Further molecular and genetic analysis of upSET, the MLL5...

Further molecular and genetic analysis of upSET, the MLL5 orthologue in D. melanogaster

by Hung Yu (Jack) Yang

B.Sc. (Hons.), Simon Fraser University, 2011

Thesis Submitted in Partial Fulfillment of the

Requirements for the Degree of

Master of Science

in the

Department of Molecular Biology and Biochemistry

Faculty of Science

Hung Yu (Jack) Yang 2014

SIMON FRASER UNIVERSITY Summer 2014

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Approval

Name: Hung Yu (Jack) Yang Degree: Master of Science (Molecular Biology and Biochemistry) Title: Further molecular and genetic analysis of upSET, the

MLL5 orthologue in D. melanogaster

Examining Committee: Chair: Dr. Dipankar Sen Professor

Dr. Barry Honda Senior Supervisor Professor

Dr. Donald Sinclair Supervisor Senior Lecturer

Dr. Nicholas Harden Supervisor Professor

Dr. David Baillie Internal Examiner Professor Simon Fraser University, Department of Molecular Biology & Biochemistry

Date Defended/Approved: July 18, 2014

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Partial Copyright Licence

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Abstract

Several histone methyltransferases are products of the MLL (Mixed Lineage Leukemia)

family of genes. Although their mechanisms of action remain elusive, these genes are

important because they are often deleted in patients with certain types of leukemia. In

most cases, MLLs function catalytically via their SET domains, and their modifications of

histones are important for chromatin structure and gene regulation. Here I report my

analysis of the fly orthologue of MLL5, upSET. In contrast to an earlier study, RNAi

inhibition of upSET gene expression did not result in female sterility but was instead

lethal. Although UpSET has a SET domain, down-regulation of upSET does not affect

global H3K4-methyl levels. Unlike human MLL5, the SET domain of UpSET does not

appear to be an OGT substrate. Additionally, I have characterized UpSET as a

suppressor of variegation; this suggests that UpSET is involved in establishment and/or

maintenance of chromatin structure and accessibility.

Keywords: upSET; Su(var); PEV; HDAC; epigenetics; MLL5

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Acknowledgements

I would like to thank my senior supervisor, Dr. Barry Honda, for the opportunity to

pursue graduate studies in his laboratory. I would also like to thank the other members

of my grad studies committee, Dr. Donald Sinclair and Dr. Nicholas Harden. My

committee has been instrumental in providing well-needed feedback, support, and

insights. They have been instrumental to my research project on characterizing a gene

that was largely uncharacterized at the beginning of my studies in 2011. Also, I would

like to thank my internal examiner, Dr. David Baillie, for examining my thesis, and Dr.

Dipankar Sen, for chairing my defense. Most importantly, my research would not have

been possible without the scientific wisdom and guidance from my unofficial supervisor,

Dr. Graham Hallson. He has provided timely constructive feedback on my experimental

designs, and data analysis. Dr. Kathleen Fitzpatrick has also provided valuable feedback

on my project progress during regular lab meetings. Both Dr. Sinclair and Dr. Fitzpatrick

have provided valuable feedback on this thesis.

The three technicians (past and current) of the Honda lab have provided

indispensable support. Shawn Cotsworth and Inho (Steve) Kim were my mentors when I

first joined the Honda laboratory. They have provided quality training on research

fundamentals that were essential to starting my research project. The SET domain of

upSET was originally PCR amplified by Jonathan Radke (former graduate student). He

had subcloned it into the pJET1.2 vector (Thermo Scientific) before donating the

construct to me. Teresa Stefanelli (current graduate student) provided introductory

training on western blot techniques for probing levels of O-GlcNAc. The first set of

upSET RNAi transgenics (upSETRNAi-Random 1M – 9M) was prepared by members of our

laboratory in 2010-2011 before I joined the lab in May of 2011. Members of the

Verheyen and Harden laboratories have also provided general assistance in regards to

experimental designs and equipment training. More specifically, Eric Hall and Jessica

Gardner from the Verheyen lab were instrumental in training me on taking high quality

images of fruit flies through the microscope photo tube.

The two members of our laboratory that had assisted me regularly were Dr.

Hallson and Kevin Beja (most current technician). Dr. Hallson was instrumental in

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training me for assaying bulk methyl-H3K4. He was also a valuable consultant in regards

to experimental design and data analysis. Further, he personally conducted a few

experiments related to the upSET project in regards to looking at bulk methyl-H3K4

levels in an sxc mutant, and an upSET RNAi. His results were discussed in greater

depth in his PhD thesis (2013). More recently, Kevin Beja provided direct assistance with

genetics-based experiments, and general technical support with experimental protocols,

reagents, supplies, and equipments. I also wish to thank Jonathan Radke and Teresa

Stefanelli for their genuine support and encouragement on pursuing graduate studies.

Last but not least, I would like to thank my life partner, Lillian Wang, for her continued

support through the ups and downs of my life.

The upSETe00365 mutant allele (originally from the Exelixis Collection at the

Harvard Medical School) was provided by Dr. Hector Rincon-Arano from the Groudine

laboratory at the Fred Hutchinson Cancer Research Center. Deficiencies and other

mutants were obtained from the Bloomington Stock Center at Indiana University. See

Appendix A for more details.

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Table of Contents

Approval .............................................................................................................................ii Partial Copyright Licence .................................................................................................. iii Abstract .............................................................................................................................iv Acknowledgements ........................................................................................................... v Table of Contents ............................................................................................................. vii List of Tables .....................................................................................................................ix List of Figures.................................................................................................................... x List of Acronyms ............................................................................................................... xii

Chapter 1. Introduction ............................................................................................... 1 1.1. Functions of Histone modifications, and the Histone Code ...................................... 1 1.2. Readers of Histone Modifications and their Mechanisms ........................................ 2 1.3. H3K4 Methyltransferases ......................................................................................... 4 1.4. Regulation of HOX genes by MLL ............................................................................ 5 1.5. Position-effect variegation (PEV) and model of heterochromatin spreading

in Drosophila ............................................................................................................ 7 1.6. Specific Single Gene Mutations Can Modify PEV .................................................... 9 1.7. Structural comparison of MLL5 to other MLLs ....................................................... 10 1.8. MLL5 and UpSET (CG9007) .................................................................................. 13

Chapter 2. Materials and Methods ........................................................................... 17 2.1. Drosophila genetic crosses .................................................................................... 17 2.2. Generation of upSET RNAi strains ......................................................................... 18 2.3. Phenotypic characterization of PEV ....................................................................... 19 2.4. Cloning of GST-SETUpSET construct ........................................................................ 21 2.5. Co-Expression and Purification of GST-SETUpSET from bacteria cell cultures ........ 21 2.6. Nuclear Protein Extraction from Adult Fruit Flies ................................................... 23 2.7. Western Blotting ..................................................................................................... 24 2.8. Quantification of Western Blots .............................................................................. 25 2.9. Quantitative PCR (qPCR) ....................................................................................... 26

Chapter 3. Characterization of upSET ..................................................................... 27 3.1. upSET is essential for viability but not fertility ........................................................ 27 3.2. The SET domain of UpSET is not modified by human OGT in bacteria

culture .................................................................................................................... 34 3.3. Global levels of H3K4 methylation is unaffected in upSET RNAi ........................... 36 3.4. Knockdown of upSET enhances Polycomb phenotypes ........................................ 38 3.5. UpSET is a suppressor of variegation .................................................................... 40

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Chapter 4. Discussion and Concluding Remarks ................................................... 52

References .................................................................................................................. 58 Appendix A. List of flies used in this study ............................................................ 70 Appendix B. Sequences of upSET RNAi construct ............................................... 73 Appendix C. List of Primers Designed for this Study ............................................. 75 Appendix D. Recipes of Protein Purification Buffers .............................................. 76

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List of Tables

Table 1 Primary Antibodies used for probing H3K4 methylation .......................... 25

Table 2 Sterility observations from genetically crossing upSETe00365 flies to wild-type (w1118) flies ................................................................................ 33

Table 3 Percentage of Stubble of upSET mutants carrying SbV........................... 50

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List of Figures

Figure 1 Positioning of Bromodomain, SET Domains, and PHD Fingers in MLL protein family in Homo sapien and Drosophila melanogaster. ........ 11

Figure 2 NCBI Protein Blast (blastp) alignment of translated amino acid sequence of the SET domain of upSET against human and mouse protein databases. ................................................................................... 12

Figure 3 NCBI Protein Blast (blastp) alignment of translated amino acid sequence of the PHD domain of upSET against human and mouse protein databases. ....................................................................... 13

Figure 4 Map showing available upSET deficiencies ............................................ 15

Figure 5 Method: Measurement of the width of the eye in D. melanogaster. ........ 20

Figure 6 upSETRNAi-Random 1M – 9M driven with tub-Gal4 with UAS-Dicer2 at 29 ⁰C. ............................................................................................................ 28

Figure 7 Relative Viability of upSETRNAi-attP2 driven with Gal4 drivers at 29 ⁰C. ............................................................................................................ 29

Figure 8 Examples of eclosion related defects resulted from driving upSETRNAi-attP2 using tub-Gal4 with UAS-Dicer2 at 29 ⁰C. ....................... 30

Figure 9 Relative Viability of upSETe00365 at 25 ⁰C. ................................................ 32

Figure 10 Human OGT (hOGT) does not modify the SET domain of UpSET in bacteria culture system. ....................................................................... 35

Figure 11 Global H3K4 methylation is unaffected by upSET RNAi (Blot) ................ 37

Figure 12 Global H3K4 methylation unaffected by upSET RNAi (Quantification) ........................................................................................ 38

Figure 13 upSET RNAi enhances Polycomb phenotype in males (Quantification) ........................................................................................ 39

Figure 14 upSET RNAi enhances Polycomb phenotype in males (Images) ........... 40

Figure 15 Percent Wild-Type Eye Pigmentation of upSET hypomorph and deficiency classes carrying white-mottled-4. ........................................... 42

Figure 16 Eye variegation of upSET hypomorph carrying white-mottled-4. ............ 43

Figure 17 Eye variegation of upSET mutants/deficiency/RNAi carrying BSV. .......... 45

Figure 18 Average width of Eye of upSET hypomorphic mutant carrying BSV. ........ 46

Figure 19 Average width of Eye of upSET deficiency carrying BSV. ........................ 47

Figure 20 Average width of Eye of upSET “protein null” mutant carrying BSV. ........ 48

Figure 21 Average width of Eye of upSET RNAi carrying BSV. ................................ 49

Figure 22 Stubble variegation of upSET hypomorph carrying SbV. ......................... 51

Figure 23 Schematic showing possible roles of the UpSET SET domain ............... 55

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Figure 24 Schematic showing possible mechanism involving UpSET, Rpd3/Sin3A, Ebi, and SMRTER .............................................................. 56

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List of Acronyms

Term Initial components of the term (examples are below)

aa dsRNA E(var) H3K4 HAT HDAC Het HMT IR OGT PCR PEV PTM RNAi SEM Su(var)

Amino Acids Double stranded RNA Enhancer of Variegation Histone H3 Lysine 4 Histone Acetyltransferase Histone Deacetylase Heterochromatin Histone methyltransferase Inverted Repeats O-linked N-acetylglucosamine (GlcNAc) transferase Polymerase Chain Reaction Position effect variegation Post-translational modifications RNA interference Standard Error of the Mean Suppressor of Variegation

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Chapter 1. Introduction

1.1. Functions of Histone modifications, and the Histone Code

Two evolutionarily conserved histone post-translational modifications (PTMs) are

histone acetylation and histone methylation. Both of these modifications commonly occur

on the N-terminal lysine tails of histone 3. Histone acetylation affects chromatin structure

directly by effectively neutralizing the positive charge on the lysine residue (GRUNSTEIN

1997, also reviewed by LEE and WORKMAN 2007). This results in less attraction/more

repulsion between the histone tail and the negatively charged DNA backbone, promoting

chromatin accessibility for the transcriptional machinery. The reversal of this modification

(i.e. histone deacetylation) would theoretically restrict chromatin access. Histone

methylation, on the other hand, does not affect chromatin structure directly, but rather it

affects the nucleosome’s interactions with effector proteins. These proteins include

chromatin remodelling complexes that are capable of “reading” these methyl

modifications, and carrying out downstream actions (ALLIS et al. 2007). Histone

methylation can be associated with either gene activation or gene repression, depending

on which lysine tail is methylated. Lysine can be mono-, di-, or trimethylated via

replacement of hydrogens of its NH3+ group with methyl moieties. As a general rule,

H3K4 and H3K36 methylation are associated with transcriptional activation, and H3K9

and H3K27 di-/trimethylation are associated with transcriptional repression. Other

possible histone methylation sites exist; however, the outcomes of the methyl marks are

less predictable, and it is thus more difficult to generalize about their function.

Since the identification of histone modifications, the histone code hypothesis has

been introduced; this hypothesis suggests that specific combinations of multiple histone

modifications can be read by effector proteins which ultimately affect chromatin structure

and gene expression (STRAHL and ALLIS 2000, JENUWEIN and ALLIS 2001). Different

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combinations of modifications can lead to unique biological outcomes. Genome-wide

studies of epigenomic profiles have supported the histone code hypothesis, such that

the cytogenetic heterochromatin border coincides with the epigenomic border (e.g. high

levels of H3K4 methylation on the euchromatin side of the border and high levels of

H3K9 methylation on the heterochromatin side of the border) (RIDDLE et al. 2011).

However, it remains unclear whether histone modifications are necessary for

establishing gene regulation, or whether they are only important for maintaining gene

regulation. What is certain is that normal histone modification patterns appear to be

important for the health of organisms as abnormalities in their patterns have been

correlated with cancer and various disease states (reviewed by MAUNAKEA et al.

2010).

1.2. Readers of Histone Modifications and their Mechanisms

Effector proteins that “read” histone modifications generally contain

bromodomains (BRD), chromodomains (CD), or plant homeodomains (PHD) capable of

recognizing and binding to specific combinations of histone modifications and/or the

chromatin landmarks provided by the modifications (ALLIS et al. 2007). The

bromodomain acts as an acetyl-lysine binding domain, and the chromodomain acts as a

methyl-lysine binding domain. PHD domain-containing-proteins (e.g. NURF, ING2,

SMCX, UpSET/MLL5) appear to bind to methylated histone H3 (e.g. H3K4me2,

H3K4me3, H3K9me3) (LI et al. 2006, PEÑA et al. 2006, IWASE et al. 2007, ALI et al.

2013). The recognition of and binding to these methyl marks can generate cross talk

between different effectors, and this can cause recruitment of other histone modifiers

such as histone acetylases (HATs), or histone deacetylases (HDACs), which directly

promote or restrict chromatin accessibility.

An important example of how the aforementioned readers function is the

extensively studied protein, heterochromatin protein 1a (HP1a) that binds to modified

histone H3, and interacts with other histone modifying proteins. In Drosophila, HP1a

(also known as Su(var)2-5), is an essential protein involved in heterochromatin assembly

and maintenance (reviewed by EISSENBERG and ELGIN 2014). The gene encoding

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this protein was identified in screens for dominant modifiers of Position-effect

Variegation (PEV; REUTER AND WOLFF 1981; SINCLAIR et al. 1983, SINCLAIR et al.

1989). HP1a possesses an N-terminal chromodomain (CD), and a C-terminal chomo-

shadow domain (CSD). HP1a binds to H3K9me2 and H3K9me3 via its CD (JACOBS

and KHORASANIZADEH 2002, NIELSEN et al. 2002), and physically interacts with a

major H3K9 methyltransferase, SU(VAR)3-9, via its CSD (SCHOTTA et al. 2002). It is

widely thought that binding of HP1a to H3K9 methyl marks provides a mechanism for

recruiting additional SU(VAR)3-9, and that this is a necessary prerequisite for the

establishment of the heterochromatin (GREWAL and ELGIN 2002).

Two major proposals have been advanced to explain how HP1a binding to H3K9

methyl marks might promote heterochromatin assembly and maintenance

(EISSENBERG and ELGIN 2014). These include: (1) Binding of HP1a to H3K9 methyl

marks prevents histone demethylation and acetylation of the same residue. Both histone

demethylation and acetylation on H3K9 typically promote chromatin accessibility; thus

HP1a binding establishes and maintains the heterochromatin environment; and (2) HP1a

is also able to form homodimers via its CSD; this is believed to promote uniform

nucleosome spacing which favours chromatin condensation (MENDEZ et al. 2013).

There are also many other examples in which a reader binds to a modified

histone to trigger downstream actions. For example, SHI et al. (2006) reported that the

PHD domain of a chromatin-associated protein, ING2 (inhibitor of growth 2), binds to

H3K4me3 in response to DNA damage. Binding of H3K4me3 by ING2 stabilizes the

binding of a histone deacetylase, HDAC1. Similarly, it has been shown that members of

the ING protein family recruit histone acetyltransferases (HATs) or histone deacetylases

(HDACs) to chromatin upon recognition and binding of H3K4 modifications (MARTIN et

al. 2006, PEÑA et al. 2006, TAVERNA et al. 2006). Another well characterized example

involves the histone demethylase, LSD1, and BHC80 (a component of a histone

deacetylase complex) (LAN et al. 2007). LSD1 completely demethylates di-methylated

H3K4. Thereafter, BHC80 binds to unmethylated H3K4 using its PHD finger; this pattern

of binding is crucial for maintaining normal gene regulation of LSD1 target genes.

Furthermore, knockdown of BHC80 resulted in de-repression of its target genes.

Therefore, it was suggested that the binding of the unmethylated state by BHC80

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prevents future methylation of the promoters of these genes; thus, this promotes gene

repression.

1.3. H3K4 Methyltransferases

Like other types of histone methylation, each H3K4 residue can carry one to

three methyl groups (H3K4me3, H3K4me2, or H3K4me1) (ALLIS 2007). In

Saccharomyces cerevisiae, it appears that active genes are saturated with H3K4me3

near their transcriptional start sites (TSS) and promoter regions, and are enriched in

H3K4me2 in regions between TSS and the gene body; on the other hand, protein coding

regions of genes are predominantly monomethylated on H3K4 (POKHOLOK et al.

2005). This pattern of H3K4 methylation is conserved in Drosophila melanogaster with

minor variations (ROY et al. 2010).

The transfer of methyl groups from S-adenosyl methionine onto lysine (or

arginine) residues of histone tails is catalyzed by histone methyltransferases

(EISSENBERG and SHILATIFARD 2010). One of the hallmarks of H3K4-specific

methyltransferases is the possession of a Su(var)3-9, Enhancer of Zeste, Trithorax

(SET) domain (ALLIS 2007); Su(var)3-9, Enhancer of Zeste, and Trithorax catalyze the

trimethylation of H3K9, H3K27, and H3K4, respectively. The SET domain is the core

catalytic component of these enzymes.

In mammals, several H3K4-specific HMTs (MLL1, MLL2, MLL3, MLL4, and

MLL5) have been identified as products of members of the mixed lineage leukemia

(MLL) family of genes (EISSENBERG and SHILATIFARD 2010). These HMTs share

significant homology to the ancestral Set1 protein; this protein is the key component of

the multi-subunit COMPASS (Complex Associated with Set1) complex, which is

significantly conserved from yeast to mammals (reviewed by SHILATIFARD 2012). In

Drosophila melanogaster, dSET1 has been shown to be the major H3K4

methyltransferase as part of the COMPASS complex (ARDEHALI et al. 2011, MOHAN

et al. 2011, HALLSON et al. 2012). Its amino acid sequence is most similar to the

mammalian Set1a and Set1b. There are homologues of many of the mammalian SET-

containing proteins in Drosophila; for instance, Drosophila Trithorax (trx) is most similar

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in sequence to the mammalian MLL1 and MLL2. Trithorax-related (trr) is most similar to

MLL3 and MLL4, and upSET (CG9007) is most similar to MLL5. Among these, only

dSET1, Trx, and Trr can catalyze H3K4 methylation. A short isoform of MLL5 was

reported to possess H3K4-specific methyltransferase activity that is dependent on an O-

GlcNAc modification by O-GlcNAc transferase (OGT) (FUJIKI et al. 2009).

1.4. Regulation of HOX genes by MLL

In Drosophila, the anterior-posterior (AP) axis of the developing embryo is first

established by maternally deposited transcripts that generate protein concentration

gradients; these gradients define the expression pattern for gap genes (reviewed by

PEEL et al. 2005). The expression pattern of gap genes then defines the expression

pattern for pair-rule genes, which code for transcription factors. The latter regulate the

expression of the segment polarity genes, the products of which ultimately define Hox

gene expression. Hox genes encode transcription factors that are involved in activating

effector genes that stimulate differentiation of segments of the developing embryo. Thus,

early in embryogenesis, the aforementioned hierarchy of gene expression establishes

the overall segmental pattern of Hox gene expression; however, the early-acting

regulatory proteins are only transiently present and two other group of factors, the

trithorax group (trxG) and Polycomb group (PcG) proteins act antagonistically to

maintain the appropriate expression patterns of HOX proteins.

To emphasize: trxG and PcG proteins are not required for the initial

establishment of the Drosophila body plan, but rather they are essential for maintaining

proper Hox gene regulation after the early-acting regulatory proteins become absent.

The key to the genetic identification of PcG genes as repressors of Hox genes is the fact

that misexpression of Hox genes causes homeosis (or homeotic transformation)

(reviewed by PEEL et al. 2005). Thus, because PcG proteins are required to maintain

the appropriate repression of Hox genes, PcG mutations cause ectopic misexpression of

Hox genes, resulting in pleiotropic homeotic phenotypes. On the other hand, most trxG

genes were identified as suppressors of PcG phenotypes and trxG mutations cause

homeotic phenotypes resulting from the lack of Hox gene expression; thus trxG proteins

are required to maintain appropriate Hox gene activation. There are also many genes

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that act as ETPs (enhancer of trithorax and Polcyomb genes); for instance, a P-element-

induced allele of Additional sex combs (Asx) displayed both anterior and posterior

transformations that appear to enhance both trxG and PcG phenotypes (SINCLAIR et al.

1998, ETP reviewed by BECK et al. 2010).

As mentioned earlier, mis-expression of Hox genes results in homeosis, which

can be observed as changes in segment identity during development. An example of this

in Drosophila is the replacement of antennae with ectopic legs due to a gain-of-function

mutant allele that overexpresses the Hox gene, Antennapedia (SCHNEUWLY et al.

2002). Thus, mutant alleles of many PcG genes display posterior homeotic

transformations, including ectopic expression of sex combs on the second and third set

of legs in males (ALLIS 2007). On the other hand, trxG mutants generally display

anterior transformations (KENNISON 1995, BREEN 1999).

Generally, trxG proteins function as components of complexes that are

categorized as histone-modifiers, or ATP-dependent chromatin remodelers, involved in

maintaining activation of Hox genes (reviewed by ALLIS 2007). At the molecular level,

TrxG proteins maintain activation of Hox genes by preserving active histone methyl

marks (H3K4me3), which are required to prevent transcriptional silencing by PcG

proteins (KLYMENKO and MULLER 2004). On the other hand, Polycomb group (PcG)

proteins function antagonistically to trxG proteins by stably maintaining gene silencing of

Hox genes.

MLL1 and Trx (Trithorax) are also members of the evolutionarily conserved trxG

of proteins because of their role as a regulator of hox genes, and their role as a H3K4-

specific methyltransferase (MILNE et al. 2002). It was originally thought that MLL5 might

also be a part of the trxG of proteins because of sequence similarities to MLL1/Trx

(EMERLING et al. 2002). Additionally, one mammalian study showed that MLL5

functions in retinoic-acid-induced granulopoiesis (FUJIKI et al. 2009). Since retinoic acid

also regulates differential expression of Hox genes along the AP axis in mammals

(DUESTER 2008), and Hox genes are partly regulated by trxG proteins (ALLIS 2007), it

was thought that MLL5 might also be a member of trxG.

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1.5. Position-effect variegation (PEV) and model of heterochromatin spreading in Drosophila

PEV is a complex phenomenon that can be observed by placing a euchromatic

gene next to a region of centromeric heterochromatin (reviewed by SCHOTTA et al.

2003, GIRTON and JOHANSEN 2008). In Drosophila, the placement of a euchromatic

gene in heterchromatin can be accomplished by chromosomal rearrangement, or P-

element insertion; this gives a variegated phenotype caused by mosaic gene repression

by heterochromatin spreading from the breakpoint such that genes are inactive in some

cells but remain active in others. This phenomenon is relevant to my interest as many

proteins associated with chromatin structure and gene regulation (e.g. SU(VAR)3-9,

HP1a) have roles in PEV.

That the chromosomal position of a gene (position-effect) can be important for its

proper expression was first demonstrated in a study of a dominant, sex-linked eye

mutation in the Bar gene, that reduces the number of facets in the adult eye of the fruit

fly (STURTEVANT 1925). Normally, females have two copies of Bar (one on each X-

chromosome, B/B) which gives a normal wild-type eye phenotype. Bar mutants carry

more than one copy of the Bar gene on the X chromosome, resulting in reduction in the

width of the eye. STURTEVANT (1925) first coined the term “position effect”, when he

noticed that the number of facets per eye in homozygous Bar females (BB/BB), and

heterozygous Bar females (BBB/B) were significantly different, even though both had the

same number of copies of Bar; this suggested that chromosomal position/structure might

be important for Bar gene expression.

An important variation on position effect is that of position-effect variegation

(PEV), first described by Muller (MULLER 1930, SCHULTZ 1950). In a study of x-ray-

induced mutations, Muller reported the discovery of mutants that display a “mottled” eye

phenotype, which is a mosaic distribution of red pigments in the fly’s eye. The variegated

phenotype is a result of a chromosomal rearrangement that moved the white (w) gene

next to heterochromatin such that white on the inverted chromosome is silenced in some

cells, and active in others. The protein coded by w functions as a transporter required for

pigment deposition in the eye and other tissues (MACKENZIE et al. 1999); thus, white

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mutations show varying degrees of loss of eye pigmentation. The classical w1 allele (and

most strong alleles) causes complete absence of eye pigmentation (MORGAN 1910). It

is widely believed that silencing of a euchromatic gene that is placed adjacent to a

heterochromatic breakpoint is caused by heterochromatin “spreading” (GIRTON and

JOHANSEN 2008). Silencing varies from cell to cell because the length of

heterochromatin spreading along the chromosome also varies from cell to cell. Thus,

PEV is an important demonstration of the fact that chromosome/chromatin structure can

influence gene expression.

The PEV effect is strongest when euchromatic genes are closest to the

breakpoint of heterochromatin (DEMEREC 1940, reviewed by GIRTON and JOHANSEN

2008). The PEV effect progressively becomes weaker as genes are further and further

away from the breakpoint, suggesting a physical “spreading” effect of heterochromatin

into euchromatin. Gene silencing due to heterochromatin spreading is generally

accepted as a linear progression such that spreading cannot jump over a gene.

DEMEREC and SLIZYNSKA (1937) observed this in their study of a variegating allele

that had roughest (rst) and white (w) rearranged next to the breakpoint. In this allele, rst

is closest to the breakpoint, followed by w. Indeed, all PEV phenotypes displayed normal

eyes, variegated rst, or variegated rst and w, but not variegated w alone.

In addition to genetic evidence, the heterochromatin spreading model is also

supported by biochemical evidence. As discussed in section 1.2, HP1a binds to both

methylated H3K9, and H3K9 methyltransferase SU(VAR)3-9, via its CD and CSD,

respectively (JACOBS and KHORASANIZADEH 2002, NIELSEN et al. 2002, SCHOTTA

et al. 2002). SU(VAR)3-9 associates with both HP1 and H3K9 deacetylase HDAC1

(CZERMIN et al. 2001). Because of these findings, it has been proposed that spreading

of heterochromatin requires the de-acetylation of H3K9, subsequent methylation of

H3K9, and the binding of HP1 to methylated H3K9 (GREWAL and ELGIN 2002). Binding

of HP1 recruits more SU(VAR)3-9 to methylate H3K9, which ultimately spreads silencing

marks, and heterochromatin.

The answer to how heterochromatin spreading is restricted remains unclear.

Because of dosage-dependent behaviour of some PEV modifiers, it was originally

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thought that spreading terminates when key components of heterochromatin are

exhausted (LOCKE 1988). More recently, studies of H3S10 phosphorylation have

provided a boundary model in which a defined heterochromatic/euchromatic boundary

exists which impedes the binding of HP1 to methylated H3K9, thereby restricting

heterochromatin spread into euchromatin (HIROTA et al. 2005, JOHANSEN and

JOHANSEN 2006). An alternative barrier model has also been proposed; in this model,

an enhancer of variegation E(var)3-93D (also known as GAGA factor), in complex with

FACT (facilitates chromatin transcription), binds to chromatin region in which H3K4

methyl levels are high and H3K9 methyl level are low. The latter modifications are

characteristic of a euchromatin/heterochromatin border (NAKAYAMA et al. 2007).

Several kinds of variegating alleles involving rearrangement of the w gene, a

duplication of Bar on the Y chromosome, or rearrangements involving other genes which

generate an easily observable/quantifiable phenotypes, are commonly used in PEV

studies. In this study, I used white-mottled-4 (wm4), Bar-of-Stone-variegated (BSV), and

Stubble-variegated (SbV). wm4 contains a paracentric inversion of the white gene that

brings it to the heterochromatic environment of the X chromosome (MULLER 1930,

SCHULTZ 1950). BSV contains duplication of Bar in the heterochromatic environment of

the Y chromosome, resulting in varying reductions of the eye width (SINCLAIR et al.

1983). Stubble-variegated was generated from the translocation of Stubble between the

second and third chromosomes (SINCLAIR et al. 1983, LLOYD et al. 1996), resulting in

varying penetrance of the Stubble (bristle) mutant phenotype.

1.6. Specific Single Gene Mutations Can Modify PEV

Several laboratories have isolated single gene mutations that dominantly modify

PEV; these are referred to as Suppressors of Variegation (Su(var)s), or Enhancers of

Variegation (E(var)s) (REUTER AND WOLFF 1981; SINCLAIR et al. 1983; REUTER et

al. 1987; ESSENBERG et al. 1990; TSCHIERSCH et al. 1994; CLÉARD et al. 1997;

CLÉARD and SPIERER 2001). These mutations have been extensively characterized

using different variegating rearrangements. This approach has proved to be an effective

way to identify candidate genes involved in heterochromatin assembly and/or

maintenance. For example, SU(VAR)3-7, an important heterochromatin associated

10

protein that interacts with HP1 and SU(VAR)3-9 to form multimeric heterochromatin

protein complexes was identified using this approach.

With respect to white-mottled-4, strong Su(var) mutations generate a pigmented

eye (i.e. similar to wild-type); in contrast, strong E(var) mutations generate eyes that are

relatively devoid of pigment (i.e. nearly white). The PEV modifying capabilities suggest

that Su(var)s affect target genes that are involved in heterochromatin formation, and/or

spreading, whereas E(var)s affect target genes that are involved in restricting the spread

of heterochromatin into euchromatin (LOCKE et al. 1988). Mutations in Su(var)

theoretically abolish heterochromatin formation. In the context of wm4, this correlates to

“releasing” white from the repressive heterochromatic environment. Mutations in E(var)

would contribute the opposite effect.

In this study, I have used the aforementioned approach to determine that UpSET

is a Suppressor of Variegation; the PEV phenotypes associated with UpSET suggests

that it is involved in heterochromatin formation and/or maintenance.

1.7. Structural comparison of MLL5 to other MLLs

MLL family genes are generally known as epigenetic regulators involved in

chromatin regulation, and hox gene regulation (reviewed by ANSARI and MANDAL

2010). They are commonly found either modified or deleted in leukemia patients. Most of

the members of the MLL family are well-conserved in relation to the original Set1

identified in yeast (reviewed by EISSENBERG and SHILATIFARD 2010, SHILATIFARD

2012). From a structural point of view, MLL1, MLL2, MLL3, MLL4, and their respective

Drosophila homologs all possess C-terminal SET domains that are responsible for

H3K4-specific methyltransferase capabilities (Figure 1). MLL1, MLL2, and their

Drosophila homolog Trx contain multiple centrally positioned PHD finger domains. In

contrast, MLL3 and MLL4 contain a much larger number of dispersed PHD fingers in

their C-terminal halves. PHD Fingers are known to be involved in recognition of histone

modifications, and protein-protein interactions (reviewed by MUSSELMAN et al. 2011).

Centrally positioned bromodomains are exclusive to MLL1 and MLL2. In addition, DNA

binding AT-hooks are present on the N-terminal end of MLL1 and MLL2, only. MLL5 is

11

by far the least conserved MLL protein; it contains only a single PHD domain and a

single SET domain. Unlike other MLLs, its SET domain is positioned near the N-

terminus. The amino acid sequence of the entire MLL5 is only 38% identical to that of

MLL1 (EMERLING et al. 2002). Furthermore, the Drosophila ortholog of MLL5, upSET

(CG9007), shows even less conservation with respect to the other MLLs i.e. it is only

29% identical to MLL5. Although the entire upSET gene is loosely conserved to MLL5,

UpSET does contains single PHD and SET domains. Both of these domains are well

conserved with those in MLL5 (see amino acid sequence alignments in Figures 2 and 3).

Unlike MLL5, the SET domain of UpSET is centrally positioned. Interestingly, UpSET

(3146 aa) is nearly twice as long as MLL5 (1858 aa).

Figure 1 Positioning of Bromodomain, SET Domains, and PHD Fingers in MLL protein family in Homo sapien and Drosophila melanogaster.

12

Figure 2 NCBI Protein Blast (blastp) alignment of translated amino acid

sequence of the SET domain of upSET against human and mouse protein databases.

Shaded grey rectangles show the conserved Threonine 440 residue. See section 2.4 for the entire amino acid sequence.

13

Figure 3 NCBI Protein Blast (blastp) alignment of translated amino acid

sequence of the PHD domain of upSET against human and mouse protein databases.

1.8. MLL5 and UpSET (CG9007)

In a number of cell culture studies, MLL5 was identified as a key component

involved in cell cycle regulation. MLL5 is associated with cell cycle regulators p21, pRb,

and CDK1 (Cdc2) (CHENG et al. 2008, LIU et al. 2010). An isoform of the MLL5 protein,

NKp44L, acts as a cellular ligand in cytotoxicity at the cell-surface level to alert the

natural killer (NK) network (BAYCHELIER et al. 2013). High levels of MLL5 appear to be

important in treating patients with myelodysplastic syndromes (MDS) using a

hypomethylating agent, decitabine (YUN et al. 2014).

In mice, MLL5 is required for normal spermatogenesis at the terminal

differentiation stage such that MLL5 deficient male mice are infertile. Deletion of the

yeast homolog of MLL5, Set3, results in meiotic failure that leads to sporulation defects

14

(PIJNAPPEL et al. 2001). More recently, a reported “protein null” mutant of upSET was

homozygous viable but female sterile (RINCON-ARANO et al. 2012). However, thorough

testing of this mutant with upSET deficiencies raised questions on whether it is a true

protein null mutant, and whether the reported female sterility might be caused by the

disruption of a second site (discussed in Chapter 3).

At the onset of my project, there was one report that a short isoform of MLL5

possessed H3K4 methyltransferase activity via its SET domain in human HL60 cells

(FUJIKI et al. 2009). According to the latter study, this catalytic capability of MLL5

required O-GlcNAc (β-N-acetylglucosamine) modification of its Threonine 440 by OGT.

In addition, these workers found that the methyltransferase activity of the human MLL5 is

important for activating a set of target genes involved in differentiating mammalian

leukocytes via a retinoic-induced pathway. Further, SHI et al. (2007) had demonstrated

in a proteome-wide analysis in Saccharomyces cerevisiae (yeast) that the yeast

homolog of MLL5, Set3, was capable of binding tri-methylated H3K4 via its PHD finger

domain. Taken together, MLL5 appeared to have roles in “binding” and/or “writing” H3K4

methylation.

Our laboratory has been successful in using Drosophila as a model system to

study the function of the global H3K4 methyltransferase, dSET1, in a multicellular

eukaryote (HALLSON et al. 2012). As part of the ongoing effort to expand this approach

to study the roles of other MLL5 homologues, I began my research with a view to

characterizing the function of upSET (CG9007), the fly orthologue of MLL5, particularly

because of its possible function as a H3K4 methyltransferase. upSET is located on

chromosome 3L (13,995,227...14,013,248) in the cytogenetic region, 70C11-70C12

(Figure 4, information from Flybase; ST. PIERRE et al. 2014). As mentioned above, the

gene contains sequences that encode for a SET domain, and a PHD finger (EMERLING

et al. 2002). The SET domain of UpSET is encoded by 225 nucleotides from the fourth

translated exon of upSET (translates to 75 aa).

15

Figure 4 Map showing available upSET deficiencies Three upSET deficiencies (Df(3L)ED4515, Df(3L)ED4529, and Df(3L)ED4536) were tested in this study.

In a recent study that was published while my work was in progress, RINCON-

ARANO et al. (2012) demonstrated that UpSET functions by recruiting the histone

deacetylase Rpd3/Sin3A (HDAC1) to promoter regions of genes in order to restrict the

extent of histone acetylation and therefore of chromatin domains in which genes are

actively expressed. Thus, in the absence of UPSET, these active domains can spread

into adjascent regions causing illegitimate activation of genes therein. Interestingly,

UPSET mutations act as enhancers of Pc, which supports the idea of illicit expansion of

active gene domains into Hox genes which facilitates their ectopic expression.

Furthermore, it was shown in S2 cell culture that the knockdown of upSET results in the

upregulation of normally silent genes. Interestingly, KIM and BURATOWSKI (2009) also

16

observed a similar mechanism with the yeast homolog of MLL5, Set3. According to their

study in yeast, Set1 catalyzes the dimethylation of H3K4, which recruits SET3 histone

deacetylase complex (KIM and BURATOWSKI 2009). The PHD Finger of Set3 is able to

bind to H3K4me2 placed by Set1. This is particularly interesting because SET3 is

analogous to the mammalian HDAC3/SMRT complex (PIJNAPPEL et al. 2001). WANG

et al. (2013) confirmed in Drosophila S2 cells using a ChIP-MS approach that UpSET

interacts with not only Rpd3, but also SMRTER and Ebi. Although not proven yet, it is

reasonable to suggest that SMRTER and Ebi function together in this mechanism as

well as they have been linked to EGF and Notch signalling in eye development (DONG

et al. 1999, TSUDA et al. 2002, TSUDA et al. 2006). RINCON-ARANO et al. (2012)

observed ectopic expression of Notch and its target genes in UPSET mutant ovarioles;

this further argues that these aforementioned proteins all function together somehow.

17

Chapter 2. Materials and Methods

2.1. Drosophila genetic crosses

All crosses were set up with 10-15 pairs of parents unless otherwise noted. F1

progeny of both male and female genders were collected for relative viability analysis,

sterility tests, and characterization of PEV. Flies were generally maintained at room

temperature days prior to the setup of genetic crosses. A standard cornmeal-yeast-

molasses medium is used as a source of diet for flies’ maintenance and crosses. Mutant

alleles and RNAi strains were used to elucidate the functions of genes. Crosses

involving the use of RNA interference were performed at 29⁰C; otherwise they were

performed at 25⁰C. Male RNAi flies were crossed to virgin females carrying Gal4 drivers

(e.g. tub-Gal4/TM3 Sb) that ultimately drive the expression of specific gene targeting

dsRNAs. These dsRNAs enter the RNA interference pathway to knockdown transcripts

of that particular gene. Female Gal4 drivers were selected to make use of the maternal

deposits of Gal4 (i.e. enhance the knockdown). To further enhance the effects of RNAi,

UAS-Dicer2 is often incorporated in these crosses to overexpress the Dicer-2

component of the RNAi pathway. Relative viability is calculated as a percentage of

number of F1 progeny from mutant or RNAi class divided by number of F1 progeny from

a reliable internal control class. Sterility tests were carried out by crossing the class of

interest (i.e. one male, or one virgin female) to one w1118 fly of the opposite gender.

Occasionally, the size of the sterility test crosses were expanded to two flies crossed to

two flies, or two crossed to three. All fly strains (see Appendix A) used in this project

were acquired from the Blooming Drosophila Stock Centre at Indiana University and The

Groudine Lab at the Fred Hutchinson Cancer Research Center, or were produced in

house (see 2.2).

18

2.2. Generation of upSET RNAi strains

Two different sets of RNAi strains were generated for this project. The first set

was made by first cloning an inverted repeat of a 257 bp DNA fragment that was PCR

amplified from the fourth translated exon of upSET (CG9007) (see Appendix B). The

inverted repeat was generated using the pGEM-Wiz vector as outlined by BAO and

CAGAN (2006). The inverted repeat was cloned into a standard P-element vector,

pUAST. The inverted repeats were randomly inserted into the Drosophila genome upon

microinjection of the plasmid construct, introduction of a transposase source, and

screening for successful transpositions. The generation of the plasmid construct was

completed in our laboratory, while the injections and the screenings were carried out by

BestGene Inc. in 2011. This procedure yielded nine different transgenic strains (1M, 2M,

3M, 4M, 5M, 6M, 7M, 8M, and 9M) under the order ID # 8302. The locations of the

insertions are unknown, but insertions tend to occur in euchromatin where chromatin is

most accessible. These strains are referred to as upSETRNAi-Random 1M – 9M (see Appendix

A for a list of flies used in this study).

Similarly, the second set of upSET RNAi strains were generated by first cloning

an inverted repeat of a 321 bp DNA fragment from the first translated exon of upSET

(see Appendix B). This 321 bp DNA fragment was PCR amplified from Drosophila

melanogaster genomic DNA using the primers listed in Appendix C. The KAPA HiFi

HotStart DNA Polymerase kit (KAPA Biosystems, KK2501) was used for PCR. Standard

protocol was carried out such that initial denaturation was at 95⁰C for 5 minutes,

denaturation at 98⁰C for 20 seconds, primer annealing at 60⁰C for 15 seconds, and

extension at 72⁰C for 1 minute. The last three steps were repeated for 40 cycles,

followed by a final extension step at 72⁰C for 5 minutes. Standard reagent volumes were

used as recommended by product manual. The amplicon was subcloned into the pGEM-

Wiz vector as before to generated the inverted repeat (BAO and CAGAN 2006), which

was cloned into an attB/attP compatible P-element vector, pUAST-attb (BISCHOF et al.

2007). The vector allowed site-specific insertion of the inverted repeat construct into the

attP2 landing site located near the cytogenetic site, 68A4. The plasmid construct was

made in our laboratory, while the subsequent procedures were carried out by BestGene

Inc. in 2012 under the order ID # 10678 (handled by Dr. Cain Yam). The process yielded

19

three identical strains. In my analysis, I conducted experiments primarily using the first of

the three (1M). This strain will be referred to as upSETRNAi-attP2.

2.3. Phenotypic characterization of PEV

Three variegating alleles were used for this position-effect variegation (PEV)

study. These were In(1)wm4, Dp(1;Y)BSCV,and T(2;3)SbV (MOORE et al. 1979, SINCLAIR

et al. 1983), hereafter referred to as white-mottled-4 (wm4), Bar-of-Stone-varigated (BSV),

and Stubble-variegated (SbV). The exact sources and genotypes of these stocks are

listed in Appendix A. For all PEV-related genetic crosses, males of the variegating

alleles were crossed to females of the mutant lines (including upSET deficiencies and

RNAi). Crosses typically resulted in two F1 classes: a mutant class (Mutant/+), and an

internal control class (Balancer/+) unless otherwise specified.

Quantification of drosopterins (red pigments) from the adult fly eye was

performed using the fluorometric technique described by Ephrussi and Herold (1944)

with a few minor modifications. Female flies were aged at 25⁰C for six days prior to

freezing in liquid nitrogen. Flies were either stored at -70⁰C, or used for eye pigments

extraction. Heads were separated from bodies by brief vortexing. A razor blade was

used to manually separate the heads from the bodies in a few instances. Ten heads per

genotype were subjected to red pigment extraction in 0.5 ml of acidified ethanol (30%

ethanol, pH 2). Heads were homogenized using a standard pestle, and incubated at

room temperature for 48 hours in the dark. This was considered a biological replicate.

Following incubation, each sample was evenly split into two separate tubes. Each of

these was considered a technical replicate. These samples were `cleaned` by

centrifugations at 10,000 rpm twice. Cleared supernatants were measured for optical

absorbance at 480 nm (A480) using the Nanodrop ND-1000 spectrophotometer. Two

biological replicates of each genotype were prepared. These translated to a total of four

technical replicated per genotype. An average A480 was calculated from A480 of the four

technical replicates. Calculated values were normalized to the average absorbance of

wild-type (OreR) eye pigments extract, and reported as a percentage of wild-type eye

pigmentation.

20

Widths of eyes were measured using a dissecting microscope (LEICA MZ6)

equipped with an ocular micrometer (Figure 5). All measurements were done at the

zoom setting of 2.0. At this zoom level, 1 arbitrary unit on the ocular micrometer was

equivalent to 0.515 mm of a standard ruler. Measurements were first recorded as an

arbitrary unit, and then converted to millimetre (mm). 50 flies per genotype were scored

unless otherwise stated. Student’s t-tests were used to validate the significance of the

difference in mean of the datasets by comparing the eye width measurements of the

control class to the mutant class. The null hypothesis was that those in the control class

are the same as the mutant class. The alternative hypothesis was that they are different.

The probability values resulted from the t-tests were calculated using the TTEST ()

function using Microsoft Excel Spreadsheets (2007). The t-test performed uses a two-

tailed distribution, comparing two datasets with unequal variance (heteroscedastic).

p<0.05 (t-test) were reported within the descriptions of their associated figures.

Figure 5 Method: Measurement of the width of the eye in D. melanogaster. Asterisk (*) indicates the width of the eye that was measured for PEV analysis involving Bar of Stone-Variegated (BSV).

For PEV analysis using Stubble-variegated (SbV), fourteen defined bristles

(anterior post-alars, posterior dorsocentrals, posterior supra-alars, and anterior and

posterior scutellars and sternopleurals) on each fly were scored as being either Stubble

(Sb) or wild-type. Percentages of Stubble ± SEM from control and mutant classes were

reported. 40 flies were scored per genotype. Out of the 40, half were males, and half

were females. The significance of the difference in datasets of the two classes was

validated using Student’s t-tests like before with the width of the eye.

21

2.4. Cloning of GST-SETUpSET construct

In order to express the SET domain of upSET as a GST fusion protein in BL21

(DE3) E. coli competent cells, the entire 225 bp of the SET domain from the fourth

translated exon of upSET was cloned into pGEX-4T1 in-frame with a 5’ sequence coding

for GST. Both the vector and the insert DNA were subjected to double digestion with

EcoRI/XhoI to allow “sticky-ends” ligation with T4 DNA ligase. The construct was verified

by restriction digest diagnostics and DNA sequencing. The entire SET domain coding

sequence is:

CAAGTTGCGCAGCAGCCACCATCCTACCTAAAGGGTCCGGAGGTGT

GTGTGGATACGCGGACCTATGGCAACGATGCACGATTCGTGCGTCG

ATCTTGCCGACCAAATGCCGAGTTGCAGCACTACTTCGAGAAGGGA

ACGCTGCATTTGTATATAGTGGCATTAACACACATACGTGCCCAGAC

GGAGATCACGATCCGCCACGAGCCACACGACTTGACGGCG

The sequence above translates to (75 aa, predicted 8.66 kDa):

QVAQQPPSYLKGPEVCVDTRTYGNDARFVRRSCRPNAELQHYFEKGT

LHLYIVALTHIRAQTEITIRHEPHDLTA

2.5. Co-Expression and Purification of GST-SETUpSET from bacteria cell cultures

In order to test if O-GlcNAc Transferase (OGT) modified the SET domain of

upSET, I obtained two pET28a constructs containing either the wild-type human OGT

(hOGT), or the catalytic inactive hOGT from the Vocaldo laboratory at Simon Fraser

University. Both of these vectors have been extensively tested and used in their OGT-

related studies (SHEN et al. 2012). Either of these hOGT constructs was co-transformed

with the upSET SET construct. They have also generously supplied a widely-used

positive control for these types of studies, namely a known hOGT substrate known as

TAB1. Note that for the positive control that was supplied, TAB1 was cloned into a

pET28a vector which enables fusion of a polyhistidine binding tag, whereas hOGT was

22

cloned into a pMAL-C2X vector which enables the fusion of a maltose-binding protein

(MBP) tag.

Once the aforementioned expression constructs were ready, three distinct co-

transformation procedures were carried out. hOGT-pET28a was co-transformed with

SET-pGEX-4T1. hOGT(catalytic inactive)-pET28a was co-transformed with SET-pGEX-

4T1 as a negative control. hOGT-pMAL-C2X was co-transformed with TAB1-pET28a as

a positive control. Each of these combinations was transformed into BL21 (DE3)

competent cells that lacked protease activity, and plated on standard agar plates

containing ampicillin and kanamycin to allow selective growth. Individual colonies were

picked to grow 3 ml bacterial overnight cultures with appropriate amounts of antibiotics in

standard LB media at 37⁰C. The cultures were topped with LB to approximately 15 ml

without further additions of antibiotics the next morning. Cultures were grown at 37⁰C

until an OD600 of approximately 0.8. Subsequently, cultures were induced with IPTG (0.5

mM) and ZnSO4 (0.1 mM). Optimal induction conditions for co-expression of hOGT and

GST-SET is 25⁰C for 4 hours. The conditions were the same for the other two co-

expressions (also optimized by SHEN et al. 2012). Cells were spun down (into pellets)

by 4,000 rpm centrifugation for 15 minutes at 4⁰C. Each cell pellet was washed once

with 1 X PBS, and spun down again as before. Each pellet was resuspended in 500 µl

of sonication buffer, and left on ice for 15 minutes. Resuspended cells were sonicated 3

times in ice for 5 minutes using the Diagenode Bioruptor UCD-200 at high power setting

(20kHz for 320W, 45s on / 15s off), and spun down by centrifugation at 10,000 rpm at

4⁰C. After each five-minute sonication, the ice in the sonicator was replaced with fresh

ice to minimize protein degradation. Following sonication, the samples were spun down

for 10 minutes at 20,000 rpm at 4⁰C to separate the insoluble proteins from the soluble

proteins.

When purifying GST-SET, soluble proteins were incubated in 60 μl of pre-

washed Glutathione Sepharose 4B Media (GE Healthcare, 17-0756-01), and rotated for

90 minutes at 4⁰C. When purifying His6-TAB1, soluble proteins were incubated in 60 μl

of pre-washed Ni-NTA Sepharose media (GE Healthcare, 17-5268-01). Beads

conjugated with fusion proteins were washed three times. Each wash consisted of

rotation for 5 minutes in 500 µl wash buffer, followed by 2 minutes spin at 8,000 rpm at

23

room temperature. The samples were the beads containing the fusion proteins were ran

directly on 8% SDS-PAGE for further analysis. Known amounts of Bovine Serum

Albumin (BSA) were run on the same SDS-PAGE. BSA was used to compare, and to

confirm that the amount of GST-SET proteins extracted and loaded onto the gel for

Western Blotting analysis was sufficient for detection (i.e. at least 4 µg). Purified His6-

TAB1 produced in bacterial cultures is approximately 44 kDa (SHEN et al. 2012),

whereas GST-SET is 45 kDa.

In GST- protein purification procedures, the sonication buffer used is the same as

the wash buffer. This buffer contained 1X PBS (pH 7.5), 10% glycerol, 0.1% Triton X-

100, 1 mM DTT, 2.5 mM MgCl2, 20 U/ml DNAse I, 0.2 mg/ml lysozyme, and 1X protease

inhibitor cocktail (Calbiochem). Contrarily, in the His6- protein purification, the sonication

buffer is different from the wash buffer. This particular sonication buffer contained 50 mM

NaH2PO4 (pH 8.0), 300 mM NaCl, 10 mM imidazole, 0.1% Triton X-100, 0.2 mg/ml

lysozyme, 20 U/ml DNase I, 10 mM β-mercaptoethanol, 10% glycerol, and 1X protease

inhibitor cocktail (Calbiochem). The wash buffer contained all of those in the sonication

buffer without the lysozyme, and DNAse I. Also, the imidazole is increased to 20 mM in

the wash buffer. See Appendix D for recipes of these aforementioned buffers.

2.6. Nuclear Protein Extraction from Adult Fruit Flies

Nuclei from adult flies (50% males, 50% females) were extracted as described in

SHAFFER et al. 1994 at 4⁰C. 100 adult flies per genotype were grinded in 1 ml low salt

buffer (20 mM HEPES (pH 7.5), 2mM dTT, 1mM EDTA, 10% glycerol, 0.1% Igebal, 1X

protease inhibitor cocktail I (Calbiochem)) using a standard glass homogenizer. Each

homogenized mix was transferred onto a sucrose cushion (low salt buffer with 0.7 M

Sucrose), spun down by centrifugation at 10,000 rpm at 4⁰C, and pellet was

resuspended in low salt buffer. This sucrose cushion step was repeated once more.

Next, each sample was spun down by centrifugation at 10,000 rpm for 5 minutes, and

resuspended in 200 – 250 μl of high salt buffer (low salt buffer with 400 mM NaCl).

Samples were sonicated in ice for 5 minutes using the Diagenode Bioruptor UCD-200 at

high power setting (20 kHz for 320W, 45s on / 15s off) in sonication buffer at 4⁰C.

24

Sonication buffer contained 20 mM HEPES (pH 7.5), 400 mM NaCl, 10% glycerol, 1 mM

EDTA, 2 mM DTT, 0.1% Triton X-100, and 1X protease inhibitor cocktail I (Calbiochem).

Samples were spun down by centrifugation at 10,000 rpm at 4⁰C. The insoluble (protein)

pellets were discarded, and the soluble protein samples (i.e. supernatant) were used for

downstream western blotting analysis.

2.7. Western Blotting

Western blots were performed following standard procedures for both O-GlcNAc

and H3K4 methylation analysis. The primary mouse monoclonal IgM O-GlcNAC

(CTD110.6) Antibody (Santa Cruz Biotechnology, sc-59623) at 1/5000 stock

concentration was used for probing O-GlcNAc levels in bacterial protein extracts. The

CTD110.6 antibody was previously characterized by COMER et al. (2001). Primary anti-

GST antibody was used to confirm the presence of GST-fusion proteins in bacterial

protein extracts (ABM, G0181). Primary antibodies and antibody concentrations used to

probe for levels of H3K4 methylation in protein extracts from adult flies are listed in Table

1. Binding specificities of each methyl-H3K4 antibody has been tested and optimized by

my colleague, Dr. Graham Hallson, in our laboratory, and also by others (EGELHOFER

et al. 2011, HALLSON et al. 2012). Detection of western blotting was performed using

Supersignal Pico (Thermo Scientific) and exposure to film. Exposed films containing

bands were scanned, and quantified using ImageJ as described in section 2.7.

25

Table 1 Primary Antibodies used for probing H3K4 methylation

Targeting Company Catalogue Number Stock Concentration Used

H3 Abcam ab1791 1/10,000

H3K4me1 Abcam ab8895 1/5000

H3K4me2 Millipore 07-030 1/5000

H3K4me3 Active Motif 39159 1/5000

2.8. Quantification of Western Blots

The ImageJ software (http://imagej.nih.gov/ij/docs/faqs.html) was used to

perform densitometry on the bands observed on my western blots for probing levels of

H3K4 methylation. The band intensity of each lane was determined using ImageJ as

described in the ImageJ tutorial at http://howtowesternblot.net/data-analysis-

3/quantification/. Baseline background noise from the film was taken into account for all

integrated measurements. Experimental band intensities (H3K4me3, H3K4 me2,

H3K4me1) were normalized according to total H3 loaded (control) such that:

Normalized Intensity of H3K4me3 = Intensity of H3K4me3 Intensity of H3



http://howtowesternblot.net/data-analysis-3/quantification/

http://howtowesternblot.net/data-analysis-3/quantification/

26

Normalized intensities were compared between upSET RNAi and w1118 (wild-type

control) as a ratio of RNAi/w1118 such that a ratio of >1 suggests an increase in

methylation levels, whereas a ratio of <1 suggests a decrease.

2.9. Quantitative PCR (qPCR)

RNA was extracted from adult fruit flies by homogenization in TRIzol reagent

(Invitrogen), chloroform extraction, and isopropanol precipitation using standard protocol.

Genomic DNA was removed from the RNA extract by treating RNA with DNAse I for one

hour at 37⁰C, followed by DNAse I inactivation at 65⁰C for 10 minutes in 50 mM EDTA.

DNAse-treated RNA used as template to synthesize cDNA using the iScript Select cDNA

synthesis kit (Biorad). Oligo-dT primers provided in the kit were used in the synthesis

reactions using standard protocol. Quantitative PCR reactions were prepared in triplicate

using the KAPA Sybr Fast Kit (1 μl cDNA, 0.5 μl primer mix, 3.5 μl ddH2O, and 5 μl

KAPA Sybr Fast Mastermix). qPCR conditions were 40 cycles of 94⁰C for 3 seconds,

60⁰C for 30 seconds and 72⁰C for 60 seconds. Fluorescence measurements were

obtained using the MBB department’s StepOne thermocycler (Applied Biosystems).

Binding efficiencies of these primers were estimated from the slope of a standard

curve generated using dilutions of adult fly cDNA. Transcript levels of upSET were

calculated relative to wild-type levels using the ∆∆ cycle threshold method with rp49, khc,

and tub56D as reference genes to normalize differences in input cDNA levels (PFAFFL

2001). qPCR Primers targeting upSET are listed in Appendix C.

27

Chapter 3. Characterization of upSET

3.1. upSET is essential for viability but not fertility

The two types of RNAi transgenes targeting CG9007 (hereafter referred to as

upSET) for knockdown are controlled by UAS sequences and are thus Gal4-inducible

(see Materials and Methods). To test whether upSET is essential for viability, I initially

drove upSETRNAi-Random 1M – 9M with the ubiquitous driver, tub-Gal4. The crosses were

performed under conditions that maximize gene knockdown. Thus, I expressed Gal4

under control of the tubulin promoter at 29⁰C with the addition of UAS-Dicer-2. If the

targeted gene is essential, one should expect changes in viability/fertility upon

knockdown. However, since the RNAi transgenes would have undergone random

insertion in the genome, it seemed likely that any effects RNAi knockdown of upSET

would have on viability and/or fertility, would be variable among the 9 different transgenic

lines; thus, an insertion in loosely packed chromatin regions could theoretically result in

higher expression of the target dsRNA, resulting in a greater knockdown. As can be

seen, expression of nearly all of the RNAi transgenes caused clear cut reductions in

viability (Figure 6): indeed, the expression of the two transgenic lines, 7M and 8 M,

caused complete lethality. When crosses were setup en masse, the occasional

“escaper” was fertile with occasional wing defects (abnormal fusion of two wing layers,

or ectopic wing vein). In addition, the 4M and 9M transgenes also caused significant

reductions in viability. One caveat of this experiment is that the RNAi construct used to

target upSET shares homology with a 19 bp segment of nAchRalpha-80B. My colleague,

Dr. Hallson, addressed this concern by performing qPCR on transcripts extracted from

adult flies of 7M driven with tub-GAL4 with UAS-Dicer2. The RNAi transgene only

depletes nAchRalpha-80B levels by approximately 50% compared to wild-type levels

(HALLSON, unpublished). Therefore, this off-target is unlikely to be the cause of the

100% lethality caused by two of the transgenes.

28

In order to ensure that knockdown of upSET was gene-specific, I used an RNAi

transgene for CG9007 in which the sequence used had no off-target homologies i.e. the

upSETRNAi-attP2 and this line was crossed to two different ubiquitous Gal4 drivers.

Importantly, upSETRNAi-attP2 displayed significant lethality when driven at 29⁰C (Figure 7).

The RNAi transgene depletes upSET levels by 35.4% in females compared to wild-type

levels, and by 71.6% in males compared to wild-type levels (see Appendix C for

sequences of qPCR primers used to quantify upSET). UAS-Dicer2/+; upSETRNAi-attP2/tub-

Gal4 flies exhibited a 90% reduction in viability, although, the reduction in viability of the

relevant genotype caused when the Act5C-Gal4 driver was used was only 60%.

upSETRNAi-attP2 were fertile when driven with either Gal4 driver. I observed that many

escapers of RNAi-induced lethality in these crosses were unable to emerge from their

pupal cases (Figure 8). Those that did survive occasionally displayed the wing defects

similar to those mentioned above.

Figure 6 upSETRNAi-Random 1M – 9M driven with tub-Gal4 with UAS-Dicer2 at 29 ⁰C. The Gal4 drivers were tub-Gal4 with UAS-Dicer2. The total sample sizes of each of these crosses were >100 F1 adults.

29

Figure 7 Relative Viability of upSETRNAi-attP2 driven with Gal4 drivers at 29 ⁰C. The Gal4 drivers were tub-Gal4, and Act5C with UAS-Dicer2. The total sample sizes (n) of each of these crosses were 376, and 221 F1 adults, respectively.

30

Figure 8 Examples of eclosion related defects resulted from driving

upSETRNAi-attP2 using tub-Gal4 with UAS-Dicer2 at 29 ⁰C. Photographs of the adult fruit fly emerging from its pupal case: 80% eclosed (A), 50% eclosed (B), 0% exclosed (C).

Since the results of my RNAi experiments suggested that upSET is essential for

viability, but not fertility, I decided to investigate this issue further using two putative

mutant upSET alleles, kindly provided by Dr. Rincon-Arano (RINCON-ARANO et al.

2012). The hypomorphic upSETMB08950 allele is associated with insertion of a Minos

transposon after the codon coding for amino acid T2599 (BELLEN et al. 2004).

Homozygous (upSETMB08950/ upSETMB08950) and heterozygous (upSETMB08950/TM3 Sb)

mutants are viable and fertile; however, this may not be surprising if the residual activity

of upSET is sufficient in these flies. I explored the functional effects of the mutant further

by generating flies that are hemizygous for the hypomorphic allele: the diagnostic

upSETDf(3L)ED4515/upSETMB08950 flies were also viable and fertile, suggesting that the lesion

in question has no upSET-specific effects on viability or fertility.

I next tested the putative amorphic allele upSETe00365; this allele is associated

with the insertion of a P-element into the second intron of the gene. The insertion is

upstream of the transcriptional start site (TSS). According to RINCON-ARANO et al.

(2012), males and females homozygous for this mutation are viable but the females are

31

sterile i.e. they lay only a few eggs and none develops. Moreover, based on Western

blot analysis using antibodies against the UPSET protein, these workers have

characterized upSETe00365 as a “protein null” mutant.

Clearly the mutant is not a recessive lethal, since homozygous flies are evident in

the stock in which the upSETe00365 third chromosome is balanced with TM3, Sb Ser Act-

GFP. However, when I crossed balanced females and males from the stock, there was

only a modest reduction in relative viability of upSETe00365 homozygotes (Figure 9). If this

lesion is a true protein null, then the latter observation is rather surprising, given my

RNAi data which suggests that the gene is essential. One possibility is that the gene is

essential, but that upSETe00365 is not a complete protein null (i.e. there is sufficient mRNA

and protein produced to ensure survival). Indeed, using qPCR, a research associate in

the lab has found that there is a minimum of 20% of upSET RNA produced in

upSETe00365 homozygotes (HALLSON, unpublished). This raises the possibility that there

may also be residual UPSET protein in the homozygoutes and that upSETe00365 may not

be a true protein null allele.

32

Figure 9 Relative Viability of upSETe00365 at 25 ⁰C. Viability scores were normalized to the internal control (upSETe00365/TM3 Sb Ser Act-GFP) and expressed as a percentage of relative viability. The total sample sizes (n) of cross is 321 F1 adults.

In agreement with the findings of RINCON-ARANO et al. (2012), I observed that

homozygous upSETe00365 females are sterile (Table 2) i.e. they lay few eggs and none of

them hatches. It is possible that the sterility reflects weakness/lack of normal mobility of

the mutant females and thus their ability to mate properly. However, it seems unlikely,

given the fact that the lesion shows only modest semi-lethality when homozygous. As

expected, my analysis showed that homozygous upSETe00365 males are fertile (Table 2).

33

Table 2 Sterility observations from genetically crossing upSETe00365 flies to wild-type (w1118) flies

Cross # Male Parent Female Parent Sterile?

1 1 upSETe00365/ upSETe00365 1 w1118 No

2 2 upSETe00365/ upSETe00365 2 w1118 No

3 1 w1118 1 upSETe00365/ upSETe00365 Yes



6 2 w1118 2 upSETe00365/ upSETe00365 No

7 2 w1118 3 upSETe00365/ upSETe00365 No


9 1 upSETDf(3L)ED4515/upSETe00365 1 w1118 No

10 2 upSETDf(3L)ED4515/upSETe00365 2 w1118 No

11 1 w1118 1 upSETDf(3L)ED4515/upSETe00365 No



34

It is possible that the observed recessive female sterility associated with

upSETe00365 is caused by disruption of one or more separate genes linked to upSET. This

might explain the fact that I observed no fertility defects in female escapers of the

aforementioned RNAi experiments. To extend this analysis, with the assistance of Kevin

Beja, I generated upSETDf(3L)ED4515/upSETe00365 flies; these showed no reduction in

relative viability and both males and females were fully fertile (data not shown). Similar

tests of two other upSET deletions upSETDf(3L)ED4529, and upSETDf(3L)ED4536 yielded

comparable results (data not shown). Taken together, these findings strongly support

the hypothesis that the fertility and reduced viability associated with homozygosis of the

upSETe00365 third chromosome is due to lesion(s) in another gene or genes on the

chromosome. However, since it is possible that upSETe00365 is not a complete protein null,

and because my RNAi data suggest that the upSET gene is essential, definitive

corroborative evidence pertaining to the biological requirements for the upSET gene

function awaits further analysis.

3.2. The SET domain of UpSET is not modified by human OGT in bacteria culture

As mentioned in the introduction, a short isoform of MLL5 is capable of catalyzing

the addition of both mono and di methyl groups to histone 3 lysine 4 (H3K4) tails in vitro

(FUJIKI et al. 2009). Specifically, OGT adds an O-GlcNAc sugar moiety to the Threonine

440 residue of the SET domain of MLL5. Since our laboratory is also interested in OGT,

I attempted to see if this modification is conserved with the SET domain of upSET.

First, I wished to determine whether the modified SET domain residue is

conserved in Drosophila. To accomplish this, I used the NCBI protein blast tool

(reviewed by JOHNSON et al. 2008) to align the translated amino acid sequence of the

SET domain of upSET (see Section 2.4) against sequences in the human and the

mouse protein databases. The purpose of this bioinformatics analysis is to see if the

modified Threonine 440 residue is conserved in upSET. Importantly, I found that

Threonine 440 residue is indeed conserved in flies (Figure 2). This raises the possibility

that this SET domain residue may also be modified by OGT in flies. In order to test this, I

co-expressed the SET domain of UpSET as a GST fusion protein and human OGT

35

(hOGT) in bacterial cells. As a positive control, I also co-expressed a known OGT

substrate, TAB1, and hOGT in the same cells at the same time (SHEN et al. 2012).

Since OGT is reportedly absent in bacteria, presumably any O-GlcNAc modifications

observed would be those catalyzed by the co-expressed hOGT. Following the

expression of these proteins, the substrate proteins (GST-SET or His6-TAB1) were

purified, and probed for O-GlcNAc (see Materials and Methods).

Figure 10 Human OGT (hOGT) does not modify the SET domain of UpSET in

bacteria culture system. Protein samples (His6-TAB1, GST-SET) were originally expressed and purified from bacteria cultures. Purified proteins were probed for O-GlcNAc using anti-O-GlcNAc (CTD110.6) antibody. His6-TAB1 co-expressed with human OGT was used as positive control. His6-TAB1 expressed without human OGT was used as negative control. TAB1 is a known OGT substrate (SHEN et al. 2012).

The results of the aforementioned experiment are given in Figure 10. Importantly,

the positive control was successful i.e. TAB1 was modified; moreover, this modification

required co-expression of hOGT (compare with lane 2). However, the data clearly show

36

that, on its own, the SET domain of UpSET does not serve a substrate for hOGT: note

the absence of target protein in lane 3 (co-expression of GST-SET and hOGT). However,

these data do not conclusively rule out the possibility that the SET domain is only OGT-

modified when it is part of the entire UpSET protein, or that Ser or Thr residues

elsewhere on the protein can be modified by OGT. Unfortunately, the full-length cDNA of

upSET is very large (>12 kb) and thus, it could not be conveniently cloned for this study.

3.3. Global levels of H3K4 methylation is unaffected in upSET RNAi

Although RINCON-ARANO et al. (2012) found that the SET domain of upSET

does not catalyze the addition of methyl groups to purified histones in vitro, it is possible

that any such catalytic activity requires the entire upSET protein. To address the

possibility that upSET plays a role in global H3K4 methylation, I have also tested the

effects of upSET RNAi on global H3K4 methylation. In this experiment, I compared

global methyl-H3K4 levels in nuclear extracts of upSET RNAi adult flies to those of

nuclear extracts from internal control flies; upSETRNAi-attP2 was driven with tub-Gal4 with

UAS-Dicer2 at 29⁰C. i.e. UAS-Dicer2/+; upSETRNAi-attP2/tub-Gal4 were the experimental

flies and UAS-Dicer2/+; upSETRNAi-attP2/TM3 Sb flies served as the control. The data

(Figures 11 and 12) show that global H3K4 methylation (mono, di, or tri) levels were

largely unchanged upon knockdown of upSET.

37

Figure 11 Global H3K4 methylation is unaffected by upSET RNAi (Blot) Nuclear proteins extracted from adult flies were probed for H3, H3K4me1, H3K4me2, and H3K4me3. The genotype of the upSET RNAi was UAS-Dicer2/+; upSETRNAi-attP2/tub-Gal4. The genotype of the internal control was UAS-Dicer2/+; upSETRNAi-attP2/TM3 Sb.

38

Figure 12 Global H3K4 methylation unaffected by upSET RNAi (Quantification) This figure shows the quantification of data from Figure 11. Global H3K4 methylation levels of upSET RNAi were normalized to those of the internal control.

3.4. Knockdown of upSET enhances Polycomb phenotypes

RINCON-ARANO et al. (2012) found that both UpSET mutants enhanced

Polycomb (Pc), thereby suggesting that wild-type function is required for full PcG

function. Because the exact nature of these alleles is unclear (e.g. upSETe00365 may

carry a second-site mutation that affects female fertility), I decided to test whether the

down-regulation of upSET using RNA interference can also enhance Pc. To accomplish

this, I generated a Polycomb mutant stock bearing Act5C (Gal4 driver). I then crossed

females of this modified strain to males of the RNAi. F1 males with the genotypes of

Act5C-Gal4/+; upSETRNAi-attP2/Pc3 and CyO/+;upSETRNAi-attP2/Pc3 were scored for number

of sex combs per fly. (The latter served as the control). Wild-type male flies exhibit two

sex combs on their prothoracic legs. However, males heterozygous for Polycomb often

exhibit one or more ectopic sex comb teeth on their mesothoracic and/or metathoracic

legs (although the penetrance is variable) (LEWIS 1978). For the purposes of this

experiment, the number of legs per male fly (n = 10 flies) with sex combs (i.e. if there

39

was at least one sex comb tooth on a given leg, it was designated as a sex comb) was

determined and the results are shown in Figure 13 (see also Figure 14). From the

sample group analyzed, it appears knockdown of upSET by RNAi also enhances

Polycomb (note the average number of sex combs per experimental male is 3.8, versus

2.6 for the control males). The difference in mean values is statistically significant

according to a Student’s t-test. P<0.05 was 0.00104.

Figure 13 upSET RNAi enhances Polycomb phenotype in males

(Quantification) upSET RNAi was driven with Act5C (Gal4) at 29⁰C under Pc3 background. The genotype of RNAi was Act5C/+; upSETRNAi-attP2/Pc3. The control lacked Act5C. It had a genotype of CyO/+;upSETRNAi-attP2/Pc3. Error bars display ± SEM. SEM are 0.249, and 0.163, respectively. p<0.05 (t-test) = 0.00104.

40

Figure 14 upSET RNAi enhances Polycomb phenotype in males (Images) upSET RNAi was driven with Act5C (Gal4) at 29⁰C under Pc3 background. RNAi enhances Polycomb phenotype as displayed by the extra sex combs on the second set of leg appendages (marked with asterisks).

3.5. UpSET is a suppressor of variegation

As shown earlier, down-regulation of upSET does not markedly influence global

methyl-H3K4 in Drosophila melanogaster. Further, the SET domain on it own is

incapable of catalyzing the transfer of methyl groups to histones in vitro (RINCON-

ARANO et al. 2012). H3K4 methylation is generally associated with active gene

expression. Enhancement of Pc by RNAi knockdown of upSET, coupled with similar

results using the upSET mutants (RINCON-ARANO et al. 2012) strongly demonstrates

that upSET is required for repression of at least some PcG target genes. Also, the fact

that upSET plays a role in recruitment of Rpd3/Sin3A histone deacetylase machinery to

promoter regions of active gene is consistent with the view that upSET’s main function

involve gene regulation or at least restriction of chromatin domains in which genes are

active. However, it is unclear whether upSET can, in some cases, be involved in gene

activation. What is clear is that the protein does influence chromatin

structure/accessibility of active promoter regions (RINCON-ARANO et al. 2012). Since is

PEV is one of the most well-studied manifestations of the importance of chromatin

structure for gene expression, it is worthwhile asking whether upSET-induced alterations

in chromatin structure can influence PEV i.e. do upSET mutations modify the expression

of genes subject to PEV? In principle, upSET mutants might have no effect on PEV, or

they could act as E(var)s (i.e. cause an increase in assembly/spread of heterochromatin)

or they could act as Su(var)s (i.e. cause a decrease in assembly/spread of

heterochromatin) (see the Introduction).

41

In my first PEV experiment, I tested the effects of upSET mutations on white (w)

gene variegation. In Drosophila, the w gene is required for pigmentation deposition in a

number of tissues including most strikingly, the large compound eyes (MACKENZIE et

al. 1999). The variegating rearrangement, white-mottled-4 (wm4) consists of an X-

chromosomal inversion which relocates the euchromatic w gene close to

heterochromatin; as mentioned in the introduction, this results in patches of red and

white eye tissues due to variegating expression of w (reviewed by GIRTON and

JOHANSEN 2008). Strong Su(var) mutations, suppress wm4 variegation i.e. the eyes of

wm4 flies have higher levels of pigmentation, often approaching those of wild-type. On the

other hand, strong E(var) mutations reduce the level of pigmentation in the eyes of wm4

flies. To determine whether upSET is a Su(var) or E(var), I crossed w-/w-

;upSETMB08950/TM3 Sb females to wm4 males and determined the levels of red

pigmentation in the eyes of the diagnostic F1 females and relevant controls (see

Materials and Methods). (F1 males were excluded from this analysis because they were

all w-). All measurements were expressed as a percentage of red pigmentation in

Oregon-R wild-type females and the results are shown in Figure 15 (bar on the left) and

a relevant image is shown in Figure 16. Clearly, the upSETMB08950 acts as a dominant

Su(var): red eye pigment levels of w-/wm4;upSETMB08950/+ flies were 52.8% of wild-type,

whereas those of w-/wm4;TM3 Sb/+ flies (the internal control class) were only 16.0% of

wild-type. I also tested the upSET deficiency, upSETDf(3L)ED4529, in a similar fashion and

the results are also shown in Figure 15 (bar on the right). Red eye pigment levels in the

diagnostic w-/wm4;upSETDf(3L)ED4529/+ flies were 31% those of wild-type, whereas those of

w-/wm4;TM3 Sb/+ control flies were 18.5%. These data show that a deletion of the upSET

gene also dominantly suppresses PEV, albeit not as strongly as the mutant allele used.

The difference between the deletion and upSETMB08950 may be explained if the deletion

also removed one or more other genes that can act as E(var)s.

42

Figure 15 Percent Wild-Type Eye Pigmentation of upSET hypomorph and

deficiency classes carrying white-mottled-4. Average A480 readings recorded from eye pigments extracts were normalized to those from wild-type (OreR), and presented as a percentage of wild-type eye pigmentation. Two biological replicates of 10 female heads of each class were used for this eye pigmentation assay. Two technical replicates of each biological extract were carried out. Average readings of each class were calculated from four distinct A480 readings. The upSET hypomorph class had the genotype of upSETMB08950/+. The upSET deficiency class had the genotype of upSETDf(3L)ED4529. Internal controls were F1 females with the genotype of TM3 Sb/+ from their respective crosses. All eyes analyzed were from F1 females as they carried the wm4 variegator.

43

Figure 16 Eye variegation of upSET hypomorph carrying white-mottled-4. All eyes analyzed were of F1 females as they carried wm4. Photographs show an upSET hypomorph fly (upSETMB08950/+, left) with significant suppression of eye variegation, and an internal control fly (TM3 Sb/+, right).

Since the transposon associated with upSETe00365 and the upSETRNAi-attP2

transgene both effectively carry w+ reporter genes, it was not possible to examine their

effects on wm4 variegation. Therefore, I extended my analysis by testing the effects of the

upSET mutants and down-regulation of upSET via RNAi on two other variegating loci,

Bar-of-StoneV (BSV) and StubbleV (SbV). I shall first describe the experiment using BSV.

Typically, the presence of a non-variegating second copy of the Bar (B) gene

dramatically narrows the flies’ compound eyes. BSV consists of an extra copy of B on the

Y chromosome that variegates (i.e. the extra B gene is variably silenced by surrounding

heterochromatin), thereby producing wider eyes (although narrower than wild-type) in

the appropriate males. In principle, variegation suppression of BSV by upSET mutants or

down-regulation of the gene should result in males with narrower eyes compared to

those of the control males carrying BSV.

In separate crosses, I tested two insertion alleles of upSET, an upSET deletion

and expressed upSETRNAi-attP2 for their effects on BSV and the results are shown in

Figures 18-21, with representative fly images shown in Figure 17. (The details of the

crosses and method of measurement of eye width are given in the Materials and

Methods).

These results clearly show that the two upSET insertional alleles, a deletion and

RNA-induced knockdown of upSET all show significant suppression of BSV. For example,

44

BSV-bearing males that were also heterozygous for upSETMB08950, showed a ~30%

relative reduction in eye width compared to that of the TM3, Sb control males (Figures

17 and 16). Similarly, BSV;upSETDf(3L)ED4529/+ exhibited a ~25% relative reduction in the

eye-width (Figures 18 and 16). Moreover, the upSETe00365 allele (Figures 19 and 16) and

the expression of upSETRNAi-attP2 (Figures 20 and 16) caused reductions of relative eye-

width of ~50% and ~40%, respectively.

These data support my conclusion from the wm4 experiment that reduced

expression of the upSET gene causes dominant suppression of PEV.

45

Figure 17 Eye variegation of upSET mutants/deficiency/RNAi carrying BSV. All eyes analyzed were of F1 males as they carried BSV. Photographs show upSET mutants/deficiency/RNAi (shown on left) flies with significant suppression of variegation, and internal controls flies (shown on right).

46

Figure 18 Average width of Eye of upSET hypomorphic mutant carrying BSV. All eyes analyzed were of F1 males as they carried BSV. 50 eyes were measured from each class of males. upSET hypomorph had the genotype of upSETMB08950/+. Internal control had the genotype of TM3 Sb/+. Error bars plotted with standard deviation of ± 0.0229 mm for upSET hypomorph, and ± 0.0208 mm for the control. SEM are 0.0323 mm, and 0.00295 mm, respectively. p<0.05 (t-test) = 1.80E-36.

47

Figure 19 Average width of Eye of upSET deficiency carrying BSV. All eyes analyzed were of F1 males as they carried BSV. 10 eyes were measured from each class of males. upSET deficiency had the genotype of upSETDf(3L)ED4529/+. Internal control had the genotype of TM3 Sb/+. Error bars plotted with standard deviation of ± 0.0172 mm for upSET deficiency, and ± 0.0210 mm for the control. SEM are 0.00543 mm, and 0.00666 mm, respectively. p<0.05 (t-test) = 6.11E-08.

48

Figure 20 Average width of Eye of upSET “protein null” mutant carrying BSV. All eyes analyzed were of F1 males as they carried BSV. 30 eyes were measured from each class of males. upSET “protein null” mutant had the genotype of upSETe00365/+. Internal control had the genotype of TM3 Sb Ser Act-GFP/+. Error bars plotted with standard deviation of ± 0.0204 mm for upSET “protein null”, and ± 0.0268 mm for the control. SEM are 0.00372 mm, and 0.00490 mm, respectively. p<0.05 (t-test) = 4.14E-29.

49

Figure 21 Average width of Eye of upSET RNAi carrying BSV. All eyes analyzed were of F1 males as they carried BSV. 50 eyes were measured from each class of males. upSET RNAi was driven with Act5C (Gal4) ubiquitous driver at 29⁰C. This class had the genotype of upSETRNAi-attP2/Act5C. Internal control had the genotype of upSETRNAi-attP2/TM6C. Error bars plotted with standard deviation of ± 0.0468 mm for upSET RNAi, and ± 0.0324 mm for the control. SEM are 0.00662 mm, and 0.00458 mm, respectively. p<0.05 (t-test) = 3.04E-21.

In my next and final experiment, I tested the insertion alleles of upSET for their

effects on the variegating Stubble (Sb) allele, SbV. When fully expressed, the dominant

third chromosome Sb mutation causes a shortening and blunting of the flies’ bristles, in

contrast to the longer and tapered bristles seen in wild-type flies. The SbV mutant

involves a translocation between the second and third chromosome in which the Sb

allele is relocated next to heterochromatin and is sporadically silenced, causing variable

lengthening and tapering of some bristles; the resulting bristles can be designated as

wild-type or Sb (LLOYD et al. 1996). In this experiment, I examined and classified

fourteen major thoracic bristles as either Sb or wild-type; these data were then quantified

and expressed as a percent of fully mutant Sb (see the Materials and Methods). The

results are given in Table 3 and representative images are shown in Figure 22. Both

upSET alleles caused a clear cut dominant suppression of SbV, as evidenced by an

approximate doubling of the percent Sb bristles in experimental flies, in comparison with

control flies. Taken together, the results of my PEV experiments clearly demonstrate that

upSET can act as a Su(var) gene.

50

Table 3 Percentage of Stubble of upSET mutants carrying SbV.

Statistics t-test performed by comparing data from the same gender of the mutant class

to the control class. E.g. t-test of % Stubble of hypomorph mutant class (males) to %

Stubble of the control class (males). Mutant classes were obtained by crossing the

mutant females to males of the control class carrying SbV.

Genotype M/F % Stubble ± SEM P<0.05 (t-test) n

______SbV______

TM3 Ser

(Control)

M 30.4 ± 2 N/A 20

F 36.1 ± 2 N/A 20

upSETMB08950

SbV

(Hypomorph Mutant)

M 66.4 ± 4 1.00E-09 20

F 60.4 ± 3 5.80E-07 20

upSETe00365

SbV

(“Protein Null” Mutant)

M 64.3 ± 2 2.10E-12 20

F 60.4 ± 2 1.13E-09 20

51

Figure 22 Stubble variegation of upSET hypomorph carrying SbV. Photographs show a control class fly with <50% Stubble (left), and an upSET hypomorph fly (upSETMB08950/ SbV) with >50% Stubble (right).

52

Chapter 4. Discussion and Concluding Remarks

In my current work, I have demonstrated that the upSET gene is important for

viability i.e. RNAi-mediated knockdown of the gene reduces viability. However, unlike the

previous report from RINCON-ARANO et al. (2012), my data suggest that female fertility

is not affected. Our laboratory is currently addressing the issue of whether upSET is an

essential gene using deletion methodologies (e.g. P-element imprecise excision).

Interestingly, the P-element insertion allele designated as a protein null by RINCON-

ARANO et al. (2012) does not affect viability when crossed to deficiencies removing the

upSET gene. In addition, there is evidence that the recessive female sterility reported for

upSETe00365 (RINCON-ARANO et al. 2012) may be due to one or more second site

mutations on the chromosome bearing this allele. Since the developmental expression

profile for the upSET gene indicates a significant maternal contribution (Flybase; ST.

PIERRE et al. 2014), it may be necessary to construct UpSET germ-line clones in order

to define more precisely the extent of the requirements for the upSET product for

development and fertility. Nevertheless, my results support the notion that the upSET

gene is very important for normal functions in the fly.

My finding that the ubiquitous knockdown of upSET (i.e. down-regulation of

upSET in all cells) causes enhancement of Pc confirms the data of RINCON-ARANO et

al. (2012); my data are important because of the aforementioned concern that

upSETe00365 may also carry one or more second-site mutations. In other words, second-

site mutations may also enhance Pc. Interestingly, the cytological location of upSET,

70C11-70C12 overlaps with a deleted segment (70C6-70C15) that was previously

shown to enhance Pc (LANDECKER et al. 1994). It is unclear whether the enhancement

of Pc associated with down-regulation of upSET is direct, or indirect. If upSET acts

directly, does upSET function in a complex that mediates PcG repression or do

expanded domains of activated gene expression caused by down-regulation of upSET

impinge on domains of PcG repression? If it acts indirectly, does reduced upSET

function change the rate of development or do the expanded domains of gene

53

expression sequester one or more proteins required for PcG repression? It is noteworthy

that mutants for several putative components of a Drosophila SET3 complex (see below)

have been shown to interact with PcG members; for instance rpd3 (CHANG et al. 2001,

Flybase; ST. PIERRE et al. 2014) and Sir2 mutants (FURUYAMA et al. 2004, Flybase;

ST. PIERRE et al. 2014) enhance PcG phenotypes. Furthermore RPD3 reportedly co-

fractionates with PcG components (CHANG et al. 2001) and SIR2 associates with an

E(Z) Histone methyltransferase complex (FURUYAMA et al. 2004). On the other hand,

ebi mutants reportedly act as suppressors of PcG phenotypes; nevertheless, it has been

recently shown that EBI interacts with Pc-containing complexes (STRUBBE et al. 2011).

Clearly, it would be worthwhile to explore further the link between SET3 complex and

PcG functions.

I have shown that the SET domain of the fly UpSET protein is not modified by

OGT and that upSET does not affect global methylation levels of H3K4. However, it is

unclear whether OGT/SXC is capable of modifying Threonine or Serine residues outside

of the SET domain on UpSET. Nevertheless, my observations are consistent with

another in vitro study showing that the SET domain of UpSET alone is incapable of

transferring methyl moieties to H3 Lysine tails, and also it does not catalyze histone

methylation in the presence of the fly OGT (RINCON-ARANO et al. 2012).

It may not be surprising if it is conclusively shown that the SET domain of UpSET

on its own has no histone methyltransferase activity. MLL5 and upSET share little

homology with other annotated histone methyltransferases. In its entirety, MLL5 is only

38% identical to MLL1 (EMERLING et al. 2002) and it is only 29% identical to upSET

(CG9007). The MLL5 product is 1858 amino acids in length, whereas upSET contains

3238 amino acids. Yet, these two homologous genes are weakly conserved. However,

they do both encode a single PHD domain and a single SET domain. In both cases, it

appears that the SET domains are incapable of catalyzing histone methylation on their

own. In the case of MLL5, OGT modification of the SET domain is required for methyl

transferase activity (FUJIKI et al. 2009), and to my knowledge, the latter findings have

not been corroborated. The position of the PHD and SET domains within upSET and

MLL5 differs from that of other MLLs (Figure 1). Furthermore, A-T hooks that are known

to mediate DNA binding are conspicuously absent in both upSET and MLL5; since

54

UpSET binds to chromatin, it likely does so via interactions with other proteins, or

epigenetic marks. Indeed, the PHD finger of MLL5 is known to interact preferentially with

H3K4me3 (ALI et al. 2013). Given that H3K4me3 is generally associated with active

transcription, whereas H3K9me3 is usually associated with gene repression, it is not

surprising that MLL5 functions in promoter regions of actively transcribed regions

(RINCON-ARANO et al. 2012; ALI et al. 2013). Interestingly, the latter study showed that

the PHD finger of upSET binds to H3K4me3 with a ten-fold stronger affinity than that of

MLL5. Another unusual aspect of MLL5 is its PHD finger binding pocket. The crystal

structure of MLL5 bound to H3K4me3 revealed that its PHD finger contains a single

aromatic residue (W141) and an acidic residue (D128) (ALI et al 2013, LEMAK et al.

2013). Normally, PHD fingers accommodate H3K4me3 with a binding pocket comprising

2 to 4 aromatic residues (VAN INGEN et al. 2008, CHAMPAGNE and KUTATELADZE,

2009, ALI et al. 2013). The latter are usually positioned perpendicular to one another to

form an aromatic cage. In contrast, MLL5 contains a corresponding planar “cage” and

both W141 and D128 are essential for binding to H3K4me3. Reported binding affinities

for H3K4me2 are contradictory. ALI et al. reported that MLL5 PHD binds H3K4me3 five-

fold more strongly than it binds H3K4me2, whereas LEMAK et al. reported a two-fold

difference. Both cage residues are conserved in upSET, suggesting that the PHD finger

of upSET interacts with H3K4me3 in a similar fashion. In short, the findings to date seem

to suggest that upSET/MLL5 function primarily through their PHD domains in a non-

canonical binding mechanism.

55

Figure 23 Schematic showing possible roles of the UpSET SET domain

The function of the apparently catalytically inactive SET domain of upSET is a

mystery. Based on the current knowledge about proteins containing SET domains, I

propose two possible functions (Figure 23). One is that the SET domain of upSET (one

monomer) can function as self regulator by interacting with the SET domain of another

monomer. Self-association of SET domains has been reported for Drosophila

TRITHORAX and ASH1 proteins (ROZOVSKAIA et al. 2000). This self-oligomerization

mechanism is unique to SET proteins as others including Drosophila E(Z) and

SU(VAR)3-9 do not self-oligomerize.

Another possibility is that the SET domain of UpSET can interact with other

proteins to indirectly regulate gene expression. Thus, RINCON-ARANO et al. (2012)

have shown via co-immunoprecipitation (co-IP) from Kc nuclear extracts that UpSET

physically interacts with Rpd3 and Sin3A as part of a histone deacetylase complex

(HDAC). Their model suggests that UpSET together with Rpd3/Sin3A restricts histone

acetylation around promoter regions through interaction with the HDAC machinery. This

could explain my identification of UpSET as a suppressor of variegation, since the

Su(var) phenotype could reflect the fact that, when there is less upSET available, active

euchromatic chromatin can spread into the regions containing variegating genes,

56

thereby activating them (RINCON-ARANO 2013). Interestingly, gain-of-function alleles of

rpd3 also act as Su(var)s (MOTTUS et al. 2000). Furthermore, it has been reported that

the Sir2 gene, which codes for another putative Drosophila SET3 complex component, is

also required for PEV (ROSENBERG and PARKHURST 2002). Our laboratory is

currently exploring whether other putative Set3 components are also required for

heterochromatic silencing.

Figure 24 Schematic showing possible mechanism involving UpSET,

Rpd3/Sin3A, Ebi, and SMRTER

Most of the evidence I have discussed suggests that upSET (and the SET3

complex) has an overall repressive effect on gene expression. Thus far, it has been

established that UpSET and Rpd3/SIN3A interact physically (RINCON-ARANO et al.

2012, WANG et al. 2013). Future work is needed to elucidate whether UpSET interacts

directly with Ebi, SMRTER, and/or Sir2, or whether the interaction is primarily through

other HDAC1 components. Ebi does not interact directly with Rpd3 (also referred to as

HDAC1) (QI et al. 2008). However, it does interact with another histone deacetylase,

HDAC3, which interacts with a recombinant form of HDAC1 in S2 cells (BARLOW et al.

2001, QI et al. 2008). Another histone deacetylase to consider is Sir2, which is a

component of the SET3 complex (ROSENBERG and PARKHURST 2002). In yeast,

deletion of Rpd3 and Sin3 enhanced silencing (SUN and HAMPSEY 1999); this raises

the possibility that Rpd3/Sin3A is not solely responsible for catalyzing histone

57

deacetylation to restrict chromatin accessibility. It is plausible that another HDAC (e.g.

HDAC3 or Sir2) is involved in this process. I propose that UpSET functions as a reader

of active H3K4me3 marks via its PHD finger. This is somewhat analogous to the

observation that MLL5 yeast homolog Set3 binds H3K4me2 via its PHD finger, and

recruits HDAC components (KIM and BURATOWSKI 2009). Binding to H3K4me3

prompts the recruitment of Rpd3 and Sin3A (outlined in Figure 24). Upon recruitment,

UpSET also interacts with Ebi/SMRTER. Ebi recruits HDAC3 (or another histone

deacetylase e.g. Sir2) that work cooperatively with HDAC1 (Rpd3/Sin3A) to establish

histone deacetylation. One of these interactions might be established via the SET

domain of UpSET.

To elucidate whether any of the aforementioned SET3 complex components are

binding partners of upSET, one can carry out a pull-down experiment from fly nuclear

extracts using an antibody that targets upSET, followed by probing of these individual

components or by mass-spectrometry. As discussed earlier, some components of the

complex have effects on PEV while others have yet to be tested. The same components

reported have roles in PcG repression. It would be of great interest to see what might

happen in terms of PEV and Pc enhancement in double mutants of upSET and each of

these other components. Further, which of these components are essential for the

complex to function? Can ectopic expression of one substitute a mutation of another?

58

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Appendix A. List of flies used in this study

Table A1 List of all Drosophila melanogaster stocks used in this study, including genotypes, alternative labels (used to describe stocks), stock source, and their descriptions

upSET (CG9007) Mutants & RNAi Stocks

Genotype Alt. Label Stock Source Description

upSETRNAi-Random 1M – 9M - Self/B. Honda Lab (via BestGene Inc., order ID #8302)

upSET RNAi

upSETRNAi-attP2 - Self/B. Honda Lab (via BestGene Inc., order ID #10678)

upSET RNAi

w1118; PBac{RB}upSETe00365 /TM3 Sb Ser Act-GFP

upSETe00365 /TM3 Sb Ser Act-GFP

Hector Rincon-Arano from the Groudine Lab, U. of Washington (originally from Exelixis at Harvard Medical School)

upSET “protein null” mutant allele (BELLEN et al. 2004)

w1118; Mi{ET1}upSETMB08950

upSETMB08950 Bloomington stock center (#26139)

upSET hypomorphic mutant allele (BELLEN et al. 2004)

upSET (CG9007) Deficiency Stocks


w1118; Df(3L)ED4515, P{3'.RS5+3.3'}ED4515/TM6C, cu1 Sb1

upSETDf(3L)ED4515 /TM6C

Bloomington stock center (#9071)

upSET deficiency – deleted segment: 70C6--70C15 (RYDER et al. 2007)

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upSET deficiency – deleted segment: 70C6--70D2 (RYDER et al. 2007)




upSET deficiency – deleted segment: 70C11--70D3 (RYDER et al. 2007)

PEV Variegator Stocks


Dp(1;Y)BSCV, y+/Inscy BSV Bloomington stock center (#4298)

Variegating allele of B on Y chromosome (MOORE et al. 1979)

T(2;3)SbV, In(3R)Mo, Sb1 sr1/TM3, Ser1

SbV/TM3 Ser Bloomington stock center (#878)

Variegating allele of Sb (SINCLAIR et al. 1983)

In(1)wm4 wm4 Bloomington stock center (#807)

Vaiegating allele of w (SINCLAIR et al. 1983)

Gal4 Drivers


y1 w*; P{tubP-GAL4}LL7/TM3, Sb1

tub-Gal4/TM3 Sb


Expresses Gal4 in the pattern of αtub84B (LEE and LUO 1999)

UAS-Dicer2/UAS-Dicer2;tub-Gal4/TM3 Sb

- B. Honda Lab Expresses Gal4 in the pattern of αtub84B with overexpression of UAS-Dicer2

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y1 w*; P{Act5C-GAL4}25FO1/CyO, y+

Act5C/CyO Bloomington stock center (#4414)

Expresses Gal4 in the pattern of act5C (ITO et al. 1997)

Act5C/CyO;UAS-Dicer2/UAS-Dicer2

- Self/B. Honda Lab Expresses Gal4 in the pattern of act5C with overexpression of UAS-Dicer2

Act5C/CyO;Pc3/TM3 Sb Ser

- Self/B. Honda Lab Expresses Gal4 in the pattern of act5C under Pc3 mutant background (GINDHART and KAUFMAN 1995)

sxc1/CyRoi; Act5C/TM6B

- Teresa Stefanelli/B. Honda Lab

Expresses Gal4 in the pattern of act5C under sxc1 mutant background (GINDHART and KAUFMAN 1995)

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Appendix B. Sequences of upSET RNAi construct

Figure B1 Inverted Repeat sequence of the RNAi construct used to make upSETRNAi-Random 1M – 9M transgenic flies.

Spacer DNA from pGEM-Wiz is bolded.

AGCGTTCCCTTCTCGAAGTAGTGCTGCAACTCGGCATTTGGTCGGCAAGATCGA

CGCACGAATCGTGCATCGTTGCCATAGGTCCGCGTATCCACACACACCTCCGGA

CCCTTTAGGTAGGATGGTGGCTGCTGCGCAACTTGTGGCACACTTCCGTCCGGC

CGAAGAGTCTGCATGGGTGCCTCCACTCCGGGCAGCTGGTAAAAGAATACAAAC

TGTCCCGGAGTCTTGTGCGCCTTGAAGCTGTTAAGGTAGTTGCTCGGCGGCGGT

GTGTTCATGTTGACGGTGGGATTCTGGGTACGGAACTGGGTGGTCAGCATATAC

TTGCCGCGTAACTCGTGGATGGGGGCATGGGGCGACAAGTCAACGCTGCTTATT

AGCACCTTGGCGCCTGCATACGGAATAAGCTGTGCCCTCTGCTCAAGCTGCTCT

GCACTGCCAGCCGTGGAGAGTTGCTGTTGAATTTGCCGCAACGCCTTATTCTCC

GTGTTTTGTATGCTCTGCAATAGGCTAGGCTCTAGGTGAGTTTCTATTCGCAGTCGGCTGATCTGTGTGAAATCTTAATGAAGGGTCCAATTACCAATTTGAAACTCAGAGCCTAGCCTATTGCAGAGCATACAAAACACGGAGAATAAGGCGTTGCGGCAAA

TTCAACAGCAACTCTCCACGGCTGGCAGTGCAGAGCAGCTTGAGCAGAGGGCA

CAGCTTATTCCGTATGCAGGCGCCAAGGTGCTAATAAGCAGCGTTGACTTGTCG

CCCCATGCCCCCATCCACGAGTTACGCGGCAAGTATATGCTGACCACCCAGTTC

CGTACCCAGAATCCCACCGTCAACATGAACACACCGCCGCCGAGCAACTACCTT

AACAGCTTCAAGGCGCACAAGACTCCGGGACAGTTTGTATTCTTTTACCAGCTGC

CCGGAGTGGAGGCACCCATGCAGACTCTTCGGCCGGACGGAAGTGTGCCACAA

GTTGCGCAGCAGCCACCATCCTACCTAAAGGGTCCGGAGGTGTGTGTGGATAC

GCGGACCTATGGCAACGATGCACGATTCGTGCGTCGATCTTGCCGACCAAATGC

CGAGTTGCAGCACTACTTCGAGAAGGGAACGCT

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Figure B2 Inverted Repeat sequence of the RNAi construct used to make upSETRNAi-attP2 transgenic flies.

Spacer DNA from pGEM-Wiz is bolded.

GCGTCAATGATGACGTTGCTGGTGCTACCGTCCGTGGTCACAATCTTGTAGATC

TGGGGATTAATTAGCTCCTGACCGGAGCTCCCCGAAGAGGCTGCCCCCGCAGC

TAAAGCTCCGGTTCCGGCTGCTGTACGCACCGCGTACTTTTGCGACTGATTGAT

CGACTGAGACTGGCTGGCGCTAAGGATCTGGCGCGTACTTATCGACATGCGAC

GTTGCATGATGCGCAGTTGTGCCTGTGTGGACAAGCTGCTGCCATCGATGACGA

TATGAGGGGGTGGCGGCTGTGGCCCACTGGCTACTCCGATGCGATTTATGCTG

AGGTGAGTTTCTATTCGCAGTCGGCTGATCTGTGTGAAATCTTAATGAAGGGTCCAATTACCAATTTGAAACTCAGCTCAGCATAAATCGCATCGGAGTAGCCAGTGG

GCCACAGCCGCCACCCCCTCATATCGTCATCGATGGCAGCAGCTTGTCCACACA

GGCACAACTGCGCATCATGCAACGTCGCATGTCGATAAGTACGCGCCAGATCCT

TAGCGCCAGCCAGTCTCAGTCGATCAATCAGTCGCAAAAGTACGCGGTGCGTAC

AGCAGCCGGAACCGGAGCTTTAGCTGCGGGGGCAGCCTCTTCGGGGAGCTCC

GGTCAGGAGCTAATTAATCCCCAGATCTACAAGATTGTGACCACGGACGGTAGC

ACCAGCAACGTCATCATTGACGC

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Appendix C. List of Primers Designed for this Study

Table C1 List of Primers designed for my research project on upSET.

Primer Sequence Purpose

upSET FWD ATG CCC ATG TCC AGT CAC qPCR

upSET REV GGC TAC TCC GAT GCG ATT TA qPCR

upSET RNAi FWD

TCT AGA CAG CAT AAA TCG CAT CGG AG

PCR to make RNAi construct for upSETRNAi-attP2

upSET RNAi REV

TCT AGA GCG TCA ATG ATG ACG TTG

PCR to make RNAi construct for upSETRNAi-attP2

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Appendix D. Recipes of Protein Purification Buffers

Table D1 Recipe of GST- Protein Purification Sonication/Wash Buffer, pH 7.5

Stock Solutions used to make buffer (10 ml recipe) Volumes

50% Glycerol 2 ml

10% Triton X-100 0.1 ml

10X PBS 1 ml

1000 mM DTT 10 µl

1000 mM MgCl2 25 µl

5 U/µl DNAse 40 µl

10.0 mg/ml Lysozyme 200 µl

100X Protease inhibitor 100 µl

100 mM ZnSO4 10 µl

ddH2O 6.5 ml

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Table D2 Recipe of His6- Protein Purification Sonication & Wash Buffers, pH 8

Stock Solutions used (5 ml recipe) Sonic. Buffer Wash Buffer

NaH2PO4 H2O (137.99 g/mol) 0.034 g 0.034 g

NaCl (58.44 g/mol) 0.088 g 0.088 g

1 M imidazole 50 µl 100 µl

10% Triton X-100 50 µl 50 µl

10.0 mg/mL lysosyme 100 µl 0 µl

5 U/µl DNAse 20 µl 0 µl

(100%, 14.3 M) β-mercaptoethanol 3.5 µl 3.5 µl

50% glycerol 1 ml 1 ml

100x Protease inhibitor 50 µl 50 µl

ddH2O (approximate) 3.73 ml 3.73 ml

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