Formation of telomeric repeat-containing RNA (TERRA) foci ... · (TERFs) in the tumor cells, but...

Journ

alof

Cell

Scie

nce

Formation of telomeric repeat-containing RNA(TERRA) foci in highly proliferating mouse cerebellarneuronal progenitors and medulloblastoma

Zhong Deng1, Zhuo Wang1, Chaomei Xiang2,3, Aliah Molczan2, Valérie Baubet2,3, Jose Conejo-Garcia2,Xiaowei Xu4, Paul M. Lieberman1,* and Nadia Dahmane2,3,*1Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, PA 19104, USA2Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA 19104, USA3Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA4Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

*Authors for correspondence ([email protected]; [email protected])

Accepted 1 May 2012Journal of Cell Science 125, 4383–4394� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.108118

SummaryTelomeres play crucial roles in the maintenance of genome integrity and control of cellular senescence. Most eukaryotic telomeres canbe transcribed to generate a telomeric repeat-containing RNA (TERRA) that persists as a heterogeneous nuclear RNA and can bedevelopmentally regulated. However, the precise function and regulation of TERRA in normal and cancer cell development remains

poorly understood. Here, we show that TERRA accumulates in highly proliferating normal and cancer cells, and forms large nuclearfoci, which are distinct from previously characterized markers of DNA damage or replication stress. Using a mouse model formedulloblastoma driven by chronic Sonic hedgehog (SHH) signaling, TERRA RNA was detected in tumor, but not adjacent normal

cells using both RNA fluorescence in situ hybridization (FISH) and northern blotting. RNA FISH revealed the formation of TERRA foci(TERFs) in the nuclear regions of rapidly proliferating tumor cells. In the normal developing cerebellum, TERRA aggregates could alsobe detected in highly proliferating zones of progenitor neurons. SHH could enhance TERRA expression in purified granule progenitorcells in vitro, suggesting that proliferation signals contribute to TERRA expression in responsive tissue. TERRA foci did not colocalize

with cH2AX foci, promyelocytic leukemia (PML) or Cajal bodies in mouse tumor tissue. We also provide evidence that TERRA iselevated in a variety of human cancers. These findings suggest that elevated TERRA levels reflect a novel early form of telomereregulation during replication stress and cancer cell evolution, and the TERRA RNA aggregates may form a novel nuclear body in highly

proliferating mammalian cells.

Key words: Telomere repeat-containing non-coding RNA, TERRA, Neuronal progenitors, Cancer, Cerebellum, Medulloblastoma, Sonic hedgehog

IntroductionTelomeres are repetitive DNA structures at the ends of linear

chromosomes required for chromosome maintenance and genome

stability (Blackburn et al., 2006; Cech, 2004). Mammalian

telomere DNA consists of variable length double stranded

TTAGGG repeats ending in a single stranded 39 overhang thatcan form complex higher-order nucleoprotein structures to cap

chromosome ends (de Lange, 2002; de Lange, 2004). The

telomere-associated proteins, termed shelterin or telosome, are

important for telomere end-protection and length regulation (de

Lange, 2005a; Liu et al., 2004; Palm and de Lange, 2008). In

proliferating cells, telomere repeat length is maintained by an

elaborate mechanism involving DNA replication machinery,

telomerase and its associated factors, and nucleolytic end-

processing enzymes (Jain and Cooper, 2010; Ye et al., 2010). In

the absence of telomerase activity, telomeres shorten by attrition,

and critically short telomeres elicit a DNA damage signal that can

lead to cell cycle arrest and replicative senescence (Sahin and

Depinho, 2010). Loss of telomere capping function leads to cell

cycle arrest in normal senescing cells, but this protective

mechanism is bypassed in normal proliferating cells and

potentially further deregulated in human cancers (Blasco, 2005;

Blasco, 2007; De Lange, 2005b).

TERRA is a heterogeneous length non-coding RNA transcribed

through the terminal telomere repeats of eukaryotic chromosomes

(Azzalin et al., 2007; Schoeftner and Blasco, 2008). In fission

yeast, transcripts can be detected for both sense and antisense

strands of the telomere repeat tract and adjacent telomere sequence

(Bah et al., 2012; Greenwood and Cooper, 2012). TERRA RNA

can interact with shelterin proteins to regulate telomere

heterochromatin formation (Deng et al., 2009) and capping by

the telomere single strand DNA binding protein Pot 1 (Flynn et al.,

2011; López de Silanes et al., 2010). TERRA can also interact with

the catalytic subunit of telomerase (TERT) and inhibit telomerase

enzyme activity in vitro (Redon et al., 2010; Schoeftner and

Blasco, 2008). TERRA RNA expression can be regulated by

several mechanisms, including developmental status, cellular

stress, and telomere epigenetic state (Azzalin et al., 2007;

Caslini et al., 2009; Deng et al., 2009; Schoeftner and Blasco,

2008). In addition, TERRA levels are elevated in both human and

mouse iPS cells, suggesting that TERRA expression correlates

with proliferative capacity and contributes to nuclear

Research Article 4383

mailto:[email protected]:[email protected]

Journ

alof

Cell

Scie

nce

reprogramming (Marion et al., 2009; Yehezkel et al., 2011).

However, the expression and function of TERRA in the context ofcancer remains poorly understood.

To investigate the regulation and function of TERRA innormal and cancer cell development, we examined TERRA

expression in human cancer biopsies and in a mouse model formedulloblastoma. Medulloblastoma is a malignancy of thecerebellum with the highest incidence of all human pediatric

malignant brain tumors (Ellison, 2010). A subset of these tumorsis thought to arise from deregulation of granule neuronprogenitors (GNPs) development in the cerebellum (Ellison,

2002; Ellison, 2010; Gilbertson, 2004; Gilbertson and Ellison,2008). GNPs are subject to very high rates of proliferation duringthe early postnatal period. Sonic hedgehog (SHH) is required forthe rapid proliferation of these neuronal progenitors during

normal development (Dahmane and Ruiz i Altaba, 1999;Wallace, 1999; Wechsler-Reya and Scott, 1999). Patched 1(Ptch1) is a receptor for the SHH ligand and an important

negative regulator of SHH signaling. Mutations in Ptch1 can leadto medulloblastoma in human (Hahn et al., 1996; Johnson et al.,1996) and mouse models (Goodrich et al., 1997) (reviewed by

Corcoran and Scott, 2001; Ruiz i Altaba et al., 2002). In thiswork, we show that normal and cancer proliferating granuleneuron progenitors express high level of TERRA and exhibit

formation of TERRA foci. These foci (TERFs) are distinct fromcH2AX DNA damage foci, but occur in cells where the telomererepeat DNA has shortened. TERRA foci can also be found inhighly proliferating progenitor cells during normal mouse

development. Finally, we show that TERRA is elevated invarious types of human cancers originating in diverse organs.

ResultsTERRA form foci in a mouse model for medulloblastoma

To analyze the expression of TERRA in a mouse model of humancancer, we employed Ptch1+/2 mice, a widely used genetic model

for human SHH-positive subtype medulloblastoma (Ellison, 2010;Goodrich et al., 1997). These tumors are composed of proliferatingGNPs marked by Math1 (also known as Atoh1) (Oliver et al.,2005), and express Gli1, a target and mediator of the SHH signaling

pathway (Goodrich et al., 1997) (Fig. 1A; supplementary materialFig. S1). Tumor can be readily distinguished from normal/non-tumor cerebellar tissue based on histology and in situ expression

analysis of various markers (Fig. 1A; supplementary material Fig.S1). To examine TERRA expression in mouse normal and cancertissue, we first employed RNA fluorescence in situ hybridization

(FISH) using methods that have been optimized for detection ofrare and unstable RNA (Deng et al., 2009; Flynn et al., 2011). ATAMRA-conjugated PNA probe was used under non-denaturingconditions to selectively distinguish telomere RNA from telomere

DNA. RNA-FISH revealed that TERRA forms discrete foci(TERFs) in the tumor cells, but not in the adjacent non-tumorcells of the same cerebellum (Fig. 1B). TERFs were not detected in

sections pre-treated with RNase A, indicating that the signaldetected with the TERRA probe indeed corresponds to RNAexpression (Fig. 1B, lower panels; supplementary material Fig.

S1B). As an additional specificity control, a FAM-conjugated PNAprobe for antisense TERRA failed to detect any distinct foci(supplementary material Fig. S2B). Quantification of multiple

RNA FISH experiments using computer imaging softwareindicated that ,80% of tumor cells have a ,7.5-fold greatermean fluorescence intensity relative to normal cells in adjacent

non-tumor tissue (Fig. 1C–E). These findings were further

confirmed by RNA FISH using a DNA oligonucleotide probe(TAACCC)7, which unlike the PNA probe, has very low capacityfor binding duplex DNA. The (TAACCC)7 DNA oligonucleotide

probe also revealed elevated TERFs in tumor cells with nodetectable signal in the normal part of the same cerebellum(Fig. 1F). No signal for TERRA expression was observed with amutated (TAACAC)7 version of this DNA oligo probe (Fig. 1F),

further indicating that these foci are TERRA-specific and thatTERRA levels are selectively elevated in tumor cells.

Elevated TERRA expression in mouse medulloblastomatissue

To confirm and validate RNA FISH results with other independenttechniques, we next examined TERRA expression by northern blot

and quantitative (q) RT-PCR using RNA isolated from dissectednormal (non visible tumor) and tumor regions of Ptch1+/2 cerebella(Fig. 2; supplementary material Fig. S3). In agreement with the in

situ data (Fig. 1; supplementary material Fig. S1), northern blotanalysis indicated that the tumor contained significantly higherlevels (,4-fold; P50.0301) of TERRA RNA than the non-tumorcounterpart (Fig. 2A,B; supplementary material Fig. S3). RNase A

treatment eliminated TERRA detection, indicating that TERRAsignal consists of RNA, and not fragmented telomere DNA(Fig. 2A, right lanes). Comparable results were observed with three

other matched non-tumor and tumor cerebella from different mice(supplementary material Fig. S3A). Quantitative RT-PCR analysisof TERRA expression from individual subtelomeres revealed an

increase of TERRA expression from various chromosomes(Fig. 2C; supplementary material Fig. S3B,C). For example, oneof the tumors presented a large increase at 11q (,6-fold), but amore moderate increase at 2q (1.8-fold; Fig. 2C, upper panel).Marker mRNAs for medulloblastoma tumor identity (Math1 andGli1) and post-mitotic neuronal marker (Map2) were used toconfirm the accuracy of dissection (Fig. 2C, bottom panel;

supplementary material Fig. S3B,C). We also observed thatmRNA for the telomerase subunit TERT was not increased intumor samples (Fig. 2C, lower panel; supplementary material Fig.

S3). These results validate findings by RNA FISH, indicating thatTERRA is elevated in mouse cancer tissue, and that some, but notall telomeres express high TERRA levels (Fig. 2B,C).

TERRA is elevated in highly proliferating progenitor cellsin the developing mouse brain

Transcriptional profiling analysis have shown that mouse postnatal

(P1–P10) GNPs resemble a subtype of both mouse and humanSHH-dependent medulloblastoma (Kho et al., 2004). Theexpression of TERRA in primary SHH-subtype medulloblastomasuggested that it might also be expressed in the rapidly dividing

postnatal GNPs. To investigate whether TERRA expressioncorrelates with normal proliferating GNPs, we first used RNAFISH on mouse 5-day postnatal (P5) cerebellar sections; this stage

corresponds to a peak of proliferation during the amplification ofthe GNPs pool, and a time when GNPs respond to mitogenic SHH(Fig. 3A) (Dahmane and Ruiz i Altaba, 1999). We detected

elevated TERRA levels in the outer external germinal layer (oEGL)containing the proliferating GNPs, while TERRA expressiondecreases as GNPs become SHH unresponsive and postmitotic in

the inner EGL (iEGL; Fig. 3B, top panel). In the adult cerebellumonly very few mature granule neurons in the internal granule layer(IGL) show low levels of TERRA expression (Fig. 3B, lower

Journal of Cell Science 125 (18)4384

Journ

alof

Cell

Scie

nce

panel). TERRA foci were not observed in RNase A-treated cells,indicating that these are indeed RNA foci (data not shown).

Northern blot and qRT-PCR analyses confirmed the RNA FISHresults and showed that TERRA RNA levels are highest during theproliferative expansion phases of GNPs development (P0 and P5;

Fig. 3C–E) and then decrease as GNPs differentiate (P15 and adult;Fig. 3C–E), in a similar manner to Gli1 and Math1 (Fig. 3F).

SHH growth factor stimulation increases TERRA levels

To determine whether the growth factor SHH contributes toelevated TERRA expression, we assayed the effect of recombinant

SHH on purified primary P5 mouse GNPs in vitro (Fig. 4). Using

northern blot analysis, we found that high level of SHH activation

(indicated by a large increase in Gli1 expression) following GNPs

treatment with recombinant SHH, results in a moderate (,1.3-fold) increase in bulk TERRA levels in GNP cells, as measured by

northern blotting (Fig. 4A,B; supplementary material Fig. S4). By

qRT-PCR analysis, we found that TERRA from several different

chromosomes increase ,2- to 3-fold (Fig. 4D). To control forSHH activity, we show that SHH treatment of GNPs leads to ,30-fold increase in Gli1 and ,2.5-fold increase in Math1 RNA levels,two bona fide targets of SHH activation in these cells (Fig. 4C).

Fig. 1. TERRA foci formation in mouse medulloblastoma. (A) (Top panel) Hematoxylin and Eosin staining of a region of a Ptch1+/2 mouse cerebellum with non-

tumor (left panel; indicated by an arrow) and tumor tissue (right panel; no. 6850). The normal morphology of cerebellar folia with the IGL (containing the mature granule

neurons) and the Purkinje layer containing the Purkinje neurons is seen in left panel. Scale bar: 200 mm. (Lower panel) Sections of cerebellum containing both normal andtumor tissue showing the results of in situ hybridizations with specific digoxigenin-labeled RNA probes for Gli1 and Math1. Note that Gli1 and Math1 are highly

expressed in the tumor part of the cerebellum. Asterisk in left panel denotes the normal expression of Gli1 in the normal Bergmann glial cells that form a layer in this, still

organized, non-tumor part of the cerebellum. Scale bars: 100 mm. (B) Confocal images of FISH analyses of TERRA expression in sections of the cerebellum of the samemouse as above containing both normal and tumor tissue. Note the presence of TERRA expression (strong red labeling) in the tumor as compared to the low or

undetectable expression in the non-tumor cerebellar tissue. Asterisk denote non-specific TERRA signal in the Purkinje layer. RNase A treatment leads to an elimination

of the tumor-specific TERRA signal (middle panels). Images were taken with 406lens (lower panels) to reveal details of TERRA localization in the nuclei. Scale bar:20 mm. Other images were taken with 206lens. (C) Histogram comparing relative TERRA fluorescence signal intensity (FU) in non-tumor (black) vs tumor (red) tissue,analyzed by RNA FISH and ImageProPlus software. (D) TERRA expression measured as total mean fluorescence intensity (FU). Values are means 6 standard error

from three independent experiments. (E) Quantification of cells with §1 TERRA signals of mean intensity .40 FU. More than 800 nuclei from at least three

independent experiments were counted for quantification. The P-value was calculated using a two-tailed Student’s t-test in all cases. (F) Confocal images of FISH

analyses of TERRA expression in tumor (top panel) or non-tumor (middle panel) using an Alexa-Fluor-488-conjugated DNA oligonucleotide probe (TAACCC)7 (top

two panels) or a control mutant probe (TAACAC)7 as a negative control (lower panel). Sections of the cerebellum of the same mouse (no. 7040) as in supplementary

material Fig. S1 containing both non-tumor and tumor tissue were used. Scale bar: 20 mm.

TERRA accumulation in highly proliferating cells 4385

Journ

alof

Cell

Scie

nce

These findings indicate that TERRA expression can be elevated by

high level of SHH signaling in normal GNPs derived from the

developing cerebellum.

TERFs form in cells with shortened telomeres

Telomere shortening has been reported to occur in highly

proliferative normal and cancer cells (Raynaud et al., 2010;

Wentzensen et al., 2011; Xu and Blackburn, 2007). To compare

the average telomere DNA length in tumor relative to normal

cerebellar tissue, we compared the mean average fluorescence

intensity of telomere DNA FISH signals. This method can be

used to assess the average length of telomere repeat DNA

(Baerlocher et al., 2006). Computer analyzed images of FISH

signals showed that in medulloblastoma, telomere repeat signal

intensity in tumor tissue was significantly reduced relative to

normal tissue (supplementary material Fig. S5). These results

indicate that TERRA foci form in tumor cells where telomere

length has shortened.

TERFs are distinct from cH2AX DNA damage foci

DNA damage foci are commonly observed in rapidly dividingand precancerous tissue (Sedelnikova and Bonner, 2006).

Telomere dysfunction-induced foci (TIFs) are thought to arisefrom the critical short length and/or uncapping of telomere ends,and can be examined by colocalization of cH2AX foci withtelomeric DNA (TelDNA) (d’Adda di Fagagna et al., 2003; Takaiet al., 2003). To determine if TERRA expression correlates withthe appearance of TIFs, we examined the immunofluorescence

staining pattern of cH2AX and in situ hybridization signals forTelDNA (Fig. 5A; supplementary material Fig. S6A, Fig. S7A)or TERRA RNA (Fig. 5B; supplementary material Fig. S6B,C,

Fig. S7B,C) in mouse medulloblastoma. Medulloblastoma tumorsaccumulate heterogeneous staining for cH2AX (with ,2.5% ofcells forming pan-nuclear foci, referred as large cH2AX foci),while non-tumor sections of the adult cerebellum show

essentially no cH2AX signals. Pan-nuclear cH2AX foci havebeen observed in certain types of DNA damage conditions,including viral infection (Fragkos et al., 2009) and inhibition of

ATR signaling (Fragkos et al., 2009; Toledo et al., 2011). TERFswere also detected in most tumor cells but not in normal adjacenttissue (Fig. 5B and Fig. 1). Remarkably, TERFs did not appear in

cells with pan-nuclear cH2AX staining (Fig. 5B; supplementarymaterial Figs S6,S7). Quantification of at least three independentexperiments indicated that ,2.5% of tumor cells contain pan-nuclear cH2AX staining (Fig. 5C). This is in contrast to the,80% of tumor cells that contain TERFs (Fig. 1E). Among the2.5% of cH2AX positive cells, we found 80% of thesecolocalized with strong telomere DNA FISH signals (Fig. 5D).

While small cH2AX foci could also be detected in tumorsections, these foci did not overlap with TERRA RNA or TelDNA to a significant extent (supplementary material Figs S6, S7,

S12). Similarly, TERFs did not overlap with smaller cH2AX fociobserved in rapidly dividing normal neurons of developingcerebellum in P6 mice (supplementary material Fig. S8). Taken

together, these findings indicate that TERFs are distinct fromTIFs and other cH2AX signals, yet form among the samepopulation of highly proliferative normal and tumor tissue.

TERFs are nuclear foci distinct from PML and Cajal bodies

To further investigate the subcellular localization of TERFs weexamined at higher magnification the confocal images of mouse

medulloblastoma tumors (Fig. 6A; supplementary material Fig.S9A) or normal P5 cerebellar (Fig. 6B; supplementary materialFig. S9B) sections after in situ hybridization with a TERRA PNA

probe. We observed that TERRA foci typically appear as one tothree bright foci in the nuclear region of tumor tissue (Fig. 6A).Fewer and less bright foci were observed in the nucleus of normalP5 cerebellar tissues known to be rapidly dividing (Fig. 6B). To

determine whether TERFs colocalized with other well-characterized nuclear structures, we performed immuno-RNA-FISH with antibodies to either promyeolocytic leukemia (PML)

protein (Fig. 6C), coilin (Fig. 6D), or phospho-histone H3(H3pS10; Fig. 6E). While TERRA can colocalize with PML insome ALT cell lines (data not shown), we found only few TERFs

that colocalize with PML in mouse medulloblastoma tumor tissue(Fig. 6C). Similarly, TERFs rarely colocalized with coilin, whichis a marker of Cajal bodies where the telomerase holoenzyme is

assembled (Fig. 6D). Additionally, TERFs were not observed inH3pS10 positive cells, which is a marker for mitotic cells. Thus,TERFs do not colocalize with several well-characterized nuclear

Fig. 2. TERRA is highly expressed in mouse primary medulloblastoma.

(A) Total RNA from dissected mouse medulloblastoma tumor and non-tumor

tissue (no. 7040) were assayed by northern blot and the blot was first probed with32P-labeled (TAACCC)4 for TERRA RNA expression. 18S RNA expression is

shown as a quantification control. Numbers on the left show the position of RNA

markers (in kb). (B) Quantification of TERRA levels from at least three

independent northern blot analyses using RNA isolated from matched non-

tumor and tumor cerebella in different mice, one of which is shown in A. Bar

graph represents TERRA signal intensity relative to 18S signal, and relative

intensity for non-tumor cerebella was set at 100. The P-value was calculated

using a two-tailed Student’s t-test. (C) Top panel: quantitative RT-PCR analysis

of TERRA using primers specific for subtelomeres of chromosome 2q, 11q, 5q

and telocentric chromosomes (Telocen). Lower panel: expression of Map2, Tert,

Gli1 and Math1 in non-tumor and tumor cerebellum (no. 7040) to validate the

accuracy of dissection. DDCT method relative to non-T cerebellum and Gapdh

were used to calculate relative RT-PCR between non-tumor and tumor

cerebellum. Bar graph represents the average value from at least three

independent PCR reactions (means 6 s.d.).


Journ

alof

Cell

Scie

nce

structures, and may represent a unique nuclear structure that

forms during the interphase of rapidly dividing cells.

TERRA expression is elevated in many different humancancer types

To explore whether TERRA levels are elevated in human

cancers, we first examined RNA derived from human ovarian

tissue diagnosed as either normal, advanced primary tumor, or

metastatic tumor (Fig. 7A–D). Northern blot analysis indicated

that most of the ovarian primary and metastatic tumors expressed

higher levels of TERRA than otherwise normal ovarian tissue

(Fig. 7A,B). Chromosome-specific qRT-PCR analysis also

confirmed that primary and metastatic tumors had higher levels

of TERRA at most, but not all subtelomeres tested relative to the

normal tissue (Fig. 7C,D). In one sample derived from normal

ovarian tissue, we observed very high levels of TERRA by

northern blotting (supplementary material Fig. S10A). However,

upon further molecular characterization of these tissues, this

sample (HR406) was found to express levels of the proliferation

marker Ki67 that were .50-fold relative to two other normalovarian tissue, and 3- to 5-fold greater than advanced primary or

metastatic tumors (supplementary material Fig. S10B), indicating

that this ovarian tissue was highly proliferative. This further

supports the correlation between cell proliferation state and

elevated TERRA levels. To further investigate whether TERRA

was elevated in other human cancer tissues, we compared

TERRA expression in primary human tumors and matched

normal tissue controls derived from various cancer biopsies,

including those derived from stomach, lung and colon (Fig. 7E–

H). Northern blot analyses showed that TERRA is expressed at

higher levels in tumor-derived tissues relative to matched control

tissues (Fig. 7E; supplementary material Fig. S10A). Antisense

TERRA was not detected when the same northern blot was

stripped and reprobed with G-rich probe, indicating that the

telomere RNA species is strand specific (Fig. 7E, right panel;

supplementary material Fig. S11). TERRA expression was also

analyzed by chromosome-specific qRT-PCR for several different

telomeres (Fig. 7F–H). The cell proliferation marker Ki67 was

used as a control for tumor dissection, and was elevated ,6- to100-fold relative to normal matched tissue RNA. Consistent with

northern blotting data, we found that TERRA RNA was elevated

in tumor tissue relative to matched control tissue for most

subtelomeres tested. However, only a few subtelomeres showed

significantly (.4-fold) higher TERRA levels in tumor tissuerelative to normal, and no particular subtelomere showed

elevated TERRA consistently for all the tumor samples. For

example, only telomere 13q expressed high (.4-fold) TERRAlevels in one stomach cancer biopsy, while 17q and 2q were

elevated in lung, and 10q and 17q elevated in a colon carcinoma

biopsy. No one subtelomere was consistently elevated for

Fig. 3. TERRA is elevated in highly proliferating

progenitor cells. (A) Schematic representation of the mouse

cerebellar cortex during the first postnatal week. Granule

neurons, their progenitors (GNPs) and Purkinje neurons

(red) are shown. Proliferation of GNPs occurs in the outer

external germinal layer (oEGL). Postmitotic GNPs

accumulate in the inner external germinal layer (iEGL) and

migrate to the internal granular layer (IGL) through the

Purkinje layer. Cycling GNPs in the oEGL respond to SHH

secreted form Purkinje neurons. (B) RNA FISH analysis of

TERRA expression on wild-type mouse P5 and adult

cerebella sagittal sections. The iEGL and outer oEGL are

indicated. Note the expression of TERRA in the P5 EGL,

with a stronger expression in the oEGL where most cells are

cycling. All nuclei are counterstained with DAPI (blue).

TERRA expression is low in mature granule neurons in the

adult IGL. Scale bar: 20 mm. (C) Northern blot analysis ofTERRA levels in normal mouse cerebellum at various stages

of development. 18S RNA expression is shown as

quantification control. Numbers on the left show the

position of markers in kb. Highest TERRA levels are

observed at the highest peak of GNPs proliferation at P5.

Levels of TERRA are downregulated as GNPs start their

differentiation program. (D) Quantification of northern blot

analyses for TERRA levels for each cerebellar stages from

three independent experiments, one of which is shown in C.

(E) Quantitative RT-PCR of TERRA RNA from different

cerebellar stages using TERRA-specific primers for

subtelomeres of chromosomes 2q, 11q, and telocentric

chromosomes (TeloCen). DDCT methods relative to P0

cerebellum and Gapdh were used to calculate relative RT-

PCR between different cerebellar stages. Bar graph

represents the average value from two independent

experiments and at least three independent PCR reactions

(means 6 s.d.). (F) Quantitative RT-PCR analysis of Gli1

and Math1 expression in the developing and adult

cerebellum as described in E.


Journ

alof

Cell

Scie

nce

TERRA expression, even when tumors were derived from the

same organ (data not shown). These results indicate that TERRA

expression is elevated in various human cancer tissues, and can

be expressed from a few chromosomes, which may vary among

cell and cancer types.

DiscussionWe show here that in mammalian cells in vivo, TERRA

expression is linked to the proliferative and tumorigenic state

of the cell. We found that TERRA levels were elevated in highly

proliferating normal and cancer cells in both mouse tissue

sections and human cancer biopsies. Using a well-established

mouse model of brain cancer (i.e. medulloblastoma), we found

that TERRA RNA was elevated in tumor, but not in normal

cerebellar tissue (Figs 1, 2; supplementary material Figs S1–S3).

RNA FISH revealed pronounced TERRA foci, termed TERFs, in

cells overexpressing TERRA RNA. TERFs were sensitive to

RNase A and not detected with control probes containing a

mutation in the telomere repeat sequence, indicating that these

were indeed TERRA-containing foci. Furthermore, TERFs

appeared only in tissues where high level TERRA expression

was validated by northern blotting and qRT-PCR (Fig. 2;

supplementary material Fig. S3). TERFs generally formed as

one or few major foci in the nuclear region of proliferating cells.

TERRA and TERFs were also elevated in rapidly dividingprogenitor cells of the developing cerebellum, indicating that

normal proliferating cells can also form TERFs (Fig. 3). TERRAlevels were also enhanced by addition of recombinant SHHgrowth factor to purified progenitor cells in vitro, suggesting thatstrong and chronic growth factor stimulation promotes TERRA

accumulation (Fig. 4; supplementary material Fig. S4). TERFsformed in the same cell populations where cH2AX DNA damagefoci form (Fig. 5; supplementary material Figs S6, S7, S12) and

telomere DNA shortening has occurred (supplementary materialFig. S5), but TERFs did not colocalize with cH2AX foci (Fig. 5),nor with PML or Cajal nuclear bodies (Fig. 6). Finally, we

provide evidence that TERRA is expressed at higher levels inmany human cancer tissues relative to matched controls (Fig. 7;supplementary material Fig. S10). These findings support themodel that TERRA expression is coupled to cellular proliferation

and that TERFs are a novel indicator of proliferative stress.

Our results are different from several previous reports thatdescribed either a decrease in TERRA expression between

normal and colon cancer (Schoeftner and Blasco, 2008),diminished TERRA in advanced astrocytoma (Sampl et al.,2012) or a lack of expression in cancer cell lines (Ng et al., 2009;

Zhang et al., 2009). The differences may reside in the use oftumor tissue compared to cancer cell lines (Ng et al., 2009; Zhanget al., 2009) as well as in the techniques used to detect TERRAexpression [a comprehensive use of RNA FISH, northern blotting

and qRT-PCR in our study as opposed to dot blotting inSchoeftner and Blasco or quantitative RT-PCR from fewchromosomes (supplemental material Figs. S13-15) (Schoeftner

and Blasco, 2008; Sampl et al., 2012)]. It is also likely thatTERRA expression in human cancers may be regulated in a morecomplex manner corresponding to the patient history, cancer

type, and tumor tissue integrity. In our experiments, human tumorRNA was purified from freshly isolated unfixed biopsies to avoidRNA degradation. Our use of a mouse cancer model has also

enabled us to examine adjacent non-tumor and tumor tissue onthe same section. In mouse model of medulloblastoma, we wereable to utilize methods developed for RNA FISH that reduce thetime of fixation and denaturation that commonly degrade or

modify RNA. Together, these methods have allowed us toobserve TERRA expression and localization in tissue sectionsand isolates that better preserve the in vivo physiology of cellular

proliferation and cancer.

The function and regulation of TERRA has been explored inseveral previous studies (Azzalin et al., 2007; Deng et al., 2009;

Redon et al., 2010; Schoeftner and Blasco, 2008). We havepreviously shown that TERRA can protect telomere ends fromDNA damage signaling by promoting telomeric heterochromatinformation (Deng et al., 2009). TERRA has also been shown to

inhibit telomerase activity in vitro (Redon et al., 2010; Schoeftnerand Blasco, 2008). TERRA levels can be elevated in cell lineslacking DNA methyltransferase 3b (DNMT3b) (Yehezkel et al.,

2008), as well as in response to developmental and stress signals(Schoeftner and Blasco, 2008). Our data suggests that TERRAexpression can be enhanced by growth factor signaling (e.g.

SHH) and cell proliferation in vivo. Moreover, chronicproliferation signals associated with carcinogenic mutations(like Ptch1+/2) lead to elevated TERRA and TERF formation.

We also observed that telomere DNA signal decreases(supplementary material Fig. S5) and that TERT mRNA levelsare not elevated (Fig. 2; supplementary material Fig. S3) in

Fig. 4. TERRA expression can be induced in purified progenitor cells

stimulated with high SHH signaling activation in vitro. (A) GNPs were

purified from wild-type mouse P5 cerebella and cultured for 12 hours with or

without SHH (600 ng/ml). TERRA RNA was analyzed by northern blotting

using a 32P-labeled (TAACCC)4 oligonucleotide probe. 18S RNA expression

is shown as an internal control for RNA loading. RNase A treatment is shown

in the right panel. (B) Quantification of TERRA levels from at least three

independent northern blot analyses using RNA isolated from GNPs treated

with SHH for 12 hours or left untreated, one of which is shown in A. Bar

graph represents TERRA signal intensity relative to 18S signal, and relative

intensity for SHH (2) was set at 100. The P-value was calculated using a two-

tailed Student’s t-test. (C) qRT-PCR analysis for expression of Gli1 and

Math1 is shown as a control for SHH activity. DDCT methods relative to

untreated GNPs and Gapdh were used to calculate relative RT-PCR by SHH

treatment. Bar graph represents mean 6 standard deviations from three

independent experiments. (D) qRT-PCR analysis of individual TERRA

expression using subtelomere-specific primers for chromosomes 2q, 11q,

TeloCen, 18q or 5q as described in C.


Journ

alof

Cell

Scie

nce

tumor tissue. We propose that TERRA is required for telomere

end processing and protection during normal cell proliferation,

and that elevated TERRA and TERF protects telomeres from

eliciting a DNA damage response during conditions of

proliferative stress (Fig. 8).

cH2AX-positive DNA damage foci have been shown to beelevated in highly proliferating cells and precancerous lesions

(Bartkova et al., 2005; Gorgoulis et al., 2005; Jackson and Bartek,

2009). We found that small cH2AX foci accumulate in mousemedulloblastoma, with ,2.5% of the tumor cells containing pan-nuclear cH2AX foci (Fig. 5). In contrast, ,80% of these cellswere TERF positive. Importantly, most cH2AX foci did notoverlap with TERFs, but did colocalize with telomere DNA foci

(TIFs). Others have shown that telomere DNA forms aggregates

in highly proliferating cancer cells (Mai and Garini, 2006).

Unfortunately, we were unable to colocalize TERFs with telomere

DNA signals, so can not exclude the possibility that TERFs are

expressed at telomere aggregates in highly proliferating cells. We

did find that telomeres were generally shorter in mouse cancer

cells that express TERRA and form TERFs (supplementary

material Fig. S5), suggesting that short telomeres may promote

TERRA transcription activation. Elevated TERRA expression in

highly proliferating cancer and normal progenitor cells may

stabilize these abnormally short telomeres, perhaps through

inhibition of the DNA damage response. This is consistent with

the observed lack of DNA damage associated cH2AX focicolocalizing with TERFs (Fig. 5; supplementary material Figs

S6 and S7). TERRA may prevent DNA damage response at short

telomeres by stabilizing the shelterin complex, heterochromatin

formation, or protection of single stranded telomeric DNA (Deng

et al., 2009; Flynn et al., 2011).

TERRA foci and telomeric transcript accumulations (Tacs) have

been described previously (Azzalin et al., 2007; Marion et al.,

2009; Schoeftner and Blasco, 2008; Zhang et al., 2009). In human

cell lines, TERRA can colocalizes to metaphase telomeres and

form variable numbered foci that can be upregulated by depleting

components of the nonsense-mediated decay (NMD) pathway

(Azzalin et al., 2007). In undifferentiated mouse ES cells, Tacs

associate with both sex chromosomes, but upon ES cell

differentiation, Tacs relocalize to the inactive X-chromosome in

females or the Y-chromosome in males (Schoeftner and Blasco,

2008). In telomerase-deficient TRF2-overexpressing (K5TRF2/

TERC2/2) mouse models of telomere dysfunction, Tacs delocalize

from the inactive X-chromosome, reflecting a novel form of

telomere dysfunction (Schoeftner et al., 2009). While we did not

directly examine the localization of TERFs with respect to the

inactive X chromosome, the TERFs that we observe in rapidly

proliferating cells are likely to be very similar to Tacs. However,

since Tacs have not been reported to be elevated in rapidly

proliferating neuronal progenitor or medulloblastoma, we refrain

from concluding that these TERFs and Tacs are identical

structures.

RNA mediated nuclear body formation has been observed for a

variety of other RNA species (Shevtsov and Dundr, 2011).

Fig. 5. Tumor-associated TERRA foci do not

colocalize with cH2AX. (A) Confocal microscopy

images of immunofluorescence of cH2AX foci

(green) combined with DNA FISH for telomere

repeat DNA (TelDNA; red) in non-tumor (non-T) or

tumor sections. DAPI stain is in blue. Arrowheads

indicate large cH2AX foci. Arrows indicate nuclei

containing intense telomere DNA signals

colocalizing with large cH2AX foci. Enlarged (56)images of the regions in the yellow boxes are shown

in the right panels. (B) Confocal microscopy images

of immunofluorescence of cH2AX foci (green)

combined with RNA FISH for TERRA (red) in non-

tumor or tumor sections of the cerebellum.

Arrowheads indicate large cH2AX foci. Enlarged

(56) images of the regions in the yellow boxes areshown in the right panels. (C) Quantification of cells

containing large cH2AX foci. More than 1000 nuclei

from at least three independent experiments were

counted for quantification. The P-value was

calculated using a two-tailed Student’s t-test.

(D) Quantification of large cH2AX foci that

colocalize with intense telomere DNA FISH signals

to form TIFs. More than 1000 nuclei from at least

three independent experiments were counted for

quantification. The P-value was calculated using a

two-tailed Student’s t-test.


Journ

alof

Cell

Scie

nce

Transcription of the tandem arrays of satellite III (sat III) repetitive

DNA is known to form nuclear stress bodies located at

pericentromeric DNA (Biamonti and Vourc’h, 2010). Recent

genome-wide sequencing studies have found that satellite repeat

transcripts were aberrantly overexpressed in various human and

mouse epithelial cancers (Ting et al., 2011). While, Sat III repeat

DNA and RNA have some intriguing similarities with TERRA

DNA and RNA, we did not observe that TERFs had similar patterns

of granulation commonly seen with SatIII nuclear stress bodies in

tissue culture cell lines. However, it will be important to determine

if TERF positive tumor tissue is also enriched in Sat III nuclear

bodies, and whether these structures share common features with

TERFs. TERFs may also share features with other RNA-nucleated

bodies, including the miRNA processing P-bodies (Parker and

Sheth, 2007) and cytoprotective aggresomes (Wileman, 2007). In

some cancer cells, telomere DNA has been shown to aggregate in

conjunction with nuclear lamina (Mai and Garini, 2006; Raz et al.,

2008). In contrast to TERFs, telomere aggregates colocalize with

cH2AX DNA damage foci, suggesting that these are distinctnuclear structures (Raz et al., 2008). Nevertheless, it is possible that

TERFs reflect a cluster of several highly transcribed telomeres that

correspond to the telomere aggregates observed in some cancer

types. TERFs did not show significant colocalization with other

well-characterized nuclear structures, including coilin-containing

Cajal bodies or PML-containing ND10 bodies (Fig. 6). Thus,

TERFs appear to represent a unique nuclear body formed by

TERRA accumulation from one or more telomeres in highly

proliferating cells.

An important unanswered question is how TERRA expression,

processing, and localization may differ in normal differentiated,

normal proliferating, and abnormal cancer cells. TERRA levels

and TERFs can be detected in both proliferating cell types,

although we did observe greater TERRA processing to several

smaller RNA species (Fig. 7; supplementary material Fig. S10)

and an average greater density of TERFs in cancer tissue relative

to progenitor cells (Figs 2,6). It may not be surprising that

progenitor cells have similar TERRA expression patterns as

cancer cells, since they are known to share common gene

expression profile as in the case of medulloblastoma (Kho et al.,

2004). We suggest that the major difference between cancer and

Fig. 6. TERFs are nuclear, interphase-associated foci

distinct from PML and Cajal bodies. (A) Confocal

microscopy images of TERRA RNA FISH on tissue

sections derived from mouse medulloblastoma (no. 6850).

(B) Same as in A, except in normal cerebellar tissue

derived from a stage P5 mouse. (C) TERRA RNA FISH

(red) combined with immunofluorescence with antibody

to PML (green) on tissue sections derived from mouse

medulloblastoma (no. 7171). All nuclei were

counterstained with DAPI (blue). Enlarged (zoomed)

merged images are shown in the right panels. (D) Same as

in C, except TERRA RNA FISH (red) combined with

coilin immunofluorescence (green).

(E) Same as in C, except TERRA RNA FISH (red)

combined with immunofluorescence with antibody to

phosphorylated histone H3S10 (green).


Journ

alof

Cell

Scie

nce

Fig. 7. TERRA is overexpressed in human tumor tissues. (A) Northern blot analysis of TERRA RNA isolated from normal ovarian tissue, advanced primary ovarian

cancer and metastatic ovarian cancer tissue. Numbers at the bottom show the average value of TERRA signals relative to 18S RNA signals in tumor versus normal tissues

from two independent northern blots. Asterisks indicate that the sample is not included in the analysis because of the degradation of the 18S signal. (B) Quantification of

average TERRA levels from many northern blot analyses using RNA isolated from tissues derived from two normal ovaries (540071 and 709152), eight primary and nine

metastatic ovarian cancers, a representative of which is shown in A. Bar graph represents TERRA signal intensity relative to the 18S signal, and average relative intensity

for two normal ovary tissues was set at 100. (C) qRT-PCR analysis of Ki-67 expression and TERRA levels in the indicated ovarian cancers and normal ovary tissue

(709152). TERRA levels were assayed using primers specific for TERRA RNA transcribed from subtelomeres of human chromosome 2q, 10q, 13q, as indicated. DDCT

methods relative to normal ovary and Gapdh were used to calculate relative RT-PCR between normal and tumor samples. Bar graph represents the average value from

three independent PCR reactions (means 6 s.d.). (D) The same as in C, except using primers specific for TERRA RNA transcribed from subtelomeres of human

chromosome XqYq, 15q, 16p, as indicated. (E) TERRA expression in dissected primary human tumor tissue from carcinoma of the stomach, lung and colon (normal and

tumor) analyzed by northern blotting. 18S RNA expression is shown as quantification control. Equal intensity of 18S* signals indicate that RNA from each sample were

subject to similar levels of degradation during the preparation process. The same northern blot was stripped, and reprobed with 32P-labeled (TTAGGG)4 probe for anti-

sense TERRA (right panel). Numbers on the left show the position of markers (in kb). Numbers at the bottom show the value of TERRA signals relative to the 18S RNA

signals in tumor versus normal tissues. (F) qRT-PCR analysis of Ki-67 expression (left panel) and TERRA levels (right panel) in the tumor and patient matched control

tissues from stomach. TERRA levels were assayed using primers specific for TERRA RNA transcribed from subtelomeres of human chromosome 2q, 17q, 10q, 13q, 15q,

16q and XqYq, as indicated. Bar graph represents the average value from at least three independent PCR reactions (means 6 s.d.). (G) The same as in F, except in the

tumor and patient matched control tissues from lung. (H) The same as in F, except in the tumor and patient matched control tissues from the colon.


Journ

alof

Cell

Scie

nce

progenitor cells is the abnormal prolonged exposure to

proliferation signals and the potential stabilization of shortened

and damaged telomeres through the action of TERRA. We also

observed TERF expression is highly elevated in human cancer

cell lines with ALT elongated telomeres, as observed in U2OS

cells (supplementary material Fig. S15). It is therefore possible

that TERRA expression in mouse medulloblastoma correlates

with the ALT phenotype, but additional experiments are needed

to confirm this possibility. While the precise function,

processing, and localization of TERFs remains unknown, these

details will be essential for understanding the regulation of

telomeres during normal and cancer cell proliferation. In

summary, our data demonstrates that TERRA accumulates and

forms TERFs in highly proliferating progenitors and cancer

tissue, and suggests that TERFs may provide a novel and

sensitive biomarker for telomere dysfunction in cancer.

Materials and MethodsMouse breeding

Medulloblastoma used in our study formed in the Ptch1+/2 colony either alone orin combination with p53+/2. The tumor 6850 (Fig. 1A) originated in aPtch1+/2;RP58fl/fl mouse obtained during the breeding between Ptch1+/2 andRP58fl/fl;nestinCreER. The RP58fl/fl does not present any abnormality (Xiang et al.,2012).

All procedures were approved by the Wistar Institute Institutional Animal Careand Use Committee.

Telomeric RNA FISH on tissue section

Tissue sections (7–12 mm) were prepared as described previously (Fernandez et al.,2010). Fresh frozen sections were fixed in 4% paraformaldehyde (PFA) for 10minutes on ice, washed twice with cold PBS, and twice with RIPA (150 mM NaCl,50 mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 1 mMEDTA) buffer for 10 mins each. The sections were fixed again in 4% PFA for 10mins at 25 C̊, washed three times with cold PBS, and acetylated in acetylation bufferfor 10 mins at 25 C̊. After acetylation, the sections were washed three times withcold PBS containing 0.05% Tween-20 (PBST), and prehybridized in hybridizationbuffer (50% formamide, 56SSC, 56Denhardts, 25 mg/ml yeast RNA, 0.5 mg/mlsalmon sperm DNA) for 1 hr at 37 C̊. RNase A treatment was performed in PBSTwith 100 mg/ml RNase A for 30-60 mins at 37 C̊ before prehybridization.Hybridization was performed overnight at 37 C̊, and the following probes were used

in the hybridization: a Tamra-(CCCTAA)3 PNA probe (Panagene Inc.) or an Alexa-Fluor-488-(TAACCC)7 oligonucleotide probe (IDT) for TERRA RNA, a Fam-(TTAGGG)3 PNA probe for TERRA antisense, and an Alexa-Fluor-488-(TAACAC)7 probe as a control for specificity. After hybridization, slides werewashed as follows: 26SSC, 50% formamide, three times at 39 C̊ for 5 mins; 26SSC, three times at 39 C̊ for 5 mins each; 16SSC, 10 mins at room temperature; 46SSC once at room temperature. Slides were counterstained with 0.1 g/ml DAPI in 46SSC, 0.1% Tween-20, washed in 46SSC, and mounted with mounting media.Images for H/E staining were taken with a Nikon E600 Upright microscope (NikonInstruments) with ImageProPlus software (media Cybemetrics) and AdobePhotoShop CS5 for image processing. Confocal images were taken with ZeissLSM510META NLO laser scanning confocal system on a Zeiss Axiovert 200Minverted microscope with Zeiss AIM Ver.4.0 software and Adobe PhotoShop CS5for image processing. For quantification purpose, the same technical settings wereapplied to the capture of all images (Ferlicot et al., 2003; Ourliac-Garnier andLondono-Vallejo, 2011). The mean fluorescence intensity of TERRA spots (FI/spot)from unmodified black and white images was automatically quantified byImageProPlus software and expressed in fluorescence units (FU). The data wasexported into an Excel worksheet for frequency and statistical analysis. P-valueswere calculated using two-tailed Student’s t-tests.

RNA preparation and analysis

Total RNA for normal ovarian tissue samples was purchased from Biochain(R1234183, lot no. 709152), Agilent Technologies (540071), and Zyagen(HR406). Total RNA (540045) for normal human breast was purchased fromAgilent Technologies. All other human tissues were collected according to theguidelines and policies of the Hospital of the University of PennsylvaniaInstitutional Review Board. Stage III–IV human ovarian carcinoma specimenswere procured through Research Pathology Services at Dartmouth–HitchcockMedical Center under institutional approval (CPHS17702). For ovarian cancersamples, primary means from the initial mass in the ovary and metastatic meansfrom anywhere else in the peritoneal cavity. Total RNA was purified with Trizolreagent (Invitrogen) as manufacturer’s instructions. The RNA samples weretreated with DNase I for 45 min at 37 C̊, followed by DNase I inactivation in thepresence of EDTA at 65 C̊ for 5 min prior to further analysis. For northern blotting,about 3–7.5 mg of total RNA were used. Hybridizations were performed in Churchbuffer (0.5N Na-phosphate, pH 7.2, 7% SDS, 1mM EDTA, 1% BSA) for 16-18 hrsat 50 C̊. The membrane was washed twice in 0.2N Na-phosphate, 2% SDS, 1 mMEDTA at room temperature, once in 0.1N Na-phosphate, 2% SDS, 1 mM EDTA at50 C̊, and analyzed by phosphor-imager (Amersham Biosciences). The blots werefirst hybridized with a 32P-labeled (TAACCC)4 probe, then stripped, and probedwith either a 32P-labeled (TTAGGG)4 or 18S probe. When indicated, RNAsamples were treated with RNase A (Roche) at a final concentration of 100 mg/mlfor 30–60 mins at 37 C̊. Images were processed with a Typhoon 9410 Imager (GEHealthcare) and quantified with ImageQuant 5.2 software (Molecular Dynamics).TERRA RNA levels were calculated as percentages relative to signals from controlsamples and 18S internal control. Reverse transcriptions were performed withSuperScript III (Invitrogen) using 1 mg of total RNA as per the manufacturer’sinstructions. A specific primer (CCCTAA)6 was used to obtain TERRA cDNA at55 C̊. Real-time PCR experiments were performed as described (Deng et al.,2009). Relative RT-PCR was determined using DDCT methods relative to controlsamples and internal control Actin and Gapdh for human samples or Gapdh formouse samples. Primer sequences used for real-time PCR are listed insupplementary material Table S1.

Telomere DNA FISH

Tissue sections were prepared essentially as described in RNA FISH methodsection. Telomeric DNA FISH was performed as followings. After acetylation,sections were subject to RNase A treatment by the incubation in PBST with100 mg/ml RNase A for 30–60 mins at 37 C̊. Sections were washed three times inPBS, and dehydrated in cold ethanol series 5 mins for each (70%, 95%, 100%).Slides with sections were denatured on a 80–85 C̊ hot plate for 5 mins in thepresence of about 120 ml of hybridization mix (70% formamide, 10 mM Tris-HCl,pH 7.2, 0.5% blocking reagent) containing telomeric PNA-Tamra-(CCCTAA)3probe, and hybridization was performed in the dark for overnight at roomtemperature. The slides were washed two times for 15 mins each in 70%formamide, 10 mM Tris-HCl, pH 7.2, 0.1% BSA followed by three washes of 5mins each in 0.1 M Tris-HCl, pH 7.2, 0.15 M NaCl, 0.08% Tween-20, stainedwith 0.1 mg/ml DAPI, and mounted in mounting medium. Confocal images weretaken with Zeiss LSM510META NLO laser scanning confocal system on a ZeissAxiovert 200M inverted microscope with Zeiss AIM Ver.4.0 software and AdobePhotoShop CS5 for image processing. Image capture and quantification processwere performed using the same methods as those used for RNA FISH.

For combined cH2AX staining and FISH analysis, we performed RNA or DNAFISH as described above. After washes, the sections were fixed again in 4%paraformaldehyde for 15 mins, blocked in blocking solution (1 mg/ml BSA, 3%goat serum, 0.1% Triton X-100, 1 mM EDTA in PBS) for at least 30 mins, andincubated with cH2AX monoclonal antibody (Upstate) diluted in blocking solution

Fig. 8. TERRA foci (TERF) formation during growth-factor-induced

proliferation in progenitor and cancer cells. Normal progenitor cells

subjected to high level of proliferation (such as granule neuron progenitors in

the mammalian postnatal cerebellum) through the activity of growth factors

(such as SHH) express high level of TERRA RNA (red dots). Elevated

chronic growth factor signaling, as occurs in the Ptch1+/2 model of

medulloblastoma, results in high TERRA expression and stabilization of

shortened telomeres in tumor cells. TERFs form at high level in early stage

cancer cells in the absence of cH2AX DNA damage foci. Stabilization of

short telomeres is predicted to promote cancer cell evolution.


Journ

alof

Cell

Scie

nce

(1:100) for 1 hr at room temperature. The sections were washed three times withPBST for 5 mins each, and incubated with Alexa-Fluor-488-conjugated secondaryantibody (2 mg/ml stock solution diluted 1:600 in PBST) in blocking solution for30 mins. The sections were further washed with PBST, counter stained with DAPI,and mounted in mounting medium before confocal microscope. Rabbit polyclonalantibodies to Coilin (H-300) and phospho-histone H3 (Ser10) were purchased fromSanta Cruz and Millipore, respectively. Monoclonal antibody to mouse PML was agift from Olga Vladimirova at the Wistar Institute.

Granule neuron progenitor purification

Granule neuron progenitors (GNPs) were purified from P5 mouse cerebella asdescribed previously (Fernandez et al., 2010). SHH (600 ng/ml, R&D) was addedto the media for 12 hours.

AcknowledgementsWe would like to thank Federico Valdivieso, Michael Feldman, theTumor tissue bank and the Abramson Cancer Center for the tumorsamples. We also thank the Department of Pathology at DartmouthUniversity for ovarian cancer tissue RNA, and Fred Keeney andJames Hayden in the Wistar Institute Microscopy Core.

FundingThis work was supported by the Wistar Institute Cancer Center CoreGrant [grant number P30 CA10815]; the American Cancer Society[grant number RSG-08-045-01-DDC to N.D.]; the National BrainTumor Society to N.D., National Institutes of Health [grant numberRO1CA140652 to P.M.L.); and a Scientist Development grant fromthe American Heart Association [grant number 11SDG5330017 toZ.D.]. Deposited in PMC for release after 12 months.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.108118/-/DC1

ReferencesArmentano, M., Filosa, A., Andolfi, G. and Studer, M. (2006). Coup-TFI is required

for the formation of commisural projections in the forebrain by regulating axonalgrowth. Development 133, 4151-4162.

Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E. and Lingner, J. (2007).Telomeric repeat containing RNA and RNA surveillance factors at mammalianchromosome ends. Science 318, 798-801.

Baerlocher, G. M., Vulto, I., de Jong, G. and Lansdorp, P. M. (2006). Flowcytometry and FISH to measure the average length of telomeres (flow FISH). Nat.Protoc. 1, 2365-2376.

Bah, A., Wischnewski, H., Shchepachev, V. and Azzalin, C. M. (2012). The telomerictranscriptome of Schizosaccharomyces pombe. Nucleic Acids Res. 40, 2995-3005.

Bartkova, J., Horejsı́, Z., Koed, K., Krämer, A., Tort, F., Zieger, K., Guldberg, P.,

Sehested, M., Nesland, J. M., Lukas, C. et al. (2005). DNA damage response as acandidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864-870.

Biamonti, G. and Vourc’h, C. (2010). Nuclear stress bodies. Cold Spring Harb.Perspect. Biol. 2, a000695.

Blackburn, E. H., Grieder, C. W. and Szostak, J. W. (2006). Telomeres andTelomerase: the path from maize, Tetrahymena, and yeast to human cancer and aging.Nat. Med. 12, 1133-1338.

Blasco, M. A. (2005). Telomeres and human disease: ageing, cancer and beyond. Nat.Rev. Genet. 6, 611-622.

Blasco, M. A. (2007). Telomere length, stem cells and aging. Nat. Chem. Biol. 3, 640-649.

Caslini, C., Connelly, J. A., Serna, A., Broccoli, D. and Hess, J. L. (2009). MLLassociates with telomeres and regulates telomeric repeat-containing RNA transcrip-tion. Mol. Cell. Biol. 29, 4519-4526.

Cech, T. R. (2004). Beginning to understand the end of the chromosome. Cell. 116, 273-279.

Corcoran, R. B. and Scott, M. P. (2001). A mouse model for medulloblastoma andbasal cell nevus syndrome. J. Neurooncol. 53, 307-318.

d’Adda di Fagagna, F., Reaper, P. M., Clay-Farrace, L., Fiegler, H., Carr, P., Von

Zglinicki, T., Saretzki, G., Carter, N. P. and Jackson, S. P. (2003). A DNA damagecheckpoint response in telomere-initiated senescence. Nature 426, 194-198.

Dahmane, N. and Ruiz i Altaba, A. (1999). Sonic hedgehog regulates the growth andpatterning of the cerebellum. Development 126, 3089-3100.

de Lange, T. (2002). Protection of mammalian telomeres. Oncogene 21, 532-540.

de Lange, T. (2004). T-loops and the origin of telomeres. Nat. Rev. Mol. Cell Biol. 5,323-329.

de Lange, T. (2005a). Shelterin: the protein complex that shapes and safeguards humantelomeres. Genes Dev. 19, 2100-2110.

De Lange, T. (2005b). Telomere-related genome instability in cancer. Cold SpringHarb. Symp. Quant. Biol. 70, 197-204.

Deng, Z., Norseen, J., Wiedmer, A., Riethman, H. and Lieberman, P. M. (2009).TERRA RNA binding to TRF2 facilitates heterochromatin formation and ORCrecruitment at telomeres. Mol. Cell 35, 403-413.

Ellison, D. (2002). Classifying the medulloblastoma: insights from morphology andmolecular genetics. Neuropathol. Appl. Neurobiol. 28, 257-282.

Ellison, D. W. (2010). Childhood medulloblastoma: novel approaches to theclassification of a heterogeneous disease. Acta Neuropathol. 120, 305-316.

El-Zaatari, M., Tobias, A., Grabowska, A. M., Kumari, R., Scotting, P. J., Kaye, P.,Atherton, J., Clarke, P. A., Powe, D. G. and Watson, S. A. (2007). De-regulation ofthe sonic hedgehog pathway in the InsGas mouse model of gastric carcinogenesis. Br.J. Cancer. 96, 1855-1861.

Ferlicot, S., Durrbach, A., Ba, N., Desvaux, D., Bedossa, P. and Paradis, V. (2003).The role of replicative senescence in chronic allograft nephropathy. Hum. Pathol. 34,924-928.

Fernandez, C., Tatard, V. M., Bertrand, N. and Dahmane, N. (2010). Differentialmodulation of Sonic-hedgehog-induced cerebellar granule cell precursor proliferationby the IGF signaling network. Dev. Neurosci. 32, 59-70.

Flynn, R. L., Centore, R. C., O’Sullivan, R. J., Rai, R., Tse, A., Songyang, Z.,Chang, S., Karlseder, J. and Zou, L. (2011). TERRA and hnRNPA1 orchestrate anRPA-to-POT1 switch on telomeric single-stranded DNA. Nature 471, 532-536.

Fragkos, M., Jurvansuu, J. and Beard, P. (2009). H2AX is required for cell cyclearrest via the p53/p21 pathway. Mol. Cell. Biol. 29, 2828-2840.

Gilbertson, R. J. (2004). Medulloblastoma: signalling a change in treatment. LancetOncol. 5, 209-218.

Gilbertson, R. J. and Ellison, D. W. (2008). The origins of medulloblastoma subtypes.Annu. Rev. Pathol. 3, 341-365.

Goodrich, L. V., Milenković, L., Higgins, K. M. and Scott, M. P. (1997). Alteredneural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109-1113.

Gorgoulis, V. G., Vassiliou, L. V., Karakaidos, P., Zacharatos, P., Kotsinas, A.,

Liloglou, T., Venere, M., Ditullio, R. A., Jr, Kastrinakis, N. G., Levy, B. et al.(2005). Activation of the DNA damage checkpoint and genomic instability in humanprecancerous lesions. Nature 434, 907-913.

Greenwood, J. and Cooper, J. P. (2012). Non-coding telomeric and subtelomerictranscripts are differentially regulated by telomeric and heterochromatin assemblyfactors in fission yeast. Nucleic Acids Res.40, 2956-2963.

Hahn, H., Wicking, C., Zaphiropoulous, P. G., Gailani, M. R., Shanley, S.,Chidambaram, A., Vorechovsky, I., Holmberg, E., Unden, A. B., Gillies, S. et al.

(1996). Mutations of the human homolog of Drosophila patched in the nevoid basalcell carcinoma syndrome. Cell 85, 841-851.

Jackson, S. P. and Bartek, J. (2009). The DNA-damage response in human biology anddisease. Nature 461, 1071-1078.

Jain, D. and Cooper, J. P. (2010). Telomeric strategies: means to an end. Annu. Rev.Genet. 44, 243-269.

Johnson, R. L., Rothman, A. L., Xie, J., Goodrich, L. V., Bare, J. W., Bonifas, J. M.,Quinn, A. G., Myers, R. M., Cox, D. R., Epstein, E. H., Jr et al. (1996). Humanhomolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272,1668-1671.

Kho, A. T., Zhao, Q., Cai, Z., Butte, A. J., Kim, J. Y., Pomeroy, S. L., Rowitch, D. H.

and Kohane, I. S. (2004). Conserved mechanisms across development andtumorigenesis revealed by a mouse development perspective of human cancers.Genes Dev. 18, 629-640.

Liu, D., O’Connor, M. S., Qin, J. and Songyang, Z. (2004). Telosome, a mammaliantelomere-associated complex formed by multiple telomeric proteins. J. Biol. Chem.279, 51338-51342.

López de Silanes, I., Stagno d’Alcontres, M. and Blasco, M. A. (2010). TERRAtranscripts are bound by a complex array of RNA-binding proteins. Nat. Commun. 1,33.

Mai, S. and Garini, Y. (2006). The significance of telomeric aggregates in theinterphase nuclei of tumor cells. J. Cell. Biochem. 97, 904-915.

Marion, R. M., Strati, K., Li, H., Tejera, A., Schoeftner, S., Ortega, S., Serrano, M.

and Blasco, M. A. (2009). Telomeres acquire embryonic stem cell characteristics ininduced pluripotent stem cells. Cell Stem Cell 4, 141-154.

Ng, L. J., Cropley, J. E., Pickett, H. A., Reddel, R. R. and Suter, C. M. (2009).Telomerase activity is associated with an increase in DNA methylation at theproximal subtelomere and a reduction in telomeric transcription. Nucleic Acids Res.37, 1152-1159.

Oliver, T. G., Read, T. A., Kessler, J. D., Mehmeti, A., Wells, J. F., Huynh, T. T.,Lin, S. M. and Wechsler-Reya, R. J. (2005). Loss of patched and disruption ofgranule cell development in a pre-neoplastic stage of medulloblastoma. Development132, 2425-2439.

Palm, W. and de Lange, T. (2008). How shelterin protects mammalian telomeres.Annu. Rev. Genet. 42, 301-334.

Parker, R. and Sheth, U. (2007). P bodies and the control of mRNA translation anddegradation. Mol. Cell 25, 635-646.

Raynaud, C. M., Hernandez, J., Llorca, F. P., Nuciforo, P., Mathieu, M. C.,Commo, F., Delaloge, S., Sabatier, L., André, F. and Soria, J. C. (2010). DNAdamage repair and telomere length in normal breast, preneoplastic lesions, andinvasive cancer. Am. J. Clin. Oncol. 33, 341-345.

Raz, V., Vermolen, B. J., Garini, Y., Onderwater, J. J., Mommaas-Kienhuis, M. A.,

Koster, A. J., Young, I. T., Tanke, H. and Dirks, R. W. (2008). The nuclear laminapromotes telomere aggregation and centromere peripheral localization duringsenescence of human mesenchymal stem cells. J. Cell Sci. 121, 4018-4028.


http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.108118/-/DC1http://dx.doi.org/10.1242%2Fdev.02600http://dx.doi.org/10.1242%2Fdev.02600http://dx.doi.org/10.1242%2Fdev.02600http://dx.doi.org/10.1126%2Fscience.1147182http://dx.doi.org/10.1126%2Fscience.1147182http://dx.doi.org/10.1126%2Fscience.1147182http://dx.doi.org/10.1038%2Fnprot.2006.263http://dx.doi.org/10.1038%2Fnprot.2006.263http://dx.doi.org/10.1038%2Fnprot.2006.263http://dx.doi.org/10.1093%2Fnar%2Fgkr1153http://dx.doi.org/10.1093%2Fnar%2Fgkr1153http://dx.doi.org/10.1038%2Fnature03482http://dx.doi.org/10.1038%2Fnature03482http://dx.doi.org/10.1038%2Fnature03482http://dx.doi.org/10.1101%2Fcshperspect.a000695http://dx.doi.org/10.1101%2Fcshperspect.a000695http://dx.doi.org/10.1038%2Fnm1006-http://dx.doi.org/10.1038%2Fnm1006-http://dx.doi.org/10.1038%2Fnm1006-http://dx.doi.org/10.1038%2Fnrg1656http://dx.doi.org/10.1038%2Fnrg1656http://dx.doi.org/10.1038%2Fnchembio.2007.38http://dx.doi.org/10.1038%2Fnchembio.2007.38http://dx.doi.org/10.1128%2FMCB.00195-09http://dx.doi.org/10.1128%2FMCB.00195-09http://dx.doi.org/10.1128%2FMCB.00195-09http://dx.doi.org/10.1016%2FS0092-8674%2804%2900038-8http://dx.doi.org/10.1016%2FS0092-8674%2804%2900038-8http://dx.doi.org/10.1023%2FA%3A1012260318979http://dx.doi.org/10.1023%2FA%3A1012260318979http://dx.doi.org/10.1038%2Fnature02118http://dx.doi.org/10.1038%2Fnature02118http://dx.doi.org/10.1038%2Fnature02118http://dx.doi.org/10.1038%2Fsj.onc.1205080http://dx.doi.org/10.1038%2Fnrm1359http://dx.doi.org/10.1038%2Fnrm1359http://dx.doi.org/10.1101%2Fgad.1346005http://dx.doi.org/10.1101%2Fgad.1346005http://dx.doi.org/10.1101%2Fsqb.2005.70.032http://dx.doi.org/10.1101%2Fsqb.2005.70.032http://dx.doi.org/10.1016%2Fj.molcel.2009.06.025http://dx.doi.org/10.1016%2Fj.molcel.2009.06.025http://dx.doi.org/10.1016%2Fj.molcel.2009.06.025http://dx.doi.org/10.1046%2Fj.1365-2990.2002.00419.xhttp://dx.doi.org/10.1046%2Fj.1365-2990.2002.00419.xhttp://dx.doi.org/10.1007%2Fs00401-010-0726-6http://dx.doi.org/10.1007%2Fs00401-010-0726-6http://dx.doi.org/10.1038%2Fsj.bjc.6603782http://dx.doi.org/10.1038%2Fsj.bjc.6603782http://dx.doi.org/10.1038%2Fsj.bjc.6603782http://dx.doi.org/10.1038%2Fsj.bjc.6603782http://dx.doi.org/10.1016%2FS0046-8177%2803%2900340-Xhttp://dx.doi.org/10.1016%2FS0046-8177%2803%2900340-Xhttp://dx.doi.org/10.1016%2FS0046-8177%2803%2900340-Xhttp://dx.doi.org/10.1159%2F000274458http://dx.doi.org/10.1159%2F000274458http://dx.doi.org/10.1159%2F000274458http://dx.doi.org/10.1038%2Fnature09772http://dx.doi.org/10.1038%2Fnature09772http://dx.doi.org/10.1038%2Fnature09772http://dx.doi.org/10.1128%2FMCB.01830-08http://dx.doi.org/10.1128%2FMCB.01830-08http://dx.doi.org/10.1016%2FS1470-2045%2804%2901424-Xhttp://dx.doi.org/10.1016%2FS1470-2045%2804%2901424-Xhttp://dx.doi.org/10.1146%2Fannurev.pathmechdis.3.121806.151518http://dx.doi.org/10.1146%2Fannurev.pathmechdis.3.121806.151518http://dx.doi.org/10.1126%2Fscience.277.5329.1109http://dx.doi.org/10.1126%2Fscience.277.5329.1109http://dx.doi.org/10.1126%2Fscience.277.5329.1109http://dx.doi.org/10.1038%2Fnature03485http://dx.doi.org/10.1038%2Fnature03485http://dx.doi.org/10.1038%2Fnature03485http://dx.doi.org/10.1038%2Fnature03485http://dx.doi.org/10.1093%2Fnar%2Fgkr1155http://dx.doi.org/10.1093%2Fnar%2Fgkr1155http://dx.doi.org/10.1093%2Fnar%2Fgkr1155http://dx.doi.org/10.1016%2FS0092-8674%2800%2981268-4http://dx.doi.org/10.1016%2FS0092-8674%2800%2981268-4http://dx.doi.org/10.1016%2FS0092-8674%2800%2981268-4http://dx.doi.org/10.1016%2FS0092-8674%2800%2981268-4http://dx.doi.org/10.1038%2Fnature08467http://dx.doi.org/10.1038%2Fnature08467http://dx.doi.org/10.1146%2Fannurev-genet-102108-134841http://dx.doi.org/10.1146%2Fannurev-genet-102108-134841http://dx.doi.org/10.1126%2Fscience.272.5268.1668http://dx.doi.org/10.1126%2Fscience.272.5268.1668http://dx.doi.org/10.1126%2Fscience.272.5268.1668http://dx.doi.org/10.1126%2Fscience.272.5268.1668http://dx.doi.org/10.1101%2Fgad.1182504http://dx.doi.org/10.1101%2Fgad.1182504http://dx.doi.org/10.1101%2Fgad.1182504http://dx.doi.org/10.1101%2Fgad.1182504http://dx.doi.org/10.1074%2Fjbc.M409293200http://dx.doi.org/10.1074%2Fjbc.M409293200http://dx.doi.org/10.1074%2Fjbc.M409293200http://dx.doi.org/10.1002%2Fjcb.20760http://dx.doi.org/10.1002%2Fjcb.20760http://dx.doi.org/10.1016%2Fj.stem.2008.12.010http://dx.doi.org/10.1016%2Fj.stem.2008.12.010http://dx.doi.org/10.1016%2Fj.stem.2008.12.010http://dx.doi.org/10.1093%2Fnar%2Fgkn1030http://dx.doi.org/10.1093%2Fnar%2Fgkn1030http://dx.doi.org/10.1093%2Fnar%2Fgkn1030http://dx.doi.org/10.1093%2Fnar%2Fgkn1030http://dx.doi.org/10.1242%2Fdev.01793http://dx.doi.org/10.1242%2Fdev.01793http://dx.doi.org/10.1242%2Fdev.01793http://dx.doi.org/10.1242%2Fdev.01793http://dx.doi.org/10.1146%2Fannurev.genet.41.110306.130350http://dx.doi.org/10.1146%2Fannurev.genet.41.110306.130350http://dx.doi.org/10.1016%2Fj.molcel.2007.02.011http://dx.doi.org/10.1016%2Fj.molcel.2007.02.011http://dx.doi.org/10.1097%2FCOC.0b013e3181b0c4c2http://dx.doi.org/10.1097%2FCOC.0b013e3181b0c4c2http://dx.doi.org/10.1097%2FCOC.0b013e3181b0c4c2http://dx.doi.org/10.1097%2FCOC.0b013e3181b0c4c2http://dx.doi.org/10.1242%2Fjcs.034876http://dx.doi.org/10.1242%2Fjcs.034876http://dx.doi.org/10.1242%2Fjcs.034876http://dx.doi.org/10.1242%2Fjcs.034876

Journ

alof

Cell

Scie

nce

Redon, S., Reichenbach, P. and Lingner, J. (2010). The non-coding RNA TERRA is anatural ligand and direct inhibitor of human telomerase. Nucleic Acids Res. 38, 5797-5806.

Ruiz i Altaba, A., Sánchez, P. and Dahmane, N. (2002). Gli and hedgehog in cancer:tumours, embryos and stem cells. Nat. Rev. Cancer 2, 361-372.

Sahin, E. and Depinho, R. A. (2010). Linking functional decline of telomeres,mitochondria and stem cells during ageing. Nature 464, 520-528.

Sampl, S., Pramhas, S., Stern, C., Preusser, M., Marosi, C. and Holzmann, K.

(2012). Expression of telomeres in astrocytoma WHO Grade 2 to 4: TERRA levelcorrelates with telomere lenght, telomerase activity, and advanced clinical grade.Transl. Oncol. 5, 56-65.

Schoeftner, S. and Blasco, M. A. (2008). Developmentally regulated transcription ofmammalian telomeres by DNA-dependent RNA polymerase II. Nat. Cell Biol. 10,228-236.

Schoeftner, S., Blanco, R., Lopez de Silanes, I., Muñoz, P., Gómez-López, G.,

Flores, J. M. and Blasco, M. A. (2009). Telomere shortening relaxes X chromosomeinactivation and forces global transcriptome alterations. Proc. Natl. Acad. Sci. USA106, 19393-19398.

Sedelnikova, O. A. and Bonner, W. M. (2006). GammaH2AX in cancer cells: apotential biomarker for cancer diagnostics, prediction and recurrence. Cell Cycle 5,2909-2913.

Shevtsov, S. P. and Dundr, M. (2011). Nucleation of nuclear bodies by RNA. Nat. CellBiol. 13, 167-173.

Takai, H., Smogorzewska, A. and de Lange, T. (2003). DNA damage foci atdysfunctional telomeres. Curr. Biol. 13, 1549-1556.

Ting, D. T., Lipson, D., Paul, S., Brannigan, B. W., Akhavanfard, S., Coffman, E. J.,Contino, G., Deshpande, V., Iafrate, A. J., Letovsky, S. et al. (2011). Aberrantoverexpression of satellite repeats in pancreatic and other epithelial cancers. Science331, 593-596.

Toledo, L. I., Murga, M., Zur, R., Soria, R., Rodriguez, A., Martinez, S., Oyarzabal,

J., Pastor, J., Bischoff, J. R. and Fernandez-Capetillo, O. (2011). A cell-basedscreen identifies ATR inhibitors with synthetic lethal properties for cancer-associatedmutations. Nat. Struct. Mol. Biol. 18, 721-727.

Wallace, V. A. (1999). Purkinje-cell-derived Sonic hedgehog regulates granule neuron

precursor cell proliferation in the developing mouse cerebellum. Curr. Biol. 9, 445-

448.

Wechsler-Reya, R. J. and Scott, M. P. (1999). Control of neuronal precursor

proliferation in the cerebellum by Sonic Hedgehog. Neuron 22, 103-114.

Wentzensen, I. M., Mirabello, L., Pfeiffer, R. M. and Savage, S. A. (2011). The

association of telomere length and cancer: a meta-analysis. Cancer Epidemiol.

Biomarkers Prev. 20, 1238-1250.

Wileman, T. (2007). Aggresomes and pericentriolar sites of virus assembly: cellular

defense or viral design? Annu. Rev. Microbiol. 61, 149-167.

Xiang, C., Baubet, V., Pal, S., Holderbaum, L., Tatard, V., Jiang, P., Davuluri, R. V.

and Dahmane, N. (2012). RP58/ZNF238 directly modulates proneurogenic gene

levels and is required for neuronal differentiation and brain expansion. Cell Death

Differ. 19, 692-702.

Xu, L. and Blackburn, E. H. (2007). Human cancer cells harbor T-stumps, a distinct

class of extremely short telomeres. Mol. Cell 28, 315-327.

Ye, J., Wu, Y. and Gilson, E. (2010). Dynamics of telomeric chromatin at the

crossroads of aging and cancer. Essays Biochem. 48, 147-164.

Yehezkel, S., Segev, Y., Viegas-Péquignot, E., Skorecki, K. and Selig, S. (2008).

Hypomethylation of subtelomeric regions in ICF syndrome is associated with

abnormally short telomeres and enhanced transcription from telomeric regions. Hum.

Mol. Genet. 17, 2776-2789.

Yehezkel, S., Rebibo-Sabbah, A., Segev, Y., Tzukerman, M., Shaked, R., Huber, I.,

Gepstein, L., Skorecki, K. and Selig, S. (2011). Reprogramming of telomeric

regions during the generation of human induced pluripotent stem cells and subsequent

differentiation into fibroblast-like derivatives. Epigenetics 6, 63-75.

Zhang, L. F., Ogawa, Y., Ahn, J. Y., Namekawa, S. H., Silva, S. S. and Lee, J. T.

(2009). Telomeric RNAs mark sex chromosomes in stem cells. Genetics 182, 685-698.

Zheng, J. L. and Gao, W. Q. (2000). Overexpresion of Math1 induces robust production

of extra hair cells in postnatal rat inner ears. Nat. Neurosci. 3, 580-586.


http://dx.doi.org/10.1093%2Fnar%2Fgkq296http://dx.doi.org/10.1093%2Fnar%2Fgkq296http://dx.doi.org/10.1038%2Fnrc796http://dx.doi.org/10.1038%2Fnrc796http://dx.doi.org/10.1038%2Fnature08982http://dx.doi.org/10.1038%2Fnature08982http://dx.doi.org/10.1038%2Fncb1685http://dx.doi.org/10.1038%2Fncb1685http://dx.doi.org/10.1038%2Fncb1685http://dx.doi.org/10.1073%2Fpnas.0909265106http://dx.doi.org/10.1073%2Fpnas.0909265106http://dx.doi.org/10.1073%2Fpnas.0909265106http://dx.doi.org/10.1073%2Fpnas.0909265106http://dx.doi.org/10.4161%2Fcc.5.24.3569http://dx.doi.org/10.4161%2Fcc.5.24.3569http://dx.doi.org/10.4161%2Fcc.5.24.3569http://dx.doi.org/10.1038%2Fncb2157http://dx.doi.org/10.1038%2Fncb2157http://dx.doi.org/10.1016%2FS0960-9822%2803%2900542-6http://dx.doi.org/10.1016%2FS0960-9822%2803%2900542-6http://dx.doi.org/10.1126%2Fscience.1200801http://dx.doi.org/10.1126%2Fscience.1200801http://dx.doi.org/10.1126%2Fscience.1200801http://dx.doi.org/10.1126%2Fscience.1200801http://dx.doi.org/10.1038%2Fnsmb.2076http://dx.doi.org/10.1038%2Fnsmb.2076http://dx.doi.org/10.1038%2Fnsmb.2076http://dx.doi.org/10.1038%2Fnsmb.2076http://dx.doi.org/10.1016%2FS0960-9822%2899%2980195-Xhttp://dx.doi.org/10.1016%2FS0960-9822%2899%2980195-Xhttp://dx.doi.org/10.1016%2FS0960-9822%2899%2980195-Xhttp://dx.doi.org/10.1016%2FS0896-6273%2800%2980682-0http://dx.doi.org/10.1016%2FS0896-6273%2800%2980682-0http://dx.doi.org/10.1158%2F1055-9965.EPI-11-0005http://dx.doi.org/10.1158%2F1055-9965.EPI-11-0005http://dx.doi.org/10.1158%2F1055-9965.EPI-11-0005http://dx.doi.org/10.1146%2Fannurev.micro.57.030502.090836http://dx.doi.org/10.1146%2Fannurev.micro.57.030502.090836http://dx.doi.org/10.1038%2Fcdd.2011.144http://dx.doi.org/10.1038%2Fcdd.2011.144http://dx.doi.org/10.1038%2Fcdd.2011.144http://dx.doi.org/10.1038%2Fcdd.2011.144http://dx.doi.org/10.1016%2Fj.molcel.2007.10.005http://dx.doi.org/10.1016%2Fj.molcel.2007.10.005http://dx.doi.org/10.1042%2Fbse0480147http://dx.doi.org/10.1042%2Fbse0480147http://dx.doi.org/10.1093%2Fhmg%2Fddn177http://dx.doi.org/10.1093%2Fhmg%2Fddn177http://dx.doi.org/10.1093%2Fhmg%2Fddn177http://dx.doi.org/10.1093%2Fhmg%2Fddn177http://dx.doi.org/10.4161%2Fepi.6.1.13390http://dx.doi.org/10.4161%2Fepi.6.1.13390http://dx.doi.org/10.4161%2Fepi.6.1.13390http://dx.doi.org/10.4161%2Fepi.6.1.13390http://dx.doi.org/10.1534%2Fgenetics.109.103093http://dx.doi.org/10.1534%2Fgenetics.109.103093http://dx.doi.org/10.1038%2F75753http://dx.doi.org/10.1038%2F75753

Fig 1Fig 2Fig 3Fig 4Fig 5Fig 6Fig 7Fig 8Ref 1aRef 1Ref 2Ref 3Ref 4Ref 5Ref 5aRef 6Ref 7Ref 8Ref 8aRef 9Ref 10Ref 11Ref 12Ref 13Ref 14Ref 15Ref 16Ref 17Ref 18Ref 18aRef 18bRef 19Ref 20Ref 21Ref 22Ref 23Ref 24Ref 25Ref 26Ref 27Ref 28Ref 29Ref 30Ref 31Ref 32Ref 33Ref 34Ref 35Ref 36Ref 37Ref 38Ref 39Ref 40Ref 41Ref 42Ref 43Ref 44Ref 45Ref 46Ref 47Ref 48Ref 49Ref 50Ref 51Ref 52Ref 53Ref 54Ref 55Ref 56Ref 57Ref 58Ref 59Ref 60Ref 61Ref 62Ref 62a

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 150 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 200 /GrayImageDepth 8 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /FlateEncode /AutoFilterGrayImages false /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 600 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly true /PDFXNoTrimBoxError false /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox false /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (Euroscale Coated v2) /PDFXOutputConditionIdentifier (FOGRA1) /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /False

/CreateJDFFile false /SyntheticBoldness 1.000000 /Description >>> setdistillerparams> setpagedevice

Formation of telomeric repeat-containing RNA (TERRA) foci ... · (TERFs) in the tumor cells, but...

Documents

Transcript of Formation of telomeric repeat-containing RNA (TERRA) foci ... · (TERFs) in the tumor cells, but...