4, or GST or Gal4 alone as a
controls. Proteins were crosslinked to DNA with 1% paraformaldehyde for 10
minutes and the nuclei were isolated by centrifugation. The DNA was sheared by
sonication to generate DNA fragments between 200 and 1000 bp. The C-terminal
fusion proteins were immunoprecipitated, washed and uncrosslinked by adding high
salt and incubating at 65° overnight. DNA was recovered by phenol/chloroform
extraction and ethanol precipitation and used as a template for PCR. Reaction
products were visualized by agarose gel electrophoresis.
Primers used analyze the immunoprecipitated DNA were to CX31.1 promoter
(Genbank accession number NM_010291):
(-30 to -201) 5’TGGGGGTGAAAGGTCAAAGTGT3’;
5’GTGTGTGTGTGGGAGGAGCTGT3’
(-201 to -351) 5’GCAATGGAGGAGGAGGGAAGAG3’;
5’AACCTTGTGTGGGGATGAAACG3’
(-455 to -610) 5’GGTGTGTGGCAGATAGGCTTCA3’;
5’CTCTCCAGTCCCTGCATTTGCT3’
(-589 to -761) 5’CCCATCCTTCATTTCCCTGGTT3’;
5’TGAAGCCTATCTGCCACACACC3’
(-859 to -1081)
5’ATGGTGGCCGTTCATACAGAGC3’;5’GCTTGGAGTTGGGAGACAGGAG3’
55
For the 3’UTR, (-1060 to -1217) 5’ CCAACCAGCCTTTCCTCTCCAT3’;
5’RGCTCTGTATGAACGGCCACCAT3’
Dendritic arborization assays
Cerebellar granules cells were imaged 24-48h post-transfection using the ImageXpress
500A system (Molecular devices). Dendrites were analyzed employing ImageJ and
NeuroJ programs.
56
FUTURE EXPERIMENTS
The findings described in this chapter beg further investigation on the molecular
mechanisms by which CCAT influences transcription. Several models can explain
how CCAT can influence gene expression. On one hand, CCAT can bind DNA
directly by binding to specific cis-regulatory DNA sequences such as the transcription
factors CREB and NFAT. The identification of this binding motif could point to
endogenous target genes and would help us to better understand CCAT’s function. If
CCAT cannot bind DNA on its own, an alternative model must be considered where
the CCAT-DNA interaction is mediated by DNA binding partners. These binding
partners could be themselves sequence-specific transcription factors or components of
the basal transcriptional machinery. Here I describe a series of experiments that would
help shed some light on these questions
Does CCAT bind DNA and what is its target sequence?
To begin to understand how CCAT regulates transcription I searched for
sequence homology with previously known DNA-binding proteins. Of the 53 DNA-
binding domains for which Pfam has Hidden Markov models none can be predicted
from CCAT’s sequence. While this suggests that CCAT does not bind DNA it is
possible that it employs a yet uncharacterized domain for interacting with DNA.
Interestingly, the C-terminus of Cav2.1, a P/Q type channel, contains several NLSs and
a predictable AT-hook motif commonly found in many nuclear proteins (Aravind and
57
Landsman, 1998). In the absence of sequence homology with previously known DNA
binders, I turned to prediction methods based on amino acid composition and
secondary structure (Ahmad et al., 2004). Such methods predict DNA-binding
properties for the c-termini of Cav1.2, 1.3 and 2.1, which is likely due to the high
concentration of basic residues within these sequences. These predictions, in the
absence of structural or sequence conservation information, need to be taken
cautiously. Together, sequence analysis although suggestive provides little definite
information on CCAT’s DNA-binding properties.
We have already identified a 148 bp region in the CX31.1 promoter to be
necessary and sufficient for CCAT-dependent transcriptional activation. In chapter 2,
we showed that CCAT regulates transcription of Cx31.1 by acting directly at the
promoter. Deletion of this sequence eliminated the ability of CCAT to activate
transcription and insertion of this domain alone was sufficient to confer CCAT
regulation on to a reporter gene (Figure 5). This sequence can be used as starting point
to investigate CCAT’s ability to bind DNA.
In future experiments the 148 bp sequence can be further narrowed down by
deletion analysis. Once the target DNA sequence has been sufficiently reduced point
mutations can be made to determine which bases are important for CCAT
transcriptional activity. Subsequently, binding of CCAT to isolated sequences can be
evaluated using mobility shift assays and labeled probes from the target sequence
identified above. In these experiments, the migration of labeled oligos is compared
between reactions containing CCAT or control proteins. Because CCAT may bind to
58
DNA non-specifically, it is important to test control oligos for which binding is not
expected and additionally, perform a competitive assay using unlabeled probe.
To provide independent evidence of binding and to demonstrate that this
binding is sufficient to confer CCAT-dependent transcription reporter genes can be
constructed of several repeats of the candidate motif by inserting them in front of the
luciferase coding sequence and a basal promoter. These vectors can be used for
transcriptional assays where luciferase expression can be compared in the presence
and absence of CCAT.
Alternatively a non-biased approach can be used. PCR-assisted binding site
selection can be used to determine the optimal DNA binding site for CCAT. In this
approach, a random library of oligonucleotides (20nt) is incubated with purified
CCAT-GST fusion proteins, the CCAT-DNA complexes are then isolated via their
GST tags and bound oligos are recovered. Our library of oligos has been constructed
so that T7 forward and T3 reverse primers flank the 5’ and 3’ ends of each oligo
respectively, and can thus be randomly amplified using PCR. After several rounds of
binding, recovery and amplification, the oligomers can be cloned and examined by
sequencing. The sequences recovered will hopefully provide the consensus DNA
binding domain for CCAT.
Assuming that the above experiments will yield a sequence or family of related
sequences for CCAT, a subsequent experiment will be to use a bioinformatic approach
to determine endogenous target genes by searching putative regulatory regions in the
genome for the presence of such motifs. Because such an approach would likely yield
many false positives (given the short size of the DNA motifs), it is prudent to restrict
59
the initial search to genes previously identified in our microarray experiments
(Gomez-Ospina et al., 2006). Confirmatory experiments involve building reporters
using upstream regulatory regions from likely candidates. Deletion studies can be
used to prove necessity.
Nuclear binding partners
The absence of predictable DNA-binding domains in CCAT’s sequence has
led us to consider that CCAT may bind DNA via a specialized DNA-binding protein
or scaffolding protein. One possible mechanism involves a motif-specific transcription
factor. Such mechanism would be reminiscent to the one employed by Notch, another
membrane protein that is cleaved and encodes a transcriptional regulator within its
intracellular domain (ICD). ICD lacks a DNA-binding ability and so uses a DNA
bound transcription factor called CSL to induce transcription of target genes
(Schroeter et al., 1998). However, these DNA-binding partners need not be sequence-
specific. It is possible that CCAT influences transcription by binding to chromatin or
basal transcription factors. CCAT could promote either their recruitment or activation
on target promoters such is the case for the RNA poly II carboxyl-terminal domain
phosphatases (Meinhart et al., 2005).
Our lab has begun to develop and utilize several proteomic approaches for the
identification of nuclear partners for CCAT. In pilot experiments, we expressed
CCAT or a mutant form lacking the nuclear localization domain in Neuro2A cells,
immunoprecipitated them via epitope tags, and identified interacting proteins by mass
60
spectrometry. This approach has identified several binding proteins. As described in
chapter 2, one protein immunoprecipitated by full length CCAT is P54/nrb/NonO
(NonO), a DNA and RNA-binding protein that appears to play roles in transcriptional
regulation as Ill as RNA processing (Shav-Tal and Zipori, 2002). We verified the
interaction by co-immunoprecipitation followed by western blotting against
endogenous NonO (Figure 4). This confirmation suggests that such biochemical
screen could be a productive approach toward identifying nuclear partners for CCAT.
The next step is to determine how these proteins relate to CCAT’s mechanism of
action.
In the case of NonO, the literature gives us some interesting clues regarding
possible mechanisms. NonO has been demonstrated to associate with the Carboxyl-
terminal domain of RNA pol II (Emili et al., 2002) and has been recently shown to
couple Neuronal Wiskott-Aldrich syndrome protein (N-WASP) with RNA polymerase
II to regulate transcription (Wu et al., 2006). Thus, one could hypothesize that NonO
could acts as an adaptor protein between CCAT and the transcriptional apparatus.
Future experiments can be designed to test this hypothesis. To begin, one can search
for the presence of RNA pol II in immunoprecipitated complexes of epitope tagged
CCAT along with NonO. Subsequent experiments can be aimed at substantiating the
need for NonO in CCAT-dependent transcription.
The question whether NonO is necessary for CCAT’s observed transcriptional
activation can be tested by knocking-down endogenous NonO and replacing it with a
NonO incompetent to bind CCAT. Towards this aim, a NonO protein unable to bind
CCAT and resistant to RNAi needs to be constructed. This modified NonO can be
61
included in transcriptional assays using CCAT-Gal4 to determine if it can block or
reduce transcription. According to my hypothesis, luciferase expression should be
decreased and this effect should be rescuable by full-length NonO insensitive to
RNAi. Considering NonO’s pleotropic role in the nucleus, it is possible that
knockdown and rescue with modified NonO will have a global negative impact on
transcription and it would not be specific for CCAT. Consequently, it is critical to
control for this possibility by co-expressing a constitutively expressed reporter, which
should report for overall decreases in transcription in the same cells.
62
Unt Unt HEK293T Cortex
240
75
Unt CytNuc
75
43
A B
C
240 anti-CCAT
anti-CREB
anti-CCATanti-II-III loop
EM ergedH oechstC C AT
II-III loop H oechst
H oechstC C AT
M erged
M ergedG AD 45
F
Mem
240 anti-II-III loop
anti-GAPDH38
C C AT Hoechst Merged
400X 600X
G H
D
M ergedH oechstC C AT
CB
IC IO
BSM ergedH oechstC C AT
I
H oechstC C AT M ergedG AD 45
400X
100X
Cav1.2 Cav1.2 Cav1.2
A
YFP-Cav1.2 Hoechst MergedHoechst Merged
B
Myocyte HEK 293TNeuron
Hoechst Mergedc280
Hoechstc330 Merged
D
F
1.0
2.0
3.0
c503 c330 c280 c330-50
c330-100
Nucle
ar/c
ytop
lasm
ic ra
tioE
C
**
Hoechst Mergedc503-133
HoechstCCAT-ConfocalCCAT-Epi
G
Nucleus
L
TC-Y
FP
Fluo
resc
ence
(A.U
.)
Time (s)
Cytoplasm
190
180
170160
50 100 150 200 250 300
t=0 s t=250 st=75 st=-50 s
H
Cav1.2-YFP
c503 YFP1642-2143 aac503-133 1642-2011 aac330 1814-2143 aac280 1864-2143 aac330-50 1841-2101 aac330-100 1814-2051 aa
YFP
YFP
YFP
YFP
YFP
63
A
Hoechstanti-CCAT Merged
5mM K+
60mM K+
C
F
E18 P1 P21 Adult
anti-CREB
75
50
E18 P1 P21 AdultP8
anti-CCAT
anti-II-III loop
anti- ß-actin
210
150
240
42
H
I
anti-CCATMembrane
Nuclear
240
EGTA
60
50
40
30
20
10
02.52.01.51.00.5
Num
ber o
f cel
ls
Nuclear/cytoplasmic ratio
Hoechstanti-CCAT MergedB
0
0.5
1.0
1.5
5 K+ EGTA
60 K+
Nuc
lear
/cyt
opla
smic
ratio
***
EGTA Nimo MK-801 NBQX
D
E
-40
-20
0
% d
ecre
ase
5 10 15 20 25 30time (min)
5 K+
60 K+
EGTA
60 K+
Glutamate
EGTA
Glutamate
Glutamate
500
1000
1500
M
ean
cell
body
fluor
esce
nce
(A.U
.)
60 K+5 K+
Nucle
ar/cy
topla
smic
ratio
0
0.4
1.2
0
0.8
60 K+5 K+
Lactacystin
60mM K+
*
G
64
a
IQ Gal4
c503
c330
c280IQ Gal4
c503
c330
c280
Gal4
Cav1.2
Cav1.2
-Gal4
0.5
1.0
3.5
3.0
2.5
2.0
1.5UA
S T
rans
crip
tion
(Fi
refly
/Ren
illa)
0
C
F
Gal4
CREB-Gal4
(+P
KA)c5
03-G
al4
c330
-Gal4
c280
-Gal4
A
Blot: α-p54/nrb
Blot: CCAT
Blot: α-p54/nrbIP CCAT
90
20
60
60
B
Cav1.2∆
133-G
al4
c503-CCAT
c280-CCAT
1
2
3
4
5
6
7
0
0
1
2
3
4
60mM K+
+++
---2.5mM EGTA
**
D
∆133
UA
S T
rans
crip
tion
(Fi
refly
/Ren
illa)
UA
S T
rans
crip
tion
(Fi
refly
/Ren
illa)
5
****
**
0.5
1.0
3.53.02.52.0
1.5UA
S T
rans
crip
tion
(Fi
refly
/Ren
illa)
0
E
Gal4
Cav1.2
c503
Gal4
Cav1.3
c537
Gal4Cav
2.1
c530
Gal4
3.5
c503∆133
-G
al4
****
**
65
Connexin 31.1 (Gjb5)
Formin (Fmn)
Peroxisomal membrane protein (Mpv17)Regulator of G-protein signaling (Rgs5)
Diflavin reductase (Ndor1)
Vomeronasal 1 receptor H13 (V1rh13)
Catenin alpha like 1 (Catna1)SET-binding protein (Sbf1)
Schlafen (Slfn)
FBJ osteosarcoma oncogene (Fos)
LIM and SH3 protein 1 (Lasp1)
GATA binding factor 6 (Gata6)SRY-box containing gene 2 (Sox3)
Phosphatase EF hand containing (Ppef2)
Paralemmin 2 (Palm2)
TRP channel (TrpV4)
Potassium channel (Kcnn3)
Forkhead box E3 (Foxe3)
Protease 26S subunit ATPase (Psmc5)
Activating transcription factor (Atf7)
Serine/threonine/tyrosine kinase (Styk1)
Myosin IG (Myo1G)
Nitric oxide synthase 1, neuronal (Nos1)
Sodium/Calcium exchanger 1 (Scl8A1)
Glutamate receptor NMDA2D (Grin2d)Artemin (Artn)
Glucokinase (Gck)
Chemokine (C-X3-C motif) ligand 1(Cx3cl1)
Leucine-rich pentatricopeptide repeat (Lrpprc)
Claudin 19 (Cldn19)
MYC-CCAT-ER MergedHoechst
Hoechst MergedMYC-CCAT-ER
IP
IP
IP
IP
C C
C C
I I
I I
350
350
250
250
150
150
50
3' UTR 3' UTR
450
0
86420
12108642
10 12-6 -4 -2Fold Change
Rel
ativ
e m
RN
A ex
pres
sion
1
0
345
-1-2-3
-4
FmnRgs
5Gjb5
nNOS1
Scl8A1
Artn Ppef2
+ 4O
HT
1h
- 4O
HT
Cx3
1.1
trans
crip
tion
Fire
fly (A
.U.)
10
3
4
5
6
2
7
2
pcDNA3 pcDNA3 CCAT
pcDNA3CCAT∆TA
**
Cx3
1.1
trans
crip
tion
(Fol
d In
duct
ion
+4O
HT/
-4O
HT)
10
0
30
40
50
60
20
ER CCAT-ER CCAT∆TA-ER
5’ promoter 3’ promoter
Cx3
1.1
trans
crip
tion
Fire
fly (A
.U.)
5
0
15
20
25
10
A B C D
E F G H
A B C D E F G H2K
A
B D F
C E G
66
A
B
C
D
E
F
G
H
0
1
2
3
4
5mM K+ 65mM K+
****
Cx3
1.1
trans
crip
tion
F
irefly
(A.U
.)
CCAT CCAT*
RCav1.2
sh65
00 MCav1.2
sh62
03 RCav1.2
sh65
00
anti-flag M2
anti-DsRed
240
30
Rel
ativ
e C
x31.
1 m
RN
A ex
pres
sion
65mM K+ 0 Ca+2
Neuro2A
Cortical
Thalamic
0
1
2
-1
-4
-3
-2
-5
Cx3
1.1
trans
crip
tion
(F
irefly
/Ren
illa)
0
****1
2
3
4
5
6
sh-scr MCav1.2 sh6203
RCav1.2 sh6410
RCav1.2 sh6500
MCav1.2
sh62
03
65
30
Cx3
1.1
trans
crip
tion
F
irefly
(A.U
.)
anti-CCAT
anti-DsRed
0
1
2
3
4
5
6
****
sh-scrVector CCAT* CCAT∆TA
RCav1.2 sh6500
Cav1.2-flag
RCav1.2
sh64
10 RCav1.2
sh65
00MCav1.2
sh62
03
Cav1.2∆TA-flag
RCav1.2
sh65
00
Cav1.2-flag Cav1.2-flag*
RCav1.2
sh65
00MCav1.2
sh62
03 RCav1.2
sh65
00 MCav1.2
sh62
03
240
30
anti-flag M2
anti-DsRed
Cx3
1.1
trans
crip
tion
F
irefly
(A.U
.)
0
1
2
3
4
5
6
7 ** **
Vector Cav1.2* Cav1.2∆TA
Vector
sh-scr RCav1.2 sh6500Vector
67
60
40
20
0
% of cells
50403020100
60
40
20
0
% of cells
50403020100Neurite length (µm)
50403020100
Number of Primary Neurites
CCA∆TA Vector CCAT0.0
0.5
1.0
1.5
2.0
2.5
CCAT∆TA Vector CCAT0.0
2.5
5.0
7.5
10.0
12.5Av
g.N
eurit
e Le
ngth
(µm
)Av
g. #
of P
rimar
y N
eurit
es
% o
f Cel
ls%
of C
ells
CCAT∆TA
Vector
CCAT
**
A B
CD
E
CCAT∆TA CCAT CCAT
**
Neurite length (µm)
Neurite length (µm)
60
40
20
0
% of cells
% o
f Cel
ls
Length of Primary Neurites
**
**
68
69
A CCAT’s nuclear localization domain is conserved among vertebrates channels CAC1C_RAT 1794 VEGHGPPLSPAVRVQEAAWKLSSKRCHSRESQGATVSQDMFPDETRSSVRLSEEVEYCSE CAC1C_MOUSE 1791 VEGHGPPLSPAVRVQEAAWKLSSKRCHSRESQGATVNQEIFPDETR-SVRMSEEAEYCSE CAC1C_HUMAN 1790 VEGHGPPLSPAIRVQEVAWKLSSNRCHSRESQAAMAGQEETSQDETYEVKMNHDTEACSE CAC1C_RABBIT 1821 VEGHGSPLSPAVRAQEAAWKLSSKRCHSQESQIAMACQEGASQDDNYDVRIGEDAECCSE CAC1C_ZEBRAFISH 1808 ----GPPLT-TIPLPRPTWCFPNKSSDSSDSRLPIIRREEASTDETYDETFLDE----RD CAC1C_RAT 1854 PSLLSTDILSYQDDENRQLTCLEEDKREIQ CAC1C_MOUSE 1851 PSLLSTDMFSYQEDEHRQLTCPEEDKREIQ CAC1C_HUMAN 1850 PSLLSTEMLSYQDDENRQLTLPEEDKRDIR CAC1C_RABBIT 1881 PSLLSTEMLSYQDDENRQLAPPEEEKRDIR CAC1C_ZEBRAFISH 1858 QAMLSMDMLEFQDEESKQLAPMVE------
B CCAT’s N-terminal transcription activation domain is conserved among vertebrates channels
CAC1C_RAT 1642 LVGKPSQRNALSLQAGLRTLHDIGPEIRRAISGDLTAEEELDKAMKEAVSAASEDDIFRR CAC1C_MOUSE 1639 LVGKPSQRNALSLQAGLRTLHDIGPEIRRAISGDLTAEEELDKAMKEAVSAASEDDIFRR CAC1C_RABBIT 1669 LVGKPSQRNALSLQAGLRTLHDIGPEIRRAISGDLTAEEELDKAMKEAVSAASEDDIFRR CAC1C_HUMAN 1687 LVGKPSQRNALSLQAGLRTLHDIGPEIRRAISGDLTAEEELDKAMKEAVSAASEDDIFRR CAC1C_ZEBRAFISH 1684 LVAKIPPKTALSLQAGLRTLHDMGPEIRRAISGDLTVEEELERAMKETVCAASEDDIFRR CAC1C_RAT 1702 AGGLFGNHVSYYQ-SDSRSNFPQTFATQRPLHINKTGNNQADTESPSHEKLVDSTFTPSS CAC1C_MOUSE 1699 AGGLFGNHVTYYQ-SDSRGNFPQTFATQRPLHINKTGNNQADTESPSHEKLVDSTFTPSS CAC1C_RABBIT 1729 AGGLFGNHVSYYQ-SDSRSAFPQTFTTQRPLHISKAGNNQGDTESPSHEKLVDSTFTPSS CAC1C_HUMAN 1747 AGGLFGNHVSYYQ-SDGRSAFPQTFTTQRPLHINKAGSSQGDTESPSHEKLVDSTFTPSS CAC1C_ZEBRAFISH 1744 SGGLFGNHVNYYHQSDGHVSFPQSFTTQRPLHISKSGS-PGEAESPSHQKLVDSTFTPSS CAC1C_RAT 1762 YSSTGSNANINNANNTALG-RFPHPAGYSSTVSTVEGH-GPPLSPAVRVQEAAWKL CAC1C_MOUSE 1759 YSSTGSNANINNANNTALG-RFPHPAGYSSTVSTVEGH-GPPLSPAVRVQEAAWKL CAC1C_RABBIT 1789 YSSTGSNANINNANNTALG-RLPRPAGYPSTVSTVEGH-GSPLSPAVRAQEAAWKL CAC1C_HUMAN 1807 YSSTGSNANINNANNTALG-RLPRPAGYPSTVSTVEGH-GPPLSPAIRVQEVAWKL CAC1C_ZEBRAFISH 1803 YSSSGSNANINNANNTAIGHRYPKP-----TVSTVDGQTGPPLT------------
C CCAT’s N-terminal transcription activation domain is homologous to GATA4’s C-terminal domain GATA4_HUMAN 313 IQTRK-RKPKNLNKSKTPAAPSGSESLPPASGASSNSSNATTSSS--EEMRPIKTEPGLS GATA4_RAT 311 IQTRK-RKPKNLNKSKTPAGPPG-ESLPPSSGASSNSSNATSSSSSSEEMRPIKTEPGLS GATA4_CHICK 264 IQTRK-RKPKNLNKTKTPAGPSSSESLTPTTSSTSSSSSATTT----EEMRPIKTEPGLS CAC1C-RAT 1722 PQTFATQRPLHINKTGNNQADTESPSHEKLVDSTFTPSSYSSTG---SNANINNANNTAL consensus ** ::* :**: : : * * :: * ::: : ::: GATA4_HUMAN 370 SHYGHSSSVSQTFSVSAMSGHGPSIHPVL----SALKLSPQGYASPVSQSPQTS GATA4_RAT 372 SHYGHSSSMSQTFST--VSGHGSSIHPVL----SALKLSPQGYPSPVSQTSQAS GATA4_CHICK 320 SHYGHPSPISQAFSVSAMSGHGSSIHPAI----SALKLSPQAYQSAISQSPQAS CAC1C-RAT 1779 GRFPHPAGYSSTVST--VEGHGPPLSPAVRVQEAAWKLSSKRCHSRESQGATVS consensus :: * : * ::* : ***: : *:: :* *** * ** :*
D CCAT’s C-terminal transcription activation domain is conserved among vertebrate channels CAC1C_RAT 2009 SSMARRARPVSLTVPSQAGAPGR-QFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQEL CAC1C_MOUSE 2005 SSMARRARPVSLTVPSQAGAPGR-QFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQEL CAC1C_RABBIT 2035 SSAARRARPVSLTVPSQAGAQGR-QFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQEL CAC1C_HUMAN 2087 SSAARRVRPVSLMVPSQAGAPGR-QFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQEL CAC1C_ZEBRAFISH 2017 NHSGRAQRPVSLTVPPVTRRDSISLAHGSAGSLVEAVLISEGLGRYAHDPSFIQVAKQEI CAC1C_RAT 2059 ADACDMTIEEMENAADNILSGGAQQSPNGTLLPFVNCRDPGQDRAVVPE-DESCVYALGR CAC1C_MOUSE 2064 ADACDMTIEEMENAADNILSGGAQQSPNGTLLPFVNCRDPGQDRAVAPE-DESCAYALGR CAC1C_RABBIT 2094 ADACDLTIEEMENAADDILSGGARQSPNGTLLPFVNRRDPGRDRAGQNEQDASGACAPGC CAC1C_HUMAN 2146 ADACDMTIEEMESAADNILSGGAPQSPNGALLPFVNCRDAGQDRAGGEE-DAGCVRARG- CAC1C_ZEBRAFISH 2077 AEACDMTMEEMENAADNILNANAPPNANGNLLPFIQCRDTGSQ-------ESRCSLSLGL CAC1C_RAT 2127 GRSEEALPDSRSYVSNL CAC1C_MOUSE 2123 GRSEEALADSRSYVSNL CAC1C_RABBIT 2153 GQSEEALADRRAGVSSL CAC1C_HUMAN 2204 APSEEELQDSRVYVSS- CAC1C_ZEBRAFISH 2130 SPATGSDGALEAELEESEGAGQRNSPLMEDEDMECVTSL
Figure S1. CCAT’s nuclear localization and transcription activation domains are conserved among CaV1.2 channels in vertebrates
70
Figure S2. CCAT derived from CaV1.2-YFP channel is regulated by depolarization
71
Supplemental Figure 3. CCAT regulates expression of endogenous genes
72
Figure S4.
7.32
18.
968
(5.6
87 to
13.
15)
1.22
5 (1
.205
to 1
.267
)G
jb5
NM
_010
291
Mus
mus
culu
s ga
p ju
nctio
n m
embr
ane
chan
nel p
rote
in b
eta
5 (G
jb5)
, mR
NA
[NM
_010
291]
3.56
93.
812
(2.9
5 to
5.2
09)
1.06
8 (1
.055
to 1
.08)
Mpv
17N
M_0
0862
2M
us m
uscu
lus
Mpv
17 tr
ansg
ene,
kid
ney
dise
ase
mut
ant (
Mpv
17),
mR
NA
[NM
_008
622]
3.55
13.
487
(2.8
67 to
3.9
91)
0.98
2 (0
.961
to 0
.998
)C
ol11
a2N
M_0
0992
6M
us m
uscu
lus
proc
olla
gen,
type
XI,
alph
a 2
(Col
11a2
), m
RN
A [N
M_0
0992
6]3.
107
3.57
6 (2
.796
to 4
.337
)1.
151
(1.1
39 to
1.1
72)
2310
021P
13R
ikBC
0265
04;
Mus
mus
culu
s R
IKEN
cD
NA
2310
021P
13 g
ene,
mR
NA
(cD
NA
clon
e M
GC
:570
92 IM
AGE:
6489
956)
, com
plet
e cd
s. [B
C04
9362
]2.
924
2.46
8 (1
.758
to 3
.041
)0.
844
(0.8
04 to
0.8
76)
Cat
nal1
NM
_018
761
Mus
mus
culu
s ca
teni
n al
pha-
like
1 (C
atna
l1),
mR
NA
[NM
_018
761]
2.73
52.
606
(2.4
15 to
2.7
12)
0.95
3 (0
.884
to 1
.03)
Slco
5a1
AK04
1736
1M
us m
uscu
lus
solu
te c
arrie
r org
anic
ani
on tr
ansp
orte
r fam
ily, m
embe
r 5A1
(Slc
o5a1
), m
RN
A [N
M_1
7284
1]2.
709
2.14
3 (1
.848
to 2
.63)
0.79
1 (0
.591
to 1
.044
)Pp
ef2
NM
_011
148
Mus
mus
culu
s pr
otei
n ph
osph
atas
e, E
F ha
nd c
alci
um-b
indi
ng d
omai
n 2
(Ppe
f2),
mR
NA
[NM
_011
148]
2.47
21.
955
(1.3
76 to
3.5
44)
0.79
1 (0
.712
to 0
.936
)Bt
g4AB
0509
83M
us m
uscu
lus
B-ce
ll tra
nslo
catio
n ge
ne 4
(Btg
4), m
RN
A [N
M_0
1949
3]2.
420
1.99
4 (1
.392
to 3
.077
)0.
824
(0.7
66 to
0.8
62)
His
t1h1
tN
M_0
1037
7M
us m
uscu
lus
hist
one
1, H
1t (H
ist1
h1t),
mR
NA
[NM
_010
377]
2.35
11.
855
(1.3
16 to
2.6
14)
0.78
9 (0
.708
to 0
.974
)Ta
f3AK
0155
34M
us m
uscu
lus
TAF3
RN
A po
lym
eras
e II,
TAT
A bo
x bi
ndin
g pr
otei
n (T
BP)-a
ssoc
iate
d fa
ctor
, mR
NA
(cD
NA
clon
e IM
AGE:
3066
0628
), pa
rtial
cds
[BC
0890
30]
2.29
313
.65
(9.9
36 to
16.
02)
5.95
4 (5
.237
to 6
.534
)Ifi
t1N
M_0
0833
1M
us m
uscu
lus
inte
rfero
n-in
duce
d pr
otei
n w
ith te
tratri
cope
ptid
e re
peat
s 1
(Ifit1
), m
RN
A [N
M_0
0833
1]2.
237
1.87
9 (1
.412
to 2
.502
)0.
84 (0
.712
to 0
.891
)BI
8554
34;
BI85
5434
603
3825
13F1
NC
I_C
GAP
_Mam
6 M
us m
uscu
lus
cDN
A cl
one
IMAG
E:54
1488
3 5'
, mR
NA
sequ
ence
[BI8
5543
4]2.
205
5.29
5 (3
.177
to 1
0)2.
401
(2.0
16 to
3.0
73)
Usp
18N
M_0
1190
9M
us m
uscu
lus
ubiq
uitin
spe
cific
pro
teas
e 18
(Usp
18),
mR
NA
[NM
_011
909]
2.17
71.
885
(1.4
16 to
3.0
9)0.
866
(0.8
6 to
0.8
71)
Cld
n19
AK03
2743
Mus
mus
culu
s cl
audi
n 19
(Cld
n19)
, mR
NA
[NM
_153
105]
2.17
61.
756
(1.5
46 to
2.1
89)
0.80
7 (0
.721
to 0
.992
)R
assf
6AK
0054
72M
us m
uscu
lus
Ras
ass
ocia
tion
(Ral
GD
S/AF
-6) d
omai
n fa
mily
6 (R
assf
6), m
RN
A [N
M_0
2847
8]2.
157
2.03
(1.6
35 to
2.4
93)
0.94
1 (0
.916
to 0
.959
)M
cf2
NM
_133
197
Mus
mus
culu
s m
cf.2
tran
sfor
min
g se
quen
ce (M
cf2)
, mR
NA
[NM
_133
197]
2.14
96.
673
(6.1
28 to
7.0
73)
3.10
5 (2
.969
to 3
.234
)Bn
ip3
NM
_009
760
Mus
mus
culu
s BC
L2/a
deno
viru
s E1
B 19
kDa-
inte
ract
ing
prot
ein
1, N
IP3
(Bni
p3),
mR
NA
[NM
_009
760]
2.10
12.
067
(1.7
34 to
2.2
96)
0.98
4 (0
.742
to 1
.566
)N
tn4
NM
_021
320
Mus
mus
culu
s ne
trin
4 (N
tn4)
, mR
NA
[NM
_021
320]
2.08
05.
608
(3.9
57 to
7.7
53)
2.69
6 (2
.529
to 2
.933
)C
cl5
NM
_013
653
Mus
mus
culu
s ch
emok
ine
(C-C
mot
if) li
gand
5 (C
cl5)
, mR
NA
[NM
_013
653]
2.07
51.
89 (1
.44
to 2
.727
)0.
911
(0.9
02 to
0.9
29)
Rgs
5BQ
9488
84;
Mus
mus
culu
s cD
NA
clon
e IM
AGE:
3710
250,
com
plet
e cd
s. [B
C00
5656
]2.
059
1.97
9 (1
.156
to 2
.622
)0.
961
(0.8
77 to
1.0
53)
Stk3
AK08
7803
;M
us m
uscu
lus
2 da
ys p
regn
ant a
dult
fem
ale
ovar
y cD
NA,
RIK
EN fu
ll-le
ngth
enr
iche
d lib
rary
, clo
ne:E
3300
23A1
5 pr
oduc
t:unk
now
n ES
T, fu
ll in
sert
sequ
ence
[AK0
8780
1]1.
998
1.90
6 (1
.704
to 2
.132
)0.
954
(0.8
57 to
1.0
61)
Cyl
n2N
M_0
0999
0M
us m
uscu
lus
cyto
plas
mic
link
er 2
(Cyl
n2),
mR
NA
[NM
_009
990]
0.15
62.
053
(0.8
03 to
3.3
6)13
.15
(10.
87 to
16.
45)
V1rh
13N
M_1
3423
8.1
Mus
mus
culu
s vo
mer
onas
al 1
rece
ptor
, H13
(V1r
h13)
, mR
NA
[NM
_134
238]
0.25
72.
286
(1.7
35 to
2.6
75)
8.89
3 (4
.627
to 1
2.7)
Egln
3N
M_0
2813
3M
us m
uscu
lus
EGL
nine
hom
olog
3 (C
. ele
gans
) (Eg
ln3)
, mR
NA
[NM
_028
133]
0.35
12.
74 (2
.015
to 3
.857
)7.
813
(5.9
65 to
9.3
03)
Cam
pN
M_0
0992
1M
us m
uscu
lus
cath
elic
idin
ant
imic
robi
al p
eptid
e (C
amp)
, mR
NA
[NM
_009
921]
0.36
10.
285
(0.1
95 to
0.4
42)
0.78
9 (0
.775
to 0
.813
)AU
0187
78BC
0134
79M
us m
uscu
lus
expr
esse
d se
quen
ce A
U01
8778
(AU
0187
78),
mR
NA
[NM
_144
930]
0.37
50.
331
(0.1
4 to
0.7
84)
0.88
3 (0
.846
to 0
.921
)BQ
1751
90U
nkno
wn
0.40
32.
698
(2.5
68 to
2.8
87)
6.69
4 (6
.483
to 6
.805
)Lm
lnAK
0304
63M
us m
uscu
lus
leis
hman
olys
in-li
ke (m
etal
lope
ptid
ase
M8
fam
ily) (
Lmln
), m
RN
A [N
M_1
7282
3]0.
411
5.24
3 (4
.109
to 8
.328
)12
.75
(11.
47 to
14.
12)
2310
001H
13R
ikAK
0307
66M
us m
uscu
lus
mR
NA
for m
KIAA
1042
pro
tein
[AK1
2242
6]0.
418
3.15
5 (2
.222
to 4
.735
)7.
553
(5.0
87 to
9.3
97)
Swap
70N
M_0
0930
2M
us m
uscu
lus
SWA-
70 p
rote
in (S
wap
70),
mR
NA
[NM
_009
302]
0.42
51.
909
(1.7
32 to
2.0
71)
4.49
(4.2
74 to
4.9
19)
LOC
4325
85BB
6675
59PR
EDIC
TED
: Mus
mus
culu
s th
yroi
d ho
rmon
e re
cept
or a
ssoc
iate
d pr
otei
n 1
(Thr
ap1)
, mR
NA
[XM
_109
726]
0.44
71.
92 (1
.55
to 2
.384
)4.
296
(3.3
93 to
6.1
76)
Pip5
k2a
NM
_008
845
Mus
mus
culu
s ph
osph
atid
ylin
osito
l-4-p
hosp
hate
5-k
inas
e, ty
pe II
, alp
ha (P
ip5k
2a),
mR
NA
[NM
_008
845]
0.46
40.
456
(0.3
65 to
0.5
77)
0.98
3 (0
.919
to 1
.052
)49
2150
9F24
Rik
AK01
4852
Mus
mus
culu
s ad
ult m
ale
test
is c
DN
A, R
IKEN
full-
leng
th e
nric
hed
libra
ry,c
lone
:492
1509
F24
prod
uct:h
ypot
hetic
al G
lyco
side
hyd
rola
se fa
mily
35 c
onta
inin
g pr
otei
n,[A
K014
852]
0.46
92.
637
(2.3
67 to
2.7
97)
5.62
2 (5
.46
to 5
.81)
BC01
0711
Mus
mus
culu
s cD
NA
clon
e IM
AGE:
5010
343,
par
tial c
ds. [
BC07
1254
]0.
479
7.83
7 (5
.104
to 9
.72)
16.3
7 (1
4.58
to 1
7.78
)Fm
n1N
M_0
1023
0M
us m
uscu
lus
form
in 1
(Fm
n1),
mR
NA
[NM
_010
230]
0.47
92.
046
(2.0
07 to
2.0
7)4.
273
(4.1
86 to
4.3
41)
Pa2g
4N
M_0
1111
9M
us m
uscu
lus
prol
ifera
tion-
asso
ciat
ed 2
G4
(Pa2
g4),
mR
NA
[NM
_011
119]
0.48
00.
426
(0.3
31 to
0.5
72)
0.88
7 (0
.801
to 1
.08)
Prss
32BC
0249
03M
us m
uscu
lus
prot
ease
, ser
ine,
32
(Prs
s32)
, mR
NA
[NM
_027
220]
0.48
41.
889
(1.6
74 to
2.0
85)
3.89
9 (3
.38
to 4
.395
)C
lasp
1AJ
2769
62M
us m
uscu
lus
13 d
ays
embr
yo fo
relim
b cD
NA,
RIK
EN fu
ll-le
ngth
enr
iche
d lib
rary
, clo
ne:5
9304
24F1
3 pr
oduc
t:CLI
P-as
soci
atin
g pr
otei
n C
LASP
1, fu
ll in
sert
seq
[AK0
3118
1]0.
493
0.42
4 (0
.345
to 0
.496
)0.
86 (0
.789
to 0
.997
)AK
0320
63M
us m
uscu
lus
adul
t mal
e m
edul
la o
blon
gata
cD
NA,
RIK
EN fu
ll-le
ngth
enr
iche
d lib
rary
, clo
ne:6
3305
68D
16 p
rodu
ct:u
ncla
ssifi
able
, ful
l ins
ert s
eque
nce.
[AK0
3206
3]0.
512
0.36
7 (0
.347
to 0
.394
)0.
717
(0.6
93 to
0.7
35)
Grin
2dN
M_0
0817
2M
us m
uscu
lus
glut
amat
e re
cept
or, i
onot
ropi
c, N
MD
A2D
(eps
ilon
4) (G
rin2d
), m
RN
A [N
M_0
0817
2]0.
519
0.47
5 (0
.255
to 0
.885
)0.
916
(0.7
96 to
1.0
57)
Muc
10N
M_0
0864
4M
us m
uscu
lus
muc
in 1
0, s
ubm
andi
bula
r gla
nd s
aliv
ary
muc
in (M
uc10
), m
RN
A [N
M_0
0864
4]0.
572
0.46
6 (0
.399
to 0
.521
)0.
815
(0.6
2 to
1.0
23)
Ccl
12N
M_0
1133
1M
us m
uscu
lus
chem
okin
e (C
-C m
otif)
liga
nd 1
2 (C
cl12
), m
RN
A [N
M_0
1133
1]0.
575
0.48
5 (0
.333
to 0
.84)
0.84
3 (0
.508
to 1
.495
)Fi
gla
NM
_012
013
Mus
mus
culu
s fa
ctor
in th
e ge
rmlin
e al
pha
(Fig
la),
mR
NA
[NM
_012
013]
0.58
60.
468
(0.2
68 to
0.6
2)0.
798
(0.7
7 to
0.8
3)Pc
dha5
NM
_009
959
Mus
mus
culu
s pr
otoc
adhe
rin a
lpha
5 (P
cdha
5), m
RN
A [N
M_0
0995
9]
Gom
ez-O
spin
a, T
suru
ta, B
arre
to-C
hang
, Hu
and
Dol
met
sch
Supp
lem
enta
ry T
able
1
CC
AT/C
CAT
TASy
nony
ms
Gen
bank
Des
crip
tion
Gen
es s
igni
fican
tly re
gula
ted
by C
CAT
CC
AT/U
ntra
nsfe
cted
CC
AT <
CC
ATTA
CC
ATTA
/Unt
rans
fect
ed
CC
AT/C
CAT
TA
CC
AT >
CC
ATTA
CC
AT/U
ntra
nsfe
cted
CC
ATTA
/Unt
rans
fect
edSy
nony
ms
Gen
bank
Des
crip
tion
Tab
le 1
. Gen
es S
igni
fican
tly u
preg
ulat
ed b
y C
CA
T o
vere
xpre
ssio
n
73
Gene name Description Fold Changenovel proteins-32 genesNAP050965-1 Unknown 12.6AK046243 adult male corpora quadrigemina cDNA, RIKEN full-length enriched library, clone:B230359I22 product:hypothetical LIM domain, Villin headpiece domain containing protein, full insert sequence. [AK046243] 2.416AK036079 16 days neonate cerebellum cDNA, RIKEN full-length enriched library, clone:9630032O13 product:unknown EST, full insert sequence [AK036079] 2.219530076H17 unknown EST [9530076H17] 2.187BC026942 RIKEN cDNA 2610036N15 gene, (cDNA clone MGC:30353 IMAGE:5011571), complete cds. [BC026942] 2.116AK032743 12 days embryo male wolffian duct includes surrounding region cDNA, RIKEN full-length enriched library, clone:6720426C15 product:CLAUDIN-19 (FRAGMENT), full insert sequence. [AK032743] 2.011D130050C24 unclassifiable [D130050C24] 2.004NM_145463 RIKEN cDNA 9430059P22 gene (9430059P22Rik), [NM_145463] 1.976NAP071275-1 Unknown 1.903NAP104086-1 for mKIAA1398 protein. [AK129349] 1.896B930011K02 unclassifiable [B930011K02] 1.892E030017C01 hypothetical protein [E030017C01] 1.872A130026M04 unclassifiable [A130026M04] 1.821NAP051804-1 Unknown 1.819B930091H02 unknown EST [B930091H02] 1.8C430019P20 unknown EST [C430019P20] 1.788A930002G02 hypothetical SAM domain (Sterile alpha motif) containing protein [A930002G02] 1.787NAP034432-1 Unknown 1.776E230011A12 unknown EST [E230011A12] 1.77BC006743 cDNA clone MGC:12079 IMAGE:3708702, complete cds. [BC006743] 1.724AK019190 10 days embryo whole body cDNA, RIKEN full-length enriched library, clone:2610510H03 product:hypothetical protein, full insert sequence. [AK019190] 1.619NM_025832 RIKEN cDNA 1300019C06 gene (1300019C06Rik), [NM_025832] 1.605AK085030 13 days embryo lung cDNA, RIKEN full-length enriched library, clone:D430026K21 product:hypothetical ARM repeat structure containing protein, full insert sequence. [AK085030] 1.594AK003565 18-day embryo whole body cDNA, RIKEN full-length enriched library, clone:1110008E08 product:hypothetical protein, full insert sequence. [AK003565] 1.556BC023450 cDNA sequence BC035291, (cDNA clone IMAGE:5054193), with apparent retained intron. [BC023450] 1.535NAP029182-1 Unknown 1.511NM_028170 RIKEN cDNA 1700030K09 gene (1700030K09Rik), [NM_028170] 1.491AK005714 adult male testis cDNA, RIKEN full-length enriched library, clone:1700007H20 product:hypothetical Heat shock hsp20 (alpha crystallin) proteins family containing protein, full insert sequence. [AK005714] 1.478C330007P06 hypothetical protein [C330007P06] 1.476NM_029532 RIKEN cDNA 6330548G22 gene (6330548G22Rik), [NM_029532] 1.457AK030240 adult male testis cDNA, RIKEN full-length enriched library, clone:4933429B21 product:zinc finger protein 13, full insert sequence. [AK030240] 1.453NAP030565-1 Unknown 1.452Transcription -8 genesNM_013926 chromobox homolog 8 (Drosophila Pc class) (Cbx8), [NM_013926] 1.499NM_010377 histone 1, H1t (Hist1h1t), [NM_010377] 1.509NM_010234 FBJ osteosarcoma oncogene (Fos), [NM_010234] 1.681NAP056339-1 S79410 nuclear localization signal binding protein {}, partial (12%) [TC1084061] 2.378E430001J03 MYC PROTO-ONCOGENE PROTEIN (C-MYC) [E430001J03] 1.51E030018P21 general transcription factor II I [E030018P21] 2.077D130023D18 TAFII140 PROTEIN (FRAGMENT) homolog [] [D130023D18] 1.532ENSMUST00000036004 HNRNP A1 (FRAGMENT). [Source:SPTREMBL;Acc:P70370] [ENSMUST00000036004] 1.499Signaling molecules- 6 genesNM_031256 pleckstrin homology domain-containing, family A (phosphoinositide binding specific) member 3 (Plekha3), [NM_031256] 1.727NM_008813 ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1), [NM_008813] 1.477NM_007913 early growth response 1 (Egr1), [NM_007913] 1.587C530047D22 transducer of ERBB2, 2 [C530047D22] 1.5BC005656 regulator of G-protein signaling 5, (cDNA clone IMAGE:3710250), complete cds. [BC005656] 3.089AB015614 for SET-binding protein (SEB), partial cds. [AB015614] 2.686Receptor and adhesion molecules - 6 genesX12905 for properdin (AA 5 - 441). [X12905] 1.471NM_010688 LIM and SH3 protein 1 (Lasp1), [NM_010688] 2.308NM_009142 chemokine (C-X3-C motif) ligand 1 (Cx3cl1), [NM_009142] 1.827NAP040979-1 CKR2_C-C chemokine receptor type 2 (C-C CKR-2) (CC-CKR-2) (CCR-2) (CCR2) (JE/FIC receptor) (MCP-1 receptor). [Mouse] {}, complete [TC968512] 1.874BC059909 gene model 793, (NCBI), (cDNA clone IMAGE:6825008), partial cds. [BC059909] 3.971AF068258 EY-cadherin precursor, , partial cds. [AF068258] 1.9229330162O16 inferred: sprouty homolog 4 (Drosophila) [9330162O16] 1.607cytoskeleton - 5 genes NM_010230 formin (Fmn), [NM_010230] 1.591NAP026121-1 MYOSIN HEAVY CHAIN, FAST SKELETAL MUSCLE, EMBRYONIC (FRAGMENT). [Source:SWISSPROT;Acc:P13541] [ENSMUST00000007302] 1.461M74753 myosin heavy chain , 3' flank. [M74753] 1.481ENSMUST00000068404 ACTIN RELATED PROTEIN 2/3 COMPLEX, SUBUNIT 5; ACTIN RELATED PROTEIN 2/3 COMPLEX, SUBUNIT 5 (165 KDA). [Source:RefSeq;Acc:NM_026369] [ENSMUST00000068404] 1.455BC014809 tropomyosin 2, beta, (cDNA clone MGC:18587 IMAGE:3497670), complete cds. [BC014809] 1.501Metabolic enzymes -3 genesNM_018788 exostoses (multiple)-like 3 (Extl3), [NM_018788] 1.556NAP045478-1 AF199509 NADPH-dependent FMN and FAD containing oxidoreductase {Homo sapiens}, partial (17%) [TC1078483] 6.414BC028276 demethyl-Q 7, (cDNA clone IMAGE:5376178), partial cds. [BC028276] 1.557Translation - 2 genesNAP108110-1 AF337055 lysyl tRNA synthetase {Methanosarcina barkeri}, partial (3%) [TC960574] 1.457NM_007906 eukaryotic translation elongation factor 1 alpha 2 (Eef1a2), [NM_007906] 1.456Proteases- 2 genesNAP031150-1 AF302077 neprilysin-like peptidase gamma {}, partial (5%) [TC1010010] 1.5182700060P05 proteasome (prosome, macropain) 26S subunit, non-ATPase, 7 [2700060P05] 1.483Ion channel and Transporters- 2 genesNM_010291 gap junction membrane channel protein beta 5 (Gjb5), [NM_010291] 14.36NM_009579 solute carrier family 30 (zinc transporter), member 1 (Slc30a1), [NM_009579] 1.496Others- 2 genesNM_008622 Mpv17 transgene, kidney disease mutant (Mpv17), [NM_008622] 8.499NAP037317-1 T2_Octapeptide-repeat protein T2. [Mouse] {}, partial (25%) [TC951701] 1.782
CCAT vs GFP upregulated genes
Table 2. CCAT versus GFP upregulated genes
74
Gene name Description Fold Changenovel proteins-175 genesC230075P17 unclassifiable [C230075P17] -7.6921700034G24 unknown EST [1700034G24] -5.348BC024416 mRNA similar to RIKEN cDNA 9030624G23 gene (cDNA clone MGC:36379 IMAGE:4988668), complete cds [BC024416] -4.2196530405F15 unknown EST [6530405F15] -3.268NM_177210 RIKEN cDNA D830044D21 gene (D830044D21Rik), mRNA [NM_177210] -3.0584631411J20 unknown EST [4631411J20] -2.9241110005G03 unclassifiable [1110005G03] -2.849NM_026690 RIKEN cDNA 0610012D14 gene (0610012D14Rik), mRNA [NM_026690] -2.762A130066H07 unknown EST [A130066H07] -2.6184732470K04 weakly similar to CDNA FLJ25135 FIS, CLONE CBR06974 [Homo sapiens] [4732470K04] -2.4884933412F11 inferred: RIKEN cDNA 4933412F11 gene [4933412F11] -2.481AK016814 adult male testis cDNA, RIKEN full-length enriched library, clone:4933415A04 product:hypothetical protein, full insert sequence. [AK016814] -2.463NAP027343-1 Unknown -2.451B930066C19 unclassifiable [B930066C19] -2.415NP580029 BAC29978.1 unnamed protein product [] [NP580029] -2.315NAP065110-1 Unknown -2.2529630032J19 unknown EST [9630032J19] -2.2171700091C04 unknown EST [1700091C04] -2.198D830035G22 hypothetical Hypothetical protein HI1434 (YbaK homologue) structure containing protein [D830035G22] -2.1653110001P10 unclassifiable [3110001P10] -2.151ENSMUST00000049565 RIKEN cDNA 9530080O11 gene (9530080O11Rik), mRNA [NM_175680] -2.151ENSMUST00000052692 RIKEN cDNA 1110006O24 gene (1110006O24Rik), mRNA [NM_021417] -2.137AK033606 adult male cecum cDNA, RIKEN full-length enriched library, clone:9130025L13 product:similar to FGF RECEPTOR 4B [Homo sapiens], full insert sequence. [AK033606] -2.137NAP037294-1 Unknown -2.1324833401H05 unclassifiable [4833401H05] -2.1327030417J21 hypothetical protein [7030417J21] -2.110NAP012952-001 Unknown -2.083AK014285 17 days embryo head cDNA, RIKEN full-length enriched library, clone:3200001I04 product:hypothetical Cysteine-rich flanking region, C-terminal/Leucine-rich repeat/Leucine-rich repeat, typical subtype containing protein, full insert sequence -2.079NM_177850 RIKEN cDNA 9230105K17 gene (9230105K17Rik), mRNA [NM_177850] -2.058ENSMUST00000056761 adult male testis cDNA, RIKEN full-length enriched library, clone:4930505N22 product:hypothetical protein, full insert sequence. [AK015708] -2.058NAP051032-1 UI-M-HB0-clk-h-02-0-UI.r1 NIH_BMAP_HB0 cDNA clone IMAGE:30619849 5', mRNA sequence [CF742218] -2.033B830034B11 unknown EST [B830034B11] -2.012BC020151 cDNA sequence BC020151, mRNA (cDNA clone MGC:28382 IMAGE:4021767), complete cds. [BC020151] -2.008D230050L11 unclassifiable [D230050L11] -2.000AK015919 adult male testis cDNA, RIKEN full-length enriched library, clone:4930527J03 product:hypothetical Alanine-rich region containing protein, full insert sequence. [AK015919] -1.9928430403D15 unclassifiable [8430403D15] -1.988BC025887 open reading frame 34, mRNA (cDNA clone IMAGE:5252967), partial cds. [BC025887] -1.976BI151098 BI151098 602917037F1 NCI_CGAP_Lu29 cDNA clone IMAGE:5067368 5', mRNA sequence [BI151098] -1.953B230379C01 unknown EST [B230379C01] -1.946C530030P08 hypothetical protein [C530030P08] -1.946ENSMUST00000051080 adult male testis cDNA, RIKEN full-length enriched library, clone:4930516E05 product:hypothetical Gram-positive cocci surface protein 'anchoring' hexapeptide containing protein, full insert sequence. [AK015803] -1.942NAP014274-001 Unknown -1.938D030041L05 unclassifiable [D030041L05] -1.934E130309O17 hypothetical protein [E130309O17] -1.927AK005010 adult male liver cDNA, RIKEN full-length enriched library, clone:1300015B04 product:similar to CDNA FLJ32009 FIS, CLONE NT2RP7009498, WEAKLY SIMILAR TO FIBULIN-1, ISOFORM A PRECURSOR [Homo sapiens], full insert sequence [AK005010]-1.923AK049933 adult male hippocampus cDNA, RIKEN full-length enriched library, clone:C630012M08 product:SNRNA ACTIVATING PROTEIN COMPLEX 50 KDA SUBUNIT(PROXIMAL SEQUENCE ELEMENT-BINDING TRANSCRIPTION FACTOR BETA -1.923C030002C18 unknown EST [C030002C18] -1.919AK077155 adult male testis cDNA, RIKEN full-length enriched library, clone:4933414G08 product:weakly similar to HYPOTHETICAL 68.7 KDA PROTEIN [Macaca fascicularis], full insert sequence. [AK077155] -1.919ENSMUST00000060815 Unknown -1.916NAP062636-1 UI-M-CG0p-bdb-d-11-0-UI.s1 NIH_BMAP_Ret4_S2 cDNA clone UI-M-CG0p-bdb-d-11-0-UI 3'. [BE981373] -1.912A930019J01 unknown EST [A930019J01] -1.905E330009A12 unknown EST [E330009A12] -1.901NAP068274-1 RIKEN cDNA 6030419C18 gene (6030419C18Rik), mRNA [NM_176921] -1.901AK029554 adult male testis cDNA, RIKEN full-length enriched library, clone:4921526F01 product:weakly similar to PROTEIN C21ORF13 [Homo sapiens], full insert sequence. [AK029554] -1.8989430062F24 unclassifiable [9430062F24] -1.8946720477H23 unknown EST [6720477H23] -1.890AK003303 18-day embryo whole body cDNA, RIKEN full-length enriched library, clone:1110002J03 product:unknown EST, full insert sequence [AK003303] -1.890AK037260 16 days neonate thymus cDNA, RIKEN full-length enriched library, clone:A130001I01 product:inferred: RIKEN cDNA 2810488G03 gene, full insert sequence. [AK037260] -1.890NP573446 BAC36495.1 unnamed protein product [] [NP573446] -1.883NM_177051 RIKEN cDNA C730014E05 gene (C730014E05Rik), mRNA [NM_177051] -1.883ENSMUST00000055270 2 days pregnant adult female ovary cDNA, RIKEN full-length enriched library, clone:E330039O16 product:unknown EST, full insert sequence. [AK087910] -1.883NAP103733-1 Unknown -1.880A430101J24 unclassifiable [A430101J24] -1.880NAP071390-1 RIKEN cDNA B830017H08 gene (B830017H08Rik), mRNA [NM_001002790] -1.876AK018840 adult male testis cDNA, RIKEN full-length enriched library, clone:1700034E13 product:hypothetical Zinc finger, C2H2 type containing protein, full insert sequence. [AK018840] -1.876NAP035983-1 Unknown -1.873NAP097891-001 Unknown -1.862AK015956 adult male testis cDNA, RIKEN full-length enriched library, clone:4930533K18 product:hypothetical protein, full insert sequence. [AK015956] -1.859ENSMUST00000059184 adult male testis cDNA, RIKEN full-length enriched library, clone:4930423F13 product:hypothetical protein, full insert sequence. [AK019581] -1.859NAP050809-1 Unknown -1.855A130057H05 unknown EST [A130057H05] -1.842C630018D16 unknown EST [C630018D16] -1.835E030043F12 RIKEN cDNA 2210414H16 gene [E030043F12] -1.828AK016492 adult male testis cDNA, RIKEN full-length enriched library, clone:4931430N09 product:unclassifiable, full insert sequence [AK016492] -1.828Gene name Description Fold Change5033423K11 hypothetical protein [5033423K11] -1.821AK003577 18-day embryo whole body cDNA, RIKEN full-length enriched library, clone:1110008I14 product:hypothetical SEA domain containing protein, full insert sequence. [AK003577] -1.818NM_026808 RIKEN cDNA 1110028A07 gene (1110028A07Rik), mRNA [NM_026808] -1.805AI467211 vd74h04.x1 Beddington embryonic region cDNA clone IMAGE:806359 3'. [AI467211] -1.802AK015063 adult male testis cDNA, RIKEN full-length enriched library, clone:4930403J07 product:hypothetical Double-stranded RNA binding (DsRBD) domain/Adenosine-deaminase (editase) domain containing protein, full insert sequence. [AK015063] -1.7921500031H18 unknown EST [1500031H18] -1.786E130307J04 hypothetical protein [E130307J04] -1.786E130318F11 unknown EST [E130318F11] -1.779AA738637 vv59h10.r1 Soares_thymus_2NbMT cDNA clone IMAGE:1226755 5'. [AA738637] -1.7731110038B08 inferred: homologue to bA12M19.1.3 (novel protein) {Homo sapiens} [1110038B08] -1.773NAP063026-1 similar to hypothetical protein FLJ38281 (LOC382019), mRNA [XM_356088] -1.773AK041206 adult male aorta and vein cDNA, RIKEN full-length enriched library, clone:A530090G11 product:R KAPPA B homolog [Homo sapiens], full insert sequence. [AK041206] -1.7732900090F08 unknown EST [2900090F08] -1.770BC021614 cDNA sequence BC021614, mRNA (cDNA clone MGC:37914 IMAGE:5102505), complete cds. [BC021614] -1.764AK011391 10 days embryo whole body cDNA, RIKEN full-length enriched library, clone:2610014F08 product:hypothetical SAM domain (Sterile alpha motif)/Modified RING finger domain/G-protein beta WD-40 repeats containing protein, full insert sequence. [A-1.764C230075A15 unclassifiable [C230075A15] -1.761AK006745 adult male testis cDNA, RIKEN full-length enriched library, clone:1700049J03 product:hypothetical protein, full insert sequence. [AK006745] -1.757E130120I19 unclassifiable [E130120I19] -1.754AK034394 adult male diencephalon cDNA, RIKEN full-length enriched library, clone:9330185P08 product:CDNA FLJ10704 FIS, CLONE NT2RP3000841 homolog [Homo sapiens], full insert sequence. [AK034394] -1.7424932416K20 hypothetical protein [4932416K20] -1.736AK035222 adult male urinary bladder cDNA, RIKEN full-length enriched library, clone:9530003A11 product:hypothetical Copper amine oxidase containing protein, full insert sequence. [AK035222] -1.736AK006033 adult male testis cDNA, RIKEN full-length enriched library, clone:1700016H13 product:hypothetical protein, full insert sequence. [AK006033] -1.736AK081177 10 days neonate cerebellum cDNA, RIKEN full-length enriched library, clone:B930096O19 product:CGI-40 PROTEIN homolog [Homo sapiens], full insert sequence. [AK081177] -1.730NM_026208 RIKEN cDNA 1700019N19 gene (1700019N19Rik), mRNA [NM_026208] -1.724AK016497 adult male testis cDNA, RIKEN full-length enriched library, clone:4931431F19 product:hypothetical Ubiquitin domain containing protein, full insert sequence. [AK016497] -1.724A730045E23 unknown EST [A730045E23] -1.721NM_021447 ring finger protein 30 (Rnf30), mRNA [NM_021447] -1.718NAP106603-1 Unknown -1.712NM_030890 open reading frame 31 (ORF31), mRNA [NM_030890] -1.712NAP029297-1 Unknown -1.709NM_206973 RIKEN cDNA A930009H15 gene (A930009H15Rik), mRNA [NM_206973] -1.704D830005K03 unknown EST [D830005K03] -1.698NM_025584 RIKEN cDNA 2410026K10 gene (2410026K10Rik), mRNA [NM_025584] -1.695NM_023258 PYD and CARD domain containing (Pycard), mRNA [NM_023258] -1.695AK006273 adult male testis cDNA, RIKEN full-length enriched library, clone:1700023F06 product:hypothetical protein, full insert sequence. [AK006273] -1.695NAP044523-1 Unknown -1.6862210403N08 hypothetical protein [2210403N08] -1.686NAP033236-1 Unknown -1.681Transcription -34 genesENSMUST00000053071 TRANSCRIPTION FACTOR GATA-6 (GATA BINDING FACTOR-6). [Source:SWISSPROT;Acc:Q61169] [ENSMUST00000053071] -2.494AK043538 10 days neonate cortex cDNA, RIKEN full-length enriched library, clone:A830006N08 product:hypothetical Ankyrin repeat region circular profile/Yeast DNA-binding domain containing protein, full insert sequence. [AK043538] -2.475NM_008814 insulin promoter factor 1, homeodomain transcription factor (Ipf1), mRNA [NM_008814] -2.353NM_183298 forkhead box E1 (thyroid transcription factor 2) (Foxe1), mRNA [NM_183298] -2.247NM_146065 activating transcription factor 7 (Atf7), mRNA [NM_146065] -2.174AY364010 NALP12 mRNA, partial cds. [AY364010] -2.165AB010307 mRNA for mszf2, partial cds. [AB010307] -2.041X51959 Brn-3 gene POU-box region. [X51959] -2.016NM_009237 SRY-box containing gene 3 (Sox3), mRNA [NM_009237] -1.992NM_015758 forkhead box E3 (Foxe3), mRNA [NM_015758] -1.972AK077696 8 days embryo whole body cDNA, RIKEN full-length enriched library, clone:5730530B02 product:weakly similar to HYPOTHETICAL 30.1 KDA PROTEIN (TGF BETA INDUCIBLE NUCLEAR PROTEIN TINP1) (HAIRY CELL LEUKEMIA -1.908NM_009576 zinc finger protein of the cerebellum 4 (Zic4), mRNA [NM_009576] -1.869AK040404 0 day neonate thymus cDNA, RIKEN full-length enriched library, clone:A430091O22 product:hypothetical RNA-binding region RNP-1 (RNA recognition motif) containing protein, full insert sequence. [AK040404] -1.845NM_152947 zinc finger protein 339 (Zfp339), transcript variant B, mRNA [NM_152947] -1.825NM_178192 histone 1, H4a (Hist1h4a), mRNA [NM_178192] -1.799AK017633 8 days embryo whole body cDNA, RIKEN full-length enriched library, clone:5730441M18 product:hypothetical Zinc finger, C2H2 type containing protein, full insert sequence. [AK017633] -1.779
CCAT vs GFP downregulated genes
Table 3. CCAT versus GFP downregulated genes
75
ENSMUST00000023456 YIPPEE-LIKE PROTEIN 1 (DGL-1) (MDGL-1). [Source:SWISSPROT;Acc:Q9ESC7] [ENSMUST00000023456] -1.748NAP048862-1 ets homologous factor (Ehf), mRNA [NM_007914] -1.742AY080897 onecut 3 mRNA, complete cds. [AY080897] -1.695E130320J01 runt related transcription factor 3 [E130320J01] -1.689Signaling molecules- 36 genesAJ539223 mRNA for erythroid differentiation regulator (edr gene). [AJ539223] -3.106NM_009362 trefoil factor 1 (Tff1), mRNA [NM_009362] -2.899NM_007445 anti-Mullerian hormone (Amh), mRNA [NM_007445] -2.469NM_008712 nitric oxide synthase 1, neuronal (Nos1), mRNA [NM_008712] -2.342NAP102169-1 oxytocin (Oxt), mRNA [NM_011025] -2.331NM_009379 thrombopoietin (Thpo), mRNA [NM_009379] -2.3201500036H07 weakly similar to ARF GTPASE-ACTIVATING PROTEIN GIT2 (G PROTEIN-COUPLED RECEPTOR KINASE- INTERACTOR 2) (TYROSINE-PHOSPHORYLATED PROTEIN CAT-2) [] [1500036H07] -2.183U49723 guanylyl cyclase C (Gucy2c) mRNA, partial cds. [U49723] -2.037D930034C02 protein tyrosine phosphatase, non-receptor type 14 [D930034C02] -2.004E130103H07 inferred: ADP-ribosylation factor-directed GTPase activating protein isoform b {} [E130103H07] -1.9926330436O20 hypothetical Sushi domain / SCR repeat / CCP module containing protein [6330436O20] -1.992ENSMUST00000053066 SIMILAR TO QUAKING II. [Source:SPTREMBL;Acc:Q8K4Y1] [ENSMUST00000053066] -1.927NM_010473 histidine rich calcium binding protein (Hrc), mRNA [NM_010473] -1.927NM_011477 small proline-rich protein 2K (Sprr2k), mRNA [NM_011477] -1.912NAP019468-001 hematopoietic cell signal transducer (Hcst), mRNA [NM_011827] -1.883Gene name Description Fold ChangeNAP106185-1 AXU1_ AXIN1 up-regulated gene 1 protein (TGF-beta induced apoptosis protein 3) (TAIP-3). [] {}, partial (33%) [TC1023468] -1.869NM_007522 Bcl-associated death promoter (Bad), mRNA [NM_007522] -1.859NM_016933 protein tyrosine phosphatase, receptor type, C polypeptide-associated protein (Ptprcap), mRNA [NM_016933] -1.848NM_199042 THAP domain containing, apoptosis associated protein 1 (Thap1), mRNA [NM_199042] -1.835NM_031192 renin 1 structural (Ren1), mRNA [NM_031192] -1.835NM_033614 phosphodiesterase 6C, cGMP specific, cone, alpha prime (Pde6c), mRNA [NM_033614] -1.783NM_011108 phospholipase A2, group IIA (platelets, synovial fluid) (Pla2g2a), mRNA [NM_011108] -1.776NM_007828 death-associated kinase 3 (Dapk3), mRNA [NM_007828] -1.770AK053554 0 day neonate eyeball cDNA, RIKEN full-length enriched library, clone:E130107I17 product:INOSITOL POLYPHOSPHATE 4-PHOSPHATASE TYPE II-ALPHA homolog [Rattus norvegicus], full insert sequence. [AK053554] -1.739NAP027959-1 similar to ERF-2 (LOC333473), mRNA [XM_285657] -1.721L33768 (clone 32D5-1) protein tyrosine kinase (JAK3) mRNA. [L33768] -1.7182610524P08 Era (G-protein)-like 1 (E. coli) [2610524P08] -1.701Receptor and adhesion molecules - 29 genesNM_146275 olfactory receptor 1402 (Olfr1402), mRNA [NM_146275] -4.237NM_031499 proline rich protein 2 (Prp2), mRNA [NM_031499] -2.494NAP000791-001 AB065580 seven transmembrane helix receptor {Homo sapiens}, partial (9%) [TC1021675] -2.331AF199614 fibroblast growth factor homologous factor 2 isoform 1U (FHF-2) mRNA, partial cds. [AF199614] -2.208NM_010704 leptin receptor (Lepr), mRNA [NM_010704] -2.174NM_023304 fibroblast growth factor 22 (Fgf22), mRNA [NM_023304] -2.155NM_146649 olfactory receptor 1160 (Olfr1160), mRNA [NM_146649] -2.1052310003H23 LEUCINE-RICH-DOMAIN INTER-ACTING PROTEIN 1 (PPARGAMMA COFACTOR 2) (PEROXISOME PROLIFERATIVE ACTIVATED RECEPTOR, GAMMA, COACTIVATOR 2) homolog [] [2310003H23] -2.062NM_001001999 glycoprotein Ib, beta polypeptide (Gp1bb), transcript variant 1, mRNA [NM_001001999] -2.016NM_015738 galanin receptor 3 (Galr3), mRNA [NM_015738] -1.980S78451 interleukin-3 receptor beta subunit [mice, D35 promyelocytic cells, mRNA Partial Mutant, 331 nt]. [S78451] -1.946NM_020291 olfactory receptor 480 (Olfr480), mRNA [NM_020291] -1.852NAP053944-1 AF093669 peroxisomal biogenesis factor {}, partial (8%) [TC1055468] -1.835BC051445 interleukin 1 receptor-like 1, mRNA (cDNA clone MGC:60556 IMAGE:30073421), complete cds. [BC051445] -1.835AF464177 protocadherin mRNA, partial cds; alternatively spliced. [AF464177] -1.783BC030075 T-cell receptor beta, variable 13, mRNA (cDNA clone MGC:41416 IMAGE:1531980), complete cds. [BC030075] -1.779NM_011174 proline rich protein HaeIII subfamily 1 (Prh1), mRNA [NM_011174] -1.727AK048080 16 days embryo head cDNA, RIKEN full-length enriched library, clone:C130033F22 product:CADHERIN FIB1 (FRAGMENT) homolog [Homo sapiens], full insert sequence [AK048080] -1.727NAP107834-1 AF195661 transmembrane protein I1 {Homo sapiens}, partial (81%) [TC1021639] -1.704cytoskeleton - 9 genes NM_172868 paralemmin 2 (Palm2), mRNA [NM_172868] -2.315NM_009711 artemin (Artn), mRNA [NM_009711] -2.304NM_016887 claudin 7 (Cldn7), mRNA [NM_016887] -2.066AK088011 2 days neonate thymus thymic cells cDNA, RIKEN full-length enriched library, clone:E430002D17 product:UNCONVENTIONAL MYOSIN 1G VALINE FORM (FRAGMENT) homolog [Homo sapiens], full insert sequence. [AK088011] -2.004NP063775 AAA79963.1 synapsin I [NP063775] -1.969ENSMUST00000055959 synaptopodin (Synpo), mRNA [XM_129030] -1.802AF205079 strain ICR 90 kDa actin-associated protein palladin mRNA, partial cds. [AF205079] -1.764Metabolic enzymes -13 genesNM_133657 cytochrome P450, family 2, subfamily a, polypeptide 12 (Cyp2a12), mRNA [NM_133657] -2.012NM_010292 glucokinase (Gck), mRNA [NM_010292] -1.992NM_175140 carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 8 (Chst8), mRNA [NM_175140] -1.927ENSMUST00000051542 SEPIAPTERIN REDUCTASE. [Source:SPTREMBL;Acc:Q62218] [ENSMUST00000051542] -1.845NM_021306 endothelin converting enzyme-like 1 (Ecel1), mRNA [NM_021306] -1.832M36289 beta-1,4-galactosyltransferase mRNA, 5' end. [M36289] -1.7838030457O12 cytosolic 5' nucleotidase, type 1A [8030457O12] -1.761NM_008437 napsin A aspartic peptidase (Napsa), mRNA [NM_008437] -1.718NM_011637 three prime repair exonuclease 1 (Trex1), mRNA [NM_011637] -1.692Gene name Description Fold ChangeProteases- 10 genesNM_008950 protease (prosome, macropain) 26S subunit, ATPase 5 (Psmc5), mRNA [NM_008950] -4.098NM_009776 serine (or cysteine) proteinase inhibitor, clade G, member 1 (Serping1), mRNA [NM_009776] -2.151NM_011458 serine (or cysteine) proteinase inhibitor, clade A, member 3K (Serpina3k), mRNA [NM_011458] -1.828NM_008407 inter-alpha trypsin inhibitor, heavy chain 3 (Itih3), mRNA [NM_008407] -1.773NM_009430 protease, serine, 2 (Prss2), mRNA [NM_009430] -1.767AB049453 tessp-1 mRNA for testis serine protease-1, complete cds. [AB049453] -1.761NAP102783-1 similar to SPI3C (LOC193403), mRNA [XM_111405] -1.712Ion channel and Transporters- 16 genesNAP019559-001 AF115505 sodium/calcium exchanger 1 splice variant NaCa10 {Homo sapiens}, partial (7%) [TC1006772] -2.604NAP029777-1 similar to potassium channel, subfamily K, member 9 (Task-3) (LOC223604), mRNA [XM_139425] -2.577NM_011773 solute carrier family 30 (zinc transporter), member 3 (Slc30a3), mRNA [NM_011773] -2.008NM_008172 glutamate receptor, ionotropic, NMDA2D (epsilon 4) (Grin2d), mRNA [NM_008172] -2.000NM_007378 ATP-binding cassette, sub-family A (ABC1), member 4 (Abca4), mRNA [NM_007378] -1.942NM_080466 potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3 (Kcnn3), mRNA [NM_080466] -1.842BC023117 solute carrier family 6 (neurotransmitter transporter, GABA), member 13, mRNA (cDNA clone MGC:28956 IMAGE:4240641), complete cds. [BC023117] -1.8322210409B01 NG22 PROTEIN homolog [Homo sapiens] [2210409B01] -1.773NM_022017 transient receptor potential cation channel, subfamily V, member 4 (Trpv4), mRNA [NM_022017] -1.770NM_205783 cholinergic receptor, muscarinic 5 (Chrm5), mRNA [NM_205783] -1.751AY255605 gamma-aminobutyric acid type B receptor mRNA, partial cds. [AY255605] -1.701BC039157 glutamate receptor, ionotropic, NMDA1 (zeta 1), mRNA (cDNA clone MGC:25375 IMAGE:4507986), complete cds. [BC039157] -1.678Extracellular matrix proteins - 7 genesNM_015784 periostin, osteoblast specific factor (Postn), mRNA [NM_015784] -1.992NM_053185 procollagen, type IV, alpha 6 (Col4a6), mRNA [NM_053185] -1.812NM_146007 procollagen, type VI, alpha 2 (Col6a2), mRNA [NM_146007] -1.730NM_139001 chondroitin sulfate proteoglycan 4 (Cspg4), mRNA [NM_139001] -1.706Others- 20 genesF830014N06 lymphocyte antigen 108 [F830014N06] -3.300AB032764 halap-X mRNA for haploid specific alanine-rich acidic protein, complete cds. [AB032764] -2.358Z31359 M.musculus (Balb/C) HTx02 mRNA. [Z31359] -2.062NAP100607-001 AE003774 CG31019-PA {Drosophila melanogaster}, partial (16%) [TC1024875] -1.996BC024677 allergen dI chain C2A, mRNA (cDNA clone MGC:19070 IMAGE:4193111), complete cds. [BC024677] -1.953BC051228 heat shock protein, alpha-crystallin-related, B6, mRNA (cDNA clone IMAGE:3471643), partial cds. [BC051228] -1.876U63712 testis-specific HMG-box protein m-tsHMG precursor mRNA, partial cds, and mitochondrial transcription factor m-mtTFA precursor mRNA, nuclear mRNA encoding mitochondrial protein, partial cds. [U63712] -1.859NM_011859 odd-skipped related 1 (Drosophila) (Odd1), mRNA [NM_011859] -1.835NM_012033 tubulointerstitial nephritis antigen (Tinag), mRNA [NM_012033] -1.783NM_013480 bone gamma-carboxyglutamate protein, related sequence 1 (Bglap-rs1), mRNA [NM_013480] -1.779NM_008995 peroxisome biogenesis factor 5 (Pex5), mRNA [NM_008995] -1.767AK046712 4 days neonate male adipose cDNA, RIKEN full-length enriched library, clone:B430320C24 product:weakly similar to HYPOTHETICAL 13.8 KDA PROTEIN [Homo sapiens], full insert sequence [AK046712] -1.761S83543 Cer-1=cerebellar-expressed gene/granule neuron differentiation-associated gene [mice, cerebellum, granule neurons, mRNA Partial, 622 nt]. [S83543] -1.727AK017618 8 days embryo whole body cDNA, RIKEN full-length enriched library, clone:5730435J01 product:hypothetical Serine-rich region containing protein, full insert sequence. [AK017618] -1.712NM_010245 Friend virus susceptibility 4 (Fv4), mRNA [NM_010245] -1.689
Table 3. CCAT versus GFP downregulated genes Page 2
76
Gen
es u
preg
ulat
ed b
y C
CAT
in a
ll ex
perim
ents
>1.
8 fo
ld
CC
AT v
rs c
ontr
olC
CAT
vrs
UN
TC
CAT
TA v
rs U
NT
Gjb
5N
M_0
1029
114
.57
(13.
39 to
15.
55)
8.96
8 (5
.687
to 1
3.15
)1.
225
(1.2
05 to
1.2
67)
Mus
mus
culu
s ga
p ju
nctio
n m
embr
ane
chan
nel p
rote
in b
eta
5 (G
jb5)
, mR
NA
[NM
_010
291]
Fmn1
NM
_010
230
1.65
3 (1
.642
to 1
.663
)7.
836
(5.1
04 to
9.7
2)16
.37
(14.
58 to
17.
78)
Mus
mus
culu
s fo
rmin
1 (F
mn1
), m
RN
A [N
M_0
1023
0]M
pv17
NM
_008
622
2.85
3 (0
.32
to 9
.876
)3.
812
(2.9
5 to
5.2
09)
1.06
8 (1
.055
to 1
.08)
Mus
mus
culu
s M
pv17
tran
sgen
e, k
idne
y di
seas
e m
utan
t (M
pv17
), m
RN
A [N
M_0
0862
2]C
x3cl
1N
M_0
0914
21.
821
(1.3
84 to
2.1
82)
3.13
6 (2
.51
to 3
.814
)1.
899
(1.7
29 to
2.0
82)
Mus
mus
culu
s ch
emok
ine
(C-X
3-C
mot
if) li
gand
1 (C
x3cl
1), m
RN
A [N
M_0
0914
2]Az
gp1
AY24
8694
;2.
031
(1.5
35 to
2.6
18)
2.88
(2.5
81 to
3.0
62)
1.84
2 (1
.558
to 2
.042
)M
us m
uscu
lus
zinc
-alp
ha-2
-gly
copr
otei
n 1
(Azg
p1) m
RN
A, c
ompl
ete
cds
[AY2
4869
4]Tr
im30
NM
_009
099
2.07
4 (1
.723
to 2
.416
)2.
611
(1.9
21 to
3.3
36)
2.25
9 (1
.842
to 2
.759
)M
us m
uscu
lus
tripa
rtite
mot
if pr
otei
n 30
(Trim
30),
mR
NA
[NM
_009
099]
Rim
bp2
XM_1
3239
61.
755
(1.7
07 to
1.8
47)
2.50
1 (2
.377
to 2
.619
)2.
543
(2.2
36 to
2.7
69)
Mus
mus
culu
s R
IMS
bind
ing
prot
ein
2, tr
ansc
ript v
aria
nt 1
Cat
nal1
NM
_018
761
3.54
8 (2
.99
to 4
.127
)2.
468
(1.7
58 to
3.0
41)
0.84
4 (0
.804
to 0
.876
)M
us m
uscu
lus
cate
nin
alph
a-lik
e 1
(Cat
nal1
), m
RN
A [N
M_0
1876
1]Se
leN
M_0
1134
51.
752
(1.2
65 to
3.1
88)
2.41
3 (1
.747
to 2
.873
)1.
947
(1.3
44 to
2.9
68)
Mus
mus
culu
s se
lect
in, e
ndot
helia
l cel
l (Se
le),
mR
NA
[NM
_011
345]
Slfn
10N
M_1
8154
21.
731
(1.4
67 to
2.1
49)
2.18
6 (1
.808
to 2
.82)
2.75
3 (1
.902
to 3
.375
)M
us m
uscu
lus
schl
afen
10
(Slfn
10),
mR
NA
[NM
_181
542]
1700
011H
14R
ikNM
_025
956
1.82
2 (1
.552
to 2
.129
)2.
148
(2.0
49 to
2.3
61)
1.11
2 (1
.038
to 1
.167
)M
us m
uscu
lus
RIK
EN c
DN
A 17
0001
1H14
gen
e (1
7000
11H
14R
ik),
mR
NA
[NM
_025
956]
AK08
0352
;1.
803
(1.7
61 to
1.8
31)
2.05
4 (1
.805
to 2
.295
)1.
709
(1.4
05 to
2.0
41)
Mus
mus
culu
s 3
days
neo
nate
thym
us c
DN
A, R
IKEN
full-
leng
th e
nric
hed
libra
ry, c
lone
:A63
0063
H21
pro
duct
:hyp
othe
tical
pro
tein
, ful
l ins
ert s
eque
nce
[AK0
8035
2]V1
rh13
NM
_134
238
3.02
6 (2
.773
to 3
.474
)2.
053
(0.8
03 to
3.3
6)13
.15
(10.
87 to
16.
45)
Mus
mus
culu
s vo
mer
onas
al 1
rece
ptor
, H13
(V1r
h13)
, mR
NA
[NM
_134
238]
Cdo
nN
M_0
2133
92.
173
(2.0
36 to
2.3
17)
2.02
6 (1
.289
to 2
.742
)1.
659
(1.0
52 to
2.2
04)
Mus
mus
culu
s ce
ll ad
hesi
on m
olec
ule-
rela
ted/
dow
n-re
gula
ted
by o
ncog
enes
(Cdo
n), m
RN
A [N
M_0
2133
9]R
gs5
BC00
5656
;3.
106
(2.5
69 to
3.8
)1.
89 (1
.44
to 2
.727
)0.
911
(0.9
02 to
0.9
29)
Mus
mus
culu
s cD
NA
clon
e IM
AGE:
3710
250,
com
plet
e cd
s. [B
C00
5656
]C
ldn1
9N
M_1
5310
51.
988
(1.4
33 to
2.6
66)
1.88
5 (1
.416
to 3
.09)
0.86
6 (0
.86
to 0
.871
)M
us m
uscu
lus
clau
din
19 (C
ldn1
9), m
RN
A [N
M_1
5310
5]
Gen
es d
ownr
egul
ated
by
CC
AT in
all
expe
rimen
ts <
0.5
fold
CC
AT v
rs c
ontr
olC
CAT
vrs
UN
TC
CAT
TA v
rs U
NT
Gck
NM
_010
292
0.19
2 (0
.131
to 0
.32)
0.23
(0.1
67 to
0.2
81)
0.18
3 (0
.138
to 0
.289
)M
us m
uscu
lus
gluc
okin
ase
(Gck
), m
RN
A [N
M_0
1029
2]St
yk1
NM
_172
891
0.25
8 (0
.174
to 0
.357
)0.
466
(0.4
05 to
0.5
36)
0.16
1 (0
.124
to 0
.222
)M
us m
uscu
lus
serin
e/th
reon
ine/
tyro
sine
kin
ase
1 (S
tyk1
), m
RN
A [N
M_1
7289
1]An
krd4
3N
M_1
8317
30.
307
(0.1
71 to
0.8
33)
0.40
4 (0
.317
to 0
.497
)0.
276
(0.2
46 to
0.2
97)
Mus
mus
culu
s an
kyrin
repe
at d
omai
n 43
Mfa
p3BC
0068
28;
0.32
8 (0
.242
to 0
.433
)0.
558
(0.4
29 to
0.6
66)
0.37
3 (0
.221
to 0
.508
)M
us m
uscu
lus
mic
rofib
rilla
r-ass
ocia
ted
prot
ein
3, m
RN
A (c
DN
A cl
one
MG
C:1
1870
IMAG
E:35
9787
8), c
ompl
ete
cds
[BC
0068
28]
Artn
NM
_009
711
0.34
5 (0
.317
to 0
.368
)0.
437
(0.4
16 to
0.4
61)
0.37
8 (0
.359
to 0
.389
)M
us m
uscu
lus
arte
min
(Artn
), m
RN
A [N
M_0
0971
1]G
rin2d
NM
_008
172
0.36
7 (0
.347
to 0
.394
)0.
505
(0.5
03 to
0.5
08)
0.71
7 (0
.693
to 0
.735
)M
us m
uscu
lus
glut
amat
e re
cept
or, i
onot
ropi
c, N
MD
A2D
(eps
ilon
4) (G
rin2d
), m
RN
A [N
M_0
0817
2]Po
stn
NM
_015
784
0.38
4 (0
.359
to 0
.432
)0.
507
(0.4
81 to
0.5
24)
0.33
2 (0
.286
to 0
.409
)M
us m
uscu
lus
perio
stin
, ost
eobl
ast s
peci
fic fa
ctor
(Pos
tn),
mR
NA
[NM
_015
784]
Ltbp
1N
M_0
1991
90.
408
(0.3
22 to
0.4
9)0.
588
(0.4
77 to
0.7
03)
0.32
7 (0
.318
to 0
.344
)M
us m
uscu
lus
late
nt tr
ansf
orm
ing
grow
th fa
ctor
bet
a bi
ndin
g pr
otei
n 1
(Ltb
p1),
trans
crip
t var
iant
1, m
RN
A [N
M_0
1991
9]In
hbc
NM
_010
565
0.41
9 (0
.382
to 0
.443
)0.
468
(0.3
97 to
0.5
21)
0.44
4 (0
.435
to 0
.457
)M
us m
uscu
lus
inhi
bin
beta
-C (I
nhbc
), m
RN
A [N
M_0
1056
5]Pt
prd
AK00
3303
;0.
423
(0.3
82 to
0.4
5)0.
535
(0.5
14 to
0.5
78)
0.43
6 (0
.391
to 0
.462
)M
us m
uscu
lus
18-d
ay e
mbr
yo w
hole
bod
y cD
NA,
RIK
EN fu
ll-le
ngth
enr
iche
d lib
rary
, clo
ne:1
1100
02J0
3 pr
oduc
t:unk
now
n ES
T, fu
ll in
sert
sequ
ence
. [AK
0033
03]
AK03
2063
;0.
424
(0.3
45 to
0.4
96)
0.48
1 (0
.359
to 0
.623
)0.
86 (0
.789
to 0
.997
)M
us m
uscu
lus
adul
t mal
e m
edul
la o
blon
gata
cD
NA,
RIK
EN fu
ll-le
ngth
enr
iche
d lib
rary
, clo
ne:6
3305
68D
16 p
rodu
ct:u
ncla
ssifi
able
, ful
l ins
ert s
eque
nce.
[AK0
3206
3]H
s6st
2N
M_0
1581
90.
426
(0.3
94 to
0.4
44)
0.49
3 (0
.379
to 0
.564
)0.
489
(0.3
03 to
0.7
55)
Mus
mus
culu
s he
para
n su
lfate
6-O
-sul
fotra
nsfe
rase
2 (H
s6st
2), m
RN
A [N
M_0
1581
9]49
3343
9C10
RikAK
0171
15;
0.44
(0.3
85 to
0.5
45)
0.50
1 (0
.37
to 0
.64)
0.29
8 (0
.216
to 0
.352
)M
us m
uscu
lus
adul
t mal
e te
stis
cD
NA,
RIK
EN fu
ll-le
ngth
enr
iche
d lib
rary
, clo
ne:4
9334
39C
10 p
rodu
ct:u
nkno
wn
EST,
full
inse
rt se
quen
ce. [
AK01
7115
]Xr
cc1
AK00
9778
;0.
45 (0
.41
to 0
.479
)0.
591
(0.5
48 to
0.6
24)
0.38
3 (0
.346
to 0
.458
)M
us m
uscu
lus
adul
t mal
e to
ngue
cD
NA,
RIK
EN fu
ll-le
ngth
enr
iche
d lib
rary
, clo
ne:2
3100
43G
11 p
rodu
ct:X
-ray
repa
ir co
mpl
emen
ting
defe
ctiv
e re
pair
in C
hine
se 1
, ful
l ins
ert s
eque
nce.
[AK0
0977
8]Am
hN
M_0
0744
50.
453
(0.4
09 to
0.4
77)
0.41
4 (0
.386
to 0
.468
)0.
473
(0.4
04 to
0.5
21)
Mus
mus
culu
s an
ti-M
ulle
rian
horm
one
(Am
h), m
RN
A [N
M_0
0744
5]So
x3N
M_0
0923
70.
461
(0.3
55 to
0.5
96)
0.50
9 (0
.474
to 0
.55)
0.42
9 (0
.409
to 0
.454
)M
us m
uscu
lus
SRY-
box
cont
aini
ng g
ene
3 (S
ox3)
, mR
NA
[NM
_009
237]
OR
F34
BC02
5887
;0.
461
(0.4
31 to
0.5
19)
0.51
8 (0
.49
to 0
.571
)0.
569
(0.5
56 to
0.5
93)
Mus
mus
culu
s op
en re
adin
g fra
me
34, m
RN
A (c
DN
A cl
one
IMAG
E:52
5296
7), p
artia
l cds
. [BC
0258
87]
Gal
r3N
M_0
1573
80.
469
(0.4
17 to
0.5
75)
0.51
4 (0
.445
to 0
.594
)0.
68 (0
.661
to 0
.706
)M
us m
uscu
lus
gala
nin
rece
ptor
3 (G
alr3
), m
RN
A [N
M_0
1573
8]Si
dt2
AK08
1177
;0.
471
(0.4
59 to
0.4
93)
0.58
3 (0
.56
to 0
.598
)0.
542
(0.5
39 to
0.5
44)
Mus
mus
culu
s 10
day
s ne
onat
e ce
rebe
llum
cD
NA,
RIK
EN fu
ll-le
ngth
enr
iche
d lib
rary
, clo
ne:B
9300
96O
19 p
rodu
ct:C
GI-4
0 PR
OTE
IN h
omol
og [H
omo
sapi
ens]
, ful
l ins
ert s
eque
nce.
[AK0
8117
7]Fi
gla
NM
_012
013
0.48
5 (0
.333
to 0
.84)
0.51
3 (0
.472
to 0
.548
)0.
843
(0.5
08 to
1.4
95)
Mus
mus
culu
s fa
ctor
in th
e ge
rmlin
e al
pha
(Fig
la),
mR
NA
[NM
_012
013]
Serp
ing1
NM
_009
776
0.49
4 (0
.453
to 0
.547
)0.
475
(0.4
45 to
0.5
29)
0.57
(0.4
87 to
0.7
73)
Mus
mus
culu
s se
rine
(or c
yste
ine)
pro
tein
ase
inhi
bito
r, cl
ade
G, m
embe
r 1 (S
erpi
ng1)
, mR
NA
[NM
_009
776]
Myo
1gN
M_1
7844
00.
496
(0.4
32 to
0.6
42)
0.50
9 (0
.481
to 0
.534
)0.
507
(0.4
91 to
0.5
19)
Mus
mus
culu
s m
yosi
n IG
(Myo
1g),
mR
NA
[NM
_178
440]
Kcnn
3N
M_0
8046
60.
504
(0.4
89 to
0.5
31)
0.55
2 (0
.54
to 0
.569
)0.
526
(0.5
2 to
0.5
37)
Mus
mus
culu
s po
tass
ium
inte
rmed
iate
/sm
all c
ondu
ctan
ce c
alci
um-a
ctiv
ated
cha
nnel
, sub
fam
ily N
, mem
ber 3
(Kcn
n3),
mR
NA
[NM
_080
466]
Wds
ub1
NM
_028
118
0.50
7 (0
.435
to 0
.563
)0.
575
(0.5
2 to
0.6
07)
0.48
2 (0
.415
to 0
.58)
Mus
mus
culu
s W
D re
peat
, SAM
and
U-b
ox d
omai
n co
ntai
ning
1Fo
xe3
NM
_015
758
0.50
7 (0
.463
to 0
.546
)0.
513
(0.5
to 0
.521
)0.
558
(0.5
55 to
0.5
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77
78
FIGURE LEGENDS
Figure 1. The C Terminus of Cav1.2 Is Found in the Nucleus of Neurons
(A) Western blot of HEK 293T cells expressing Cav1.2 probed with anti-II-III loop
antibody (left gel) or anti-CCAT (right gel). The first lane of each gel contains lysate
from untransfected cells (Unt), and the second contains lysate from cells expressing
CaV1.2.
(B) Western blots of membrane (Mem), nuclear (Nuc), and cytoplasmic (Cyt) extracts
from the cortex of P7 rats analyzed with the anti-CCAT (upper gel), anti-CREB (second
gel), anti-Cav1.2 II-III loop (third gel), and anti-GAPDH (bottom gel) antibodies. The
first two lanes contain extracts from HEK 293T cells expressing Cav1.2. CREB, and
CCAT immunoreactivity are detected only in the nuclear fraction, Cav1.2 probed with
anti-II-III loop antibody and GAPDH are found in the membrane and cytoplasmic
fractions confirming the efficacy of the fractionation.
(C) Immunocytochemistry of cortical neurons grown 6 days in vitro. Anti-CCAT
staining is shown in green and nuclei is shown in blue.
(D) High-power image shows strong anti-CCAT staining (green) of nuclei (blue) and
lighter staining of dendrites.
(E) Staining with anti-CaV1.2 II-III loop antibody (green) reveals strong membrane and
ER staining but little nuclear staining (blue).
(F) Costaining with anti-CCAT antibody (green) and anti-GAD65 antibody (red)
reveals that CCAT is strongly nuclear in a subpopulation of GAD65-positive neurons.
(G and H) Immunohistochemistry of P30 rat-brain sagittal sections reveals strong
nuclear staining with the anti-CCAT antibody (green) in the inferior colliculus (G:IC)
and inferior olivary nucleus (H:IO). The cerebellum is labeled C and the brain stem is
labeled BS.
(I) High-power images of anti-CCAT (green), anti-GAD65 (red), and nuclear (blue)
staining of rat olfactory lobe neurons shows that only a subpopulation of neurons have
nuclear CCAT and that many of the cells are positive for GAD65.
79
Figure 2. Ectopically Expressed CCAT Localizes to the Nucleus of Neurons via a
Nuclear Retention Domain
(A) Neurons expressing Cav1.2 tagged at the C terminus with YFP (Cav1.2-YFP) show
pronounced nuclear and cytoplasmic fluorescence. Nuclei are shown in red in all panels.
(B) Neurons expressing the Cav1.2 tagged at the N terminus with YFP (YFP-Cav1.2)
show membrane and ER fluorescence but little nuclear fluorescence.
(C) Full-length CCAT-YFP (c503-YFP) is nuclear when expressed in neurons, cardiac
myocytes, and HEK 293T cells and forms prominent punctae.
(D) Epifluorescence (left panel) and confocal (right panel) image of neurons stained
with anti-CCAT antibody showing endogenous nuclear punctae.
(E) Schematic representation of YFP-tagged CCAT-deletion mutants (top). Top panel
and second panels show nuclear punctae in HEK 293T cells expressing CCAT
containing a deletion of 133 aa from the carboxyl terminus (c503–133) and a truncated
CCAT lacking aa 1642–1814 of Cav1.2 (c330) respectively. The third panel shows cells
expressing a truncated CCAT lacking aa 1814–1864 (c280). The domain important for
nuclear localization is highlighted in red in the schematic.
(F) Mean nuclear to cytoplasmic fluorescence ratios for all the CCAT deletions
constructs schematized in part (E) (means ± SEM; n > 75). ** p < 0.001 versus c503.
(G) Time-lapse images showing fluorescence recovery after photobleaching (FRAP) of
Cav1.2-YFP in the nucleus of Neuro2A cells. The area outlined in the red circle was
bleached for 300 ms with a high-intensity pulsed 488 nM laser beam. Images were
collected every 300 ms.
(H) Time course of recovery of nuclear fluorescence (solid symbols) and time course of
loss of cytoplasmic fluorescence (empty symbols) following bleaching. Nuclear
fluorescence recovered with a time course faster than 300 ms in cells expressing YFP
alone. Shown is a representative example of eleven experiments.
Figure 3. The Nuclear Localization of CCAT Is Regulated by Intracellular
Calcium and by Developmental Processes in the Brain
80
(A) Cortical neurons stained with anti-CCAT antibody (green) and Hoechst (red)
treated with 5 mM K+ (1 hr), 65 mM K+ (1 hr), 100 µM glutamate (30 min), or 2.5 mM
EGTA (1 hr).
(B) Higher magnification image of neurons treated with 2.5 mM EGTA reveals strong
and punctate anti-CCAT staining.
(C) Histograms of the ratio of nuclear to cytoplasmic anti-CCAT fluorescence in
neurons treated with 65 mM K+, glutamate, or EGTA. Treatment with 100 µM
glutamate (blue) and 65 mM K+ (green) reduce the amount of CCAT in the nucleus of
neurons, while EGTA (red) increases the amount of CCAT in the nucleus. Control cells
in 5 mM K+ are shown in black (n = 375 per condition).
(D) Time course of the decrease in anti-CCAT nuclear fluorescence following
stimulation with 65 mM K+ (green line; n = 200) or 100 µM glutamate (blue line; n =
200).
(E) Mean nuclear to cytoplasmic fluorescence ratio of CCAT in neurons treated for 1 hr
with 2.5 mM EGTA (bar 2), 65 mM K+ (bar 3), and 65 mM K+ in the presence of 2.5
mM EGTA, 10 µM nimodipine, 10 µM MK-801 or 10 µM NBQX (bars 5–8; n = 200).
(means ± SEM) * p < 0.001
(F) Mean anti-CCAT cell body fluorescence in neurons treated with 5mM or 65mM K+
(n=50).
(G) Mean nuclear to cytoplasmic fluorescence ratio of CCAT in neurons treated with
5mM and 65mM K+ in the presence of 5µM lactacystin (n=50).
(H) Western blot analysis of membrane (top) and nuclear fractions (middle) obtained
from E18, P1, P21, and adult-brain cortex probed with the anti-CCAT antibody. CREB
was used as a loading control (lower gel).
(I) Western blot analysis of cortical membrane extracts obtained from E18, P1, P8, P21,
and adult rats analyzed with anti-CaV1.2 II-III loop antibody (top gel) and anti-β-actin
(bottom) as a loading control. Adult lane exposed 4X relative to other lanes.
Figure 4. The C Terminus of Cav1.2 Binds to Nuclear Proteins and Activates
Transcription
81
(A) CCAT immunoprecipitates endogenous p54(nrb)/NonO. Upper panel shows
p54nrb/NonO levels in the lysates. The middle panel shows immunoprecipitated c503-
Gal4 and c280-Gal4 that lacks a nuclear localization domain. The lower panel shows
endogenous p54(nrb)/NonO coimmunoprecipitated by c503-Gal4 but not by c280-Gal4.
(B) Schematic representation of the CaV1.2-Gal4 fusion and CCAT deletion proteins.
c503 CCAT is the full-length C terminus of CaV1.2 downstream of the IQ motif. c330
CCAT lacks the N-terminal transcriptional activation domain (shown in red). c280 lacks
the nuclear retention domain of CCAT and c503Δ133 lacks the C-terminal
transactivation domain (shown in blue).
(C) Reporter gene activity of Neuro2A cells expressing a UAS-luciferase-reporter
plasmid along with either the Gal4-DNA binding domain, full-length CaV1.2, full-
length CaV1.2-Gal4, or CaV1.2-Gal4 channel lacking 133 amino acids from the
carboxyl terminus. Cells were cotransfected with a Renilla driven by the thymidine
kinase promoter to control for cell number and transfection efficiency. The results are
given as the ratio of Firefly to Renilla-luciferase activity. (means ± SD) ** p < 0.0001
versus Gal4.
(D) Luciferase activity of neurons transfected with the UAS-luciferase-reporter gene
and either Gal4 alone, CREB-Gal4, or four different CaV1.2 C-terminal fragments
c503, c330, or c280 and C503Δ133 fused to Gal4. The two domains identified as
important for transcriptional activation are highlighted in B. (means ± SD; ** p < 0.005)
(E) Luciferase activity of neurons transfected with Gal4-DNA binding domain alone or
Gal4 fused to the C terminus of CaV1.2, CaV1.3, or CaV2.1. (means ± SD) ** p <
0.0001 versus Gal4.
(F) Luciferase activity of Neuro2A cells expressing CaV1.2-Gal4 treated with 5 mM, 65
mM, and 65 mM K+/2.5 mM EGTA. (means ± SD; ** p < 0.005 versus untreated).
Figure 5. CCAT Regulates Endogenous Genes
(A) A subset of mRNAs identified in microarray experiments that were upregulated (red
bars) or downregulated (green bars) by overexpression of CCAT relative to CCATΔTA
or GFP. For a full list of genes, see Tables S1–S4.
82
(B) RT-PCR analysis of mRNA levels from Neuro2A cells overexpressing CCAT
confirming changes in gene expression for a subset of these genes identified in A. Bars
represent mean fold changes relative to CCATΔTA or GFP and were normalized to β-
actin and GAPDH levels. Data are means ± SD of three independent experiments
performed in triplicate.
(C) Luciferase activity of neurons expressing a Cx31.1-luciferase-reporter gene along
with empty vector, full-length CCAT, or CCAT lacking the C-terminal transactivation
domain (CCATΔTA) (means ± SD; ** p < 0.0001).
(D) 4OHT-induced nuclear translocation of a CCAT-ER fusion. Immunocytochemistry
of Neuro2A cells expressing myc-CCAT-ER (green) before (top panels) and after
(bottom panels) addition of 5 µM 4OHT for 1 hr. Nuclei are shown in blue.
(E) Transcription of a Cx31.1-luciferase-reporter gene in Neuro2A cells expressing a
Cx31.1-luciferase reporter along with ER alone, CCAT-ER, or CCATΔTA-ER before
and after nuclear translocation by addition of 5 µM 4OHT for 6 hr.
(F) Mean luciferase activity (mean ± SEM; n = 3) of Neuro2A cells expressing CCAT
and deleted forms of the Cx31.1 promoter. The 125 bp 3′ region of the promoter that
binds CCAT is shown in red.
(G) Representative ChIP assay showing that CCAT immunoprecipitates the endogenous
Cx31.1 promoter. Agarose gel electrophoresis of PCR products amplified from either
input DNA (I) or from DNA that was immunoprecipitated by GST-CCAT (IP) or by
GST alone (C). The upper gel shows the PCR products using primers that recognize two
regions in the Cx31.1 promoter (5′ promoter and 3′ promoter). The lower gel shows
PCR products using primers that recognize the 3′ region of the Cx31.1 gene several KB
from the start site (n = 3).
Figure 6. Endogenous CCAT Regulates Transcription Driven by the Cx31.1
Promoter
(A) Depolarization of neurons with 65 mM KCl decreases the activity of the Cx31.1
reporter relative to unstimulated cells (5 mM KCl; mean ± SD; n > 3; ** p < 0.0001).
(B) RT-PCR analysis of endogenous Cx31.1 mRNA in Neuro2A cells and cortical and
thalamic neurons treated with 65 mM KCl or 0 calcium showing that depolarization
83
causes a pronounced decrease in Cx31.1 expression. mRNA levels are normalized to
mRNA levels in unstimulated cells (bars represent mean ± SEM; n = 5). (C) Western
blots showing reduced expression of FLAG-tagged rat CaV1.2 expressed in Neuro2A
cells by expression of two rat shRNAs (RCaV1.2 sh6410 and RCaV1.2 sh6500; lanes 1
and 2, top panel) but not by a mouse shRNA (MCaV1.2 sh6203) that differs by two
base pairs (lane 3, top panel). CaV1.2ΔTA is resistant to RCaV1.2 sh6500 (lane 4),
which targets the TA domain of CaV1.2. Ds-Red, expressed from the shRNA vector,
was used as a loading control (bottom panel). (D) Mean luciferase activity (± SD) of rat
neurons transfected with the Cx31.1-luciferase-reporter gene and either a scrambled
control shRNA (sh-scr), shRNA constructs targeting the mouse CaV1.2 (MCaV1.2 sh-
6203), or the rat CaV1.2 mRNAS (shRNA RCaV1.2 sh-6500, RCaV1.2 sh-6410; ** p <
0.001 versus scrambled (sh-scr) or MCav1.2 sh-6203).
(E) CCAT* is resistant to knockdown by an shRNA targeting CaV1.2 (CaV1.2 sh6500).
Western blot analysis of lysates from Neuro2A cells expressing CCAT or an RNAi-
resistant CCAT* along with the RCaV1.2 sh-6500 shRNA vector that targets rat
CaV1.2 (upper gel). Ds-Red, expressed from the shRNA vector, was used as a loading
control (lower gel). (F) Expression of RNAi-resistant CCAT* reversed the effect of
CaV1.2 sh6500 on Cx31.1 expression (bar 3), but CCATΔTA, which lacks the C-
terminal transactivation domain, does not (bar 4; means ± SD; n = 3; ** p < 0.001
versus sh-scr).
(G) CaV1.2-FLAG* is resistant to knockdown by RCav1.2 sh6500. Western blot
analysis of lysates from Neuro2A cells expressing CaV1.2-FLAG or RNAi-resistant
CaV1.2-FLAG*, and RCaV1.2 sh6500 or MCaV1.2 sh-6203 (upper gel). Ds-Red,
expressed from the shRNA vector, was used as a loading control (lower). (H)
Expression of RNAi-resistant CaV1.2-Flag* partially reversed the effect of RCaV1.2
sh6500 on Cx31.1 expression (bar 3), but CaV1.2-ΔTA*, which lacks the C-terminal
transactivation domain, did not (bar 4; means ± SD; n = 3). ** p < 0.0001 versus sh-scr.
Figure 7. CCAT Regulates Neurite Growth in Primary Neurons
84
(A and B) Representative low- and high-magnification images of cerebellar granule
cells grown in vitro for 5 days transfected with a vector expressing GFP and either
CCATΔTA (A) or CCAT (B).
(C) Histograms of mean neurite length for granule neurons expressing CCATΔTA (top
graph), a vector with GFP alone (middle graph), or CCAT (bottom graph).
(D) Average neurite length for cells expressing CCATΔTA, GFP alone, or CCAT
(means ± SEM, n = 200; ** p < 0.005 versus vector control).
(E) Average number of primary dendrites for granule neurons expressing CCATΔTA,
empty vector, or CCAT.
Figure S1. CCAT’s nuclear localization and transcription activation domains are
conserved among CaV1.2 channels in vertebrates
(A-B) and (D) Multiple alignment of the nuclear localization domains (A), N-terminal
transactivation domains (B) and C-terminal transactivation domains of homologous
CaV1.2 channels (CAC1C) from different species. Accession numbers: CAC1C_RAT
AAA18905, CAC1C_MOUSE, Q01815, CAC1C_RABBIT P15381,
CAC1C_HUMAN Q13936, CAC1C_ZEBRAFISH: NM_131900.1. (B) Multiple
alignment of the N-terminal transcriptional activation domain of rat CCAT with
GATA4 sequences from different species. N-terminal transcriptional activation domain
is 42% homologous and 27% identical to a conserved C-terminal domain of the
transcription factor GATA4. Consensus symbols indicate identity (*) and similarity (:).
All alignments were generated using the ClustalW program. Black and gray shading
show identical and similar amino acid residues respectively.
Figure S1. CCAT’s nuclear localization and transcription activation domains are
conserved among CaV1.2 channels in vertebrates
(A-B) and (D) Multiple alignment of the nuclear localization domains (A), N-terminal
transactivation domains (B) and C-terminal transactivation domains of homologous
CaV1.2 channels (CAC1C) from different species. Accession numbers: CAC1C_RAT
AAA18905, CAC1C_MOUSE, Q01815, CAC1C_RABBIT P15381,
CAC1C_HUMAN Q13936, CAC1C_ZEBRAFISH: NM_131900.1. (B) Multiple
alignment of the N-terminal transcriptional activation domain of rat CCAT with
85
GATA4 sequences from different species. N-terminal transcriptional activation domain
is 42% homologous and 27% identical to a conserved C-terminal domain of the
transcription factor GATA4. Consensus symbols indicate identity (*) and similarity (:).
All alignments were generated using the ClustalW program. Black and gray shading
show identical and similar amino acid residues respectively.
Figure S2. CCAT derived from CaV1.2-YFP channel is regulated by depolarization
(A) Neuro2A cells expressing the C-terminally tagged CaV1.2-YFP treated with either
5mM K+ (upper panels) or 65mM K+ (lower panels).
(B) Histogram of mean nuclear fluorescence of cells in part A treated with 5mM K+
(gray; n=50) and 65mM K+ (black; n= 50).
Supplemental Figure 3. CCAT regulates expression of endogenous genes
(A) Graph of the expression level of mRNAs isolated from cells expressing either the
intact CCAT (X axis) or the CCAT∆TA (Y axis) that lacks the transcriptional activation
domain. Points below the diagonal green lines line correspond to genes that are down-
regulated in the CCAT relative to the CCAT∆TA by more than three standard
deviations. Data is derived from Agilent mouse two color arrays and the expression of
each gene was normalized to the mRNA levels in untransfected Neuro2A cells. The
color of each spot represents the expression level of each mRNA relative to
untransfected cells.
(B) Graph of the expression level of mRNAs isolated from cells expressing the CCAT
(Y axis) or GFP (X axis). Points that are above the diagonal green lines correspond to
genes that are expressed at least three standard deviations higher in CCAT expressing
cells than in GFP control cells. Points that are below the diagonal (blue) correspond to
genes that are repressed by the CCAT. Only the mRNAs whose expression was more
that 100 intensity units (three SD over the background) were analyzed. Genes are color
coded according to the ratio of expression in CCAT over GFP containing cells.
(C-D) Pie charts showing the functional distribution of 66 genes that are up-regulated
(C) and 206 genes that were down-regulated (D) by CCAT relative to GFP. See
Supplemental tables 2 and 3 for additional analysis and for a complete list of genes.
86
Figure S4.
(A) RCaV1.2 sh-6500 knockdown of endogenous CaV1.2 in neurons decreases CREB
activation induced by K+. The luciferase activity of rat neurons expressing a CREB
reporter gene is reduced by coexpression of an shRNA that targets rat CaV1.2 (RCaV1.2
sh6500; black) relative to coexpression of a control shRNA (MCaV1.2 sh6203; gray).
Cortical neurons were stimulated with 65mM K+ or 65mM K+ in the presence of 10µM
nimodipine. RCaV1.2 sh6500 reduces the activation of CREB in response to KCl in a
manner that is similar to the effect of the LTC blocker nimodipine, suggesting that it is
reducing the expression of endogenous CaV1.2 in neurons. Data represents means ± SD
of at least three independent experiments performed in quadruplicate; ** p<0.0001.
(B) Blockade of CaV1.2 channel pore by nimodipine does not affect expression from the
Cx31.1 reporter in the presence or absence of CCAT. Data represents means ± SD of at
least three independent experiments performed in quadruplicate; ** p<0.0001.
87
CHAPTER 3:
An independent promoter in the CACNA1C channel gene generates short CCAT
88
SUMMARY
Thus far we have discussed how the c-terminus of the voltage-gated calcium
channel Cav1.2 encodes a transcription factor within its c-terminal domain. This
protein, which we termed CCAT, is found in the nucleus of neurons, has a potent
transcription activation domain and its intracellular localization is regulated by
intracellular calcium levels. Here we show that a short CCAT is generated from a
second transcript from both exogenous channel cDNA and the endogenous channel
gene. Consistent with this, we find that CCAT expression is independent of full-length
channel protein, suggesting that this protein is not released from proteolysis of full-
length channel. Transcription of the CCAT message is driven by an exonic promoter
whose transcriptional activity is demonstrated in the channel’s cDNA. Activity at this
promoter, and consequently CCAT expression, is regulated spatially and temporally in
the brain having highest expression during embryonic stages and predominantly in
regions of the brain rich in inhibitory neurons. We also provide evidence for two other
endogenous transcripts containing the C-terminus. Analysis of 5’ ends from
CACNA1C derived transcripts showed two additional transcriptional start sites one of
which we propose is CCAT’s transcriptional start site in vivo. The second transcript is
predicted to encode a membrane bound CCAT containing a voltage sensor. These
findings uncover another unexpected detail of CCAT’s biology and provide a unique
example in which two proteins with distinct biologic functions can be derived from a
single gene. Furthermore, we provide an example in which exonic promoters can be
used to contribute to transcriptional and protein complexity.
89
INTRODUCTION
Voltage-gated calcium channels are large complexes composed of an α1C
subunit which forms the pore, and accessory subunits β and α2δ which play a role in
targeting, tethering and regulation of the biophysical properties of these channels
(Catterall et al., 2005). Calcium influx through L-type calcium channels, encoded by
the Cav1.2, Cav1.3 α1C subunits, has been shown to be particularly effective at
regulating gene expression in response to depolarization (Morgan and Curran, 1986;
Murphy et al., 1991). This has been thought to occur mainly via the activations of
calcium-regulated transcription factors such as CREB, NFAT and MEF (Graef et al.,
1999; Hardingham et al., 1999; Mao et al., 1999; Sheng et al., 1990; Zafra et al.,
1990). Specifically for Cav1.2 and Cav1.3 signaling to CREB has been shown to be
dependent on calcium influx through the channels, calmodulin-binding motifs in the
channel’s sequence and activation of the MAP kinase pathway (Deisseroth et al.,
1998; Dolmetsch et al., 2001; Weick et al., 2003). We have recently described a
calcium channel influx-independent mechanism in which Cav1.2 containing channels
can regulate gene expression (Gomez-Ospina et al., 2006). In this model the carboxyl-
terminal domain of the channel translocates to the nucleus and directly influences
transcription. In our previous study we showed this Calcium Channel Associated
Transcription regulator or CCAT is able to bind to nuclear proteins, associate with
endogenous promoters and both augment and repress the transcription of a wide
variety of endogenous genes. In the adult brain, CCAT nuclear localization is
90
restricted to inhibitory neurons in the thalamus, inferior colliculus and several brain
stem nuclei.
One of the most important questions remaining regarding CCAT’s biology is
how this C-terminal fragment is generated. We know based on its restricted nuclear
localization that the mechanism involved would be cell-type and stimulus specific.
One possibility is that CCAT is a c-terminal fragment released after a proteolytic event
from channels at the membrane. Such a mechanism would directly link electrical
activity, i.e., activation of voltage-gated channels, to the liberation of a transcriptional
regulator during periods of increased activity. Early biochemical studies of the pore-
forming unit of L-type channels including Cav1.1, Cav1.2, and Cav1.3 found that, in
most native tissues, these channels exist as truncated proteins lacking the c-terminus
(De Jongh et al., 1994; De Jongh et al., 1991; Gerhardstein et al., 2000). It was also
reported that NMDA stimulation increased the appearance of the Cav1.2 truncated
channel in hippocampal slices (Hell et al., 1996). Calpain protease inhibitors blocked
this effect, suggesting that increased neuronal activity could lead to the activation of
calcium-regulated proteases, which could then release CCAT.
A second possibility is that CCAT is made as an independent protein expressed
either from an alternative promoter hidden within the channel’s gene, as an
independent transcript, or from an internal ribosomal entry site in the channel’s
mRNA. Regardless of the exact mechanism, this would be the first example in which
alternative transcription or translation initiation leads to the expression of two proteins
with completely separate biological functions: a calcium channel and a transcription
factor. Alternative first promoters are commonly used for tissue specific expression.
91
In fact, alternative selection of P1 promoters in the CACNA1C gene results in Exon1a
being predominantly expressed in the heart and Exon1b in smooth muscle and brain
(Saada et al., 2005). Hitherto, most alternative promoters described include 5’
promoters that lead to alternative 5’ UTRs and identical proteins or different N-termini
resulting in protein isoforms with the same general biologic function (Davuluri et al.,
2008). Examples of this alternative promoter usage include the p53, TCF/LEF and
CREM/ICER families of isoforms (Arce et al., 2006; Mioduszewska et al., 2003;
Murray-Zmijewski et al., 2006). An alternative transcript based mechanism would be
especially plausible given our most recent understanding of the eukaryotic
transcriptome. Genome-wide analyses of the mouse and human transcriptomes have
revealed that almost one half the protein-coding genes contain alternative promoters
and, not uncommonly, genes have additional internal transcriptional start sites
(Carninci et al., 2006; Frith et al., 2008; Gustincich et al., 2006).
In these study we investigated how CCAT is generated and have identified that
a short CCAT is expressed from an alternative promoter within the channel’s coding
sequence. Consistent with this, we report that in vivo and in vitro, CCAT is made
independently of existing channel protein. Using Northern blots and 5’ RACE
experiments we demonstrate that other 3’ transcripts are present in cells containing
exogenous channel and in brain mRNA extracts. In addition, we have found that an
exonic promoter residing within the penultimate exon of the rat and mouse genes is
responsible for CCAT message expression and is sufficient to promote transcription of
a reporter gene. Using minigenes we show that this exonic promoter activity is
preserved in the context of large intron sequences. As predicted by an alternative
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transcript mechanism, a single methionine mutation can abolish CCAT expression
from both exogenous cDNA and from minigenes. We find that the level of expression
of the endogenous transcript as well as the abundance of CCAT nuclear protein is
highest in embryonic stages and decreases in post-natal life. Early in development
both CCAT transcript and CCAT protein have highest expression in the thalamus and
lowest in the cortex. Accordingly, we find that reporter gene expression driven by the
exonic promoter is higher in thalamic than cortical neuronal cultures. Finally, our 5’
RACE and northern blot experiments suggest the existence of an additional transcript.
This message would encode for the C-terminus of the channel as well as 7
transmembrane helices including S6 of domain III and S1-S6 of domain IV, predicting
a membrane bound CCAT with a voltage sensor.
These data identify an unexpected alternative transcription initiation mechanism
responsible for CCAT expression and a novel promoter residing within the coding
sequence of the channel.
RESULTS
CCAT is not generated by Proteolytic Cleavage of Exogenously Expressed
Channels
We have previously described that Cav1.2 channels tagged with the Gal4 DNA
binding domain at its c-terminus led to constitutive and strong luciferase expression
from a UAS-luciferase reporter gene when expressed in N2A cells and myocytes
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(Gomez-Ospina et al., 2006). These data demonstrated to us that a c-terminal fragment
was produced which was free from the membrane and able to translocate to the
nucleus, and that this fragment contained a potent transactivation domain. We used
this assay to examine how CCAT was released from Cav1.2 channels at the membrane
by measuring the generation of CCAT protein from mutant channels expressed in cells
(Figure 1A). We first expressed Cav1.2 -Gal4 channels in Neuro2A cells (N2As) along
with accessory subunits and a UAS-luciferase reporter gene. We followed the
appearance of CCAT in two ways: we monitored protein expression using
immunoprecipitation and western blots and we measured its transcriptional activity
and abundance using transcription assays. When expressed in N2As cells wild-type
Cav1.2–Gal4 channels (WT) generated two Gal4 tagged proteins: a full-length channel
and a smaller protein of approximately 250 KDa and 40 KDa respectively (Figure 1b).
This 40 KDa band is a fusion of part of the c-terminus and Gal4 DBD thus predicting
a molecular weight for CCAT of approximately 20 KDa. In the transcription assays
WT channels evoked strong luciferase expression, one hundred fold better than Gal4
alone (Figure 1C). In the previous study, we determined that the transactivation
domain (TA) resides within the last 133 amino acids of the channel and consistent
with this, deletion of TA completely abolished transcriptional activation and the
CCAT band (Figure 1B and 1C).
We then examined whether Cav1.2 channels needed to be at the plasma
membrane to produce CCAT. We deleted a region of 150 amino acids between the last
transmembrane domain (TM) and the IQ domain. This region has been previously
shown to be required for expression of functional channels at the membrane (Wei et
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al., 1994). We observed that this deletion had no effect in the size or abundance of
CCAT protein. Additionally, incubation of the cells in brefeldinA, which inhibits
anterograde protein transport from the endoplasmic reticulum (ER) to the Golgi
apparatus, yielded similar results (Sup Fig1). These data could only be reconciled with
a mechanism where cleavage occurs co-translationally or within other compartments
of the secretory pathway such as the endoplasmic reticulum or the Golgi apparatus.
We next tested whether a reported cleavage site of Cav1.1 was necessary for the
production of CCAT protein. Mass spectrometry analysis of Cav1.1 (the skeletal
isoform of Cav1.2) purified from skeletal muscle identified the site of proteolytic
processing as Alanine 1664 (Hulme et al., 2005). The surrounding sequence,
including the Alanine is conserved in Cav1.2, corresponding to Alanine 1773,
suggesting that this could constitute a cleavage site in these channels as well. We
found that deletion of 50 amino acids surrounding the proposed cleavage site had no
effect on the size or abundance of CCAT protein (Figure 1B and 1C) indicating that
for Cav1.2 this sequence was not necessary for CCAT production. Because it seemed
difficult to explain how cleavage of channel within intracellular membrane
compartments would be employed as a way to link channel activity to CCAT
production we asked whether channel protein was at all necessary for the generation of
CCAT. We introduced a stop codon at amino acid 1910, upstream of the TA domain.
As expected, this construct when expressed in cells could no longer make full-length
Gal4-tagged channel but surprisingly Gal4 tagged CCAT protein was intact. This
result implied that channel protein was not necessary for CCAT production and that
CCAT was made as an independent protein.
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We next designed a series of experiments to determine how this channel-
independent protein was made. One possibility is that CCAT is translated from the
channel mRNA via an IRES or as a distinct message transcribed from the same cDNA.
Regardless of which model is at play, it can be predicted that an initiating methionine
would be necessary for the production of CCAT. To test this prediction, we mutated
two methionine residues in the channel sequence selected based on predicted CCAT
size of ~20 KDa and the predicted strength of the initiating AUG (Algire and Lorsch,
2006). Mutation of Methionine 2011 to Isoleucine and not Methionine 2078
completely prevented the expression of the 40KDa CCAT band and abolished
transcriptional activation. Instead of the 40KDa protein a smaller 32KD protein was
made (Figure 1D and 1E). The appearance of this smaller band can be explained by
the scanning mechanism by which the ribosome determines the starting ATG. The
translational start site in the M2011I mutant then likely becomes M2073, 62 amino
acids downstream. Additionally, a significant increase in the amount of the smaller c-
terminal protein could be seen in the western blot and is better quantified in the
luciferase assay (Fig 1E). This effect can be explained by the competitive inhibitory
effect that surrounding Methionines have on translation initiation (Kozak, 2000).
Cav1.2 Channel Protein is not necessary for CCAT Expression In Vivo
To test whether channel protein is necessary for the production of CCAT in
vivo. We looked for endogenous CCAT in Cav1.2 knockout mice (kindly provided by
Dr Jean Pierre Kinet at Harvard). The Cav1.2 conditional null allele was generated by
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homologous recombination with a construct designed to delete exons 14 and 15
(Supplementary Figure 1B and 1C). CMV-CRE-transgenic mice were used to produce
ubiquitous deletion of the floxed exon and create the Null allele. Cav1.2 deletion is
lethal and mouse embryos do not survive past 13.5 days post conception (dpc).
Consequently, we examined the embryos from heterozygous Null crosses at 11.5 and
12.5 dpc for the presence and localization of CCAT. At the 11.5 and 12.5 dpc Cav1.2
knockout embryos were anatomically and functionally normal. We first used a c-
terminal antibody (anti-CCAT) to probe for the presence of full-length channel in
heterozygous and homozygous Null 11.5 dpc embryos from the same litter. We used
biochemical fractionation to separate microsomal and nuclear fractions from N/+ and
N/N and embryos. In membrane fractions, anti-CCAT antibody recognized a 240KDa
band corresponding to the full-length channel in wild type and heterozygote embryos.
This band was absent in the knockout embryos confirming the efficacy of the
knockout strategy (Figure 1F). We have previously reported that this antibody shows
nuclear staining in a subset of neurons in the brain as well as in N2A cells. This
nuclear localization of CCAT is further supported by the fact that exogenous c-
terminus tagged with YGP localizes to the nucleus in cells (Gomez-Ospina et al.,
2006). To examine whether nuclear CCAT staining was preserved in the knockout
embryos, we probed sections of wild type and Cav1.2 KO 11.5 dpc embryos with anti-
CCAT antibody. At this age, in wild type animals, cytoplasmic and membranous anti-
CCAT immunoreactivity was seen in the developing neural tissue, heart muscle and
major blood vessels. Strong nuclear staining was noted in somites and mesenchymal
tissue. No nuclear CCAT staining was seen in the developing brain at this stage (Sup
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Fig1D). Cav1.2 null embryos showed intact nuclear staining in somites suggesting that
anti-CCAT reactive nuclear protein was still present in cells lacking full-length
channel (Figure 1G).
CCAT is translated from a Separate Transcript from the cDNA
Our data thus far suggested that an alternative mechanism independent of
channel protein was giving rise to CCAT protein. CCAT could be translated from the
Cav1.2 mRNA via an IRES or from a separate transcript. Given our observations using
channel constructs, it was clear that such mechanism is preserved in the context of the
cDNA, which supported the idea of an IRES within the channel’s message. We used
northern blots to probe for the last exon, i.e. Exon47 (Acc# AAA18905), of the
channel gene to examine transcripts from N2A cells expressing Cav1.2 -Gal4 channels.
We observed two transcripts, one of approximately 8 Kb corresponding to full-length
channel and a second transcript of approximately 1.4 Kb (Figure 2A). Even though
this strongly suggested that a second transcript was involved, it did not rule out the
possibility of an IRES within the full-length channel transcript. To test this, we deleted
the CMV promoter in the pcDNA4 Cav1.2 -Gal4 construct and checked for CCAT
expression. As expected, deletion of the CMV promoter led to the loss of the
channel’s transcript and protein. However, neither the 1.4 Kb transcript nor CCAT
protein are affected by the deletion of the promoter (Figure 2A and 2B). Luciferase
expression from the UAS reporter in the presence of the promoterless Cav1.2 -Gal4
plasmid was not only preserved but was significantly increased compared to wild-type
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plasmid. This increase, we speculate, is due to decreased transcriptional quenching in
the absence of the potent CMV promoter (Figure 2C). Together these data
demonstrated that, in the cDNA, CCAT is translated from a separate transcript and
suggested that the sequences required to drive the transcription of this message reside
within the coding sequence of the Cav1.2 gene.
An Exonic Promoter Drives CCAT Expression
To find the sequences involved in transcriptional activity, we cloned fragments
of the coding sequence of Cav1.2 in front of luciferase and tested for luciferase
expression (Figure 2D). The full coding sequence of the channel can robustly drive
expression of luciferase when taking the position of the promoter (Figure 2E). This
activity can only be observed when the channels sequence is placed in frame of the
luciferase sequence and any intervening stop codon is mutated to code for amino acid,
thus maintaining the open reading frame. Consistent with our hypothesis, this finding
suggested that translation and consequently transcription was initiated within the
channel’s coding sequence. Using truncation analysis, we found that a substantial part
of the promoter activity resided within the penultimate exon of the channel, i.e. Exon
46. Specifically, the 238 base pair region within Exon 46 upstream of M2011 was
sufficient for promoter function (Figure 2E).
We next examined whether this promoter activity was present in the context of
the gene. For this purpose we constructed several CCAT minigenes containing 4 Kb of
genomic sequence comprising the last two introns and last two exons of the
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CACNA1C gene fused to Gal4 (Figure 2F). In the control minigene, both the 238 bp
promoter and the M2011 reside within Exon 46 more than 2 Kb upstream of Gal4
(4Kb-0). TA and, more precisely, the sequence between M2011 and M2073, which is
required for transcriptional activation, straddle Exon 46 and Exon 47 (Figure 1D and
Sup Fig 2). We predicted that, if such promoter activity was present and could be
recapitulated in the minigene, it should lead to a transcript whose appropriate splicing
would create a fusion of CCAT’s transcriptional activation domain to Gal4. The
abundance of this protein could be sensitively measured by its ability to promote
luciferase expression from the UAS-luciferase reporter. As a negative control we built
a minigene where the reading frame between CCAT and Gal4 was frameshifted by
introducing a single deoxyguanosione (4Kb-1). When expressed in N2A and 293
cells, 4Kb-0 had 10 fold more activity than 4Kb-1. Deletion of the 238 bp promoter
(∆238 bp) decreased protein expression by 75% confirming that this sequence is
necessary to drive full expression of CCAT-Gal4. The M2011I mutation led to a
similar decrease in protein expression. There are three possible methionines within
this putative transcript: M2011, M2073 and M2078. To test the possibility that
translation was initiated at the methionines downstream, we built three more
minigenes where only one of the afore mentioned methionines was intact in the
context of the control construct (4Kb-0). In these experiments, only M2011 was able
to produce CCAT-Gal4 protein (Figure 2H).
We next compared the strength of the identified promoter to that of the full-
length channel’s promoter. To this end we cloned a 4 Kb genomic fragment upstream
of methionine M2011 in front of the firefly luciferase coding sequence.
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Concomitantly, a 4 Kb genomic fragment upstream of the putative methionine for
neuronal Cav1.2 transcript (rbcII) was cloned in the same manner. We introduced
neuronal/ Cav1.2 –luciferase and CCAT/Cav1.2-luciferase reporter genes into N2As
and HEK cells and measured luciferase expression. In both cell types, the genomic
region upstream of M2011 showed 2.5X higher protein expression than promoterless
vector alone. In N2As cells the promoter activity of this region was comparable to the
neuronal promoter/enhancer region (Sup Fig2A). Together, these results confirm our
hypothesis that the 238 bp region within Exon 46 of CACNA1C gene has promoter
activity. The resulting transcript is spliced as endogenous full length channel transcript
giving rise to a protein that encodes the transcription activation domain of CCAT. Our
data also strongly points at M2011 as the starting methionine for the transcript in vivo.
We compared sequences from Cav1.2 channels from Zebrafish to Human to look at
the conservation of the promoter region and M2011. This amino acid sequence and the
exon-exon borders within the c-terminus are highly conserved (Sup Fig 2B).
Alignment of these sequences reveals that the M2011 is conserved in primates,
rodents: including rat mouse and guinea pig and in Zebrafish (Sup Fig 2C).
Interestingly, M2011 is not conserved in all species even if the amino acid sequence
and exon organization is preserved. However the downstream Methionine 2073 is
conserved in all species and if alternative transcripts were made, translation would
initiate at this methionine. The sequence upstream, which includes the 238 bp
promoter region, is also highly conserved at the amino acid level. This would suggest
that Exon n-1 in other channels could function as a promoter. However, it is likely that
key differences at the nucleotide level dictate promoter activity and so without a better
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understanding of the DNA elements required for transcription, it is difficult to
speculate regarding promoter activity in other channels.
CCAT is Translated from a Separate Transcript In Vivo whose Expression is
Spatiotemporally Regulated in the Brain
To investigate the presence of independent transcripts in vivo we extracted
mRNA from the cerebral cortex, midbrain and cerebellum of rats at different
developmental stages and analyzed them using northern blots. Because our
experiments predict a transcript whose transcriptional start site (TSS) is in the coding
sequence of the channel no distinct sequence could be used for precise quantification
with RT-PCR and other hybridization methods. Thus, the main distinction is in
transcript size. Hybridization of a probe designed to recognize CCAT-TA domain
revealed two additional transcripts of approximately 4 Kb and 2.2 Kb (Figure 3A).
These transcripts were absent when using a probe that recognized S3 of domain 3 of
the full-length channel (Figure 3B). For the three transcripts we plotted the normalized
signals of the 18S ribosomal RNA from three independent experiments (Figure 3C).
The Full-length channel message was expressed at higher levels in the adult brain.
Conversely, the 2.2 Kb message was most abundant in cerebellum and midbrain of P1
rats and in E18 whole brain compared to these structures in the adult brain. The
normalized signal of 4.0 Kb band was most abundant in E18 whole brain. These data
suggests that subpopulations of neurons express at least two other CCAT-TA
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containing transcripts and their expression level is inversely correlated to the full-
length channel’s.
In parallel experiments we looked at the amount of CCAT nuclear staining in
brains during development using immunohistochemistry with anti-CCAT antibody. In
the brain, the number of cells showing CCAT nuclear staining also decreased with
age. In E18 embryos most nuclear CCAT reactive cells were found in the developing
striatum, thalamus, brain stem and cerebellar bud (Figure 3D and 3E). Nuclear
staining was almost nil in the cortex (Figure 3D and Sup Figure 3A and B). At P1 the
nuclear staining became more restricted to subpopulations of cells in the cerebellum to
the developing Purkeji layer, the thalamus and to 20-30% of the cells in the striatum.
In 3wk brains, most anti-CCAT reactivity came from membranous full length Cav1.2
channels which localized to the cell bodies and dendrites as has been reported
previously (Hell et al., 1993). This was particularly evident in the hippocampus and
cortex. Nuclear staining at this age was sparse and found mostly in the inferior
colliculus, and brain stem nuclei with scattered cells in the thalamus (Figure 3E and
(Gomez-Ospina et al., 2006)). Together, these results showed that the distribution and
abundance of CCAT nuclear protein was consistent with the distribution of the 2.2 Kb
transcript. Both transcript and protein coincide with highest expression in E18 brain,
P1 cerebellum and thalamus and decreasing levels in the adult.
The observed distribution of CCAT expression implied tissue specific CCAT
promoter usage. Specifically, we learned that the level of endogenous transcript and
nuclear staining was higher in the thalamus than in the cortex in E18 brains. Hence,
we predicted that there would be a higher level of promoter activity in thalamic
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neurons compared to cortical neurons. We tested this by comparing UAS-luciferase
expression from cultured thalamic and cortical neurons transfected with Cav1.2-Gal4
channels knowing that sufficient part of the promoter is preserved in the cDNA. As
Figure 3F shows, in thalamic cultures, 40 ± 10% of neurons have robust nuclear
CCAT compared to less than 5± 5% in cortical neuron cultures. Furthermore, in the
transcription assays thalamic neurons replicated what we have reported in N2As:
Cav1.2 -Gal4 channels upregulate the UAS promoter independently of channel protein
and rely on M2011 for expression. In cortical neurons, Cav1.2-Gal4 channels did not
lead to quantifiable differences in luciferase expression compared to untagged Cav1. 2
channels, suggesting this promoter is not transcriptionally active in these cells. This
would predict less nuclear CCAT in these neurons consistently with what we have
observed.
To better understand the expression of CCAT protein the developing brain, we
stained for CCAT at earlier embryonic stages. Our data indicated that early in the
developing mouse brain, nuclear CCAT expression was largely confined to cells in the
ventral telencephalon. We have observed expression of nuclear CCAT as early as
embryonic day 12 in parts of the ventral telencephalon corresponding to the
developing caudate and putamen (striatum), as well as in the developing thalamus,
distinct domains of periventricular nuclei in the hypothalamus, and in the developing
cerebellum (Supplementary Figure 3B and 3C). Expression of nuclear CCAT in these
regions persisted through embryonic neural development but began to decline early in
postnatal life and subsequently disappears into adulthood. Notably, in the developing
cortical plate and ventricular zones immunofluorescence staining with the C-terminal
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Cav1.2 antibody revealed primarily cytoplasmic expression, corresponding to the
presence of the full length Cav1.2 calcium channel but no CCAT expression.
CACNA1C has Multiple TSS Predicting Multiple Proteins
To provide independent evidence for the existence of an alternative transcript
encoding for CCAT in vivo we employed Rapid Amplification of cDNA Ends (5’
RACE) analysis of CACNA1C derived transcripts. Moreover, 5’ end analysis could
help us identify the TSS, corroborate the role of methionine 2011 and locate the cis-
acting element, which we predict include the sequence herein identified. We used the
RNA ligase-mediated (RLM) and oligo-capping approaches to selectively ligate an
RNA oligo adapter to previously capped full-length mRNAs. cDNA synthesis was
performed with a primer complementary to a region of Exon 47 as a gene specific
primer or an oligo dT primer. cDNA’s were PCR amplified using a primer within the
5’ oligo adapter sequence as a forward primer and a nested primer in Exon 47 as the
reverse primer. The PCR products were cloned, sequenced and the 5’ 20 bp sequences
mapped onto the gene. First, to test our approach and to determine the transcriptional
start site from the exonic promoter within the channels cDNA, we used 5’ RACE on
mRNA extracted from cell expressing Cav1.2 -Gal4 channels. For this experiments a
Gal4 specific primer was used for cDNA synthesis to select only transcripts from
exogenous construct. Cells transfected with Cav1.2-Gal4 and Cav1.2-gal4
promoterless constructs expressed a second transcript whose TSS mapped 50 bp
upstream of M2011 (Figure 4A and 4B).
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We next isolated transcripts from cortex and thalamus of E18 rats. We focused
on this stage given the greatest difference in abundance of nuclear protein between
these two brain regions and the greatest abundance of short transcript as suggested by
the northern blots. We performed five independent RACE experiments to really
convince ourselves that the 5’ ends we found corresponded to true ends of capped
RNA's and not to truncated transcripts that had escaped dephosphorylation during the
chemical selection step. Transcriptional start sites were considered equivalent if they
mapped within 20bp. The most reproducible 5’ starts and the corresponding
chromosomal location of the 20 upstream nucleotides on the mouse genome are listed
in Table 1. Three additional TSS’s were expressed from the CACNA1C gene in
addition to the already established TSS’s for full-length channel transcripts. All three
sites were within exons. For all three possible transcripts we used three available
translation initiation algorithms to predict whether and what proteins could be
translated from them (Nadershahi et al., 2004). The start site for transcript variant 4
was found to reside within Exon 46, 119 bp upstream of M2011 and 60 bp upstream
of the TSS found expressed from the channel cDNA. In both cases, the UAG codon
for M2011 is predicted to be the initiating codon and the predicted molecular weight
for CCAT is 15KDa. Thus, transcript variant 4 would presumable encode for CCAT in
vivo. Furthermore the promoter is localized at least in part to the upstream sequence in
Exon 46. In order to predict its full-length size, we thought it would be reasonable to
expect that all additional transcripts share the same 3’ termination signals and
therefore the 3’UTR. For the majority of CACNA1C-derived transcripts found in the
databases the 3’ UTR is roughly 1.6 Kb. Consequently, the estimated size for
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transcript variant 2a is 2.1 Kb. Notably, this approximates the size of the 2.2 Kb seen
our northern blot assays (Figure 3A).
Our 5’ RACE experiments also discovered two other TSS’s. Transcript 2a
resides within Exon 27 of CACNA1C. For this transcript the first and strongest
starting AUG codons were in frame with the channel’s sequence. This predicted a C-
terminal channel protein of approximately 110KDa. This predicted protein would
encompass Cav1.2’s sixth transmembrane helix of ion pore domain III, all of domain
IV and the cytoplasmic c-terminus. Hence, this protein was predicted to be membrane
bound and would have a voltage sensor. The predicted full-length transcript size for
his message was roughly 4.4 Kb, which also corresponded to the size of the higher
molecular weight transcript found in the Northern blot experiments. Lastly, transcript
3 has a start site within Exon 42 of the channel. Sequence analysis of the 5’ UTR
predicts it to be non-coding.
Systematic and genome-wide 5’end analysis of the mouse and human
transcriptomes has been carried out using cap analysis of gene expression (CAGE)
technology (Carninci et al., 2006; Kawaji et al., 2006; Kodzius et al., 2006). The basis
of this approach is the chemical targeting of 5’-capped RNA's and the formation of
short, 20-21 bp sequences of the 5’ ends of the cDNA's. These tags have been mapped
onto the mouse genomic sequences. To look for independent evidence for these
transcription start sites, we searched the CAGE tag library looking for co-clustering
with our TSS’s (http://fantom3.gsc.riken.jp/). We found 2 tags that mapped within 100
bp of the TSS’s found (Table 1). In support of transcript variant 4, we found one tag
that maps 77 bp downstream of the TSS found. The predicted starting AUG was also
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M2011. The second tag and its expected TSS mapped 92 bp downstream of the one
we identified for transcript variant 3. No protein was predicted to be translated from
this transcript. We found no CAGE tags in the vicinity of the 5’ end of transcript
variant 2a on Exon 27. Interestingly, in the CAGE database the biggest tag cluster for
CACNA1C was not the for the full-length channel but for a TSS located between
Exons 28 and 29 of the channel
(http://gerg01.gsc.riken.jp/cage/mm5/SummaryTss.php?tss_id=T06R07183F39 and
Table 1, transcript 2b). Without additional splicing of the intron sequence, this
transcript is not predicted to be protein coding. However the best, though weak,
initiator AUG is in frame with the channel and would generate a 100KDa protein with
similar localization and properties of transcript variant 2b.
Our experiments thus far suggested that alternative promoter sequences within
CACNA1C promote expression of alternative transcripts some of which could encode
for non-channel proteins including CCAT and a 110KDa, membrane bound channel
fragment or mem-CCAT (Figure 4C). To characterize the subcellular localization of
these proteins we cloned their predicted coding sequence in front of an N-terminal
GFP tag. When expressed in N2As, mem-CCAT was localized to the endoplasmic
reticulum and to a smaller extent to the plasma membrane. CCAT appeared to be
soluble, distributed between the cytoplasm and nucleus, with increased nuclear signal
(Figure 4D). Analysis of the untagged proteins using SDS-Page reveal a running
molecular weight of 120KDa for mem-CCAT and 20KDa for CCAT (data not shown).
We next sought to determine whether proteins of predicted molecular weights
could be found in brain protein extracts. First, we assumed that mem-CCAT and
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CCAT share their C-terminal sequences and therefore both proteins should be detected
using the anti-CCAT antibody. Second, both proteins were predicted to be expressed
from independent transcripts and consequently their expression should remain in our
Cav1.2 KO mice. As previously described, we fractionated membrane and nuclear
proteins from wild type and Cav1.2 null 11.5 dpc embryos separated them using SDS-
page and probed with the anti-CCAT antibody. In membrane fractions two other
additional bands in addition to the full-length channel could be detected corresponding
to MW 160 and 120KDa (Figure 1F). In nuclear fractions of WT and KO embryos,
anti-CCAT antibody recognized several bands including two small 22K and 15 KDa
bands. Thus, proteins of predicted molecular weights can be detected in the
appropriate subcellular fraction. The presence of these CCAT reactive bands in protein
extracts from embryos lacking full-length cav1.2 would be consistent with the idea
that the endogenous CCAT fragment and mem-CCAT can be generated in the absence
of Cav1.2 channel. However, the possibility that some of these bands are the result of
cross-reactivity of anti-CCAT with other nuclear proteins must also be considered.
DISCUSSION
The results of the present study demonstrate that a short CCAT is translated
from an alternative transcript whose expression is driven by an exonic promoter.
These findings provide a unique example in which two proteins with distinct biologic
functions can be derived from a single gene. They also highlight what has been
recently discovered about the eukaryotic transcriptome: many more start sites, many
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more RNAs, much more complexity than initially predicted. Here we provide an
example in which an exonic promoter can be used to contribute to transcriptional as
well as protein complexity. Such transcriptional phenomena may be at play in many
other genes throughout the genome and has far reaching implications for prediction of
gene products and interpretation of phenotypes in gene mutations and knockout
studies.
Functional Consequences of Exonic Promoters
Genome-wide analyses of the mouse and human transcriptomes have revealed
that our initial predictions greatly underestimated the number of transcripts expressed
in cells (Yasuda and Hayashizaki, 2008). The genomic sequences transcribed are more
extensive than we originally thought. The distribution of CAGE tags clearly shows
many additional promoters and common exonic transcription start sites, especially in
3’ UTR sequences (Carninci et al., 2006). Many of these exonic promoters are
conserved between species. These additional start sites can give rise to new proteins or
variations in protein sequence, thereby increasing proteome complexity. More than
half of these start sites are predicted to generate a wealth of non-coding RNAs for
which we have no function, thereby increasing RNA and regulatory complexity.
Furthermore, these additional exonic promoters will contribute to cell-type, tissue-type
and developmental gene regulation. Thus, mutations thought to be in coding
sequences may actually impact gene transcription. It can be predicted that aberrant use
of these promoters could be implicated in disease as it is known for many alternative
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promoters such as abnormal expression of c-myc in Burkitt’s lymphoma and
expression of short isoform p53 in several human cancers (Davuluri et al., 2008).
There are few examples of exons as promoter sequences in the literature. For
instance, in D. Melanogaster, the gene encoding for the sperm specific axonemal
dynein subunit Sdic, has a promoter derived from a protein-coding region from the
gene encoding the cell adhesion protein annexin (AnnX, (Nurminsky et al., 1998)).
The gene is posited to have evolved from the fusion AnnX exon 4 with Cdic intron 3
(cytoplasmic dynein intermediate chain). Transcription is initiated from promoter
elements within the AnnX Exon 4 region, and translation is initiated within the
sequence derived from Cdic Intron 3. Another example, also in flies, is NonA, a gene
important for vision, courtship song and viability in flies. NonA has upstream
regulatory regions embedded within the coding regions of adjacent gene dGpi1
(Sandrelli et al., 2001). While these two examples share the motif of coding
sequences having dual roles as promoters, the transcription of CCAT is unique in that
both promoter and coding sequences reside within a that also happens to code for a
calcium channel. Furthermore, by maintaining the reading frame CCAT is in fact a
channel fragment and has, as we have previously shown, a separate function in the
nucleus.
Our findings also beget the question of how expression from channel and
CCAT promoters is regulated. From our studies in N2A cells we surmise expression
of both proteins is not mutually exclusive. In the gene, these promoters are separated
by millions of nucleotides and could represent two very different chromatin
environments. Furthermore, the cell and tissue type specific expression of CCAT and
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the different patterns in mRNA expression during development argue that regulation
of each promoter occurs independently. However, given the evidence that the c-
terminus may also inhibit channel function, controlling their promoter elements would
be a necessary mechanism to establish an adequate expression ratio between channel
and putative negative regulator (Dzhura et al., 2003; Gao et al., 2001; Hulme et al.,
2006a). It is also conceivable that the transcript generated from the upstream channel
promoter impedes the formation of the transcription complex at the downstream
CCAT promoter. This type of relationship between these adjacent promoters is
exemplified by the human dihydrofolate reductase gene (DHR) (Martianov et al.,
2007). This mechanism would be consistent with the observed reverse expression
patterns of full-length channel and CCAT transcripts. Future experiments aimed at
understanding the DNA elements responsible for regulation of both promoters will
help understand how signaling cascades impinge on expression at both promoters.
CCAT Promoter Architecture
Our understanding of mammalian promoter architecture and evolution is
increasing rapidly. Still, present algorithms aimed at promoter or TSS prediction have
proven unsatisfactory even with the addition of phylogenetic conservation. Promoter
prediction algorithms such as Promoter 2.0 and Promoter inspector failed to predict
the CCAT promoter. Mammalian promoters can be separated in two classes,
conserved TATA-box rich promoters and CpG rich promoters. Several CpG islands
are found within the 3’end of the mouse CACNA1C gene. Two reside downstream at
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1.3 Kb and 2 Kb from the TSS and a third CpG island is 2.5 Kb upstream. However, it
is unclear whether any of these would influence transcription at the CCAT promoter
given than transcription driven by CpG islands usually occurs within 1.0 Kb (Saxonov
et al., 2006). The PolII core eukaryotic promoter is frequently observed to contain
TATA, TFII recognition, downstream core and initiator elements as well as CCAAT
and GC boxes (Carey et al., 2009). The 238 bp sequence identified here lacks a TATA
box but has a CCCAT and 2 Sp1 GC boxes at -140 bp, -170 bp and -40 bp
respectively from the TSS's found using 5’RACE (Sup Fig 4A). Promoters with
similar architecture have been reported in several brain specific genes (Christensen et
al., 2004; Ross et al., 2002; Schmitt et al., 2003; Schwarzmayr et al., 2008; Skak and
Michelsen, 1999). TATA-less promoters have broader distributions in the TSS's and
can vary by 100 bp. This can explain the range observed in our experimentally found
TSS's and CAGE tags. Future analysis of these sequences may help narrow down the
DNA elements required for CCAT promoter activity.
CCAT Promoter Evolution
Voltage-gated channels are multidomain proteins whose domains evolved separately
and consequently individual domains can be found as independent proteins in lower
organisms. It is reasonable to hypothesize that CCAT and its promoter constitute an
independent transcriptional unit that was acquired as a module during the channels’
evolution. One way to explore this is to look for sequences similar to the last two
exons of the channel while preserving the exon-exon boundary in the genome of
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ancient organisms using Blast. We found no independent proteins in lower organisms
with significant similarity to CCAT suggesting the CCAT promoter may have evolved
from sequential modifications to an ancestral channel and that the CCAT
transcriptional unit was not added to the channel gene in single step.
The evolutionary history of the C-terminus of Cav1.2 can be glimpsed by
looking at the evolution of the voltage gated channel family. The Cav 1 families of
channels diverged early during evolution from other Cav channels and have as earliest
ancestor the L-type channel (LTC) in worms and flies (http://www.treefam.org/cgi-
bin/TFinfo.pl?ac=TF312805#description). We selected channel proteins derived from
transcripts containing the longest c-terminus from human, mouse, Zebrafish and
compared to the ancestral worm and fly channels for all L-type families. Predictably,
the alignments show that the regions of greatest variation are the N and C-terminal
cytoplasmic regions. All channels in the LTC family are conserved to 20-30 amino
acid segment beyond the calmodulin binding motif, highlighting the importance of this
domain in LTC function (Sup Fig 4C and D). Multiple sequence alignment of the
mouse Cav 1.1, 1.2, 1.3 and 1.4 and the worm and fly LTC sequences highlight a
second region of conservation, a modified leucine zipper domain (LZ) (Figure). The
LZ is entirely contained in the last exon of the channel and moreover the E n-1/E n
boundary is also conserved in all channels including C. elegans. This suggests that the
last exon and therefore a significant part of the CCAT sequence was present early in
the evolution LTC genes. The sequence for E n-1, which contains the 238 bp promoter,
is on the other hand highly variable at the amino acid and nucleotide level. This
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suggests that the new regulatory sequence might have evolved around the divergence
of Cav1.2 channels.
How does a sequence that has no prior function in regulating gene expression
can become a fortuitous promoter? One hypothesis is that new regulatory sequences
can be inserted through transposition and many specific examples of mammalian
genes regulated by promoters donated by endogenous transposable elements have
been reported (Cohen et al., 2009; Ferrigno et al., 2001; Oei et al., 2004). A search for
interspersed repeats in the Mouse CACNA1C gene using RepeatMaster failed to
revealed any repetitive sequences near CCAT’s promoter. However, these sequences
can be hard to recognize, especially if the integration occurred in a distant past.
Alternatively, de novo regulatory sequences can be created through, small-scale, local
mutations which modify and create transcription binding sites.
Recent evidence has shown that LZ is required for the targeting of PKA to
Cav1.2 channels and functional regulation of the channels by PKA signaling which is
an important mechanism of cardiomyocyte regulation in response to β-adrenergic
stimulation (Hulme et al., 2003; Hulme et al., 2004). Interestingly, we found that
mutation of the I-F-I-L residues within the rat or mouse CCAT sequence is sufficient
to abolish transcriptional activation (Sup Fig 4B). This highlights the importance of
this domain in channel and CCAT function.
Alternative Transcript vs. Channel Cleavage
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Several laboratories have reported that LTCs are cleaved at their c-terminus
and the site of cleavage of Cav1.1, the skeletal isoform of Cav1.2, has been recently
identified (De Jongh et al., 1994; De Jongh et al., 1989; De Jongh et al., 1991; Hulme
et al., 2005). Thus far, our experiments on heterologously expressed Cav1.2 channels
have failed to show cleavage under conditions of low and high electrical activity and
in different cell types. We have used sensitive methods including luciferase assays and
immunoprecipitation assays to demonstrate that, in the absence of this alternative
protein, or conditions where the channel is degraded such as in 293t cells, no
additional c-terminal fragments can be observed (Figure 1). Nevertheless, our results
do not preclude the possibility that endogenous full-length channels proteins are
proteolytically processed and that we have yet to determine the exact combination of
cell type and stimulus that lead to cleavage of channels. The phenomenon of channel
proteolysis and its mechanism, as it is described until now, does necessitate further
experimental validation. For Cav1.2, the exonic promoter precludes cleavage studies in
heterologous channels. Future experiments would require mutation of all Methionines
downstream of the TSS. Perhaps studies in Cav1.1 where a cleavage site has been
identified can help elucidate the conditions that trigger cleavage of channels, the
channel sequences involved, and the exact nature of the fragment.
Other CCAT Proteins
Our results also put forward the possibility that other proteins are expressed
from the CACNA1C gene. Transcript 2a has its TSS within Exon 27 of the Mouse
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gene. The existence of this protein is supported by the presence of a transcript of
appropriate size in mRNA extracts from brain detected with a c-terminal probe and a
protein of predicted molecular weight in tissue extracts detected with a c-terminal
antibody. The predicted protein encodes for Cav1.2’s sixth transmembrane helix of
ion pore domain III, all of domain IV and the cytoplasmic c-terminus. Thus, this
membrane protein would have a plausible voltage sensor. There are several intriguing
possibilities for the role of such a protein in excitable cells. Such protein may affect
the function of individual channels or channel complexes. It could also influence
channel transport along the secretory pathway and provide a regulatory step for
channels at the membrane. This protein could also function independently to release
the transcription factor CCAT with the appropriate voltage stimulus or as an oligomer
to form functional channels or signaling complexes.
We provide strong evidence that CACNA1C encodes in addition to a calcium
channel a transcription factor within its 3’ region. We show that at least in mice, the
expression of the transcription factor CCAT is driven by a promoter nestled within the
penultimate Exon of the gene. Transcription at this promoter is regulated in a cell-type
and developmental specific ways. In the brain, regulated expression from this
promoter results in highest level of expression in subpopulations of inhibitory neuron
before postnatal life. These findings reveal another unexpected chapter of CCAT’s and
CACNA1C’s biology. The gene products from the many exonic transcriptional start
sites found in vivo will be a rich area of study in all fields of biology.
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EXPERIMENTAL PROCEDURES
Materials
Brefeldin A was purchased from Calbiochem and used at 10µM.
The generation of Anti-CCAT was described earlier (Gomez-Ospina et al., 2006). The
antibody was used 1:500-1:1000 for western blots. Anti-gal4 SC-510 was used at
1:200 for western blots and 4 µg for immunoprecipitations and was purchased from
Santa Cruz Biotechnology. Anti-Map2 was used at 1:1000 for IHC and was obtained
from Chemicon/Millipore.
Cell culture and Transfection
Neuro2A cells were cultured in Dulbecco's Minimal Essential Media (DMEM)
containing 10% fetal bovine serum (FBS; 15% for PC12s), penicillin, streptomycin
(P/S), and L-glutamine (LQ).
Cortical neurons were dissociated from E17-19 Sprague Dawley rats as described (Xia
et al., 1996) and maintained for 6–14 days in culture in Basal Medium Eagle with 5%
FBS, P/S, LQ, and 1% glucose or in Neurobasal medium containing B27 supplement
(Invitrogen).
Thalamic neurons were dissected from E17-19 Sprague Dawley rats in ice-cold Hank's
Balanced Salt Solution without Ca++ and Mg++ (HBSS, Gibco). Thalami were
enzymatically digested using trypsin (Worthington, 10mg/ml), DNase (Sigma, 200
U/ml) in HBSS at room temperature for 10 min. Thalami were washed 3x in Basal
Medium Eagle with 5% FBS, P/S, LQ and 1% glucose and gently triturated in the
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same media. Neurons were plated at 25,000/cm2. Arabinosylcytosine was added 24 h
after plating to inhibit glial cell growth.
Neuro2As (24 hr), cortical and thalamic neurons (96 hr) were transfected using
lipofectamine 2000 according to manufacturer's instructions.
Plasmid Construction
Note: All mutations were generated using the Quick Change XL mutagenesis
kit according to manufacturer’s instructions. All amino acid and base pair positions in
Cav1.2 refer to Acc# AAA18905.1
Construction of the dihydropyridine resistant Cav1.2 -Gal4 and ∆TA channels
has been previously described (Dolmetsch et al., 2001; Gomez-Ospina et al., 2006).
Construction of ∆TM-IQ was achieved in a three-step process. First, we inserted a
KpnI site immediately downstream of the IQ at position 4929 of the Cav1.2 -Gal4
channel’s coding sequence. The was accomplished by PCR amplification with the
following primers: 4930-4965 Fwd 5’-
GGCAAGCCCTCGCAGAGGAATGCACTGTCTCTGCAG-3’ and IQ reverse
(4893-4929)
5’-GGTACCGACCAGCCCCTGCTCTTTTCGCTTCTTGAATTTCCTG-3’. The
amplicon was a linearized channel vector with the KpnI site added to the reverse
primer. The PCR product was then DpnI digested, blunt ligated and transformed.
The second step was to insert another KpnI site at 4477-4482 nucleotide position
taking care of maintaining the channel’s reading frame. The primers used were: (4477-
4482) KpnI fwd 5’-
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GGATTGGTCTATCCTTGGTACCCATCACCTGGATGAATTCAAGAG-3’
(4477-4482) KpnI rev 5’-
CTCTTGAATTCATCCAGGTGATGGGTACCAAGGATAGACCAATCC-3’
Finally to obtain ∆TM-IQ, the mutant plasmid was subjected to KpnI digestion, gel
extraction, ligation and transformation.
The delta cleavage site channel was generated by deleting 150 a.a. downstream of the
IQ motif. The Cav.12-Gal4 channels was amplified using the following primers:
5371-5406 Fwd GTCAGCACTGTGGAGGGCCATGGGCCTCCCTTGTCC
IQ reverse (4893-4929)
GGTACCGACCAGCCCCTGCTCTTTTCGCTTCTTGAATTTCCTG. This linear
construct was then DpnI treated, blunt ligated and transformed.
The translational stop channel was created after deletion of 193 bp between
nucleotides 5588 and 5781 using the double KpnI site strategy using the following
primers:
5588 KpnI fwd 5’-
GCTCTCCACAGATATACTCTGGTACCAGGACGATGAAAACCG-3’
5588 KpnI rev 5’-
CGGTTTTCATCGTCCTGGTACCAGAGTATATCTGTGGAGAGC-3’ and
5781 KpnI fwd 5’-GCCTTGCCCTTGCATCTGGTACCTCACCAGGCATTGG-3’
5781 KpnI rev 5’-CCAATGCCTGGTGAGGTACCAGATGCAAGGGCAAGGC-3’
After KpnI digestion and excision of the intervening sequence there was a frameshift
in the sequences creating an early stop codon at a.a. 1910.
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Methionine mutations of Cav1.2-Gal4 channels and minigenes and CCAT
minigenes were generated using the primers:
M2011I: F 5’-TTGGCAGTGGCAGGGATCCCCCGGAGAGCCCGG-3’
R 5’-CCGGGCTCTCCGGGGGATCCCTGCCACTGCCAA-3’
(Primers add a silent Cspc site used for screening mutants).
M2073I: F 5’-CTGGCTGACGCCTGCGATATCACAATAGAGGAGATGGAG-3’
R 5’-CTCCATCTCCTCTATTGTGATATCGCAGGCGTCAGCCAG-3’
(Primers add a silent EcorV site used for screening mutants).
M2078I:
F: 5’CGACATGACAATAGAGGATATCGAGAACGCCGCAGACAACATC-3’
R: 5’-GATGTTGTCTGCGGCGTTCTCGATATCCTCTATTGTCATGTCG-3’
(Primers add a silent EcorV site used for screening mutants).
Promoterless constructs were generated by MfeI digestion and self-ligation of
the pcDNA4 Cav1.2 -Gal4 plasmid. This removed additional 407 base pairs from the
channel’s 5’ end.
The Cav1.2 CDS as promoter constructs except for the full length channel
sequence were built by PCR amplification of : Ion Pore: 4 – 445 nt, E46-E47: 5794 –
6429 nt, E46: 5794-6129 and E47: 6130-6429 and insertion via KpnI digestion into
pGL3 basic luciferase reporter vector (Promega). The full length channel sequence
was subcloned from PA1-Cav1.2 plasmid which contains the full length channel’s
sequence in between two KpnI sites in the PA1 expression vector originally obtained
from Dr. Michael Lin and described earlier (Gomez-Ospina et al., 2006). The
channel’s termination codon and on additional stop codon in between the channel’s
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and luciferase’s CDS were mutated using the following primers:
Pgl3- Cav1.2 6490 stop
F 5’-GAGATCTGCGATCTAAGGAAGCTTGGCATTCCGGTACTG-3’
R 5’-CAGTACCGGAATGCCAAGCTTCCTTAGATCGCAGATCTC-3’
Pgl3- Cav1.2 6436 stop
F 5’-CTATGTCAGCAACCTGTACGGTACCGAGCTCTTACGC-3’
F 5’-GCGTAAGAGCTCGGTACCGTACAGGTTGCTGACATAG-3’
Minigenes were generated using a multistep cloning process: 1. An NcoI site
was mutated to a MluI site in the pGL4.10 vector (Promega) with the purpose of
creating a compatible site where the genomic sequence could be inserted into. 2. To
make the construct usable for stable cell line creation, we inserted an amplified Zeocin
resistance cassette from pCDNA4 into the BamI/Sal1 sites of pGL4.10. 3. To create
4Kb-0/luciferase, a 4 Kb genomic region was PCR amplified using NcoI/MluI sites
and the BAC clone RP23-158O9 3’ as template which contains the 3’ end of
CACNA1C. 5. The Gal4 based minigene was finally generated by removing the
luciferase and subcloning a PCR amplified sequence of Gal4-DBD using the
restriction sites MluI and Xba1. 4Kb-1 was created by using the mutagenesis kit to
insert a G between the channel’s and luciferase’s coding sequence in the 4Kb-0
construct. ∆238 was created by inserting a CspcI site at 1579 bp of the 4Kb construct
within Exon 46. The sequence already had a CspcI site at the 5’ end of Exon 47.
Subsequent digestion with CspcI removed 238 bp sequence.
The neuronal channel 4Kb promoter was cloned by PCR amplification of 4Kb
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upstream of the initiating methionine for the neuronal channel from a BAC clone
RP23-117C19 that contained the 5’ end of CACNA1C. The PCR product had
EcoRV/HindIII sites added which were used for cloning into the pGL4.10 vector.
CCAT TA promoter was generated by PCR amplification of 4Kb upstream of the
2011 methionine from BAC clone RP23-158O9. This was inserted into the XhoI/MluI
sites in pGL4.10 .
The plasmids encoding the N-terminal tagged YFP-mem-CCAT and YFP-
CCAT were generated using Gateway technology (Invitrogen) by first cloning coding
the sequences from a.a. 1160-2144 for mem-CCAT and 2011-2144 for CCAT
amplified from a pCDNA4.0 Cav1.2 plasmid into the TOPO sites of the pCR8 entry
vector and subsequently transferring the Cav1.2 coding sequence into a destination
vector called pDEST-pGWYFP that contains a CMV promoter and an N-terminal YFP
in frame with the ATTR (Cox and Emili, 2006) acceptor sequences.
Subcellular Fractionation
Note: For biochemical experiments all protein samples were kept at or bellow -4°C.
Complete protease inhibitor tablets were added to all solutions fresh before each
experiment.
For Cav1.2 knockout mice and control pubs, pregnant females at 11 days post plugging
were anesthetized using CO2 and embryos were dissected in cold PBS. The tails were
cut and separated for DNA genotyping. Tissue subcellular fractionation and protein
extraction was performed as described (Cox and Emili, 2006). Briefly, after dissection
embryos were cut and washed in cold 250-STMPBS, homogenized and centrifuged at
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800g for 15 min. The nuclear pellet was resuspended and homogenized again.
Supernatants were pooled to form the cytosolic fraction I and the pellet constituted
nuclear fraction I. The cytosolic fraction was centrifuged 1h at 100,000 g to pellet the
microsomal fraction which was solubilized in ME buffer for 1h and finally centrifuged
for 30min at 9000g. This supernatant constituted the solubilized membrane fraction.
The nuclear fraction I was resuspeded in 2M-STMDPS buffer and layered on a 2M
STM cushion in a centrifuge tube. Samples were spun at 80,000g for 30-45 min. The
pelleted nuclei were solubilized in NE buffer, then passaged through an 18 gauge
needle and spun at 9000g for 30 min. Supernatant was used as salt soluble nuclear
proteins.
Immunoprecipitation and Western Blotting
N2A cells were lysed 24 hrs post-transfection using lysis buffer containing 1.5%
TritonX-100, 50 mM TRIS-HCL pH 7.5, 150mM NaCl amd 10mM EDTA and
protease inhibitor tablets (Roche). Immunoprecipitations were carried out using 4 µg
of Gal4 antibody and Protein A/G beads (Santa Cruz).
Western blotting was conducted using standard protocols. Antibodies and dilutions are
included in Supplemental Experimental Procedures. Protein concentration was
measured by the BCA method (Pierce).
Immunoflurescence
Cortical and thalamic neurons were fixed in 4% paraformaldehyde/2% sucrose and
10mM EDTA in PBS for 10 minutes followed by permeabilization with 0.025%
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TritonX-100 and blocking with 3% bovine serum albumin (BSA) in phosphate-
buffered saline (PBS). Cultured neurons were stained primarily with rabbit anti-CCAT
(1:500) and secondarily with 1:500 dilutions of Alexa 594-conjugated or Alexa 488-
conjugated anti-rabbit antibodies (Molecular Probes). Nuclei were stained using
Hoechst 33258 or DAPI (Molecular Probes).
Brains from embryonic and postnatal rats and wildtype C57/bl6 mice were
dissected and fixed in 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB, pH
7.4). Brains from animals younger than embryonic day 18 (E18) were fixed in PFA for
30 minutes, whereas brains from postnatal mice were fixed overnight at 4°C.
Subsequently brains were transferred to a 30% sucrose solution overnight for
cryoprotection, embedded in Tissue-Tek OCT compound, and cut (10µm for
embryonic brains, 25-30µm for postnatal brains) on a freezing cryostat (Leica,
CM3050). All tissue was stored at -80°C until further use. For immunofluorescence
analysis of CCAT expression, slides were washed in PBS containing 0.1% bovine
serum albumin (BSA) to remove excess OCT. Sections were blocked in 10% normal
goat serum (NGS; Gibco) containing 0.25-3% Triton X-100 for 1 hour at 25°C.
Primary antibody (anti-CCAT, 1:150) was applied overnight in 10% NGS with 0.1%
Triton X-100 at 4°C followed by the appropriate fluorochrome conjugated secondary
antibody (Alexa conjugates; Molecular Probes) for 1 hour at 25°C. Slides were then
washed in PBS with 0.1% BSA, counterstained with Hoechst or DAPI, and mounted
in Aqua Poly/Mount (Polysciences, Inc.) for fluorescence microscopy.
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Slides were visualized by conventional epifluorescence microcopy using a cooled
CCD camera (Hamamatsu) coupled to an inverted Nikon Eclipse E2000-U
microscope.
Luciferase Assay Transfections
Cav1.2-Gal4 channels were transfected in a ratio of 2:1:1:0.5 Cav1.2, β1b subunit,
firefly luciferase and Renilla luciferase reporters.
Minigenes were transfected as a 1:1:0.25 ratio of Minigen to UAS-firefly luciferase to
Renilla luciferase.
Channel coding sequence as promoter constructs were transfected at a ratio of 2:1
pGL3 based vector to Renilla luciferase.
Most luciferase assays were performed 24 hr after transfection using the Dual-Glo
luciferase assay kit from Promega. A Veritas 96-well luminometer (Turner
Biosystems) was used to measure light emission. PFA-CMV (Gal4 alone) and UAS-
luciferase constructs and were obtained as part of the PathDetect transreporting system
(Strategene). Data sets were analyzed using Igor Pro and Prism4 software. Two-paired
t tests were performed between relevant conditions.
Northern Blots
RNA isolation: mRNA was extracted from rat brains and N2A cells using the Fast-
Track mRNA isolation kit according to manufacturer’s instructions (Invitrogen).
Probe: Exon 47-pGL3 plasmid was used as template to PCR amplify Exon 47’s
sequence. After gel extraction, 25ng of PCR template were used for labeling using
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prime-it II random primers labeling kit (Stratagene) and 5µl of {α-32P]dCTP at 3000
Ci/mmol (Amersham) by following the manufacturer’s protocol. For loading control a
900bp region of the 18S RNA was amplified using the following primers:
18S RNA L 5’-GAGGGAGCCTGAGAAACGGCTA-3’
18S RNA R 5’-AACTAAGAACGGCCATGCACCA-3’ and used as template for
random labeling as described above. The full length channel probe used contained
sequence nt 2659 to 2966 in domain III of the channel’s coding sequence and was
amplified using the following primers:
IIIS1 F 5’-GCGAAGCTTagcccaaacaacaggttc-3’
IIIS3 R 5’-gcgAAGCTTatgccaaaggagatgagg-3’
Blots: Northern blots were carried out using the NorthernMax Kit solutions and
followed the protocol as recommended by the Manufacturer (Ambion). Briefly, 5µg
of mRNA were loaded into 1% RNAse free agarose gel. Electrophoresis was carried
out at ~5 V/cm. RNA was then transferred to Ambion’s BrightStar-Plus membranes
by downward capillary transfer. The RNA was crosslinked to the membrane using a
commercial crosslinker. Membranes were prehybridized at 68°C for 5-6h in
prehybridization solution plus 100µg of salmon sperm DNA. Labeled probe was
denatured before adding to hybridization solution and incubated at 55-65°C overnight.
Membranes were washed using low and high stringency washes as outlined in
Northern Max kit. Films were exposed form 5-48h at -80°C.
5’ RACE experiments
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5’ RACE was carried out using Invitrogen’s GeneRacer kit according to
manufacturer’s instructions with the following specifications:
1) 250ng of each mRNA template were dephosphorylated using calf intestinal
phosphatase (CIP) at 50°C for 1h. 2) After precipitation the cap structure was removed
using Tobacco acid pyrophosphatase (TAP) at 37° C for 1h. 3) After precipitation the
RNA oligo :
5’-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3’
was ligated to the 5’ end using T4 RNA ligase by incubating at 37C for 1 h. 4)
Reverse transcription followed using RACE outer primer: 5’-
CTACAGGTTGCTGACATAGGACCTGCT-3’ encompassing sequence including
the channel’s termination codon or olido-dT primer. 5) PCR amplification was carried
out using the primers: GeneRacerTM 5’-CGACTGGAGCACGAGGACACTGA-3’
primer and 5’RACE reverse inner primer 5’-
CACAAAAGGTAAGAGGGTGCCGTTG-3’. To increase abundance of longer
transcripts a reverse primer closer to 5’ end (5970nt of cDNA) was used for CDNA
synthesis: 5’-GAAGCTGCTGTTGAGTTTCTCACTGGACTC-3’ and the nested 5'-
CTGGTGATGAACCAGATGCAAGGGCA-3’ (5793 nt) was used for amplification.
GeneRacerTM 5′ Nested Primer 5’-GGACACTGACATGGACTGAAGGAGTA-3’
5’ RACE of the second transcript from the Cav1.2-Gal4 channel was accomplished
using the following primers: for cDNA synthesis Cav1.2-Gal4 6865 (up to stop) R:
5’-TGACCGGCGATACAGTCAACTGTCTTTG-3’ and for PCR the Cav1.2-Gal4
6565 R 5’-TCAGCGGAGACCTTTTGGTTTTGGG-3’ and GeneRacerTM 5’ as
forward primer.
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Generation of conditional knockout mice for the Cav1.2 calcium channel gene
Mice lacking the L-type a1c (Cav1.2) calcium channel gene were generated by
homologous recombination mediated gene targeting (Supplementary Figure 1B and
1C). The targeting construct was designed to delete exons 14 and 15 of the a1c gene
under the control of CRE recombinase. To facilitate Southern screening, a new BamHI
site was generated 5’ to Exon 16, and the original BamHI site 5’ to Exon 14 was
eliminated in the targeting construct (Fig X). The a1c targeted mice were maintained
in 129/sv-C57BL/6 mixed background. The a1c floxed (Neo deleted) mice were
generated by crossing a1c targeted mice with FLP transgenic mice (kindly provided by
Dr.Dymecki). Ubiquitous deletion of the floxed exon was achieved using CMV-CRE-
transgenic mice.
Sequence Analysis and Multiple Sequence Alignments
The following accession numbers were used for alignments:
Cav1: Drosophila Melanogaster NP_602305.1, Caenorhabditis elegans
NP_001023079.1
Cav1.1: Human NP_000060.2, Mouse NP_055008.2, Zebrafish NP_999891.1
Cav1.2: Human NP_001123312.1 Mouse NP_001153006.1 Zebrafish NP_571975.1
Rat P22002.1, Chimpanzee XP_522315.2, Rhesus monkey XP_001117926.1, Guinea
pig dbj|BAA34185.2, Rabbit NP_001129994.1, Horse XP_001490707.1, Dog
XP_534932.2,
Bovine XP_001255123.2, Chicken XP_416388.2
Cav1.3: Human NP_001122312.1, Mouse NP_001077085.1, Zebrafish NP_982351.1
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Cav1.4: Human NP_005174.2, Mouse NP_062528.2
Sequences were aligned with ClustalW and MAFFT multiple sequence alignment
programs. The alignments were edited using Jalview and colored using percentage
identity with a conservation color increment set to 20.
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FUTURE EXPERIMENTS
Experiments over the last five years have been aimed at characterizing basic
properties of CCAT. We first showed that such a fragment was present in cells. We
then were able to demonstrate that this channel fragment played a role in nucleus and
shared characteristics of many transcription factors. Later, we were able to discern the
way in which CCAT was made in cells. Now, we are finally in a position to ask what
is perhaps the biggest question remaining regarding CCAT’s biology: what is CCAT’s
function in vivo? In the following paragraphs I’ll be discussing several experiments
that are underway that will help us understand CCAT’s function in vivo.
The best way to ask whether CCAT is necessary for the function or
development of cells is to use loss of function studies. In order to be able to design
experiments to investigate CCAT’s role in cells using a “knockout” approach we
needed to understand how CCAT was being produced. Loss of function experiments
with CCAT are complicated by the fact CCAT is a fragment of a channel who is vital
to survival and the function of excitable cells. Consequently, any perturbations to the
CACNA1C gene sequence should be carefully designed so that they disrupt CCAT
production/function while maintaining the properties of the channel intact. We have
shown that CCAT is expressed from an alternative message and we predict that, as we
have shown in vitro, a single methionine mutation should be able to knockout the
production of the 15 KDa CCAT protein in vivo. One future experiment is to generate
mice using homologous recombination, where only M2011 has been disrupted. Our
preliminary studies suggest this mutation has no effect on the calcium conduit or
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signaling to CREB properties of the full-length channel. We are particularly interested
in the role that CCAT has in development of the brain. Our data regarding the very
specific and temporally regulated expression of CCAT in inhibitory neurons, suggest a
role for this protein in their development. We plan to study the number, migration and
behavior of inhibitory neurons in these mice, using immunohistochemical and
electrophysiology techniques as well as genetic mouse reporters.
Understanding the precise timing and location of CCAT expression will help
understand CCAT’s role in development. Toward this end, we plan to create a BAC
transgenic mouse that reports on expression from the exonic promoter identified in
chapter 3. We have obtained a BAC containing 120 Kb of the mouse CACNA1C gene.
This BAC contains the last 37 exons and the 3’ UTR of the channel thus lacking the
full-length channel’s promoter including the first ten exons. We have introduced a
GFP reporter at the 3’ end of the channel’s coding sequence immediately before the
termination codon using bacterial recombineering. Expression of GFP tagged protein
from this BAC would strongly support the presence of internal promoter elements
within the gene. Transgenic mice generated using this construct can be used to study
the activity of this promoter in vivo by analyzing the tissue and cellular distribution of
GFP at different developmental stages.
Another approach towards understanding CCAT’s function is to perform gain
of function studies. In these experiments, CCAT can be added exogenously using
transfection, electroporation or even expression from a transgene. These experiments,
especially under controlled conditions, can be used to ask very exact questions about
the effect of CCAT on specific cellular processes. One confounding factor as it
132
pertains to CCAT specifically is that CCAT overexpression may perturb the channel’s
function by inhibiting the channel or by competing for interacting proteins that may be
important for targeting or function of the channel. We have already reported the effect
of CCAT on neurite growth in cultured neurons. In the same manner, this approach
can be used to study CCAT’s role in neuronal differentiation by expressing CCAT in
precursor neuron cultures and quantifying the number of differentiated neurons and
the percentages of specific subpopulations. A version of CCAT with a mutated leucine
zipper differs by four amino acids from the wild type protein but completely lacks
transcriptional activation, making it an optimal negative control for these studies.
Finally, one can also envision more in vivo approaches where the effect of CCAT
expression is studied in developing brains of mouse embryos or newborn pubs after
introduction of the CCAT transgene using in vivo electroporation.
B C
250
40
Cav1.2
CCAT
anti-Gal4
WT∆TM-IQ
∆C.S.STOP
∆TA
UAS
Tra
nscr
iptio
n (
Fire
fly/R
enilll
a)
Gal4 WT ∆TM-IQ ∆C.S. STOP ∆TA
0.00
0.25
0.50
0.75
1.00
1.25
Cav1.2 channels
ED
WT M2011I M2078I
Anti Gal4
Cav1.2
CCAT
250
40
0.00
0.25
0.50
0.75
1.00
1.25
UAS
Tra
nscr
iptio
n (
Fire
fly/R
enilll
a)
WT M2011I M2078IGal4
Cav1.2-Gal4 channels
A
IQ TACleavage Site
Translational
Stop
Gal4TM
DAPI MERGED
100X
600X
CCATN/+ N/+ N/+ N/N N/N N/N
250
60
F G
32
150
100
*
*
*
*
133
0.0
0.5
1.0
1.5
UAS Transcription (Firefly/Renillla)
Gal
4
Cav
1.2-
Gal
4
∆Pro
mot
er+P
rom
oter
01234567
Luciferase expression (Firefly/Renillla)
Full
leng
thIo
n Po
reE4
6-47
E46
E47
M20
11AAGCTA...STOP
TTCGAT ...STOP
AA
CC
TG
TT
GG
AC
..
..
..
..
..
..
AT
GT
AC
Gal
4
AAGCTA...STOP
TTCGAT ...STOP
AA
CC
TGG
TT
GG
AC
..
..
..
..
..
..
AT
GT
AC
C AAGCTA...STOP
TTCGAT ...STOP
AA
CC
TG
TT
GG
AC
..
..
..
..
..
..
AT
GT
AC
AAGCTA...STOP
TTCGAT ...STOP
AA
CC
TG
TT
GG
AC
..
..
..
..
..
..
ATC
TAG
E47
4Kb-
0
4Kb-
1
∆238
bp
M20
11I
0.00
0.01
0.02
0.03
0.04
UAS Transcription (Firefly/Renillla)
4Kb-0
4Kb-1
∆238b
p
M2011
I
M20
11IQ
Ion
Pore
Luci
fera
se
TACGGT...ACC
GGTGGC ...GTA
...
...
ATG
TAC
AAC
CTG
TTG
GAC
..
..
..
ATG
TAC
..
..
..
Full
leng
th
TACGGT...ACC
GGTGGC ...GTA
...
...
ATG
TAC
Ion
Pore
AAC
CTG
TTG
GAC
..
..
..
ATG
TAC
..
.TACGGT...ACC
GGTGGC ...GTA
...
...
ATG
TAC
E46-
47
ATG
TAC
TACGGT...ACC
GGTGGC ...GTA
...
...
ATG
TAC
E46
AGC
CTG
TCG
GAC
..
..
..
TACGGT...ACC
GGTGGC ...GTA
...
...
ATG
TAC
E47
A
7.6
Kb
2.4
Kb
1.4
Kb
Cav
1.2-
Gal
4
Unt
+Pro
mot
er ∆Pro
mot
er
9.5
Kb
Anti-
Gal
4
Cav
1.2
CC
AT
250
Prom
oter
+
40
BC
DE
FG
H
4Kb-0
4Kb∆
MM20
11I
M2073
IM20
78I
0.00
0.01
0.02
0.03
0.04
0.05
UAS Transcription*
*
**
***
*
134
E18 P1 AdultCtxMid
9.57.5
4.4
2.2
1.8
18S
A C
).U. A( S81/ noi sser pxe dezil a
mr oN
CB Ctx MidCB
Cav1.2 E47
18S
1234
0
Full length channel5
123456
0
2.2 Kb
1
2
0E18
P1 AdultCtxCBMid CtxCB Mid
4 .0 Kb
E
3
Coronal Sagital
CbS
BS V
Th
C
V
S
CV
VC
S
0.5
1.5
2.5
3.5
0.5
1.5
2.5
3.5
U
AS T
rans
crip
tion
(N
orm
alilz
ed to
Gal
4)
UAS
Tra
nscr
iptio
n (
Nor
mal
ilzed
to G
al4)
Cav1.2
Gal4 Stop
Gal4 M2011
I
Gal4
Cav1.2
Gal4 Stop
Gal4 M2011
I
Gal4
Cor
tical
Neu
rons
Thal
amic
Neu
rons
CCAT
CCAT
DAPI
DAPI
F
***
Cerebellum Thalamus
P1
D
E18 E18
P1
3W 3W
CCAT CCAT
CCATMap2
18S
E18 P1 AdultCtxMidCB Ctx MidCB
9.57.5
4.4
2.2
1.8
Cav1.2 IIIS3
B
135
5’ MetG AAAAAA3’
AAAAAA3’
cDNA synthesis
cDNA
Exon 46 Exon 47 Gal4
M2011
RNA oligo
PCRF R
A B
Predicted Proteins
CACNA1Cgene
Chr6 119200000 119100000 119000000 118900000 118800000 118700000 118600000 118500000
CalciumChannelCaV1.2
Membrane-AnchoredC-term with Voltage Sensor
CCAT
Mem-CCAT CCAT
Cav1.2-Gal4Promoter+ -
2.01.61.0
0.5
5983 nt 6432 nt 6874 nt
N/N N/N N/+ N/+40
35
25
15
C
D
E
136
C
A CCAT DAPI Merged
Brain
Heart
Bloodvessels
Somites
HeadMesenchyme
Liver
0.00
0.25
0.50
0.75
1.00
Brefeldin AGal4Cav1.2 Gal4
+_
UA
S Tr
ansc
rip
tio
n
(Fir
efly
/Ren
illa)
C
V
HM
C
M
Cav1.2 Targeting Strategy
Chr6
Targeting Construct
Targeting Allele
NULL
FLOXED
B
Null/+
FLox/+
+/+ Targe
ted
12.1 Kb9.5 Kb8.3 Kb
D
CRE
FLP
Supplentary Figure 1
137
0.0
0.1
0.2
0.3
0.4
EmptyVector
Neuronal 4kb
CCAT 4Kb
A B
Cav1.2_HumanCav1.2_ChimpCav1.2_RhesusCav1.2_MouseCav1.2_RatCav1.2_Guinea pigCav1.2_RabbittCav1.2_HorseCav1.2_DogCav1.2_BovineCav1.2_ChickenCav1.2_Zebrafish
Consensus
305305303304305305305305305306306300
Q A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q P V P T L R L E G V E S S E K L N S S F P S I H C G S WA - E T T P G G G G S S A A R R V R P V S L MV P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q L V P T L R L E G V E S S E K L N S S F P S I H C G S WA - E T T P G G G G S S A A R R A R P V S L MV P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q P I P T L R L E G A E S S E K L N S S F P S I H C G S WA - E T T P G G G D S N T T R R A R P V S L MV P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P T T F P R P C P T P P V T P G S R - - - G R P L R P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G S S S MA R R A R P V S L T V P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P S T F P R P R P T P P V T P G S R - - - G R P L Q P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G G S S MA R R A R P V S L T V P S Q A G A P G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P T A I P R P C A T P P A T P G S R - - - GWP P K P I P T L R L E G A E S C E K L N S S F P S I H C S S W S E E P S P C G G G S S A A R R A R P V S L MV P S Q A G A P G R Q F - H G S A S S L A E AQ A L A V A G L S P L L Q R S H S P T S L P R P C A T P P A T P G S R - - - GWP P Q P I P T L R L E G A D S S E K L N S S F P S I H C G S W S G E N S P C R G D S S A A R R A R P V S L T V P S Q A G AQ G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S R A P T T C P Q P W- - - - A T P S S Q - - - GWP P R P I P T L R L E G A E S S E K L N S S F P S I H C G S W S G E P T A C G G G S S A L R R A R P V S L T V P S R A G A P G R Q L - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P G T L P R P C A T P P A T P G S R - - - GWP P Q P I P T L R L E G A E S N E K L N S S F P S I H C S S W S E E P T P C G G G D S T I R R A R P V S L T V P S Q A G A R G R Q F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H P P G T L P P P R L T P P A T P G P - - - - AWP P R P V P T L R L E G A E S S DK L T S S F P S I H C DP H I G E P T P C - G V V G T P R R A R P V S L T V P S P A G P Q G R P F - H G S A S S L V E AQ A L A V A G L S P L L Q R S H S P T T F S R L C A T P P A T P C S R - - - GWP QQ P I P T L R L E G A E S S E K L N S S F P S V H C S S R F P D S S DC G - - - - S P R R A R P V S L T V P S P T A G S S R Q F - H G S A S S L V E AQ A L A V A G L S P L L R R S H S P T L F T R L C S T P P A S P S G R S G G G P C Y Q P V P S L R L E G S G S Y E K L N S S MP S V N C S S WY S D S N- - - - G N H S G R AQ R P V S L T V P P V T R R D S I S L A H G S A G S L V E A
Q A L A V A G L S P L L Q R S H S P T T F P R P C A T P P A T P G S R - - - GWP P Q P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T P C G G G S S A A R R A R P V S L T V P S Q A G A P G R Q F - H G S A S S L V E A
417417415417418418418414418417415413
V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N G A L L P F V N C R DA GQ DR A G G E E - DA G C V R A R G R - - P S E E E L Q D S R V Y - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N G A L L P F V N C R DA GQ DR A G G E E - DA G C V R A R G R - - L S E E E L Q D S R V Y - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N G A L L P F V N C R DA GQ DR A G G E E - DA G C A R A R G R - - L S E E E L Q D S R V Y - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T L L P F V N C R DP GQ DR A V A P E - D E S C A Y A L G R - G R S E E A L A D S R S Y - - - - - - - - - - - - - - - V S N LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T L L P F V N C R DP GQ DR A V V P E - D E S C V Y A L G R - G R S E E A L P D S R S Y - - - - - - - - - - - - - - - V S N LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I G EM E N A A DN I L S G G A P Q S P N G T L L P F V N C R DP GQ DR A G G D E - D E G C A C A L G R - GW S E E E L A D S R V H - - - - - - - - - - - - - - - V R S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C D L T I E EM E N A A DD I L S G G A R Q S P N G T L L P F V N R R DP G R DR A GQ N EQ DA S G A C A P G C - GQ S E E A L A DR R A G - - - - - - - - - - - - - - - V S S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G T QQ S A N G T L F P F V N C R DP GQ DR A G G E E - N E T C A P A L E R - G K S E G E P Q D S R A C - - - - - - - - - - - - - - - G G S LV L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T L L P F V N C R DP GQ DK A G G H V - G DA C T A A L A C - Q K S E E E L Q D S R A H - - - - - - - - - - - - - - - T G S LV L I S E G L GQ F AQ DP R F L E A T T Q E L A DA C DMT I E EM E S A A DD I L S G G A GQ S P N G T L L P C A N C R DP G P DR A G G V E - DA AWA P S A E P - R Q G A E E P R D S R A F - - - - - - - - - - - - - - - A S G LV L I S E G L MQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L N G N S KQ S P N G N L L P F V N C R DA GQ D S A G E E E - E E VQ N P - - DC - X K S Q E E L K D S R I Y - - - - - - - - - - - - - - - I S S LV L I S E G L G R Y A H DP S F I Q V A KQ E I A E A C DMT M E EM E N A A DN I L N A N A P P N A N G N L L P F I Q C R DT G S Q E S R C S L - S L G L S P A T G S DG A L E A E L E E S E G A GQ R N S P L M E D E DM E C V T S L
V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G A P Q S P N G T L L P F V N C R DP GQ DR A G G E E - D+ G C A P A L G R - G K S E E E L Q D S R + Y - - - - - - - - - - - - - - - V S S LConsensus
MM
M
M
M
MMM
Cav1.2_HumanCav1.2_ChimpCav1.2_RhesusCav1.2_MouseCav1.2_RatCav1.2_Guinea pigCav1.2_RabbitCav1.2_HorseCav1.2_DogCav1.2_BovineCav1.2_ChickenCav1.2_Zebrafish
C
IQ
LZ
Exon n-1
Exon n
Supplementary Figure 2
138
CCAT
CCAT
CCAT
DAPI
DAPI
DAPI
CCAT/DAPI
E18
LV
VZ/SVZ,GE
CP
striatum(cp)
E18 Striatum
E18 Cortical plate
CCAT/DAPI
E14
IIIV
IIIV
striatum(cp)
hypothalamicnuclei
thalamus(DM, CM,rhomboid)
CP
VZ/SVZ,GE
LV
CCAT/DAPI
E12.5
LV
VZ/SVZ,GE
striatum(cp)
P1
CCAT/DAPI
striatum(cp)
cortex CCLV
septalnuclei
B C
A
Supplementary Figure 3
139
Cav1.2_HumanCav1.2_MouseCav1.2_ZebrafishCav1.4_HumanCav1.4_MouseCav1.3_HumanCav1.3_MouseCav1.3_ZebrafishCav1.1_HumanCav1.1_MouseCav1.1_ZebrafishCav1.-_C. elegansCav1.-_D. Melanogaster
I Q E Y F R K F K K R K EQ G L V G K P S Q R N - - A L S L Q A G L R T L H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N H V S Y Y Q - S DG R S A F P Q T F T T Q R P L H I N K A G S S - - - - - - - - - - - - - - - - - - Q G DT E S P S H E K L V D S TI Q E Y F R K F K K R K EQ G L V G K P S Q R N - - A L S L Q A G L R T L H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N H V T Y Y Q - S D S R G N F P Q T F A T Q R P L H I N K T G N N - - - - - - - - - - - - - - - - - - Q A DT E S P S H E K L V D S TI Q E Y F R K F K K R K EQ G L V A K I P P K T - - A L S L Q A G L R T L H DMG P E I R R A I S G D L T V E E E L E R AMK E T V C A A S E DD I F R R S G G L F G N H V N Y Y HQ S DG H V S F P Q S F T T Q R P L H I S K S G S - - - - - - - - - - - - - - - - - - - P G E A E S P S HQ K L V D S TI Q DY F R K F R R R K E K G L L G N DA A P S - T S S A L Q A G L R S L Q D L G P EMR Q A L T C DT E E E E E E - - - - - - - - - - GQ E G V E E E D E K D L E T N K A T MV S Q P S A R R G S G I S V S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R K F R R R K E K G L L G R E A P T S - T S S A L Q A G L R S L Q D L G P E I R Q A L T Y DT E E E E E E E - - - - - - E A V GQ E A E E E E A E N N P E P Y K D S I D S Q P Q S R WN S R I S V S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R K F K K R K EQ G L V G K Y P A K N - T T I A L Q A G L R T L H D I G P E I R R A I S C D L Q DD E P E E - - - - - - T K R E E E DDV F K R N G A L L G N H V N H V N S D- R R D S L QQ T N T T H R P L H VQ R P - - - - - - - - - - S I P P A S DT E K P L F P P A G N S V C H N H H NHI Q DY F R K F K K R K EQ G L V G K Y P A K N - T T I A L Q A G L R T L H D I G P E I R R A I S C D L Q DD E P E D- - - - - - S K P E E E D- V F K R N G A L L G N H V N H V N S D- R R D S L QQ T N T T H R P L H VQ R P - - - - - - - - - - S MP P A S DT E K P L F P P A G N S G C H N H H NHI Q DY F R K F K K R K E E G L V G V H P AQ N N T A I A L Q A G L R T L H D I G P E I R R A I S C D L Q DD E L V D- - - - - - F I P E E D E E I Y R R N G G L F G N H I N H I N G DP R R S S G HQ T N A T Q R P L Q VQ P P P H Y V HM EQ P V G R L G R A N AMAQQ N H H R H H H H H H H H H H HI Q E H F R K F MK R Q E E - Y Y G Y R P - K K D I VQ I Q A G L R T I E E E A A P E I C R T V S G D L A A E E E L E R - - - AMV E A AM E E G I F R R T G G L F GQ V DN - - F L E R T N S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q E H F R K F MK R Q E E - Y Y G Y R P - K K DT VQ I Q A G L R T I E E E A A P E I H R A I S G D L T A E E E L E R - - - AMV E A AM E E G I F R R T G G L F GQ V DN - - F L E R T N S L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q E H F R K F MQ R Q E E - L Y G Y R P T K K N A D E I K A G L R S I E E E A A P E L H R A I S G D L I A E D EM E R - - - AM E S G - - E E G I Y R R T G G L F G L N A DP F S S E P S S P L S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R R F K K R K EM E A K G V L P AQ T P Q AMA L Q A G L R T L H E I G P E L K R A I S G N L E T D F N F D E - - - - - - - - - - P E P Q H R R P H S L F N N L V H R L S G A G S K S P T E H E R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -I Q DY F R R F K K R K EQ E G K E G H P D S N - - T V T L Q A G L R T L H E V S P A L K R A I S G N L D E L DQ E P E - - - - - - - - - - - - P MH R R H H T L F G S VW S S I R R H G N G T F R R S A K A T A S Q S N G A L A I G G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
F T P S S Y S S T G S N A N I N N A N N T A L G R L P R P A G Y P S T V S T V E G H G P P L S P A I R VQ E V AWK L S S N R C H S R E SQ A AMA GQ E E T S Q D E T Y E V KMN H DT E A C - - - - - - - - - - - - - - - - - - - - - - - - - - - - S E P S L L S T EM L S Y Q DD E N R Q L T - - - -F T P S S Y S S T G S N A N I N N A N N T A L G R F P H P A G Y S S T V S T V E G H G P P L S P A V R VQ E A AWK L S S K R C H S R E SQ G A T V N - Q E I F P D E T R S V R M S E E A E Y C - - - - - - - - - - - - - - - - - - - - - - - - - - - - S E P S L L S T DM F S Y Q E D E HR Q L T - - - -F T P S S Y S S S G S N A N I N N A N N T A I G H R Y P K P - - - - T V S T V DGQ T G P P L T T I P L P R P T WC F P N K S S D S S D S R L P I I R R E E A S T D E T Y D E T F L D E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R DQ AM L S MDM L E F Q D E E S KQ L A P M- - V G DR L P D S L S F G P S DDDR - - - - - - - - - - - - - - - G T P T S SQ P S V P Q A G S N T H R R G S G A L I F T I P E E G N S Q P K G T K GQ N KQ D E D E E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V P DR L S Y L D EQ A G T P P C S V L L P P- - V K E K L P D S L S T G P S DDDG - - - - - - - - - - - - - - - L A P N S R Q P S V I Q A G S Q P H R R S S G V F M F T I P E E G S I Q L K G T Q GQ DNQ N E EQ E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V P DWT P D L D EQ A G T P S N P V L L P PN S I G KQ V P T S T N A N L N N A NM S K A A H G K R P S I G N L E H V S E N G H H S S H K H DR E P Q R R S S V K R T R Y Y E T Y I R S D S G D EQ L P T I C R E DP E I H G Y F R DP H C L G EQ E Y F S S E E C Y E DD S S P T W S R Q N Y G Y Y S R Y P G R N I D S E R P R G Y H H P Q G F LN S I G KQ A P T S T N A N L N N A NM S K A A H G K P P S I G N L E H V S E N G H Y S - C K H DR E L Q R R S S I K R T R Y Y E T Y I R S E S G D EQ F P T I C R E DP E I H G Y F R DP R C L G EQ E Y F S S E E C C E DD S S P T W S R Q N Y N Y Y N R Y P G S S MD F E R P R G Y H H P Q G F LN N S Y N K S P K S T N I N L N N A N V S S X P N G G H N - - R Y Y E H A P A N G Y P G S Y Y G E Y DK P R T P H GQ R R R Y Y E T Y I R S Q G S DR R R P T I R R E E E Y E E DR Y S G - - - - - - - E Y Y S G E E F Y E DD S M L S G - - - - - - - - DR Y P N S DQ E Y E T P R G Y H H P D S Y Y- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P VMA NQ R P L Q F A E I EM E EM E S P - - - V F L E D F P Q DP R T N P L A R A N T N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A N A N V A Y G N S N H S N S H V F S S V H- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P VMA NQ R P L Q F A E I EM E E L E S P - - - V F L E D F P Q N P G T H P L A R A N T N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A N A N V A Y G N S S H R N N P V F S S I C- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T Q V T S Q R P L Q F A E N R P E DA G S P P D S V F L P N T E F F P DNMP T T S N T N N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N A N F I E E F T F E S E S - - - L S A S R N- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I E K G S T L L P F Q P R S F S P T H S L A G A E G S P V P S QMH R G A P I NQ S I N L P P V N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G S A R R L P A L P P Y A N H I H D E T DDG P- - - - - - - - - - - - - - - - - - - - - - - - - - S A S A A L G V G G S S L V L G S S DP A G G DY L Y DT L N R S V A DG V N N I T R N I MQ A R L A A A G K L Q D E L Q G A G S G G E L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T F G E S I S MR P L A K N G G G A A T V A G T
- L P E E DK R D I R Q S P K R G F L R S A S L G R R A S F H L E C L K R Q K DR G G D I S Q K - - - - - T V L P L H L V H HQ A L A V A G L S P L L Q R S H S P A S F P R P F A T P P A T P G S R - - - GWP P Q P V P T L R L E G V E S S E K L N S S F P S I H C G S WA E T T P G G G G S S - A A R R- C P E E DK R E I Q P S P K R S F L R S A S L G R R A S F H L E C L K R Q K DQ G G D I S Q K - - - - - T A L P L H L V H HQ A L A V A G L S P L L Q R S H S P T T F P R P C P T P P V T P G S R - - - G R P L R P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G S S S MA R RA E V G E E R R P WQ S P R R R A F L C P T A L G R R S S F H L E C L R K H N R P - - DV S Q K - - - - - T A L P L H L V H HQ A L A V A G L S P L L R R S H S P T L F T R L C S T P P A S P S G R S G G G P C Y Q P V P S L R L E G S G S Y E K L N S S MP S V N C S S WY S D S N G - - - - N H S G R AH R AQ R Y MDG H L V P R R R L L P P T P A G - R K P S F T I Q C L Q R Q G S C E D L P - - - - - - - - - - - - - - - - - - - - - - - I P G T Y H R G R N S G P N R AQ G S WA T P P - - - - - Q R G - - - R L L Y A P L L L V E E G A A G E G Y L G R S S G P L R - - - - - - - - - - - - - - - - - - -HW S QQ H V N G H H V P R R R L L P P T P A G - R K P S F T I Q C L Q R Q G S C E D L P - - - - - - - - - - - - - - - - - - - - - - - I P G T Y H R G R T S G P S R AQ G S WA A P P - - - - - Q K G - - - R L L Y A P L L L V E E S T V G E G Y L G K L G G P L R - - - - - - - - - - - - - - - - - - -DD S P V C Y D S R R S P R R R L L P P T P A S H R R S S F N F E C L R R Q S S Q E E V P S S P I F P H R T A L P L H L MQQQ I MA V A G L D S S K AQ K Y S P S H S T R S WA T P P A T P P Y R DW- - - T P C Y T P L I Q V EQ S E A L DQ V N G S L P S L H R S SWY T - - - - - D E P D I S Y R TDD S P T G Y D S R R S P R R R L L P P T P P S H R R S S F N F E C L R R Q S S Q DDV L P S P A L P H R A A L P L H L MQQQ I MA V A G L D S S K AQ K Y S P S H S T R S WA T P P A T P P Y R DW- - - S P C Y T P L I Q V DR S E S MDQ V N G S L P S L H R S SWY T - - - - - D E P D I S Y R TD EQ P L Y H D S H R S P K R R L L P P T P Q G N R R P S F N F E C L R R Q S S Q DD L P - - - - - HQ R T A L P L H L MQ HQ VMA V A G L D S S R A H R L S P T R S T R S WA S P P P T P A S K DR - - - T P Y Y T P L I R V DR - P L R D S A S S S H S S I R K S S WY T - - - - - DDP E Y QQ R NR E F P E E T E T P A T R G R A L GQ P C R V L G P H S K P C V EM L K G L L T Q R AMP R GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A P P A P CQ C P R V E S S MP E DR K S S T P G S L H E E T P - - - - - - - - - - - - - - - -R E F L G E A DMP V T R E G P L S Q P C R A S G P H S R S H V DK L K R P MT Q R GMP E GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V P P S P CQ L S Q A E H P VQ K E G K G P T S R F L E T P N S R - - - - - - - - - - - - - - -Y E D I R D S S L Y V G G - - - - - - - - - - - - - - - - - - - - - - A S N V N DR R L S D F N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V K T N S T Q F P Y N P S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -R Y R D T G DR A G Y DQ S S R MV V A N R N L P V DP D E E EQWMR S G G P S N R S DR R N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P H L R E P M L V A R G A A L A L A GM S S E A Y E G T Y R P V G - - - - - - - - - - - - - -L P P E A N A I N Y DN R N R G I L L H P Y N N V Y A P N G A L P G H E R M I Q S T P A S P Y D- - - - - - - - - - - - - - - - - - - - - - - - - - - Q R R L P T S S DMN G L A E S L I G G V L A A E G L G K Y C D S E F V G T A A R EMR E A L DMT P E EMN L A A HQ I L S N E H S L S L I G S S N
V R P V S L MV P S Q A G - A P G R Q F H G S A S S L V E A V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E S A A DN I L S G G A P Q S P N - G A L L P F V N C R DA GQ DR A G G E E DA G C V R A R G R - P S E E E L Q D S R V Y V S S L - - - - - - - - - - - - - - - -A R P V S L T V P S Q A G - A P G R Q F H G S A S S L V E A V L I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N - G T L L P F V N C R DP GQ DR A V V P E D E S C A Y A L G R G R S E E A L A D S R S Y V S N L - - - - - - - - - - - - - - - -Q R P V S L T V P P V T R R D S I S L A H G S A G S L V E A V L I S E G L G R Y A H DP S F I Q V A KQ E I A E A C DMT M E EM E N A A DN I L N A N A P P N A N - G N L L P F I Q C R DT G S Q E S R C S L S L G L S P A T G S DG A L E A E L E E S E G A GQ R N S P L M E D E DM E C V T S L- T F T C L H V P G T H S - DP S H G K R G S A D S L V E A V L I S E G L G L F A R DP R F V A L A KQ E I A DA C R L T L D EMDN A A S D L L A - - - - - - - - - - - - - - - - - - - - - - - Q G T S S L Y S D E E S I L S R - - F D E E D L G D EMA C V H A L - - - - - - - - - - - - - - - -- T F T C L Q V P G A H P - N P S H R K R G S A D S L V E A V L I S E G L G L F AQ DP R F V A L A KQ E I A DA C H L T L D EMD S A A S D L L A - - - - - - - - - - - - - - - - - - - - - - - Q R T T S L Y S D E E S I L S R - - F D E E D L G D EMA C V H A L - - - - - - - - - - - - - - - -F T P A S L T V P S S F R - N K N S DKQ R S A D S L V E A V L I S E G L G R Y A R DP K F V S A T K H E I A DA C D L T I D EM E S A A S T L L N G N V R P R A N - G DV G P L S H R Q DY E L Q D F G P G Y S D E E P DP G - - - R D E E D L A D EM I C I T T L - - - - - - - - - - - - - - - -F T P A S L T V P S S F R - N K N S DKQ R S A D S L V E A V L I S E G L G R Y A R DP K F V S A T K H E I A DA C D L T I D EM E S A A S T L L N G S V C P R A N - G DMG P I S H R Q DY E L Q D F G P G Y S D E E P DP G - - - R E E E D L A D EM I C I T T L - - - - - - - - - - - - - - - -X S P V H L Q V P P E Y R - NQ Y L Q K R G S A T S L V E A V L I S E G L G R Y A K DP K F V A A X K H E I A DA C EMT I D EM E S A A S H X L N G G I T P V V N G V N V F P I L G H R E Y E L Q DV S A S Y S D E E P E P E P R P R Y E E D L A D EM I C I T T L - - - - - - - - - - - - - - - -- - - - - - - H S R S T R E N T S R C S A P A T A L L I Q K A L V R G G L G T L A A DA N F I MA T GQ A L A DA CQM E P E E V E I MA T E L L K G - - - - - - - - - - - - - - - - - - - - - - R E A P E GMA S S L G C L N L G S S L G S L DQ HQ G S Q E T L I P P R L - - - - - - - - - - - -- - - - - N F E E H V P R N S A H R C T A P A T AM L I Q E A L V R G G L D S L A A DA N F VMA T GQ A L A DA CQM E P E E V E V A A T E L L KQ - - - - - - - - - - - - - - - - - - - - - - E S P E G G A V P W E P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - C E E S K NQ R S A A E A S P A T DK L I QQ A L R DG G L E S L A E DP Q F V S V T R K E L A E A V N I G L Q D I E S V AQ G I V N G - - - - - - - - - - - - - - - - - - - - - - Q S G K V T K R K R R P I P V P P S K T K E A T S A V - - - - - - - - - - - - - - - - - - - - - - -- - - E G K S V R L P F S S R P V L R P A E D S R P V DR L I GQ S L G L G R Y A - DA R I V G A A R R E I E E A Y S L G EQ E I D L A A D S L A P L MQ H V GMH - - - - - - - - - D I R D I N E N S R S A L L R P A E N S S R Q H D S R G G S Q E D L L L V T T L - - - - - - - - - - - - - - - -G S I F G G S A G G L G G A G S G G V G G L G G S S S I R N A F G G S G S G P S S L S P Q HQ P Y S G T L N S P P I P DN R L R R V A T V T T T N N N N K S Q V S Q - - - - - - - - N N S N S L N V R A N A N S QMNM S P T GQ P VQQQ S P L R GQ G NQ T Y S S - - - - - - - - - - - - - - - -
V E
E DE DE DY EY E
I Q E Y F R K F K K R K EQ G L V G K P S Q R N - - A L S L Q A G L R T L H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N
C
Cav1.2_HumanCav1.2_MouseCav1.2_ZebrafishCav1.4_HumanCav1.4_MouseCav1.3_HumanCav1.3_MouseCav1.3_ZebrafishCav1.1_HumanCav1.1_MouseCav1.1_ZebrafishCav1.-_C. elegansCav1.-_D. Melanogaster
Cav1.2_HumanCav1.2_MouseCav1.2_ZebrafishCav1.4_HumanCav1.4_MouseCav1.3_HumanCav1.3_MouseCav1.3_ZebrafishCav1.1_HumanCav1.1_MouseCav1.1_ZebrafishCav1.-_C. elegansCav1.-_D. Melanogaster
Cav1.2_HumanCav1.2_MouseCav1.2_ZebrafishCav1.4_HumanCav1.4_MouseCav1.3_HumanCav1.3_MouseCav1.3_ZebrafishCav1.1_HumanCav1.1_MouseCav1.1_ZebrafishCav1.-_C. elegansCav1.-_D. Melanogaster
IQ
Leucine Zipper
A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster
I Q E Y F R K F K K R K EQ G L V G K - P S Q R - N A L S L Q A G L R T L - H D I G P E I R R A I S G D L T A E E E L DK AMK E A V S A A S E DD I F R R A G G L F G N H V T Y Y Q S D S R G N F P Q T F A T Q R P L H I N K T G N NQ A - DT E S P S H E K L V D S - - - T F T P S S Y S S - - - - -I Q DY F R K F K K R K EQ G L V G K Y P A K N - T T I A L Q A G L R T L - H D I G P E I R R A I S C D L Q DD E P E D- - - - - - - S K P E E E DV F K R N G A L L G N H V N H V N S DR R D S L QQ T N T T H R P L H VQ R P S MP P A S DT E K P - - - - - - - - - - - L F P P A G N S G C H N H HI Q DY F R K F R R R K E K G L L G R E A P T S - T S S A L Q A G L R S L - Q D L G P E I R Q A L T Y DT E E E E E - - - - - - - - - - - - E E E A V GQ E A E E E E A E N N P E P Y K D S I D S Q P Q S R WN S R I - - - - - - - - - - - - S V S L P V K E K L - - - - - - - - P D S L S T G P S DDDI Q E H F R K F MK R Q E E - Y Y G Y R P - K K - DT VQ I Q A G L R T I E E E A A P E I H R A I S G D L T A E E E L E R AM- - - V E A AM E E G I F R R T G G L F GQ V DN F L E R T N - - S L P P VMA NQ R P L Q F A E I EM E - - - E L E S P V F L E D F P Q N P G T H P L A R A N T - - - - -I Q DY F R R F K K R K EM E A K G V L P AQ T P Q AMA L Q A G L R T L - H E I G P E L K R A I S G N L E T D F N F D - - - - - - - - - - E P E P Q H R R P H S L F N N L V H R L S G A G - - - - S K S P T E H E R I E K G S T L L P F Q P R S F S P - - - - - - - - - - - T H S L A G A E G - - - S PI Q DY F R R F K K R K EQ E G K E G H P - D S - N T V T L Q A G L R T L - H E V S P A L K R A I S G N L - - - D E L DQ - - - - - - - - - E P E P MH R R H H T L F G S VW S S I R R H G N G T F R R S A K A T A S - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q S N G A L A I G - - - - -
- - - - - - - - - - T G S N A N I N N A N N T A L G R F P H P A - - - - - G Y S S T V S T V E G H G P P L S P A V R VQ E A AWK L S S K R C H S R E S Q G A T V - - - - - - - - - - - - - - - - - - - - - - - - NQ E I F P D E T R S V R M S E E A E Y C S E P S L L S T DM F S Y Q E D E H R Q L T CN H N S I G KQ A P T S T N A N L N N A NM S K A A H G K P P S I G N L E H V S E N G H Y S C K H DR E L Q R R S S I K R T R Y Y E T Y I R S E S G D EQ F P T I C R E DP E I H G Y F R DP R C L G EQ E Y F S S E E C C E DD S S P T W S R Q N Y N Y Y N R Y P G S S MD F E R P R G Y H H P Q G F LG L A P N S R Q P S V I Q A G S Q P H R R S S G V F M F T I P E E G S I Q L K G T Q GQ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - DNQ N - - - - - - - - - - - - - - - - - - - - - - - - - - - E EQ E V P DWT P D- - L D EQ A G T P S N P V L L P P HW S QQ H V N G H H - - - -- - - - - - - - - - N N A N A N V A Y G N S S H R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N N P V F S S I C Y E - - - - - - - - - - - - - - - - - - - - - R E F LV P S QMH R G A P I NQ S I N L P P V N G S A R R L P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A L P P Y A N H I H D E T DDG P R Y R D T G DR A G Y DQ S S R MV V- - - - - - - - - - G S A S A A L G V G G S S L V L G S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S DP A G G DY L Y DT L N R S V - - - - - - - A DG V N N I T R N I M
P E E DK R - - - E I Q P S P K R S F L R S A S L G - R R A S F H L E C L K R Q K DQ G G - - - - - D I S Q K T A L P L H L V Q AL A V A G L S P L L Q R S H S P T T F P R P C P T P P V T P G S R G R P L R P I P T L R L E G A E S S E K L N S S F P S I H C S S W S E E T T A C S G S S S MA R RE DDD S P T G Y D S R R S P R R R L L P P T P P S H R R S S F N F E C L R R Q S S Q DDV L P S P A L P H R A A L P L H L MQ Q QI M A V A G L D S S K AQ K Y S P S H S T R S WA T P P A T P P Y R DW S P C Y T P L I Q V DR S E S MDQ V N G S L P S L H R S S WY T D E P D I S - - - - - Y R T- - - - - - - - - - - - - V P R R R L L P P T P A G - R K P S F T I Q C L Q R Q G S C E D- - - - - - - - - - - - L P I P G T Y H R - - - - - - - - - - - G R T S G P S R A QG S WA A P P - - - - Q K G R - L L Y A P L L L V E E S T V G E G Y L G K L G G P - - - - - - - - - - - - - - - - - - L R TG E A DMP - - - V T R E G P L S Q P C R A S G P H - S R S - - H V DK L K R P MT Q R G - - - - - - - - - - - - - - - - - - - - - - - MP E GQ V P - - - - - - - P S P C QL S Q A E H P VQ K E G K G - - - - - - P T S R F L E T P N S R N F E E H V P - - - - - - - - - - - - - - - - - - - - - - -A NRNL P - - - - V DP D E E E QWMR S G G P S - N R S DR R N P H L R E - - - - - - - - - - - - - - - - - - - P M L V A R G A A L A L A GM- - - - - - - - - - S S E A Y E G T Y R P V G E G K S V R - - - - - - - L P F S S R P V L R P A E D S R P - - - - - - - - - - - - - - - - - - - - - - -
Q A R L A A - - - - A G K L Q D E L Q G A G S G G E - L R T F G E S I S MR - - - - - - - - - - - - - - - - - - - - P L A K N G G G A A T V A G T L P P E A N A I N Y DN R N R G I L L H P Y N N VY A P N G A L P G H E R M I Q S T P A S P Y DQ R R L P - - - - - - - - - - - - - - - - - - - - - - -
A R P V S L T V P S Q A G A P G R Q F H G S A S S - - - L V I S E G L GQ F AQ DP K F I E V T T Q E L A DA C DMT I E EM E N A A DN I L S G G AQQ S P N G T - - - - - - - - - - - - - - - - - - - - L L P F V N C R DP G - - - Q DR A V V P E D E S C A Y A L G R G R - S E E A L A D SF T P A S L T V P S S F R N K N S DKQ R S A D S - - - L V I S E G L G R Y A R DP K F V S A T K H E I A DA C D L T I D EM E S A A S T L L N G S V C P R A N G D- - - - - - - - - - - - - - - - - - - - MG P I S H R Q DY E L - - Q D F G P G Y S D E E - - - - P DP G R - E E E D L A D EF T - - C L Q V P G A H P N P S H R K R G S A D S - - - L V I S E G L G L F AQ DP R F V A L A KQ E I A DA C H L T L D EMD S A A S D L L A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q R T T S L Y S D E E - - - - S I L S R F D E E D L G D E- - - - - - - - - - - - - - R N S A H R C T A P A T AM L I V R G G L D S L A A DA N F VMA T GQ A L A DA CQM E P E E V E V A A T E L L K - - - Q E S P E G G - - - - - - - - - - - - - - - - - - - - A V P W E P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - V DR - - - L I GQ S L - - - G A - DA R I V G A A R R E I E E A Y S L G EQ E I D L A A D S L - - - - - - - A P L MQ - - - - - - - - - - - - - - - - - - - - H V GMH D I R D I N E N S R S A L L R P A E N S S R Q H D S R G G - S Q E D- - - -- - - - - - - - - - - - - - T S S DMN G L A E S - - - L I A E G L G K Y C - D S E F V G T A A R EMR E A L DMT P E EMN L A A HQ I L S N E H S L S L I G S S N G S I F G G S A G G L G G A G S G G V G G L G G S S S I R N A F G G S G S G P S S L S P Q HQ P Y S G T L N S P P I P DN
R S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y V S N LM I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C I T T LMA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C V H A L- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -L L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L V T T LR L R R V A T V T T T N N N N K S Q V S Q N N S N S L N V R A N A N S QMNM S P T GQ P VQQQ S P L R GQ G NQ T Y S S
A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster
A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster
A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster
A1C_MouseA1D_MouseA1F_MouseA1S_MouseA1D_C. elegansA1D_D Melanogaster
E
G
E
A V LE A V LE A V LQ A L
GL R YG V A A
HH
CAGGCATTGGCAGTGGCAGGCTTGAGCCCCCTCCTGCAGAGAAG
CCATTCTCCTACCACATTCCCCAGGCCGTGCCCCACACCCCCTG
TCACTCCAGGCAGCCGGGGCAGACCCCTACGGCCCATCCCTACC
CTACGGCTGGAGGGGGCAGAGTCCAGCGAGAAACTCAACAGCAG
CTTCCCATCCATCCACTGCAGCTCCTGGTCTGAGGAGACGACAG
CCTGTAGTGGGAGCAGCAGC
1
45
91
136
180
225
CCATT Box
Sp1/GC Box SRY
GAPGRQFHGSASSLVEAVLISEGLGQFAQDPKFIEVTTQELADACDMTIEEMGAPGRQFHGSASSLVEAVLISEGLGQFAQDPKFAEVTTQEAADACDMTIEEM
GAPGRQFHGSASSLVEAVLASEGLGQAAQDPKFAEVTTQEAADACDMTIEEM
0
1
2
3
4
5
6
7
8
UA
S T
ran
scri
pti
on
(F
ire
fly/
Re
nil
la)
C C A T ∆T A
C C A T I F I L
C C A T A A I L
C C A TA A A A
A B
D
140
Transcript variant
Tissue 5’RACE# of TSS
5’RACE StartSite*
CAGEDatabase# of tags$
CAGE StartSite
PredictedProtein Size
1abrain, heart,
smooth muscle,liver, lung
NT NT 76Ch6:119,058,607-
119,058,62613240KDa
1b brain NT NT 53Ch6:119,036,852-
119,036,871240KDa
2a thalamus 2Ch6:118,604,374-
118,604,396Not found 110KDa
2b liver, lung, brain Not found 87Ch6:118,588,084-
118,588,103? /95KDa
3 thalamus, cortex 6Ch6:118,552,082-
118,552,1031
Ch6:118,552,184-118,5
52,205NC
4 thalamus. cortex 2Ch6:
118,545,992-118,546,013
1Ch6:
118,546,069-118,546,090
15KDa
Table 1. Summary of Transcriptional Start Sites and Nearby CAGE TagsChromosomal addresses for experimental TSS are given as the corresponding location in the Mouse July 2007 genome assembly. NT (Not tested), NC (non-coding).
141
142
FIGULE LEGENDS
Figure 1. CCAT is Not Generated by Proteolytic Cleavage of Exogenously
Expressed or Endogenous Cav1.2 Channels.
(A) Schematic representation of the Cav1.2 -Gal4 fusion and channel mutants. Four
mutations are depicted: deletion from the TM to IQ motif renders the channel unable to
traffic to he membrane, deletion of conserved cleavage site for Cav1.1, a translational
stop at 1910 a.a. and deletion of TA.
(B) Western blot of N2As expressing Cav1.2 -Gal4 channels depicted in A (upper panel)
and Gal4-tagged C-terminal fragments (bottom panel) probed with an antibody to Gal4.
(C) Reporter gene activity of N2As expressing a UAS-luciferase reporter plasmid along
with Cav1.2 - Gal4 channels depicted in A or Gal4 alone as a control. Cells were co-
transfected with a Renilla luciferase construct driven by the thymidine kinase promoter
to control for cell number and transfection efficiency. Results are given as a ratio of
Firefly to Renilla luciferase activity. (Means ± SD; * < 0.0001 vs. Gal4).
(D) Western blot of N2As expressing WT, Methionine 2011 to Isoleucine and
Methionine 2078 to Isoleucine Cav1.2 -Gal4 channels (Upper Panel). Bottom panel
shows Gal4-tagged C-terminal fragments. Proteins were detected with a Gal4 antibody.
Large molecular protein in channel western represents unsolubilized, multimeric
channel proteins.
(E) Luciferase activity of N2As expressing either Gal4 alone, WT, M2011I and M2078
Gal4 tagged channels. (Means ± SD; * < 0.0001 vs. Gal4).
143
(F) Western blot analysis of membrane fractions obtained from 11.5 dpc heterozygous
(N/+) or homozygous (N/N) Cav1.2 knockout embryos probed with the anti-CCAT
antibody. Bottom panel shows loading control.
(G) Immunohistochemistry of 11.5 dpc Cav1.2 Null embryos reveals strong nuclear
staining with the anti-CCAT antibody (red) in the developing somites. Nuclei are shown
in blue.
Figure 2. CCAT is Translated from a Separate Transcript Driven by an Exonic
Promoter
(A) Northern blot analysis of mRNA extracted from N2A cells expressing Cav1.2 -gal4
channel constructs with or without CMV promoter. The first lane contains mRNA
extracted from untransfected cells. The membranes were hybridized with a
radioactively labeled RNA probe to Exon 47 of the channel.
(B) Western blot of N2As expressing Cav1.2 -gal4 channel constructs with or without
CMV promoter. Upper Panel shows full-length channels. Bottom panels shows Gal4
tagged CCAT. Membranes were immunoblotted with a Gal4 antibody.
(C) UAS reporter activity of N2As expressing Cav1.2 -gal4 channel constructs with or
without CMV promoter. (Means ± SD; * < 0.0001 vs. Gal4).
(D) Schematic representation of Firefly reporter constructs design to map the region
within the coding sequence of the channel responsible for the promoter activity. Full-
length construct has the complete sequence of the channel in place of any upstream
promoter. Ion pore construct includes all the transmembrane domains up to the IQ
motif. E46-47 contains exon 46 and 47 upstream of luciferase.
144
(E) Luciferase activity as a surrogate measure of luciferase expression levels from
constructs depicted in D. Graph compares expression level in N2As transfected with
either full-length, Ion pore, E46-47, E46 or E47 constructs. (Means ± SD).
(F) Schematic representation of the minigenes. A 4 Kb genomic segment containing the
last two exons and introns of CACNA1C was fused to the CDS of Gal4. 4Kb-0 is the
wild type minigene. 4Kb-1 is a negative control where a G has been inserted between
exon47 and Gal4. ∆238bp is the 4K-0 minigene where 238bp identified in E was
removed. M2011I is the 4Kb-0 where Met 2011 has been mutated to isoleucine.
(G) Mean luciferase activity (± SD) in N2A expressing 4Kb-0, 4Kb-1, ∆238bp and
M2011I Gal4 minigenes along with a UAS-luciferase reporter. (* < 0.0001 vs. 4Kb-0).
(H) Mean luciferase activity (± SD) in N2A expressing minigenes where either all or
two of three possible methionines were mutated. 4Kb-0 is the wild type sequence.
4Kb∆M has all three methionines mutated to isoleucines. M2011I has only this
methionine intact where M2073 and M2078 have been mutated to isoleucines. M2073I
and M2078I have only these respective methionine intact.
Figure 3. CCAT is Translated from a Separate Transcript whose Expression is
Cell-type and Developmentally Regulated In Vivo
(A) and (B) Northern blot analysis of mRNA extracted from Cortex, Midbrain and
cerebellum from E18, P1 and Adult rats. The membranes were hybridized with a
radioactively labeled DNA probe to Exon 47 of the channel (A) or to domain III
transmembrane S3 of the channel. Bottom panel shows the same membrane labeled
with an DNA probe to the 18S ribosomal RNA as loading control.
145
(C) Graphs showing the normalized expression of the full-length, 4.0Kb and 2.2Kb
band in the cortex, midbrain and cerebellum at 3 developmental stages: E18, P1 and
Adult. Each line represents an independent experiment. The northern blot shown in A
corresponds to the blue tracing. Signals were normalized to the 18S RNA signal.
(D) Imunohistochemistry of E18 coronal and sagital brain sections labeled with rabbit
anti-CCAT (red) and mouse Map2 antibodies (green). Secondary antibodies were
conjugated to Alexa fluorescent dyes. C (developing cortex), V (ventricle), S
(striatum), Th (thalamus), BS (brain stem), Cb (cerebellum).
(E) Imunohistochemistry of E18, P1 and 3-week-old rat cerebellum and thalamus
showing developmental variation in the amount and distribution of CCAT nuclear
staining. Anti-CCAT is shown in red and nuclei in blue.
(F) Immunocytochemistry of cortical and thalamic neuron grown 5 days in vitro stained
with anti-CCAT. Transcriptional assays of cortical (top) and thalamic (bottom)
neuronal cultures transfected with the UAS-luciferase reporter along with the Gal4
tagged channels described in Figure 1. Bars represent normalized transcription to Gal4
alone. (Means ± SD; * < 0.005 and ** <0.0001 vs. M2011I-Gal4).
Figure 4. CACNA1C has Multiple Transcriptional Start Sites Predicting Multiple
Proteins Including CCAT
(A) Schematic representation of the 5’RACE approach to determine the TSS for the
CCAT transcript generated from Cav1.2 -Gal4. Briefly, two sequential phosphatase
treatments are used to inactivate truncated or non-mRNAs and prepare intact, originally
capped mRNAs for ligation of an RNA oligo to the uncapped 5’ end. In this experiment
146
reverse transcription was performed with a Gal4 reverse primer. Nested primers within
the 5’ tag and the Gal4 coding sequence were used for PCR. The bands were then
cloned and sequenced.
(B) Agarose gel of PCR products amplified after performing 5’ RACE as described in A
of N2As expressing Cav1.2-Gal4 channels with and without CMV promoter. Predicted
PCR size band was 805 bp.
(C) Schematic representation of CACNA1C showing the location of TSS found and
depictions of the proteins predicted to be expressed from these transcripts.
(D) Epifluorescence images of N2A cells expressing YFP-mem-CCAT (left panels) or
YFP-CCAT (right panels).
(E) Western blot of nuclear extracts from 11.5 dpc Cav1.2 null and heterozygote
embryos probed with anti-CCAT.
Table 1. Summary of Transcriptional Start Sites and Nearby CAGE Tags
Chromosomal addresses for experimental TSS are given as the corresponding location
in the Mouse July 2007 genome assembly. NT (Not tested), NC (non-coding).
Supplementary Figure 1
(A) Mean luciferase expression in N2A cells transfected with the UAS-luciferase
reporter and either Gal4 alone or Cav1.2-Gal4 channels. Channel expressing cells were
treated with 10µM Brefeldin A for 3-6 h.
(B) Schematic of the Cav1.2 knockout strategy.
147
(C) Southern blot showing the efficacy of recombination and the expected molecular
weight of the BamHI digested genomic fragments after recombination.
(D) Immunohistochemistry of heterozygote 11.5 dpc embryos stained with anti CCAT
antibody. Membranous staining is seen in the developing cortex and heart muscle wall
(HM). Staining is noticeably nuclear in somites and mesenchymal cells (M). CCAT
staining of the liver is not detected.
Supplementary Figure 2
(A) Normalized luciferase activity of N2As expressing empty pgl4 vector or constructs
containing 4Kb of genomic sequence upstream of the neuronal Cav1.2 channel
transcript starting AUG or upstream of M2011 AUG in the 3’ end of the gene. Cells
were co-transfected with Renilla luciferase construct driven by the thymidine kinase
promoter to control for cell number and transfection efficiency. Results are given as a
ratio of Firefly to Renilla luciferase activity. (Means ± SD; * < 0.005 vs. empty vector).
(B) Schematic representation of the alignment of the c-termini of Cav1.2 channels from
various species. Color was assigned based on similarity, darker colors being identical.
The exon-exon boundaries are depicted by double arrows.
(C) Alignment of the last two exons of Cav1.2 channels from various species. Possible
initiator methionines are labeled in red.
Supplementary Figure 3
(A) Northern blot analysis of mRNA extracted from Cortex, Midbrain and cerebellum
from E18, P1 and Adult rats. The membranes were hybridized with a radioactively
148
labeled DNA probe to Domain III transmembrane S3 of the channel. Bottom panel
shows the same membrane labeled with a DNA probe to the 18S ribosomal RNA as
loading control.
(B) Imunohistochemistry of E18, P1 and 3 week old rat cortex showing mostly cell
body and dendritic staining of CCAT in the three developmental stages. Anti-CCAT is
shown in red and nuclei in blue.
(C) Nuclear CCAT is highly expressed in subpallial regions during early embryonic
neural development and is not found in all cells expressing Cav1.2.
Immunofluorescence staining with CCAT antibody green) in the developing mouse at
embryonic days 12.5, 14 and 18 and postnatal day 1 (P1) demonstrates strong nuclear
expression of CCAT in subpallial regions corresponding to the developing caudate and
putamen (striatum), in addition to cells of the rhomboid, dorsomedial, posterior and
midline nuclear groups of the thalamus, the ventral pallidum, and periventricular
hypothalamic nuclei. CCAT expression becomes progressively restricted over the
course of embryonic development and is abolished several weeks into postnatal life.
(D) Nuclear expression of CCAT is restricted to the striatum (lower panel), thalamus
and developing cerebellum by E18 and postnatal ages. In contrast, cortical neurons
display mainly cytoplasmic expression when stained with the Cav1.2 C-terminus
antibody (upper panel), indicating the presence of the full-length channel but little or no
CCAT expression.
Supplementary Figure 4
149
(A) A schematic of the 238 bp promoter sequence indicating the location of
transcription factor binding sites. Sites were searched using TFSearch, MatInspector
and rVista and only common binding sites are reported. Default thresholds were used in
all instances.
(B) Mean luciferase activity (± SD) in N2A expressing CCAT-Gal constructs along
with the UAS-luciferase reporter and TK-Renilla luciferase construct as control. CCAT-
∆TA serves as negative control. CCAT-IFIL corresponds to WT sequence. Sequences
are included to show the mutations used. CCAT lacking two of the conserved LZ
residues has <50% activity. Mutation of all four a.a to Alanines completely abrogates
transcriptional activity.
(C) Sequence alignment of the c-termini of L-type calcium family. Sequences for Cav1
channels from Zebrafish, Mouse and human were aligned to the ancestral C. elegans
and D. Melanogaster L-type channels. Colors represent similarity based on percentage
identity. Two regions of conservation are identified the IQ with extended a.a and the
modified Leucine Zipper domain.
(D) Sequence alignment focused on Mouse Cav1 channels and C. elegans and D.
Melanogaster L-type channels.
150
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