ROLE OF HSWI/SNF ASSOCIATED PRMT5 AND MSIN3A/HDAC IN THE CONTROL OF EXPRESSION AND CANCER

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the

Graduate School of The Ohio State University

By

Sharmistha Pal, M.Sc.

* * * * *

The Ohio State University

2007

Dissertation Committee: Approved by

Sa¨ıd Sif, Ph.D., Adviser Tsonwin Hai, Ph.D. Adviser Christoph Plass, Ph.D. Graduate Program in David Saffen, Ph.D. Molecular, Cellular and Developmental Biology

ABSTRACT

Gene expression is associated with dynamic changes in chromatin structure that allow activator and repressor proteins to access the highly compact DNA. The BRG1 and BRM based human SWI/SNF (hSWI/SNF) complexes represent one of the chro- matin remodeling activities, which have been linked to transcriptional activation as well as repression. hSWI/SNF complexes have been purified in association with co-repressor mSIN3/HDAC, but the role of mSIN3/HDAC containing hSWI/SNF complexes in gene repression is not well documented. The goal of this study is to understand the role of hSWI/SNF complexes in transcriptional repression, to identify other hSWI/SNF associated factor(s), and to elucidate their function in vivo.

The work presented here shows that both BRG1- and BRM-based hSWI/SNF complexes that contain mSIN3A/HDAC are associated with the protein , PRMT5, which methylates H3 and H4. Moreover, inter- action studies indicate that c- as well as mSIN3A and PRMT5 interact directly with BRG1, BRM, BAF57, and BAF45/INI1 subunits of hSWI/SNF complexes, and that the interaction of c-MYC and PRMT5/mSIN3A with hSWI/SNF is mutually exclusive. To understand the in vivo relevance of these interactions, expression of a c-MYC target gene, CARBAMOYL-PHOSPHATE SYNTHASE-ASPARTATE

CARBAMOYLTRANSFERASE-DIHYDROOROTASE (CAD) was analyzed in the presence of catalytically inactive BRG1 and HDAC inhibitor. The results presented in

ii chapter 2 show that BRG1 is involved in both transcriptional activation and repression of CAD. When BRG1 is in complex with c-MYC, it activates CAD transcription.

However, BRG1 complex containing PRMT5 and mSIN3A/HDAC is involved in re- pressing CAD expression. Further studies focused on characterizing the activity and function of the newly discovered hSWI/SNF associated factor, PRMT5. In vitro his- tone methyltransferase assays indicate that PRMT5 methylates histones H3 and H4, and the sites of methylation on the N-terminal tails are H3R8 and H4R3.

To address the role of PRMT5 in vivo, a cell line expressing antisense PRMT5 (AS-

PRMT5) was established. Characterization of the AS-PRMT5 cell line shows that

PRMT5 is critical to maintain normal cell growth and proliferation. Comparative mi- croarray analysis revealed that PRMT5 acts as a transcriptional repressor, because

227 were upregulated, while 43 genes were downregulated in AS-PRMT5 cells.

The genes repressed by PRMT5 included a number of tumor suppressors and cell cycle regulator genes.

To analyze the role of PRMT5 in transcription, expression of SUPPRESSOR

OF TUMORIGENECITY 7 (ST7 ), which was identified by microarray analysis, was studied. Chromatin immunoprecipitation assays show that PRMT5 recruitment to the promoter of ST7 correlates with symmetric dimethylation of H3R8 and H4R3 at the promoter. Moreover, reducing the levels of PRMT5 in NIH3T3 cells results in hypoproliferation, while overexpression of PRMT5 leads to hyperproliferation and anchorage independent growth. To further evaluate the role of PRMT5 in transfor- mation, expression of PRMT5 was analyzed in a panel of leukemia and lymphoma patient derived cell lines and mantle cell lymphoma (MCL) clinical samples. The re- sults from these experiments show that PRMT5 expression is elevated in cancer cells,

iii and that overexpression of PRMT5 correlates with increased global H3(Me2)R8 and

H4(Me2)R3 methylation, as well as ST7 promoter specific methylation. Furthermore, similar to the findings in NIH3T3 cells, knockdown of PRMT5 expression in the Raji

Burkitt’s lymphoma and JeKo mantle cell lymphoma cell lines resulted in decreased cell growth and proliferation.

Since PRMT5 expression was increased in cancer cells, the regulation of PRMT5 expression was studied in normal and transformed B cells. Analysis of PRMT5 mRNA levels show that despite elevated PRMT5 protein levels, PRMT5 mRNA expression was reduced in transformed cells suggesting that PRMT5 mRNA is efficiently trans- lated in cancer cells. Further analysis of micro RNAs that can bind to PRMT5

3’UTR indicated that miR-96 is involved in translational inhibition of PRMT5 .

This work shows that hSWI/SNF complexes are involved in both transcriptional activation and repression, and the latter is mediated through association with core- pressors such as mSIN3/HDAC and PRMT5. In addition, PRMT5 is an oncogene, which methylates H3R8 and H4R3, and modulates expression of tumor suppressors like ST7 to regulate cell growth and proliferation.

iv Dedicated to my parents

v ACKNOWLEDGMENTS

I would like to thank my advisor Dr. Sa¨ıd Sif for giving me the opportunity to work under his supervision. I felt that my time in graduate school has been a great learning experience, which would have not been possible without his constant and

timely help, guidance, encouragement, and support. He has always been there to discuss new challenges and ideas, and have given me enough freedom to develop my analytical skills. I have been really inspired by his passion and enthusiasm for science, and the stimulating intellectual conversations.

I would like to extend my gratitude to my committee members, Dr. Tsonwin Hai,

Dr. Christoph Plass and Dr. David Saffen for their time, help, and critical review of the dissertation thesis. I would like to thank Rommy, Li, and Sheethal for their help and support in the laboratory. You all have made this a great experience. I would like to acknowledge summer student Will, for his help with cloning and mammalian two hybrid experiments.

Finally, I would like to specially thank my family and friends. My parents have been my greatest emotional support all these years. Though they were far away, they gave me the strength to go through all the hardships and failures. I truly thank my husband, who has always been there for me. I appreciate the constant encouragement from my brother, sister-in-law, and my in-laws. I would like to express my deep

gratitude to my closest friend, Sunethra, who has helped me in all possible ways.

vi Lastly, I would like to thank my group of friends specially Antara and Suparna for their constant support and cheer.

vii VITA

April 15, 1977 ...... Born - Calcutta, West Bengal, India

June 1998 ...... B. Sc. (Honors) in Biochemistry, University of Delhi, India June 2000 ...... M. Sc. in Biochemistry, University of Delhi, India September 2000-present ...... Graduate Research Associate, The Ohio State University, Columbus, Ohio, USA

PUBLICATIONS

Dacwag CS., Ohkawa Y., Pal S., Sif S., Imbalzano AN. 2007. The protein arginine methyltransferase, PRMT5 is required for myogenesis because it facilitates ATP- dependent chromatin remodeling. Mol. Cell. Biol. 27:384-394.

Harikrishnan K, Pal S., Yarski M, Baker EK, Chow MZ, de Silva MG, Okabe J, Wang L, Jones PL, Sif S, El-Osta A. 2006. Reply to ”Testing for association between MeCP2 and the brahma-associated SWI/SNF chromatin-remodeling complex”. Nat Genet. 38:964-967.

Wang L., Baiocchi RA., Pal S., Mosialos G., Caliguiri M., Sif S. 2005. The BRG1 and hBRM-associated factor BAF57 induces apoptosis by stimulating expression of the cylindromatosis tumor suppressor gene. Mol. Cell. Biol. 25:7953-7965.

Harikrishnan KN1., Chow MZ1., Baker EK1., Pal S1., Bassal S., Brasacchio D., Wang L., Craig JM., Jones PL., Sif S., El-Osta A. 2005. Brahma links the SWI/SNF chromatin remodeling complex with MeCP2-dependent transcriptional silencing. Na- ture 37: 254-264. (1 These authors contributed equally to this work).

viii Pal S., Vishwanath SN, Edjument-Bromage H, Tempst P, Sif S. 2004. Human SWI/SNF-associated PRMT5 methylates histone H3 arginine 8 and negatively reg- ulates expression of ST7 and NM23 tumor suppressor genes. Mol. Cell. Biol. 24:9630-9645.

Pal S., Yun R, Datta A, Lacomis L, Edjument-Bromage H, Kumar J, Tempst. P, Sif S. 2003. mSin3A/HDAC2 and PRMT5-containing Brg1 complex is involved in transcriptional repression of the Myc target gene CAD. Mol. Cell. Biol. 23:7475-7487.

FIELDS OF STUDY

Major Field: Molecular, Cellular, and Developmental Biology

ix TABLE OF CONTENTS

Page

Abstract ...... ii

Dedication ...... v

Acknowledgments ...... vi

Vita ...... viii

List of Tables ...... xvi

List of Figures ...... xviii

Abbreviations ...... xxii

Chapters:

1. Introduction ...... 1

1.1 Chromatin remodeling complexes ...... 2 1.1.1 Bromodomain containing SWI/SNF remodeling complexes . 3 1.1.2 Chromodomain containing Mi-2 based NURD complexes . . 6 1.1.3 SANT domain containing ISWI remodeling complexes . . . 8 1.1.4 INO80 complex ...... 9 1.2 Histone acetyltransferases and deacetylases ...... 10 1.2.1 GNAT family ...... 11 1.2.2 MYST family ...... 12 1.2.3 p300/CBP ...... 13 1.2.4 Nuclear coactivator family ...... 14 1.2.5 family ...... 15 1.2.6 Histone Deacetylases ...... 15 1.3 Histone ...... 18

x 1.3.1 Lysine methyltransferases ...... 18 1.3.2 Arginine methyltransferases ...... 23 1.4 Histone Demethylation ...... 31 1.4.1 Lysine demethylases ...... 32 1.4.2 Arginine demethylase ...... 33 1.5 DNA methylation ...... 34 1.6 Other histone modifications ...... 37 1.7 Cross-talk between various chromatin modifications ...... 39 1.8 Chromatin modifying activities and cancer ...... 40 1.8.1 Chromatin remodeling proteins ...... 41 1.8.2 Histone acetylases and deacetylases ...... 41 1.8.3 Histone methyltransferases ...... 42 1.8.4 DNA methyltransferases ...... 44 1.9 Thesis overview ...... 44

2. SIN3A/HDAC2 and PRMT5 are associated with human SWI/SNF complexes and are involved in CAD transcriptional repression . 58

2.1 Abstract ...... 58 2.2 Introduction ...... 59 2.3 Materials and Methods ...... 61 2.3.1 Plasmid constructions, DNA digestion, PCR reaction, liga- tion, and transformation ...... 61 2.3.2 Coupled in vitro transcription and translation ...... 62 2.3.3 GST pull-down assay ...... 63 2.3.4 Co-in vitro translation and immunoprecipitation ...... 64 2.3.5 Site directed mutagenesis ...... 65 2.3.6 Expression of wild type and mutant FL-PRMT5 in Sf9 cells 66 2.3.7 Purification of flag-tagged recombinant PRMT5 and BRG1 and BRM based hSWI/SNF complexes and protein identifi- cation by mass spectrometry ...... 69 2.3.8 Mammalian-two hybrid experiment and transfection using Lipofectamine ...... 70 2.3.9 β-Galactosidase and Chloramphenicol acetyl (CAT) assays ...... 71 2.3.10 Histone methyltransferase assay ...... 73 2.3.11 MBP-MORF expression, purification and HAT assay . . . . 74 2.3.12 Acetylation of N-terminal histone peptides ...... 75 2.3.13 Histone deacetylation assay ...... 76 2.3.14 Northern blot analysis ...... 77 2.3.15 Radiolabeling of DNA probes ...... 79 2.3.16 Metabolic labeling and double immunoprecipitation assay . 80

xi 2.3.17 Chromatin immunoprecipitation assay ...... 81 2.3.18 Cell culture and transformation assay ...... 83 2.3.19 Antibodies and Western blot analysis ...... 84 2.4 Results ...... 85 2.4.1 mSIN3A and mSIN3B interact directly with BRG1, BRM, BAF57 and BAF45/INI1 hSWI/SNF subunits ...... 85 2.4.2 Protein arginine methyltransferase, PRMT5 is associated with flag-tagged BRG1-and BRM-based hSWI/SNF complexes . 86 2.4.3 PRMT5 interacts directly with components of hSWI/SNF complexes and mSIN3A ...... 88 2.4.4 PRMT5 interacts with mSIN3 and hSWI/SNF complex com- ponents through both N and C- terminal amino acid se- quences ...... 89 2.4.5 PRMT5 methylates histones H3 and H4 ...... 90 2.4.6 CAD expression is upregulated in the presence of catalyti- cally inactive BRG1 or HDAC inhibitor ...... 92 2.4.7 BRG1, mSIN3A/HDAC2, and PRMT5 are recruited to the CAD promoter ...... 93 2.4.8 c-MYC directly interacts with hSWI/SNF complexes that lack mSIN3A and PRMT5 ...... 95 2.4.9 Expression of catalytically inactive BRG1 or BRM inhibits NIH3T3 co-transformation by c-MYC and Ha-RAS . . . . . 97 2.5 Discussion ...... 98 2.5.1 Role of BRG1 based hSWI/SNF complex in repression . . . 99 2.5.2 hSWI/SNF complexes and histone deacetylation and methy- lation ...... 100 2.5.3 Role of hSWI/SNF complexes in cell growth ...... 101

3. Human SWI/SNF-associated PRMT5 methylates histones H3R8 as well as H4R3 and repress transcription of ST7 and NM23 tu- mor suppressor genes ...... 125

3.1 Abstract ...... 125 3.2 Introduction ...... 126 3.3 Materials and Methods ...... 128 3.3.1 Purification of flag-tagged INI1 complex ...... 128 3.3.2 Histone methyltransferase assay using HeLa core histone and histone N-terminal peptides as substrates ...... 128 3.3.3 Cell culture and establishment of NIH3T3/WT-PRMT5 and NIH3T3/AS-PRMT5 cell lines...... 129 3.3.4 Reverse transcriptase-polymerase chain reaction (RT-PCR) and microarray analysis ...... 129

xii 3.3.5 BrdU incorporation assay and cell cycle profile analysis . . . 132 3.3.6 Proliferation, and anchorage independent and dependent growth assays ...... 133 3.3.7 Dot blot analysis and chromatin immunoprecipitation (ChIP) assay ...... 134 3.4 Results ...... 135 3.4.1 Recombinant and hSWI/SNF-associated PRMT5 methylates H3R8 and H4R3 ...... 135 3.4.2 H3K9 and K14 acetylation interferes with PRMT5-mediated H3 methylation while H3R8 and H4R3 methylation do not inhibit acetylation ...... 137 3.4.3 Identification of genes regulated by PRMT5 ...... 138 3.4.4 PRMT5 stimulates cell proliferation and induces anchorage independent growth ...... 139 3.4.5 PRMT5 directly regulates expression of ST7 and NM23 . . 141 3.4.6 PRMT5 methylates H3R8 at the ST7 and NM23 promoter, and opposes H3K9 acetylation ...... 142 3.4.7 ST7 and NM23 tumor suppressors are differentially targeted by BRG1 and BRM- based hSWI/SNF complexes ...... 143 3.5 Discussion ...... 144 3.5.1 PRMT5 methylates specific arginine residues in H3 and H4 N-terminal tails ...... 145 3.5.2 PRMT5 regulates cell growth and proliferation by modulat- ing expression of ST7 and NM23 tumor suppressor genes . 147

4. PRMT5 overexpression is associated with global as well as ST7 promoter specific H3R8/H4R3 symmetric methylation in mantle cell lymphoma ...... 162

4.1 Abstract ...... 162 4.2 Introduction ...... 163 4.3 Materials and Methods ...... 165 4.3.1 Cell culture and B cell isolation ...... 165 4.3.2 B cell activation ...... 166 4.3.3 RIPA cell lysate preparation and Western blot analysis . . . 167 4.3.4 Immunofluorescence analysis ...... 167 4.3.5 5Azacytidine, depsipeptide and DRB treatment ...... 168 4.3.6 Real time RT-PCR ...... 169 4.3.7 Nuclear run on assay ...... 169 4.3.8 Genomic DNA isolation, construction of PRMT5 promoter driven luciferase plasmids and transfection of HeLa and JeKo cells ...... 171

xiii 4.3.9 Polyribosome profiling ...... 173 4.3.10 RNase protection assay (RPA) ...... 173 4.3.11 Construction of double stranded (ds) RNA and dsRNA trans- fection in JeKo and Raji lymphoma cells...... 175 4.3.12 Transfection of normal and transformed B lymphocytes, and luciferase assays ...... 177 4.3.13 In vitro transcription, capping, polyadenylation, and trans- lation ...... 178 4.3.14 ChIP assay ...... 179 4.3.15 Generation of lentiviral particles and infection of JeKo cells. 180 4.3.16 Statistical analysis ...... 181 4.4 Results ...... 182 4.4.1 PRMT5 is overexpressed and symmetric methylation of hi- stones H3R8 and H4R3 is increased in lymphoid cancer cell lines ...... 182 4.4.2 PRMT5 expression is not induced by cell proliferation . . . 183 4.4.3 PRMT5 mRNA level and stability are reduced in Mino and JeKo MCL cells ...... 184 4.4.4 Aberrant expression of miR-96 is associated with enhanced PRMT5 translation ...... 187 4.4.5 miR-96 can inhibit PRMT5 translation in vivo, and its bind- ing site is critical for translational regulation ...... 189 4.4.6 ST7 is repressed in lymphoid cancer cell lines that overex- press PRMT5 ...... 191 4.4.7 PRMT5 is overexpressed and its target gene, ST7 is repressed in MCL clinical samples ...... 192 4.4.8 Knocking down PRMT5 alters the growth characteristics of transformed lymphoid cell lines ...... 194 4.5 Discussion ...... 195 4.5.1 Reduced miR-96 expression is associated with enhanced PRMT5 translation in MCL ...... 196 4.5.2 PRMT5 overexpression enhances global H3R8 and H4R3 methy- lation, and is accompanied by ST7 suppression ...... 198 4.5.3 Role of PRMT5 in mantle cell lymphomagenesis ...... 200

5. Synopsis and future work ...... 228

Appendices:

A. Plasmid constructions ...... 235

xiv B. Primer sequences ...... 270

C. Antibodies ...... 283

D. Genes affected by knockdown of PRMT5 in NIH3T3 cells . . . . 285

Bibliography ...... 297

xv LIST OF TABLES

Table Page

1.1 Classification of HATs, and their expression and substrate specificity. 53

1.2 Classification of human Histone Deacetylase (HDAC) . . . . . 54

1.3 Classification of histone methyltransferases...... 54

1.4 Protein arginine methyltransferases are conserved in evolution. . . . . 55

1.5 Methylation of histone lysine and arginine residues by various methyl- transferase and their function...... 56

1.6 Known histone demethylases and their substrate specificity...... 57

A.1 pBluescript (KS+)-Ampr based plasmids ...... 235

A.2 pGex2TK-Ampr (Pharmacia) based plasmids for GST fusion protein expression ...... 241

A.3 pFastbac - Ampr (Gibco BRL) based plasmids for protein expression in Sf9 cells ...... 244

A.4 Retroviral expression plasmids (all plasmids carry - Ampr) for mam- malian expression...... 248

A.5 Lentiviral, pRRLsin IRES GFP based expression plasmids...... 252

A.6 pBXG1– Ampr based plasmids for Gal4 fusion protein expression. . . 255

A.7 pACT– Ampr (Promega) based plasmids for VP16 fusion protein ex- pression...... 258

xvi A.8 pGL2basic- Ampr (Promega) based plasmids for scoring promoter ac- tivity...... 259

A.9 pCMV-Luc- Ampr based plasmids for monitoring 3’UTR activity. . . 264

A.10 pEGFP C1- Kanr (Clontech) based plasmids for GFP fusion protein expression in mammalian cells...... 266

A.11 Reporter and normalization plasmids...... 268

A.12 Miscelleneous plasmids...... 268

B.1 Primers for RT-PCR and ChIP assays ...... 270

B.2 Primers used for cloning and mutagenesis ...... 277

C.1 List of antibodies and sources ...... 283

D.1 Genes up-regulated in anti-sense PRMT5 NIH3T3 using affymetrix high density expression arrays ...... 285

D.2 Genes down-regulated in anti-sense PRMT5 NIH3T3 using affymetrix high density expression arrays ...... 295

xvii LIST OF FIGURES

Figure Page

1.1 Nucleosome core particle...... 45

1.2 ATP-dependent chromatin remodeling complexes...... 46

1.3 Members of the Swi2/Snf2 superfamily...... 47

1.4 Covalent modifications of core histones...... 48

1.5 Reversible covalent modifications of lysine...... 49

1.6 Arginine methylation by the PRMT enzymes...... 50

1.7 Protein arginine methyltransferases expressed in humans...... 51

1.8 Cross-talk of various histone modifications...... 52

2.1 Both isoforms of mSIN3 can interact with hSWI/SNF subunits. . . . 102

2.2 Interaction of mSIN3 isoforms with hSWI/SNF components do not require BRG1 and BRM bromodomain or BAF57 HMG domain. . . . 104

2.3 Flag-tagged BRG1 and BRM complexes contain PRMT5...... 106

2.4 PRMT5 is associated with mSIN3A containing BRG1 and BRM com- plexes...... 107

2.5 PRMT5 can specifically interact with hSWI/SNF subunits and mSIN3A.109

2.6 PRMT5 interacts with hSWI/SNF components in vivo through both N and C-terminal regions...... 111

xviii 2.7 Recombinant and hSWI/SNF-associated PRMT5 can methylate his- tones H3 and H4...... 113

2.8 MBP-MORF acetylates histones H3 and H4 N-terminal peptides, which are deacetylated by hSWI/SNF complex...... 115

2.9 BRG1 chromatin remodeling and histone deacetylation are essential for efficient repression of the MYC target gene, CAD...... 117

2.10 Cell cycle dependent recruitment of wild type and mutant BRG1, mSin3A/HDAC2 and PRMT5 to the CAD promoter...... 119

2.11 c-MYC can directly interact with BRG1 and BRM complexes. . . . . 121

2.12 Expression of catalytically inactive BRG1 and BRM reduces the trans- forming activity of c-MYC and Ha-RAS...... 123

3.1 PRMT5 co-elutes with flag-tagged BAF45/INI1 complexes...... 150

3.2 Recombinant and hSWI/SNF-associated PRMT5 can specifically methy- late H3R8 and H4R3...... 152

3.3 Interplay of H3R8 and H4R3 methylation with histone N-terminal tail lysine acetylation...... 153

3.4 Characterization of sense and anti-sense (AS) PRMT5 cell lines. . . . 154

3.5 PRMT5 induces cell growth and proliferation...... 155

3.6 Overexpression of PRMT5 stimulates anchorage dependent and inde- pendent growth...... 157

3.7 BRG1 and BRM-associated PRMT5 is directly involved in transcrip- tional repression of ST7 and NM23...... 158

3.8 Symmetric H3R8 methylation and H3K9 acetylation at the ST7, NM23 and MYT1L promoter are inversely related...... 159

3.9 BRG1 and BRM are differentially recruited to methylated ST7 and NM23 promoters...... 160

xix 4.1 PRMT5 is overexpressed in lymphoma and leukemia cell lines. . . . . 202

4.2 B cell proliferation does not induce PRMT5 expression...... 204

4.3 Expression of PRMT5 is regulated at transcriptional level...... 205

4.4 AML1 sites in the promoter region of PRMT5 are critical for its activity.207

4.5 PRMT5 mRNA is more stable in B cells...... 209

4.6 PRMT5 mRNA is translated more efficiently in MCL cell lines. . . . 210

4.7 Differential expression of PRMT5-specific miRNAs in normal and trans- formed B cells...... 211

4.8 PRMT5 expression is downregulated by miR-96 in transformed B cells. 212

4.9 Effect of modified miR-96 and PRMT5 3’UTR on translation in vitro and in vivo...... 213

4.10 ST7 is repressed in transformed lymphoid cell lines...... 214

4.11 ST7 is silenced in lymphoid cell lines...... 215

4.12 Recruitment of BRG1 associated PRMT5 correlates with enhanced H3R8 and H4R3 methylation at ST7 promoter...... 216

4.13 PRMT5 mRNA expression in MCL clinical samples...... 217

4.14 MCL patients overexpress PRMT5 protein...... 218

4.15 Expression of miR-96 is reduced in MCL clinical samples 6 and 7. . . 220

4.16 Overexpression of PRMT5 correlates with ST7 silencing in MCL clin- ical samples...... 221

4.17 Recruitment of BRG1, PRMT5, and methylation of H3R8 and H4R3 at the ST7 promoter in MCL clinical samples...... 222

xx 4.18 Knocking down PRMT5 expression affects growth of transformed MCL JeKo cell...... 223

4.19 Comparison of cell cycle profile and BrdU incorporation between JeKo cells infected with either mock or AS-PRMT5 lentivirus...... 224

4.20 PRMT5 knockdown alters growth of Raji cells...... 225

4.21 PRMT5 is overexpressed in adherent cancer cell lines compared to immortalized cells...... 226

4.22 Effect of 5-Azacytidine on ST7 expression...... 227

5.1 PRMT5 localization is altered by the mutation of potential ERK2 phosphorylation sites and ERK2 phosphorylates PRMT5 in vitro. . . 233

xxi ABBREVIATIONS

A ...... Adenosine

aa ...... amino acid

Ac ...... Acetyl group

Amp ...... Ampicillin

APS ...... Ammonium Per Sulphate

AS-PRMT5 ...... Anti-sense PRMT5

ATP ...... Adenosine Triphosphate

βgal ...... β-galactosidase

BAF ...... BRG1- or BRM- associated factors

Bis ...... Bis-acrylamide bp ......

BrdU ...... 50 Bromodeoxyuridine

BRG1 ...... Brahma related gene 1

BRM ...... Brahma

BSA ...... Bovine serum albumin

Bsd ...... Blasticidin

xxii CAD ...... Carbamoyl-Phosphatesynthase- Aspartate CAT ...... Chloramphenicol Acetyl Transferase cDNA ...... Complementary DNA

ChIP ...... Chromatin Immunoprecipitation

Ci ...... Curies

CIP ...... Calf Intestinal Phosphatase cpm ...... counts per minute

Ctrl ...... Control

δ-NH2 ...... delta amino group

DBD ...... DNA Binding Domain

DEPC ...... Diethyl pyrocarbonate

Depsi ...... Depsipeptide

DMEM ...... Dulbecco’s Modified Eagle’s medium

DNA ...... Deoxyribonucleic acid dNTP ...... Deoxyribonucleoside 50-triphosphate

DTT ...... Dithiothreitol

-NH2 ...... epsilon amino group

EDTA ...... Ethylenediamine tetraacetic acid

EGFP ...... Eukaryotic Green Flourescent Protein

FBS ...... Fetal bovine serum

Fl ...... Flag tag

xxiii FL-PRMT5 ...... Flag-tagged PRMT5 g ...... gram

GST ...... Glutathione-S-transferase h ...... hour

H2A ...... Histone H2A

H2B ...... Histone H2B

H3 ...... Histone H3

H3(Me2)R8 ...... Symmetrically dimethylated arginine 8 on histone H3 H4 ...... Histone H4

H4(Me2)R3 ...... Symmetrically dimethylated arginine 3 on histone H4 HAT ...... Histone Acetyl transferase

HDAC ...... Histone Deacetylase

HEPES ...... N-2-hydroxyethylpiperazine-N0-2- ethanesulphonic acid HSV TK ...... Herpes Simplex Virus Thymidylate Ki- nase hSWI/SNF ...... human SWI/SNF complex

Hyg ...... Hygromycin

IPTG ...... Isopropyl β-D-thio-galactosidase

K ...... Lysine

Kan ...... Kanamycin

Kbp ...... Kilo base pair

xxiv KDa ...... Kilodalton

LB ...... Luria Broth

Luc ...... Luciferase gene

µg ...... Microgram

µL ...... Microliter m ...... Meter mA ...... Milliamp mM ...... Millimolar

M ...... Molar

MBP ...... Maltose Binding Protein min ...... Minute miR ...... micro RNA

MTA ...... Methyl-thio adenosine

Mut ...... Mutant

NaB ...... Sodium Butyrate

Neo ...... Neomycin

ng ...... Nanogram

nm ...... nanometer nM ...... Nanomolar

NM23 ...... Non-metastatic protein 23

No Ab ...... No Antibody

xxv NP-40 ...... Nonidet P-40

NUC ...... Nucleolin

OD ...... Optical density

ODC ...... Ornithine decarboxylase

ONPG ...... Ortho-Nitrophenyl-β-D- galactopyranoside PAGE ...... Polyacrylamide gel

PAH ...... Paired ampiphathic helix

PCR ...... Polymerase chain reaction

PI ...... Pre-immune pmol ...... Picomole

PMSF ...... Phenylmethylsulphonyl Fluoride

PRMT ...... Protein Arginine Methyltransferase

Puro ...... Puromycin

R ...... Arginine

RNA ...... Ribonucleic Acid

RNasin ...... Ribonuclease inhibitor rpm ...... Revolutions per minute

RT ...... Reverse transciptase

SAM ...... S-Adenosyl-L-Methionine sec ...... second

xxvi SD ...... Standard deviation

SDS ...... Sodium dodecyl sulphate

SDS-PAGE ...... Sodium dodecyl sulphate - polyacry- lamide gel SNF ...... Sucrose non-fermenting

ST7 ...... Suppressor of tumorigenecity 7

T ...... time

TAD ...... Transcriptional Activation Domain

TAE ...... Tris Acetate EDTA

TBE ...... Tris Borate EDTA

TEMED ...... N,N,N0,N0-tetramethylethylenediamine

TLC ...... Thin layer chromatography

Ub ...... Ubiquitin

UTR ...... Untranslated Region

ω-NH2 ...... omega amino group

WT ...... Wild type

xxvii CHAPTER 1

INTRODUCTION

Proper and controlled expression of genes and other nuclear processes like replica-

tion, DNA repair, and recombination is essential for normal cell growth, development and differentiation. Cells have evolved a variety of mechanisms to oversee the tight regulation of these processes. One of the challenges faced by a eukaryotic cell is the packaging of its genome. Genomic DNA is very long (2m) and needs to be compacted efficiently to fit in the nucleus. This is achieved by packaging the DNA into a highly ordered chromatin structure, which inhibits all processes involving DNA.

The basic unit of chromatin structure is a nucleosome, which contains 146 bp of

DNA wrapped around a histone core, comprised of two copies of histones H2A, H2B,

H3 and H4 (Figure 1.1) [173]. The nucleosomes are further compacted by association

with linker histone H1 and other non-histone proteins to form the higher order 30 nm filament structure of chromatin [97, 238]. Levels of DNA organization can be

visualized by staining DNA with basic dye, which show that chromatin is organized

as lightly stained regions called euchromatin and heavily stained loci referred to as

heterochromatin. Euchromatin is less compact and contains actively transcribed

genes, while densely stained heterochromatin, is more compact and contains DNA

regions that are transcriptionally silent including centromeres and telomeres.

1 A consequence of packaging DNA into chromatin is that DNA becomes inaccessible to cellular factors, thus inhibiting DNA based processes. To overcome this constraint, cells have developed various mechanisms to alter chromatin structure and maintain genomic fluidity. Chromatin structure is very dynamic and it changes during progres- sion through cell cycle, which is a highly regulated process. Three mechanisms have been discovered that modulate chromatin structure, (1) ATP-dependent chromatin remodeling; (2) Post-translational modification of histones; and (3) DNA methyla- tion at CpG rich sequences. The various proteins involved in modulating chromatin structure are described below.

1.1 Chromatin remodeling complexes

Chromatin has to be restructured to allow transcription, replication, repair, and recombination. Precise coordination of events involved in opening and closing chro- matin are crucial to maintain the normal chromatin structure. A number of proteins have been identified in eukaryotes that use the energy of ATP hydrolysis to non- covalently alter nucleosome structure, which in turn facilitates access to both tran- scriptional activator and repressor proteins. Chromatin remodeling proteins were

first identified in yeast by genetic screen, where their mutation affected mating type switching and sucrose fermentation [307]. Currently, a number of different chromatin remodeling proteins have been identified in yeast, Drosophila and humans, and they exist as multisubunit complexes in vivo (Figure 1.2) [261]. Central to each chromatin remodeling complex is an ATPase subunit, which binds and hydrolyzes ATP in a

2 DNA dependent manner. Nucleosome remodeling via ATP hydrolysis basically con- sists of disrupting DNA-histone contacts, by either sliding the histone octamer (nu- cleosome sliding) or displacing the histone octamer (histone transfer in trans), such that nucleosomal DNA becomes accessible to DNA binding factors [16]. The various chromatin remodeling complexes generate different remodeled chromatin structure and also show differences in substrate preference in vitro [1, 73, 200]. In vitro ex- periments have shown that ISWI-based complexes remodel nucleosomes by a sliding mechanism; while the BRG1-based SWI/SNF complex can catalyze both nucleosome sliding and histone transfer in trans [1, 160, 219]. Difference in substrate preference is reflected by the observations that ISWI-based NURF complex requires histone H4

N-terminal tails to remodel nucleosomes; whereas SWI/SNF complexes can remodel tailless nucleosomes [45, 111, 172]. In another instance, NURF and NURD activity is stimulated efficiently only by nucleosomes; whereas both naked and nucleosomal

DNA can stimulate SWI/SNF activity [287, 200]. Chromatin remodeling ATPases can be classified into five subfamilies based on the of their AT-

Pase domain and by the presence of either a bromodomain, two chromodomains, a

SANT domain, lack of all the above domains, or a 450-550 amino acid insertion in the

ATPase domain (Figure 1.3). Four of these subfamilies have been well characterized and they are described below.

1.1.1 Bromodomain containing SWI/SNF remodeling com- plexes

SWI2/SNF2 related ATPases possess a central ATPase domain and a C-terminal bromodomain, which includes 110 amino acid motif known to mediate protein-protein

3 interaction and bind acetylated histone lysine residues [178, 333]. SWI/SNF chro-

matin remodelers are highly conserved and are represented by the Swi2/Snf2-based

ySWI/SNF and Sth1-based RSC complexes in yeast. In Drosophila, the two BRM-

based dSWI/SNF complexes, BAP (Brahma associated proteins) and PBAP (Poly-

bromo associated BAP) are distinguished by the presence of OSA in BAP, and poly-

bromo and BAP170 in PBAP. Similarly in humans, the SWI/SNF complexes are

referred to as BAF (BRG1- or BRM- associated factors), and PBAF (polybromo

associated BAF), which are characterized by the presence of BAF250 and poly-

bromo/BAF180, respectively [191]. Studies in yeast have shown that despite the structural similarity between ySWI/SNF and RSC complexes, each complex per- forms different functions in vivo. While ySWI/SNF is not essential for viability and

regulates a small number of genes, RSC is more abundant and essential for cell vi-

ability [34, 272]. RSC is required for G2/M progression and it regulates expression

of ribosomal and cell wall proteins [204]. However, both ySWI/SNF and RSC com-

plexes are involved in double strand DNA break repair [38]. Similarly, Drosophila

BAP and PBAP complexes are involved in regulating distinct functions. This is evident from the immunolocalization of BAP specific subunit, OSA, and PBAP com-

ponent, polybromo, on polytene , which shows distinct but overlapping

distribution of the BAP and PBAP complexes [5, 190]. Knockdown of OSA using

RNAi in Drosophila S2 cells depletes BAP complex and this results in G2/M block,

while cells lacking PBAP show no cell cycle defects. Further studies show that BAP

complex controls cell cycle progression by directly regulating STRING/CDC25 ex-

pression, which is required for entry into mitosis [193]. The functional distinction of

human BAF and PBAF complexes is not clear, however, some functional differences

4 exist between BRG1 and BRM ATPases. Knockout studies in mice indicate that ho- mozygous deletion of BRG1 is lethal, while BRM deficient mice are viable and show increased body weight along with overexpression of BRG1 [31, 234, 274]. These ex- periments suggest that BRG1 and BRM ATPases serve distinct roles in vivo, and that

BRG1 can partially substitute for BRM in vivo. Also, analysis of BRG1 and BRM ex- pression in normal tissues shows that BRG1 is highly expressed in cells that undergo constant proliferation, while BRM expression is more prominent in non-proliferating cells [232]. In addition, BRM expression can be induced by serum starvation that does not affect BRG1 levels [197]. BRG1 and BRM are also differentially regulated by phosphorylation during the course of the cell cycle. During mitosis, phospho- rylation of BRM leads to its degradation while BRG1 phosphorylation renders it inactive [259]. Biochemical characterization of BRG1 and BRM-based hSWI/SNF complexes showed that both complexes can be separated by affinity chromatogra- phy, and that the BRG1- based hSWI/SNF complex can be further fractionated into two distinct complexes (BRG1 complexes I and II), which differ by the presence of mSIN3/HDAC and PRMT5 corepressor proteins in BRG1 complex I [259, 260, 214].

Chromatin remodeling assays showed that the three hSWI/SNF complexes remodel nucleosomes differently [260]. Although, BRG1 and BRM complexes can interact with common transcription factors like c-MYC and MYOD, reflecting redundancy in their functions, both complexes also exhibit specific interactions that differentially target them to cellular genes [214, 58, 138]. Through its N-terminal region BRG1 interacts specifically with zinc finger containing transcription factors like KLF, Sp1, GATA, and hormone receptors like RAR and RXR; while BRM can interact with CBF-1 and ICD22 ankyrin repeat proteins, which are involved in Notch signaling [138]. In

5 addition, BRG1 and BRM are targeted differently to the PRMT5 target genes, ST7 and NM23 -H1 [215]. Thus, though hSWI/SNF complexes show some redundancy in their function, they alter chromatin structure and regulate distinct pathways.

1.1.2 Chromodomain containing Mi-2 based NURD complexes

Chromodomain-containing ATPases (CHD, Chromo-Helicase/ATPase DNA bind- ing proteins) possess two N-terminal chromodomains, a central DNA dependent AT-

Pase domain and a C-terminal DNA binding motif (Figure 1.3). CHD proteins are evolutionary conserved and have been characterized in various organisms from yeast to mammals where they are involved in the regulation of normal development. In

S. pombe, mutants of the CHD protein, Hrp1, show a defective anaphase phenotype, as Hrp1 is required for proper chromatin condensation and chromosome segregation during mitosis [329]. In Drosophila, CHD mutants arrest in the first or second lar- val stage of development, suggesting that these ATPases are essential for normal fly development [145]. Genetic studies have established a link between the Drosophila

Mi-2 complex and hunchback, which can repress HOX [144]. Sim- ilarly, in C .elegans CHD proteins, LET-418 and CHD-3, play a crucial role during vulval development by antagonizing RAS signaling and preventing a multiple vulva phenotype, while in humans CHD4 has been shown to be important for normal T cell development [295, 305]. These results suggest that CHD proteins are involved in transcriptional repression during development.

In humans, five members of the CHD family (CHD 1-5) are known which are grouped into two classes. Class I (CHD 1 and 2) differs from class II (CHD 3, 4 and

5) by the absence of two copies of a PHD finger [280, 312]. CHD1 was identified as

6 a DNA binding protein with affinity for immunoglobulin promoters, and CHD2 was

discovered based on its homology to CHD1. CHD 3 and 4, also referred to as Mi-2 α

and β, were initially identified as autoantigens in dermatomyositis patients, whereas

CHD5, which is specifically expressed in the nervous system was discovered during mapping of chromosome 1p36.3, a region commonly deleted in neuroblastomas [280,

321, 337].

Of all the CHD proteins, CHD 3 and 4 have been purified as components of mul- tisubunit nucleosome remodeling and deacetylase (NURD) complexes [321, 337]. In addition to possessing DNA dependent chromatin remodeling activity attributed to

CHD 3 and 4 ATPases, NURD complexes can deacetylate histones and bind to methy- lated DNA. Apart from histone deacetylase proteins, NURD complexes also contain metastasis associated proteins, MTA1, 2, 3 and methyl-CpG binding proteins, MBD2 and 3 [27, 74, 88, 338]. Unlike Xenopus MBD3, the mammalian counterpart cannot bind methylated DNA. However, MBD2, which can bind methylated DNA, targets

NURD complex to remodel and deacetylate histones at methylated CpG islands. The

MBD2-containing NURD complex has been shown to associate with PRMT5, which symmetrically methylates MBD2 as well as histones at the promoter of NURD target genes and negatively regulates MBD2 activity and transcription [107, 278]. While both Mi-2α and Mi-2β -based NURD complexes have been implicated in transcrip- tional repression of cell cycle regulator genes, P14 ARF and P16 INK4a, both complexes are also differentially recruited in vivo [107, 136, 247]. For example, the KAP-1 corepressor represses transcription by recruiting the Mi-2α-based NURD complex, whereas the Mi-2β-based NURD complex is involved in repression of ROR-γ target

7 gene [247, 136]. Hence, NURD complexes combine different activities including chro- matin remodeling, binding to methylated DNA, histone deacetylation, and arginine methylation to efficiently repress transcription.

1.1.3 SANT domain containing ISWI remodeling complexes

ISWI (Imitation switch) chromatin remodeling complexes contain the highly con- served ISWI ATPase, which includes a C-terminal SANT domain found in a number

of transcriptional regulators like Swi3, Rsc8, Ada2, BAF155, and BAF170 (Figure

1.3) [28, 51]. The ISWI complexes were initially found in Drosophila embryo extracts

as NURF (Nucleosome remodeling factor), ACF (ATP dependent chromatin assembly

and remodeling factor) and CHRAC (Chromatin accessibility complex) complexes.

Currently ISWI complexes have been found in all organisms from yeast to mammals

(Figure 1.2) [132, 286, 287, 291]. Genetic studies in yeast have indicated that ISWI

complexes are involved in transcriptional repression of meiotic genes and mating type

locus. In Drosophila, localization of the ISWI protein on polytene chromosome shows a lack of colocalization with the bulk of RNA polymerase II, indicating that ISWI

complexes are involved in processes other than transcriptional activation [60]. In ad-

dition, genetic studies have shown that ISWI is required for expression of the GATA

target genes, Ultrabithorax (UBX) and (ENG), as well as formation of

higher order chromatin structure [10, 60]. In humans, the ISWI-based hACF/WCRF

complex is required for DNA replication through highly condensed heterochromatin

regions. RNAi depletion of ACF1 impairs replication of pericentromeric heterochro-

matin, while depletion of SNF2h decreases the rate of DNA replication [49]. Further-

more, in mouse, ISWI containing NoRC (Nucleolar remodeling complex) complex

8 represses transcription of rRNA genes by recruiting mSIN3/HDAC [271, 343]. Taken

together, it seems that ISWI complexes are involved in diverse functions including transcription, replication, chromatin assembly, and maintainence of high order chro- matin structure [62]. Further support for the essential role of ISWI complexes is provided by the observation that homozygous null mutation of the ISWI gene in

Drosophila, Xenopus, and mice but not in yeast is lethal, suggesting that ISWI com- plexes perform essential functions in higher multicellular organisms [62].

1.1.4 INO80 complex

Genetic screens for mutants that show defects in ICRE (Inositol/Choline Respon- sive Elements) dependent gene activation, identified Ino80 as a gene involved in the process, and is part of a large multisubunit complex [65]. The Ino80 complex pos- sesses chromatin remodeling activity as well as a 30→50 helicase activity. The purified

Ino80 complex contains 12 polypeptides, which include actin, actin related proteins

(Arp), and Ruv 1 and 2 proteins [254]. Ruv proteins are similar to the bacterial

RuvB protein, which recognizes holiday junctions during bacterial recombination.

Yeast cells that lack either Ino80 or one of the core subunits (Arp5 and Arp8) ex- hibit hypersensitivity to DNA damaging agents suggesting that the Ino80 complex is

involved in DNA repair [288, 192]. Furthermore, it has been shown that the Ino80 complex is recruited to double strand DNA break (DSB) at the HO locus through interaction with phosphorylated H2A, and that Ino80 chromatin remodeling activity

is required for the processing and repair of DSB. Thus, the Ino80 complex is involved

in regulating transcription as well as repair of DNA damage.

9 Why does a cell expresses and requires different complexes to remodel chromatin?

The answer lies in the various observations about the function and regulation of these complexes. Deletion of some of the remodeling proteins causes lethality indicating that these ATPases perform specific essential functions that can not be replaced by other remodeling proteins in the cell. This is supported by findings, which show that the Ino80 complex is involved in DNA repair, whereas ISWI complexes are required for chromatin assembly. Furthermore, distinct complexes target different cellular genes and their remodeling activities are differentially regulated as examplified by the regulation of BRG1- and BRM-based hSWI/SNF complexes during mitosis.

In addition, expression of chromatin remodeling proteins can be tissue-specific and differentiation state-dependent as in case of CHD5 which is expressed only in the nervous system, while BRM expression is enhanced in non-proliferating differentiated cells.

1.2 Histone acetyltransferases and deacetylases

Chromatin structure is highly dynamic and one way it is modulated is by covalent modification of histone tails (Figure 1.4). Histones are highly basic proteins that contain multiple lysine and arginine residues, which are post-translationally modified by acetylation, methylation, ubiquitination and sumoylation. Histone acetylation is catalyzed by histone acetyltransferases (HAT), which transfer an acetyl group from acetyl-CoA to the -NH2 group of lysine side chains (Figure 1.5). Lysine acetylation partly neutralizes the positive charge on histones, and this weakens histone-DNA and nucleosome-nucleosome interactions, resulting in increased accessibility to DNA in chromatin [77, 123]. Furthermore, biochemical studies have shown that acetylation

10 of histones increases accessibility of transcription factors to their DNA binding site and is linked to transcriptional activation [161, 293]. Since the discovery of histone

acetylation, a number of HATs have been identified in various organisms. HATs are classified as A-type or B-type enzymes based on their localization and substrate

specificity. A-type HATs are nuclear enzymes that acetylate nucleosomal histones

and are involved in transcriptional regulation. On the other hand, B-type HATs

are cytosolic and acetylate newly synthesized histones prior to their assembly into

nucleosomes. Based on the protein sequence homology, HATs are grouped into five

families with different substrate specificities (Table 1.1) [240, 269].

1.2.1 GNAT family

HATs like Gcn5, PCAF (p300/CREB binding protein associated factor), Hat1,

Elp3 and Hpa2 constitute the GNAT family. The histone acetyltransferase ac-

tivity of Gcn5, which functions as a transcriptional coactivator, was first charac-

terized in Tetrahymena thermophila Gcn5 homolog, p55 using in-gel activity as-

say [106, 154, 296]. Gcn5 is an evolutionarily conserved protein and homologs of

yeast Gcn5 have been identified in Drosophila and humans. In vitro studies using

recombinant protein show that Gcn5 acetylates free histones H3 and H4, but it is

unable to acetylate nucleosomal histones. In vivo Gcn5 exists as part of multisub-

unit complexes like the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex where it

can efficiently acetylate nucleosomes, suggesting that other proteins can modulate its

substrate specificity [241].

PCAF is a mammalian HAT that is highly related to Gcn5 and interacts with the

p300 and CBP coactivators. Unlike Gcn5, recombinant PCAF acetylates both free

11 and nucleosomal histones H3 and H4. PCAF functions as a transcriptional activator by acetylating histones as well as transcription factors including , MYOD, TFIIE,

and TFIIF, which stimulates the activity of transcription factors [269]. Knockout

studies in mice have indicated that homozygous deletion of GCN5, p300, and CBP

is lethal while PCAF deficient mice are normal [326, 325]. In addition to possess-

ing a HAT catalytic domain, Gcn5, PCAF, CBP, and p300 contain a C-terminal

bromodomain that mediates interaction with acetylated histones.

Hat1 is a B-type that acetylates newly synthesized histone H4 on lysine 12;

while Hat2 has been found in association with Hat1 in the nucleus where it acetylates

free histones [216]. Elp3 is the smallest subunit of the yeast transcription elongator

complex. Genetic studies have shown that Elp3 mutants exhibit slow growth and

gene activation, as well as hypersensitivity to salt and temperature changes [308].

1.2.2 MYST family

Another group of conserved histone acetyltransferases is represented by the MYST

family which has been named based on the founding members human MOZ, yeast

Ybf2/Sas3 and Sas2 (Somethingh about silencing 2 and 3), and TIP60 (Tat- interact-

ing protein 60). New members have been added to the family, which includes human

MORF (MOZ related factor) and HB01 (Histone acetyltransferase bound to ORC),

Drosophila MOF (Males absent on the first), and yeast Esa1 (Essential sas family

acetyltransferase 1) [269, 240]. MYST proteins are involved in diverse cellular pro-

cesses and function as component of multisubunit HAT complexes like SAGA, NuA3,

NuA4, and TIP60 complexes. Yeast Sas2 was found in a genetic screen of sir1 mutants

that retained mating ability, and was shown to be involved in silencing of the mating

12 type locus and telomeres. Sas3, a homolog of Sas2 is also involved in silencing of the mating type locus [230]. Esa1 another homolog of Sas2 was found to be essential for viability of yeast cells [46]. Further studies using temperature sensitive mutants of

Esa1 showed that it is required for cell cycle progression through G2/M. Drosophila

MOF mediates histone H4K16 acetylation and is required for X-chromosome dosage compensation [266]. Human MOZ is associated with leukemogenesis and is translo- cated with CBP and TIF2 in acute myeloid leukemia [23, 37]. MORF, a MOZ homolog, has been shown to acetylate free and nucleosomal histones [39]. Other human MYST proteins, TIP60 and HBO1 were identified by yeast two hybrid exper- iments as TAT interacting and an ORC (origin recognition complex) binding protein, respectively. TIP60 is involved in cell cycle progression, DNA repair, and apopto- sis [142, 244], and HBO1, which interacts with ORC1 and MCM2 is required for the assembly of the pre-replicative complex in humans and Xenopus [32, 130].

1.2.3 p300/CBP

p300 was identified through its ability to interact with E1A, while CBP (CREB

binding protein) was isolated as a CREB interactor. p300 and CBP are ubiqui-

tiously expressed and highly homologous proteins (80-90 % conserved), which act as

global coactivators [101]. Genetic studies have shown that homozygous null muta-

tion of either CBP or p300 is lethal, indicating that both p300 and CBP are essential

genes [209, 326]. In addition, in vitro studies have shown that both p300 and CBP acetylate all four free or nucleosomal histones, as well as a number of transcription fac- tors including p53, GATA, ACTR, SRC-1, and EKLF (erythroid kruppel-like factor).

13 p300 and CBP are involved in activating transcription mediated by tumor suppres- sor proteins like p53, BRCA1 and oncogenic transcription factors like c-FOS, c-JUN, and c-MYB. Furthermore, mutations in CBP as well as p300 have been associated with cancer indicating the importance of both proteins in normal cell growth and proliferation [101].

1.2.4 coactivator family

Histone acetylation has been shown to be important for hormone induced tran-

scriptional activation. The nuclear coactivator proteins, SRC1

(p160, steroid receptor coactivator 1), SRC2 (TIF2, transcription intermediary factor

2 or GRIP1, interacting protein 1), and SRC3 (acetyltrans-

ferase ACTR or p300/CBP interacting protein) constitute a different family of HAT

proteins [319]. These coactivator proteins interact with nuclear hormone receptors in

a ligand dependent manner and have been shown to harbor HAT activity, which is re-

quired for ligand-induced transactivation. SRC proteins contain an N-terminal DNA

binding and dimerization domain, a central nuclear receptor interacting domain, two

activation domains (AD1 and AD2), and a C-terminal HAT domain. Activation do-

main 1 mediates interaction with CBP, p300, and P/CAF, while activation domain 2

recruits PRMT1 and PRMT4 histone methyltransferases. SRC1, 2, and 3 have been

knocked out in mice individually with no effect on viability suggesting that there are

redundancies in SRC coactivator function [96, 302, 317, 318]. SRC1 null mice are

normal, while SRC2 null mice display low fertility, and SRC3-deficient mice show

retarded growth and decreased body mass. In addition, when both SRC1 and SRC2

are knocked out, most mice die after birth and the few survivors show resistance

14 to thyroid hormone indicating a role for SRC1 and 2 in thyroid receptor mediated gene expression. Furthermore, it has been shown that some hormone receptors show specificity towards one of the SRC coactivators. For instance, SRC1 enhances GR de- pendent gene expression, while ERα uses SRC3 as the coactivator [273]. The findings

that SRC coactivators recruit other HATs like p300 and CBP to hormone receptor

target genes, and that p300 and CBP can also interact directly with nuclear hor-

mone receptors, suggest that histone acetylation is important for hormone receptor

mediated gene expression.

1.2.5 Transcription factor family

The importance of histone acetylation in gene transcription is further supported

by the identification of HAT activity in general transcription factors (Table 1.1).

RNA polymerase II transcription intiation factor, TFIID, and RNA polymerase III

transcription initiation factor, TFIIIC, have been shown to acetylate histones [153,

188]. TFIID is a general transcription factor for RNA polymerase II that contains

TBP (TATA binding protein) and TBP associated factors (TAFs). TAF1 (TAFII 250)

is the 250 KDa subunit of TFIID, which acetylates histones H3 and H4. Similarly,

TFIIIC is a multisubunit complex that mediates transcriptional initiation by RNA

polymerase III, and three of the TFIIIC subunits (220 KDa, 110 Kda, and 90 KDa)

can acetylate histones H2A, H3, and H4.

1.2.6 Histone Deacetylases

HAT activity in general transcription factors as well as transcriptional coactiva-

tors provides a strong link between histone acetylation and gene activation. How-

ever, acetylation is reversed by histone deacetylases (HDACs), which remove acetyl

15 groups from histones to restore DNA-histone contacts and re-establish transcriptional repression. HDACs are evolutionarily conserved proteins found in all organisms from yeast to humans, and are classified into three distinct groups based on their similarity to yeast histone deacetylases Rpd3 (Class I), Hda1 (Class II), and Sir proteins (Class

III) (Table 1.2) [59, 105]. HDACs in yeast were identified by either genetic screens, or

during biochemical purification of HDAC complexes [68]. While Rpd3 was discovered

in a genetic screen for mutants with reduced potassium dependency, Sir mutants show

defective mating type switching, and Hda1 was found during purification of HDAC

activity.

In Drosophila, six distinct HDACs (DHDAC 1-4, X and DSIR2) are known, while

in mammals there are 18 different HDAC enzymes (HDAC 1-11, SIRT1-7), which

are grouped as Class I, II and III enzymes (Table 1.2) [79, 59]. Class I HDACs

are ubiquitously expressed and localized in nucleus, whereas expression of class II

HDACs is more tissue specific and they undergo nucleo-cytoplasmic shuttling in re-

sponse to external stimuli. For example, HDAC 5 and 7, which are nuclear in pro-

liferating myoblast, are relocalized to the cytoplasm upon induction of muscle cell

differentiation. Class I and II HDACs are inactive when they are expressed as re-

combinant proteins, suggesting that they require additional factors to be active. In

vivo, HDACs 1 and 2 have been isolated as components of protein complexes such

as SIN3, NURD and CoREST, which show deacetylase activity and are involved in

transcriptional silencing of target genes [321, 331, 336, 338]. Sequence specific DNA

binding transcription factors like MAD-MAX, MAD-MXI and unliganded nuclear re-

ceptor RXR-RAR interact with SIN3 or N-CoR/SMRT corepressor proteins, and thus

16 bring HDACs to target genes to deacetylate core histones and promote transcriptional repression [158, 119, 336, 120, 198].

Class III HDACs consists of the Sir2 family of proteins, which are different from the other two classes of histone deacetylases in that they require NAD+ to catalyze deacetylation of histones H3 and H4 [21]. Sir proteins in yeast are responsible for silencing in telomeres, rDNA, and mating-type loci. In yeast, Sir2 activity has been shown to increase the life span, which is measured by the number of cell divisions a mother cell undergoes before it dies. Mutation of Sir2 decreases life span by approxi- mately 50%; whereas overexpression of Sir2 causes a 30% increase in the longevity of yeast cells [139]. Work by the Guarente group showed that yeast cells grown in low glucose media (caloric restriction) have a longer life span, however, under low glucose condition Sir2 activity is enhanced, which inhibits recombination of rDNA repeats and extends life expectancy of yeast cells.

In Drosophila, SIR2 interacts with the E(Z) histone methyltransferase-containing complex, PRC2, and is required for polycomb mediated silencing [91]. In mammals, seven SIR proteins have been identified (SIRT 1-7), and SIRT1 is the most studied member (Table 1.2). While knockout of SIRT1 in mice shows that it is important for embryonic development, overexpression of SIRT1 in myoblasts inhibits muscle differ- entiation implying that SIRT1 affects muscle specific gene expression [181, 44, 89].

SIRT2 is cytoplasmic and its expression is cell cycle dependent because its levels are enhanced during mitosis [207, 64]. In contrast, SIRT3 is localized to the mito- chondrial matrix where proteolytic cleavage of its N-terminus renders it active [250].

Recent studies have shown that SIRT4 downregulates insulin secretion, while SIRT6 deficient mice are prone to DNA damage and exhibit genomic instability [113, 194].

17 However, currently it is unknown if Sir2 orthologs in mammals are involved in the regulation of longevity as observed in yeast.

Why does a cell need so many different HDACs? The answer to this question is provided by the study where inactivation of six different HDACs in yeast shows only a small overlap between the sets of genes controlled by each histone deacetylase [239].

Furthermore, the HDAC expression pattern in humans suggests that even though there is some redundancy in HDAC function, various HDACs regulate transcription of different set of genes during growth and differentiation.

1.3 Histone methyltransferases

Another modification found on histones is methylation of lysine and arginine residues and unlike acetylation, histone methylation can either activate or repress gene transcription (Figure 1.4). The transcriptional outcome of histone methylation depends on the residue modified and the status of methylation. Histone methylation involves transfer of a methyl group from the methyl donor S-adenosyl-L-methionine to either the -NH2 group of lysine or the ω- or δ-NH2 of arginine residues (Figures 1.5 and 1.6). Lysine can be modified by the addition of one, two, or three methyl groups, while arginine residues are either mono-methylated or di-methylated. A number of histone methyltransferases have been identified and they are primarily classified into either lysine-specific or arginine-specific methyltransferases (Table 1.3).

1.3.1 Lysine methyltransferases

Histone methyltransferases (HMTases) with specificity towards lysine residues can be classified into SET domain-containing or DOT1 like methylases (Table 1.3). The

SET domain is a conserved 130 amino acid motif first recognized in three Drosophila

18 position effect variegation modifier proteins, which include Suppressor of Variegation

3-9, Enhancer of Zeste, and Trithorax. SET domain methylases are classified into seven different families based on the similarity of the SET domain and the neighbor- ing motifs (Table 1.3) [61]. Drosophila SUV39 and fission yeast Clr4, are important for silencing of heterochromatin and in the case of Clr4 silencing of the mating type locus [134]. Mammalian homologs of SUV39 in mice and humans were shown to methylate histone H3 lysine 9 (H3K9) [229]. Methylation of H3K9 by SUV39 mem- bers is associated with the formation of heterochromatin and transcriptional silencing, whereas methylation of H3K4 by SET1 members mediates transcriptional activation

(Table 1.5) [61]. Unlike the SET family, little is known about the non-SET domain enzymes, yeast Dot1 and human DOT1L. Dot1 was identified in a genetic screen as a disrupter of telomeric silencing that methylates nucleosomal H3K79 [75, 205, 157].

Methylation of H3K79 has been shown to be important for marking boundaries of silencing by restricting the spread of Sir complexes and histone deacetylation, which establish transcriptional silencing [289, 206]. Further insight in the biological role of

H3K79 methylation came from studies, which showed that 53BP1 binds to methylated

H3K79 at damaged DNA sites through its tudor domain [129]. Taken together, these results suggest that H3K79 methylation has anti-silencing effects on gene expression and a role in DNA repair.

Histone lysine methylation and transcriptional repression

Methylation of H3K9, H3K27 and H4K20 is carried out by a number of differ- ent HMTases, and is involved in transcriptional repression and gene silencing (Table

1.5). In Drosophila, mutations of either SUV39 or SUV4-20 impairs heterochro- matin formation, indicating that tri-methylation of H3K9 and H4K20 by Su(var) 3-9

19 and Su(var) 4-20 HMTases, respectively, is important for the formation of repressive heterochromatin. Studies in yeast indicate that heterochromatin formation involves tri-methylation of deacetylated H3K9, which creates a binding site for the heterochro- matin binding protein1 (HP1). Binding of HP1 promotes spreading and propagation of H3K9 methylation as well as further HP1 binding [125]. In addition, studies in mice show that SUV4-20h1/ SUV4-20h2 can directly interact with HP1 protein and in the absence of SUV39h1/SUV39h2, the H4K20 tri-methylation mark is lost in pericentric regions suggesting that H4K20 tri-methylation follows H3K9 tri-methylation during the formation of hetrochromatin [151]. Recent studies in yeast have shown that the

RNAi machinery recruits the SUV39 homolog, Clr4, at the site of heterochromatin formation to mediate H3K9 methylation and is essential to initiate heterochromatin formation [294]. Heterochromatin provides an example wherein the RNAi pathway along with H3K9 and H4K20 methylation, and HP1 converge to establish epigenetic transcriptional silencing.

Unlike tri-methylation, mono- and di-methylation of H3K9 is restricted to euchro- matin region in mammals. A marked decrease in mono- and di-methylation of H3K9 in euchromatin is observed in mice that are deficient in either G9a or the G9a related protein, GLP [276, 277]. These results suggest that H3K9 methylation in euchro- matin is mostly mediated by G9a and GLP HMTases. Moreover, H3K9 methylation by G9a, GLP, Suv39h1 and Suv39h2 has been linked to transcriptional repression of specific genes like CYCLINE and A2 .

20 Another lysine methylation that has been well studied is methylation of H3K27 by the polycomb group (PcG) protein EZH2, which is associated with transcriptional si- lencing of homeotic genes (HOX) and X- chromosome inactivation. EZH2 is a compo- nent of polycomb repressive complex 2 (PRC2) that also includes embryonic ectoderm development protein (EED), suppressor of zeste 12 (SUZ12), and retinoblastoma asso- ciated protein 48 (RbAp48). Studies in Drosophila have shown that PRC2 complex is recruited to the Ultrabithorax (UBX) promoter through interactions with Pleio- homeotic (PHO) and Pleiohomeotic-like (PHOL), which bind to PRE sites (Polycomb

Response Element), and methylates H3K27 [299]. Methylated H3K27 is recognized and bound by the PRC1 complex through the chromodomain of the Polycomb (Pc) protein, establishing transcriptional silencing of the UBX homeotic gene [36, 299].

Similarly, X-chromosome inactivation also requires H3K27 tri-methylation as loss of

EED, which is required for EZH2-mediated methylation, results in defective inacti- vation of X-chromosome. Recruitment of PRC2, which is dependent on XIST (X-

Inactivation specific transcript) RNA occurs during the initiation of X- chromosome inactivation and is correlated with methylation of H3K27 [141, 220].

Histone lysine methylation and transcriptional activation

Besides mediating transcriptional repression and silencing, lysine methylation has been linked to transcriptional activation through histones H3K4, H3K36 and H3K79 methylation (Table 1.5). In S.cerevisiae, both Set1 and Set2 HMTases, which medi- ate H3K4 and H3K36 methylation, respectively, are found associated with the elon- gating RNA polymerase II (RNAP II) linking methylation of these sites to active transcription. H3K4 mono-, di- and tri-methylation are localized to active chromatin

21 with tri-methylation enriched at the 50 end of genes, while di-methylation is pre- dominantly found in the middle, and mono-methylation peaks at the 30 end of the

transcribed gene [222]. Similar to the case of methylated H3K9 and H3K27 repres-

sive marks, methylated H3K4 is bound by the methyl-binding protein, Chd1 in yeast,

and WDR5 in mammals [224, 313]. Chd1 is a component of the SAGA (Spt-Ada-

Gcn5 Acetyltransferase) HAT complex and it binds to methylated H3K4 through its

chromodomain, thereby recruiting HAT activity to help potentiate transcription. In

mammals, WDR5 (WD40 repeat domain containing protein 5) is a component of the

MLL1 (Mixed Lineage Leukemia gene 1) complex, which possesses H3K4 methylase

activity. WDR5 binds to mono- as well as di-methylated H3K4 and promotes tri-

methylation of H3K4, while knockdown of WDR5 decreases tri-methylation of H3K4

and interferes with transcriptional activation. Unlike H3K4 methylation, little is

known about the role of H3K36 and H3K79 methylation in transcriptional activation.

The histone methyltransferase Set2 is associated with elongating RNAP II through-

out the body of the gene during transcription, indicating that H3K36 methylation

might be a mark for transcriptional elongation [152, 227]. In the case of H3K79

methylation, it has been shown in S.cerevisiae that ubiquitination of H2BK123 is re-

quired for efficient methylation of H3K79 by Dot1 [205]. H2B ubiqutination has been

linked with transcriptional activation, and prior requirement of H2BK123 ubiquiti-

nation for H3K79 methylation couples these modifications to transcriptional activa-

tion [121, 143, 205, 314]. Additionally, H3K79 methylation has been associated with

upregulation of HOXA9 and HOXA5 expression by the MLL-AF10 and CALM-AF10

fusion proteins, respectively, in mixed lineage leukemia (MLL) [210, 211].

22 1.3.2 Arginine methyltransferases

Methylation of arginine in cellular proteins has been known for a long time, but

the enzymes that catalyze these modifications (PRMT) have only been identified in

the past decade [17, 140, 233]. To identify endogenous proteins containing methy-

lated , Stephane Richard’s group has generated antibodies ASYM 24, ASYM

25, SYM10, and SYM11 that can recognize peptides containing either asymmetri- cally or s ymmetrically di-methylated arginine residues in the context of the RG motif. Immunoprecipitation experiments performed using HeLaS3 extracts identified approximately 200 proteins that are involved in diverse processes like RNA process- ing, transcription, signal transduction and DNA repair, highlighting the role played by protein arginine methyltransferases (PRMTs) in various cellular pathways [22].

Despite this large number of target proteins, the list of PRMT substrates is only partial, because proteins that are methylated at non-RG motifs like histones were not identified using this assay.

PRMTs are evolutionarily conserved and are found in various organisms from yeast

to mammals (Table 1.4). PRMTs are classified either as type I, II, or III based on the

nature of the methylation produced (Figure 1.6, Table 1.3). Type I PRMTs catalyze

ω-NG -monomethylation and ω-NG, NG-asymmetric dimethylation (ADMA), type II

0 form ω-NG -monomethylation and ω-NG, NG -symmetric di-methylation (SDMA), and

type III enzymes generate δ-NG- mono-methylated arginines. To date, eleven PRMTs

have been discovered in humans, and except for PRMTs 2, 10, and 11, all PRMTs

have been shown to catalyze arginine methylation, with PRMTs 1, 4, 5, 7, 8 and 9 also

methylating histones (Figure 1.7 and Table 1.4). PRMTs 1, 3, 4, 6, and 8 are type I

PRMTs, while PRMTs 5, 7, and 9 catalyze reactions carried out by type II enzymes.

23 In S.cerevisiae, there are three PRMTs and Rmt1 (Hmt1) is the predominant arginine methyltransferase because in yeast cells lacking Rmt1 asymmetric di-methylation is

reduced to less than 15% and mono-methylation is reduced to 30% [95]. Rmt2 is

the only known type III PRMT, whereas Hsl7 and Skb1 represent type II PRMT in

S.cerevisiae and S. pombe, respectively [344, 174]. Similarly, A. nidulans has three

PRMTs that methylate histones in vitro, whereas in Drosophila nine PRMTs have

been found that are homologous to mammalian PRMTs (Table 1.4) [282, 25].

The crystal structure of the rat PRMT3 catalytic domain shows that there are

two domains, a 5-adenosyl-methionine binding domain (contains motif I, post I, II,

III) and a barrel-like domain. The catalytic site is situated between the two domains

and the amino acid residues, two glutamate, one histidine, and one aspartate that

participate in the methylation reaction are conserved in the PRMT family [340].

All PRMTs contain a conserved catalytic core domain, and differ in their N- and

C- terminal regions (Figure 1.7). Different studies have revealed that PRMTs are

involved in regulating different aspects of cellular metabolism like signaling, cytokine

production, differentiation, transcription, RNA processing, and nucleocytoplasmic

transport. The function of each human PRMT and its homolog is described below.

PRMT1

Yeast Rmt1 is recruited to transcriptionally active genes during transcriptional

elongation, and its activity is important for heterogenous nuclear ribonucleoprotein

(hRNP) mediated mRNA processing and export. This is evident from the loss of

Rmt1, which results in accumulation of mRNA transcript in the nucleus and slows

the process of mRNA maturation and export, because methylation of the hRNPs,

Np13 and Hrp1 is required for their nuclear export [253, 332].

24 In humans, PRMT1 was identified by homology to yeast Hmt1/Rmt1, and it was shown that PRMT1 can methylate yeast hRNPs, Np13 and hnRNAP1, and sup- press lethality caused by Rmt1 /Np13 double mutation [251]. PRMT1 methylated substrate analysis indicate that PRMT1 is a type I enzyme that preferentially methy- lates arginine residues at RGG and RGRG consensus sites. Homozygous deletion of

PRMT1 causes embryonic lethality at E6.5 stage, though PRMT1 is not required for the viability of PRMT1 ES cells. PRMT1 null cells retain only 16% of total PRMT activity and 46% of asymmetric arginine methylation [279, 217]. These findings indi- cate that PRMT1 is an essential gene and the predominant arginine methyltransferase in the cell. PRMT1, which methylates H4R3 is involved in diverse processes like tran- scriptional activation, protein localization, and signal transduction [270, 297]. While

PRMT1 interacts with the p160 family of nuclear receptor coactivators and facilitates transcription driven by through H4R3 methylation; PRMT1 also enhances gene transcription indirectly. For example, PRMT1 positively modulates cytokine production from Th cells by methylating N-terminal arginines in NIP45

(NFAT interacting nuclear protein 45) [196, 297]. Methylated NIP-45 heterodimer- izes with NFAT and enhances NFAT driven cytokine production upon TCR signal- ing [196]. In addition, PRMT1 is also required for the antiviral and anti-proliferative effect of IFNα/β, where PRMT1 modulates IFNα/β signaling by methylating STAT1 and inducing transcription of IFNα/β responsive genes [3, 195]. IFNα/β induce gene transcription through the transcription factor STAT1, which is sequestered in the nu- cleus by inhibitor PIAS1 unless STAT1 is methylated at the conserved Arg31 residue.

25 Furthermore, PRMT1 regulates protein localization in the cell, for instance, methy- lation of Sam68, a RNA binding protein, and RNA helicase A by PRMT1 is required for their nuclear localization [52, 267].

PRMT2

This protein differs from other members of the PRMT family by the presence of a

SH3 (Src homology) domain at the N-terminus (Figure 1.7). PRMT2 was tested in vitro for its ability to methylate Rmt1 substrates such as Np13, hnRNAP1 and MBP.

PRMT2 was unable to methylate any of these substrates, and unlike PRMT1, it is un- able to substitute for Hmt1 and rescue Rmt1/NP13 double mutants [251]. Although the methyltransferase activity of PRMT2 has not been demonstrated, PRMT2 has been implicated in transcriptional regulation in vivo. PRMT2 is unable to stimulate transcription as a Gal4-PRMT2 fusion, but it is able to bind a number of hormone receptors including ERα, ERβ, PR, TRβ, and RARα in a ligand independent man- ner and coactivate transcription [226]. In contrast, PRMT2 interacts directly with

RB and represses the transcriptional activity of by forming a ternary com- plex with E2F1 through RB [330]. Additionally, PRMT2 inhibits NF-κB dependent transcription and promotes apoptosis indirectly by retaining IκBα in the nucleus [93].

PRMT3

It is the only cytosolic type I enzyme containing an N-terminal zinc finger do- main that confers substrate specificity (Figure 1.7). PRMT3 methylates proteins in

Rat1a extracts, which is inhibited by RNA suggesting that PRMT3 targets RNA binding protein [83]. In vitro PRMT3 can methylate GST-GAR (GST fusion protein

26 containing glycine and arginine rich N-terminal region of fibrillar), Sam68 (Src associ-

ated mitosis protein 68KDa), and PABP-2 (polyA- binding protein N1), while in vivo

PRMT3 has been shown to associate with 40S, 60S, and 80S ribosomes and methylate

the rpS2 (ribosomal protein S2) 40S protein [8, 265]. Using various deletions of rpS2,

it was shown that PRMT3 could methylate RG repeats in the N-terminus of rpS2,

and disruption of the PRMT3 gene in S. pombe resulted in viable cells with an in-

creased ratio of free 60S ribosome subunit, suggesting a role for PRMT3 in ribosomal assembly [8, 275]. Additionally, PRMT3 also interacts with the tumor suppressor

DAL-1/4.1B (differentially expressed in adenocarcinoma of the lung), which nega- tively modulates PRMT3 activity [263].

PRMT4

Also known as CARM1 (Coactivator Associated Arginine Methyltransferase 1),

PRMT4 was identified in a yeast two-hybrid screen designed to identify proteins that interact with the AD2 region of p160 coactivator [248]. Other studies showed that

PRMT4 interacts and methylates transcriptional coactivators like p300 and GRIP1.

PRMT4 has also been shown to methylate both N-terminal (R2, 17, and 26) and

C-terminal (R128, 129, 131, and 134) arginines in histone H3, and enhance GRIP1

mediated as well as driven transcriptional activation [40, 13]. Fur-

thermore, genetic studies indicate that PRMT4 is an essential gene because knockout

mice undergo normal development but die at the perinatal stage. At stage 12.5 days,

PRMT4 -/- embryos look normal and by stage 18.5 to 19.5 days they appear 60%

smaller compared to normal embryos. MEFs isolated from PRMT4 -/- 12.5 days

embryos do not exhibit any methylation of PRMT4 substrates like PABP1, p300,

and histone H3R17. In addition, PRMT4 -/- MEFs are defective in activation of

27 estrogen responsive genes [322]. PRMT4 is also required for normal T cell develop-

ment and muscle differentiation [146, 41]. Using a cell line expressing flag-tagged

PRMT4, a multisubunit complex named ”NUMAC” (nucleosomal methylation acti- vator complex) was purified, which contains subunits of the BRG1-based hSWI/SNF

remodeling complex [320]. These studies indicate that PRMT4 promotes transcrip-

tional activation by methylating coactivators as well as histone H3.

PRMT5

PRMT5 homolog in S. pombe, Skb1 is a Shk1 kinase binding protein, which is an

important component of the Ras and Cdc42 dependent signaling cascade that controls

cell viability and morphology [98]. Skb1 null mutants are less elongate, while over-

expression of Skb1 results in hyperelongated cells compared to wild type cells. The

Skb1 null phenotype is rescued by overexpression of the Shk1 kinase suggesting that

PRMT5 is a positive modulator of Shk1 kinase activity. Additionally, Skb1 has been

shown to inhibit mitosis through cdc2, and is involved in maintaining cell polarity as

well as mediating hyperosmotic stress response [9, 304, 99]. As work on skb1 pro-

gressed, others were able to identify PRMT5 in a yeast two hybrid experiment as Jak2

interacting protein highlighting a role for PRMT5 in signaling [223]. PRMT5 was

also implicated in somatostatin receptor (SSTR1) signaling, because both PRMT5

and SSTR1 were found to colocalize in human embryonic kidney cells [249].

Biochemical characterization has revealed that PRMT5 is a type II methyltrans-

ferase that symmetrically methylates histones H3 and H4 along with other cellular

proteins like MBD2 and the Sm proteins, SmD1 and SmD3 [4, 29, 107, 214, 215, 278].

In vivo, PRMT5 has been found in multiple complexes where it mediates diverse

functions including RNA processing, transcriptional regulation, and muscle as well as

28 germ line differentiation [4, 55, 71, 107, 214, 215]. As a component of the 20S methy-

losome complex, PRMT5 mediates methylation of Sm D1 and Sm D3 proteins at the

RG domains. Once methylated these proteins can interact with other components

to promote assembly of the small nuclear riboprotein particles (UsnRNPs), which

are involved in pre-mRNA splicing [185, 85]. Another way PRMT5 participates in

RNA processing is through its association with fibrillarin (FIB), the RNA methyl- transferase found in the nucleolus and cajal bodies, which is involved in pre-rRNA processing and snRNP biogenesis. Despite its interaction with FIB, the function of PRMT5 in this complex remains unknown. PRMT5 has also been purified in association with the chromatin remodeling complexes, hSWI/SNF and NURD, and has been implicated in transcriptional repression of cell cycle regulator and tumor

suppressor genes [214, 215, 107]. PRMT5 represses transcription by methylating histones at the promoter regions of the MYC target gene CAD, cell cycle regula- tors CYCLINE, P14 ARF , and P16 INK4a , and the tumor suppressor genes, ST7 and

NM23 [71, 107, 214, 215]. The work in this thesis demonstrates the association of

PRMT5 with hSWI/SNF complexes and its role in transcriptional repression of MYC target gene, CAD and tumor supressor genes, ST7 and NM23 through methylation of histones H3R8 and H4R3. Another way PRMT5 interferes with gene expression is by methylating transcription elongation factor, SPT5, which inhibits transcriptional elongation as seen on IκBα and IL-8 promoters [156].

To understand the in vivo function of PRMT5, studies were performed using cell

lines that either overexpressed or had reduced levels of PRMT5. Overexpression of

PRMT5 in immortalized fibroblast results in increased cell proliferation and anchor-

age independent growth, indicating that PRMT5 is an oncogene [215]. In agreement

29 with this result, PRMT5 is over-expressed in gastric carcinoma, and reducing PRMT5 expression slows down cell growth and proliferation [108, 215]. All together these studies suggest that PRMT5 regulates normal cell growth by modulating expression of cell cycle regulators and tumor suppressor genes.

PRMT6

PRMT6 is another type I methyltransferase that was characterized based on its activity towards GST-GAR and NPl3 in vitro. PRMT6 is the only PRMT family

member that undergoes auto-methylation [84]. Cellular targets of PRMT6 include

HMGA1a, HMGA1b, DNA polymerase β, and HIV TAT [69, 187, 25, 252]. PRMT6 methylates TAT at the arginine rich transactivation motif (ARM), and reduces its transactivation ability, which impairs HIV replication. The effect of methylation on HMGA function is not known, but methylation of DNA polymerase β, which is involved in DNA excision repair, enhances its polymerase activity by increasing DNA binding and processivity.

PRMT7

PRMT7 contains two copies of the S-adenosyl-methionine (SAM) binding domain

and deletion of either SAM binding motif leads to loss of methylase activity (Figure

1.7). PRMT7 is a type II PRMT enzyme that methylates myelin basic protein, H2A,

and H4 [163]. Recent work has shown that PRMT7 activity is enhanced through

interaction with the testis specific factor, CTCFL. PRMT7 mediates H4R3 methy-

lation at the H19 and GTL2 loci in embryonic male germ cells contributing to the

imprinting of H19 and GTL2 gene [133].

30 PRMT8

PRMT8 is unique because it is the first PRMT member that is expressed exclu-

sively in brain, and is localized to the plasma membrane. PRMT8 is very similar to

PRMT1 except at the N-terminus where it carries a myristoylation signal that local-

izes it to the plasma membrane [162]. The physiological role of PRMT8 is currently

unknown.

PRMT9

Also known as FBXO11, it is the most recent member of the PRMT family, which

belongs to the type II class of enzymes [50]. Four PRMT9 splice variants have been identified, and the smallest isoform 4 has been shown to possess methyltransferase

activity. Structurally, PRMT9 is distinct from the other PRMTs, and it is able to

methylate histones H2A and H4. The biological function of PRMT9 is yet to be

determined.

PRMT 10 and 11

PRMT 10 and 11 are the most recent members of the PRMT family that were

identified based on their homology to PRMT7 and PRMT9, respectively [109]. Like

PRMT7, PRMT10 contains a second pseudo catalytic domain at the C-terminus.

Methyltransferase activity has not been demonstrated for either PRMT10 or PRMT11,

and their role in vivo is unknown.

1.4 Histone Demethylation

Since the discovery of histone methyltransferases, histone methylation was consid-

ered an irreversible mark because no enzyme was identified that could remove methyl

31 groups from lysine or arginine residues. Recently, this notion was rendered obsolete by the breakthrough discovery of the first histone demethylase, LSD1 (Lysine Specific

Demethylase 1) and the finding that PADI4/ PAD4 (Peptidyl Arginine Deiminase 4)

could interfere with histone arginine methylation by converting non-methylated and

mono-methylated arginine to citrulline (Figure 1.6, Table 1.6) [54, 255, 300]. Since

then, additional lysine demethylases have been identified which differ in their sub-

strate specificity (Table 1.6).

1.4.1 Lysine demethylases

LSD1 has been purified as a component of multiple corepressor complexes includ-

ing CtBP corepressor, CoREST, a subset of HDAC complexes, and Mi-2 based NURD

complexes. LSD1 is an amine oxidase, which catalyses the conversion of methyl-lysine

to lysine accompanied by release of formaldehyde and reduction of FAD to FADH2.

Due to the nature of the catalytic reaction carried out by LSD1, this enzyme is ca-

pable of only removing methyl groups from mono- and di-methylated H3K4 [255].

Recently, LSD1 was also shown to be involved in demethylating H3K9 when it is in

complex with the androgen receptor (AR) [186].

Another class of lysine demethylases is the jumonji domain (Jmj) containing pro-

teins, which are Fe2+ and α-ketoglutarate (α-KG) dependent oxygenases (or hydrox-

ylases) that are capable of removing methyl groups from mono-, di-, tri-methylated

lysine residues [47, 78, 149, 258, 285, 303, 324]. While JHDM1 (JmjC domain

containing histone demethylase 1) demethylates mono- and di-methylated H3K36,

JHDM3A/JMJD2A (JmjC domain containing histone demethylase 3A/JmjC domain

protein 2A) is capable of removing methyl groups from tri-methylated H3K9 and

32 H3K36 to generate di-methylated residues [303]. Thus, demethylases seem to show specificity not only towards the lysine residue being modified, but also the level of demethylation being established.

Demethylation of H3(Me2)K4 by LSD1 has been linked to transcriptional down- regulation of AchR (acetylcholine receptor), sodium channel genes (SCN1A, SCN2A, and SCN3A), and p57, while demethylation of H3(Me2)K9 by either LSD1 or

JHDM2A/JMJD1A results in transcriptional activation of PSA (prostate specific anti- gen) in the presence of androgen receptor activation [186, 255, 324].

1.4.2 Arginine demethylase

Peptidyl arginine deiminase (PADI4/PAD4) is the only known enzyme that an- tagonizes histone arginine methylation by converting either arginine or mono-methyl- arginine to citrulline in a Ca2+ and DTT dependent reaction. PADI4/PAD4 is the only peptidyl arginine deiminase family member that is localized in the nucleus and functions as a transcriptional repressor of the estrogen responsive gene, pS2, in MCF7 cells [54, 300]. Enhanced recruitment of PADI4/PAD4 to pS2 promoter in MCF7 cells correlates with increased H3 citrulline at the promoter, and decreased H3R17 methy- lation and pS2 transcription. Moreover, treatment of HL 60 granulocyte cells with

DMSO and Ca2+ ionophore has been shown to induce expression of PADI4/PAD4, and to correlate with increased levels of citrulline in total histones. Mass spectrom- etry analysis of histones following incubation with PADI4/PAD4 shows that non- methylated and mono-methylated H3R2, 8, 17 and 26, H2AR3, and H4R3 are deim- inated by this enzyme [54, 112, 300]. However, this enzyme cannot remove methyl

33 groups from symmetrically or asymmetrically di-methylated arginine residues, sug-

gesting that PAD4 exerts its effect on transcription by preventing di-methylation of

histone arginines [54].

1.5 DNA methylation

DNA methylation is an epigenetic mark that is absent in lower eukaryotic organ-

isms like S.cerevisiae and C .elegans indicating that this modification has evolved

later than histone acetylation and methylation [237]. DNA methylation mediates

long-term silencing and genomic stability by repressing transposable elements and

repetitive sequences. DNA is methylated by a class of enzymes referred to as DNA

methyltransferases (DNMTs) at cytosines in the CpG dinucleotide to generate 5-

methyl cytosine. The CpG dinucleotides occur at a lower frequency in the genome

and DNA regions that contain clusters of CpG sequences are known as CpG islands,

found mostly in promoters and first exons. DNMTs are classified as de novo methy-

lases or maintainence methylases. De novo enzymes efficiently methylate cytosines in

unmethylated DNA whereas maintainence methylases use hemimethylated DNA as

substrate. CpG methylation has been linked to transcriptional repression and silenc- ing, which opposes gene expression through recruitment of transcriptional repressors.

A number of methyl binding proteins are known to associate with methylated CpG

residues and recruit transcriptional repressors like mSIN3/HDAC.

Five DNMTs have been identified in mammals: DNMT1, DNMT2, DNMT3A,

DNMT3B, and DNMT3L. DNMT3A and DNMT3B are de novo methyltransferases,

while DNMT1 is the maintainence methylase. DNMTs contain an N-terminal regu-

latory domain, which contains the nuclear localization signal, a C-terminal catalytic

34 domain, and a central linker domain containing multiple GK dipeptides. Unlike other DNMTs, DNMT2 lacks N-terminal and central linker domains and does not show any effect on growth because homozygous null DNMT2-/- mice are viable and normal [212, 328]. In addition, though DNMT2 contains a very well conserved cat- alytic domain, it lacks DNA methyltransferase activity [212].

DNMT1 is the first purified and cloned DNA methyltransferase, which preferen- tially methylates hemimethylated DNA. DNMT1 has a diffuse nuclear localization in G1 cells and during S phase it is located at the replication fork where it methy- lates newly synthesized DNA strands to establish the parental DNA methylation pattern [167]. Homozygous deletion of DNMT1 in mice causes embryonic lethality, which is accompanied by severe demethylation of the genome, biallelic expression of imprinted genes, and inactivation of X-chromosome caused by upregulation of Xist

RNA [15, 169, 170]. Also ES cells lacking DNMT1 undergo apoptosis when they are induced to differentiate. These data suggest that DNMT1 is the major DNA methyltransferase of the cell.

DNMT3A and 3B are highly homologous proteins and are responsible for de novo methylation of CpG sequences during embryogenesis [213]. Their levels are reduced in ES cells upon differentiation and they are expressed at low levels compared to

DNMT1 in adult somatic tissues [164, 316]. DNMT3A is ubiquitously expressed whereas DNMT3B is predominantly expressed in testis, thyroid, and bone marrow.

While DNMT3A deficient mice die in early adulthood with signs of aganglionic mega- colon and loss of germ cells in males, DNMT3B knockout mice are not viable [213].

DNMT3B deficient mice lack methylation of pericentromeric minor satellite, which

35 is normal in DNMT3A knockout mice indicating that minor satellite regions are tar-

geted by DNMT3B. Further evidence in support of the role of DNMT3B in methy- lation of minor satellite DNA is found in humans with mutations of DNMT3B that

cause the rare autosomal disorder, ICF (immunodeficiency, centromere instability,

and facial anomalies). ICF results from loss of methylation of satellite DNA at the

pericentromeric region of 1, 9, and 16. Furthermore, DNMT3A -/-

and DNMT3B -/- double knockout mice die at 8.5 days with global loss of DNA

methylation of their genome, but the extent of demethylation is much lower than

that observed in DNMT1 -/- mice [213].

DNMT3L lacks active site motifs and hence does not possess any CpG specific

methyltransferase activity. DNMT3L has been shown to bind to the C-terminal

domain of DNMT3A and 3B, which stimulates their activity and increase their DNA

binding affinity [102]. DNMT3L is exclusively expressed in germ cells and is essential

for the establishment of methylation patterns in these cells. Genetic studies have

shown that DNMT3L null mice are viable; however, both male and female mice are

sterile [26].

CpG methylation is read by methyl-CpG binding proteins, which establish tran-

scriptionally repressed chromatin upon DNA methylation [225]. Currently, six methyl-

CpG binding proteins are known in mammals: MeCP2, MBD1, MBD2, MBD3,

MBD4, and KAISO. Except KAISO, which binds methyl-CpG through a zinc finger

motif, all methyl-CpG binding proteins contain a conserved DNA binding domain,

called MBD (methyl-CpG binding domain). Although, MBD3 contains a DNA bind-

ing domain, it has lost the ability to bind methylated CpG dinucleotides. Addition-

ally, apart from MBD4, which is implicated in DNA repair, all methyl-CpG binding

36 proteins act as transcriptional repressors through recruitment of histone deacetylase containing complexes. The mammalian DNA methylation machinery is composed of two components, DNMTs and MBD proteins, which establish, maintain, and read

DNA methylation.

1.6 Other histone modifications

Other post-translational modifications known to modulate chromatin structure

are histone phosphorylation, ubiquitination, sumoylation, and proline isomerization

(Figure 1.4). Phosphorylation of histone H3S10, H3S28 and H3T11 occurs during

mitosis and meiosis and is required for proper chromosome condensation and segrega-

tion in mammals [184]. In addition, H3S10 phosphorylation has been associated with

mitogen induced transcriptional activation of c-FOS and c-JUN [176, 11]. Further-

more, phosphorylation of H3S10 enhances Gcn5-mediated acetylation of H3K14 in

vitro and in vivo [171]. In Drosophila, phosphorylated H3S10 is confined to actively

transcribed interband regions of polytene chromosome, providing further evidence

that H3S10 phosphorylation is involved in transcriptional activation [208].

Histones are also modified by the addition of small proteins like Ubiquitin (Ub,

76 aa) and SUMO (Small Ubiquitin related Modifier protein, 100 aa), which are co-

valently linked through an isopeptide bond that is formed between Ub or SUMO

C-terminal carboxyl group and -amino group of histone lysine residues by the con- secutive action of activating E1 protein, conjugating E2 protein, and ligating E3 protein [342, 100]. In S.cerevisiae, histone ubiquitination is linked to both tran- scriptional activation and repression, while histone sumoylation is associated with gene repression. Deletion of the E2 Ub conjugating enzyme, Rad6, interferes with

37 telomere and mating type locus silencing, suggesting that ubiquitination is required to establish silencing [127]. In contrast, sequential ubiquitination and deubiquitina- tion is required for transcriptional activation of the SAGA target genes, Gal1 and

Alcohol dehydrogenase 2 [121]. Moreover, H2BK123 ubiquitination is required for

efficient H3K4 and H3K79 methylation, which are involved in transcriptional activa-

tion [63, 30, 205].

Unlike ubiquitination, which occurs only on H2BK123, all four histones undergo

sumoylation in S.cerevisiae [201]. While mutation of H4 sumoylation sites causes

derepression of Gal1, Trp3 and Suc2 genes, expression of SUMO conjugated H2B

or H4 inhibits induction of Gal1 expression by galactose. In addition, sumoylation

opposes histone acetylation at the Gal1 promoter, indicating that sumoylation is

a negative regulator of transcription. Though both ubiquitination and sumoylation

have been shown to regulate transcription in yeast, their role in modulating chromatin

structure in other organisms is not clear.

Besides the covalent modifications described above, recently proline isomeriza-

tion in histones carried out by proline isomerase enzymes has been identified as a

transcriptional regulator [202]. Peptidyl proline can exist either in a cis or trans

conformation, and most prolines exist in trans conformation because it has the lowest

free energy. The proline isomerase, Fpr4, interacts with histones H3 and H4, and

catalyzes cis ↔ trans isomerization of H3P30 and H3P38. Deletion or mutation of

Fpr4 in S.cerevisiae causes hypermethylation of H3K36 at repressed loci like Hsp12 ,

His4 , and Met16 , and delayed kinetics of gene induction indicating that Fpr4 reg-

ulates H3K36 methylation. Thus, proline isomerization represents another histone

modification that can modulate gene expression.

38 1.7 Cross-talk between various chromatin modifications

Multiple residues in histones can be modified in various ways thereby generat- ing a number of different combinations of histone tail modifications (Figure 1.4).

Each modification is catalyzed by a specific enzyme(s), and the presence of multiple modifications on a histone tail suggests that these enzymes specify the transcriptional outcome. The observations that certain histone modifications result in transcriptional activation, while others lead to repression indicate that these two groups of modifi- cation are mutually exclusive. Jenuwein and Allis have proposed a ”histone code”

hypothesis, which suggests that histone modifications provide binding sites for effec- tor proteins, and that various histone modifications on the same or different histone tails within a nucleosome are interdependent and act together to govern transcrip- tion [135]. Cross-talk between various chromatin modifications is well documented

(Figure 1.8). For example, phosphorylation of H3S10 promotes acetylation of H3K9 and H3K14, and inhibits methylation of H3K9 [229]. Chromatin modification on one histone can influence modifications on another histone, for instance, ubiquitination of

H2BK123 is required for efficient methylation of H3K4 and H3K79 [63, 30, 205]. Simi- larly, methylation of H3K9 is required for H4K20 methylation during heterochromatin

formation [151]. Cross-talk between histone acetylation and arginine methylation has also been observed. As shown in Chapters 2 and 3, histone deacetylation is a prerequi-

site for PRMT5-mediated H3 and H4 arginine methylation, and methylation of H3K9 interferes with symmetric dimethylation of H3R8 in vitro and in vivo [214, 215]. In

contrast, histone acetylation enhances H3R17 methylation by PRMT4 [56]. Further

support for the existence of cross-talk among different modifications is provided by

the observation that multiple modifying activities co-exist in multiprotein complexes.

39 For example, NURD and hSWI/SNF chromatin remodeling complexes are associated

with repressor enzymes such as, HDACs, PRMT5, and DNA methyl-binding proteins,

providing a link between chromatin remodeling, histone deacetylation and methy-

lation, and DNA methylation [107, 118, 214, 215, 260, 338]. Likewise, the H3K4

methyltransferase, MLL1, has been isolated in a large complex that contains nucleo-

some remodeling, histone acetylation, and histone methylation activities involved in

transcriptional activation [199].

Studies addressing gene expression and chromatin structure have found that the

milieu of histone and DNA modification marks governs the structure and transcrip-

tional state of chromatin. The reversibility of these modifications makes chromatin a

very dynamic structure and provides the required plasticity to perform all DNA-based

cellular functions.

1.8 Chromatin modifying activities and cancer

Cancer is associated with inactivation or lack of tumor suppressor genes and over-

expression of oncogenes caused by mutations, amplifications, or aberrant epigenetic

histone and DNA modification. Chromatin-modifying enzymes impact expression of

tumor suppressors and oncogenes either directly by altering histone modification of

target promoters or by modulating the activity of transcriptional regulators. For example, acetylation of c-MYC by GCN5/PCAF or TIP60 increases its stability, and acetylation of SMAD2/SMAD3 by CBP and p300 increases their DNA binding ac-

tivity [262]. Thus, altered HAT activity in cancer can impact acetylation, which acts

as a positive modulator of transcription by not only acetylating histones, but also by enhancing the activity of DNA binding factors.

40 1.8.1 Chromatin remodeling proteins

Inactivating mutations and deletion of hSWI/SNF subunits such as, INI1, BRG1,

BRM, and BAF57 have been found in various cancers. INI1 undergoes biallelic inac- tivating mutation in malignant rhabdoid tumors, which are lethal childhood tumors that affect kidneys and brain [292]. Loss of INI1 has also been frequently found in pediatric choroids plexus carcinoma indicating that inactivation of INI1 is not specific for rhabdoid malignancy. In addition, homozygous knockout of INI1 in mice causes embryonic lethality and heterozygous mice are prone to development of sarcoma and lymphoma [110]. Also, re-expression of INI1 in rhabdoid cancer cell lines causes cell cycle arrest by inducing the expression of cell cycle regulator like INK4A [20]. Simi- larly, mutations and loss of BRG1 and BRM expression has been found in tumors of breast, lung, pancreas and prostate, while mutation of BAF57 has been observed in breast cancer cell lines [57, 94, 147, 231, 311]. Like INI1 deficient mice, homozygous deletion of BRG1 is lethal and heterozygous BRG1 mice are prone to epithelial can- cer [31]. Taken together, these findings indicate that chromatin remodeling proteins act as tumor suppressor genes, which are required to maintain normal cell growth and proliferation.

1.8.2 Histone acetylases and deacetylases

Histone acetylation is linked to transcriptional activation, while histone deacety- lation leads to transcriptional repression. Alterations in histone acetylation patterns have been observed in cancer, for example, loss of H4K16 acetylation is associated with lymphoma and colorectal carcinoma indicating that acetylation of H4K16 is required for normal cell growth [80]. In acute leukemia, various chromosomal translocations

41 of HAT genes including MLL-CBP, MLL-p300, MOZ-CBP, MOZ-p300, MOZ-TIF2 and MOZ-NCOA2 have been found to be leukemogenic. Albeit rare, mutations in

p300 and CBP HATs have also been found in leukemia and epithelial cancers of breast, pancreas and colorectal mucosa [256]. These findings suggest that alterations

of acetyltransferase activity in a cell contribute to tumorigenesis.

Unlike HATs, translocations or mutations of HDACs have not been found in hu- man cancer. However, treatment of cancer cells with HDAC inhibitor triggers dif- ferentiation, growth arrest, and apoptosis, indicating an essential role of HDACs in tumorigenic cell growth and proliferation [53]. Mistargeting of HDACs is responsible for their tumorigenic activity. For instance the N-terminal DNA binding domain of

RUNX1 is frequently translocated and fused in frame to ETO in leukemia. Since ETO interacts with HDAC corepressor complexes, RUNX1-ETO fusion leads to transcrip- tional repression of RUNX1 target genes in leukemic cells. In another case, RAR-APL translocation (- acute promyelocytic leukemia gene), which oc- curs in acute promyelocytic leukemia, constitutively binds SIN3/HDAC and prevents retinoic acid-induced gene activation of RAR target genes and differentiation. These

findings indicate that mistargeting of HDACs through oncogenic DNA binding fusion proteins alters gene expression and contributes to tumorigenesis.

1.8.3 Histone methyltransferases

There is growing evidence that aberrant expression and activity of histone methyl- contributes to tumorigenesis. The H3K4 methyltransferase, MLL1 (mixed lineage leukemia gene1), which is required to maintain proper expression of homeotic

HOXA and C cluster genes, is frequently translocated in AML (60%) and ALL (80%)

42 patients, and fused to about 50 different partners generating leukemogenic fusion proteins [122]. The MLL fusion protein MLL-AF10 upregulates HOXA9 expression by recruiting the H3K79 specific methyltransferase, DOT1L, and this is required for

MLL-AF10 mediated leukemogenesis [210]. In addition, the methyltransferase of

PRC2, EZH2, is upregulated in a variety of cancers including hepatocellular, col-

orectal, breast, prostate, and pancreatic carcinomas. In prostate and breast can-

cer, enhanced expression of EZH2 is associated with aggressiveness of the disease,

whereas increased expression of EZH2 in lymphoma causes increased cell prolifera-

tion [148, 180, 290]. Similarly, another H3K4 methyltransferase, SMYD3, which is

involved in activation of oncogenes and cell cycle regulators, is overexpressed in hepa-

tocellular and colorectal carcinomas, and decreasing SMYD3 expression in these cells

inhibits cell growth and proliferation [116].

Like lysine methylation, arginine methyltransferases have recently been associ-

ated with cancer. PRMT4/CARM1 is overexpressed in primary prostate cancer and

knockdown of PRMT4/CARM1 in the LNCaP prostate cancer cell line inhibits cell

growth and proliferation [177]. Similarly, in Chapter 4, I have shown that PRMT5

is overexpressed in a variety of lymphoma and leukemia cells including mantle cell lymphoma, MCL, and consequently global symmetric methylation of H3R8 and H4R3 is enhanced. When PRMT5 expression was reduced in the JeKo MCL and the Raji

Burkitt’s lymphoma cell lines, proliferation was decreased. These findings indicate that alteration of normal histone methylation patterns is associated with cancer in

humans.

43 1.8.4 DNA methyltransferases

Comparison of normal and cancer cells have revealed that the bulk genome is hypomethylated in cancer cells at the normally hypermethylated repetitive DNA.

In contrast, CpG islands of various tumor suppressor genes are hypermethylated in transformed cancer cells, which cause transcriptional repression of tumor suppressor genes [14, 137]. In addition, enhanced expression of the DNA methyltransferase,

DNMT1, has been observed in cancer cells. While DNMT1 is overexpressed in var- ious leukemias, colon, and breast cancers, reducing DNMT1 expression in mice that are predisposed to colonic adenomas significantly reduces the occurrence of colon tu- mors [18]. Thus, DNA methylation promotes tumorigenecity by repressing tumor suppressor genes and increasing chromosomal instability through demethylation of repressed repetitive DNA elements.

1.9 Thesis overview

The work in this thesis shows that hSWI/SNF chromatin remodeling complexes are associated with histone deacetylase (mSIN3A/HDAC) and PRMT5 proteins which together mediate transcriptional repression. The results presented here identify the epigenetic marks generated by PRMT5 and their role in repressing expression of tumor suppressor genes. The role of PRMT5 in normal cell growth and proliferation has been evaluated, and my findings associate PRMT5 overexpression with human cancers.

44 Figure 1.1: Nucleosome core particle. Ribbon traces for the 146-bp DNA phosphodiester backbones (brown and turquoise) and eight histone protein main chains (blue: H3; green: H4; yellow: H2A; red: H2B). The views are down the DNA superhelix axis for the left particle and perpendicular to it for the right particle. For both particles, the pseudo-twofold axis is aligned vertically with the DNA center at the top. (Adapted from [173])

45 Figure 1.2: ATP-dependent chromatin remodeling complexes. Chromatin remodeling complexes and their associated subunits are shown. SWI2/SNF2-related ATPases are indicated in purple, ISWI-related ATPases are de- picted in red, Mi-2 related ATPases are colored orange, and DOMINO-like ATPases are shown in white. Subunits conserved between SWI/SNF complexes are shown in pink, while subunits specific to each complex are indicated in peach. Actin and actin-related proteins (Arp) are shown in green. Yellow indicates the subunits that are conserved in Drosophila (BAP111) and humans (BAF57). RVB1/RVB2 and TAP54α/TAP54β DNA helicases are shown in blue. Gray shows the highly con- served Drosophila and human ACF1 subunits found in CHRAC. Light green depicts the three subunits (MBD2, p66 and p68) found in MeCP1. bBAF indicates brain specific SWI/SNF subunits found in association with BRG1 and BRM. Arrows show the interactions of individual subunits with their respective complexes. (Adapted from [261])

46 Figure 1.3: Members of the Swi2/Snf2 superfamily. All members of the family contain a DNA-dependent ATPase domain (green) and either a bromodomain (BRD in red), two copies of chromodomain (CHD in pink), or a SANT domain (yellow). Members of the Mi-2 family, which contain a C-terminal DNA binding domain (DBD, in light blue), a partial region of homology within the DBD (black box), or two copies of a plant homeodomain (PHD in dark blue) are also shown. ATPases that have been purified and shown to be part of a complex are indicated by a bracket. (Adapted from [261])

47 Figure 1.4: Covalent modifications of core histones. Histones H2A, H3B, H3 and H4 are covalently modified by the addition of acetyl (red), methyl (blue), ubiquitin (orange) and sumo (brown) to specific lysine (K) residues as indicated. In addition, specific arginine (R) residues are methyated (blue) and serines (S) are modified through phosphorylation (purple). Recently, H3 prolines (P) at position 30 and 38 have been shown to undergo isomerization with respect to the polypeptide (green). Furthermore, few lysine residues can be modified by acetylation, methylation and sumoylation.

48 Figure 1.5: Reversible covalent modifications of lysine. Lysines can be modified by the transfer of either one to three acetyl groups from acetyl-CoA or one to three methyl groups from S-adenosyl methionine (SAM) to the -amino group (NH2) of lysine. These modifications are removed by histone deacetylases (HDACs) and demethylases respectively.

49 Figure 1.6: Arginine methylation by the PRMT enzymes. Both type I and type II PRMTs catalyze mono-methylation at the ω- NH2 of argi- nine, whereas type III enzyme found only in yeast catalyze mono-methylation at the δ- NH2. In addition, type I and II PRMTs also catalyze di-methylation of arginine residues, but differ in their ability to add the second methyl group either symmetrically or asymmetrically. Di-methylation of arginine is prevented by the ac- tion of peptidyl deiminase (PAD4/ PADI4), which modifies either arginine or mono- methylarginine to citrulline.

50 Figure 1.7: Protein arginine methyltransferases expressed in humans. The conserved catalytic motifs of PRMTs (PRMT 1-11) along with their specific domains are shown with respect to each other. PRMT7 and 10 are unique as they carry two catalytic sites each. Also the classification and histone substrate specificity of each PRMT is indicated. The specific methylation catalyzed by PRMT2 and PRMT9 isoforms 1, 2, and 3 is yet unknown.

51 Figure 1.8: Cross-talk of various histone modifications. Specific modifications on histones H2B, H3 and H4 modulate each other in either positive or negative fashion. Two-sided arrows indicate that one modification affects the other and vice versa; whereas one-sided arrow means that the specific modification from where the arrow is originati ng affects the one towards which the arrow is pointed. Presence of + (green) indicates a positive effect, while − (red) represents a negative regulation of the specified modification.

52 HAT Organisms known Histone specificity to express HAT GNAT Family HAT1 Various eukaryotes H4 (K12) GCN5 Various eukaryotes H3 (K14), H4 (K8, 16) PCAF Humans, mice H3 (K14), H4 (K8) Hat2 Yeast H3 (K14) ELP3 Yeast, humans H3, H4 Hpa2 Yeast H3 (K14), H4 MYST family Sas2 Yeast ND Sas3 Yeast H3, H4, H2A Esa1 Yeast H4 (K5), H3, H2A MOF Drosophila, humans H4 (K16), H3, H2A TIP60 Humans H4, H3, H2A MOZ Humans H3, H4 MORF Humans H3, H4 HBO1 Humans H3, H4 p300/CBP Various muticellular H2A(K5), H2B (K12, 15), organisms H3 (K9, 14, 18), H4 (K5,K8) Nuclear receptor coactivator family SRC-1 (p160) Humans, mice H3 (K14), H4 SRC-2 (ACTR) Humans, mice H3, H4 SRC-3 Humans, mice ND (TIF2/GRIP1) Transcription fac- tor family TAF1 (TAFII250) Various eukaryotes H3 (K14), H4 TFIIIC Humans H2A, H3, H4 BRCA2 Humans, mice H3, H4 ATF-2 Humans, mice H2B (K5, 12, 15), H4 (K5, 8, 16)

Table 1.1: Classification of HATs, and their expression and substrate specificity. Proteins with histone acetyltransferase activity are classified based on their sequence homology and function into five distinct families. The major histone substrates are indicated in bold.

53 Class I Class II Class III HDAC 1, 2, 3, 8 HDAC 4, 5, 6, 7, 9a, 9b, 9c, 10 SIRT 1, 2, 3, 4, 5, 6, 7

Table 1.2: Classification of human Histone Deacetylase (HDAC) enzymes. HDACs are classified based on their sequence homology to yeast histone deacetylases. No deacetylase activity has been reported for SIRT 4, 6, and 7.

Lysine SET SUV SUV39H1, SUV39H2, G9a, methyl domain 39 family GLP1 (EuHMT1), SETDB1 transferases proteins (ESET), SETDB2 (CLLL8) SET1 family MLL1 (HRX, ALL1), HRX2 (MLL4), ALR (MLL2), MLL3, SET1 (ASH2), SET1L SET1 family MLL1 (HRX, ALL1), HRX2 (MLL4), ALR (MLL2), MLL3, SET1 (ASH2), SET1L SET2 family WHSC1 (NSD2), WHSC1 (NSD3), NSD1, HIF1 (HYPB), ASH1 RIZ family RIZ (PRDM2), BLIMP1 (PRDM1) SMYD fam- SMYD1, SMYD3 ily EZ family EZH1, EZH2 SUV4-20 SUV4-20H1, SUV4-20H2 family Others SET7/9, SET8 (PR-SET7) Non-SET DOT1L domain proteins Arginine Type I PRMT1, PRMT3, PRMT4, methyl PRMT6, PRMT8 transferases Type II PRMT5, PRMT7, PRMT9 Type III Yeast RMT2

Table 1.3: Classification of histone methyltransferases. Histone methyltransferases methylate either lysine or arginine residues. Lysine spe- cific methyltransferases are further classified on the basis of the presence of SET domain and sequence homology. However, arginine methyltransferase are classified based on the reaction catalyzed and the type of product generated.

54 Human Yeast A. nidulans Ascidinas (C. Drosophila Zebrafish intestinalis) PRMT1 Rmt1/Hmt1 RMTA Ci1 DART 1, 2, Zf1 3, 6, 8, 9 PRMT2 Zf2 PRMT3 Ci3 Zf3 PRMT4 Ci4 DART4 Zf4 PRMT5 Hsl7, Skb1 RMTC Ci5 DART5 Zf5 PRMT6 Ci6 Zf6 PRMT7 Ci7 DART7 Zf7 PRMT8 PRMT9 PRMT10 PRMT11 Rmt2 RMTB

Table 1.4: Protein arginine methyltransferases are conserved in evolution. The identified PRMT genes in various organisms are organized based on their homol- ogy to human PRMT genes.

55 Histone site Histone Function methyltransferase H1K26 EZH2 Transcriptional repression H3K4 SET1, MLL1, MLL2, Transcriptional activation MLL3, SET7/SET9, SMYD3 H3K9 SUV39H1, Hetrochromatin silencing, SUV39H2, G9a, RIZ, euchromatin transcriptional GLP1, SETDB1, repression EZH2 H3K27 EZH1, EZH2, G9a Euchromatin gene silencing, X chromosome inactivation, gene imprinting H3K36 NSD1 Transcriptional elongation, heterochromatin silencing H3K79 DOT1L Transcriptional activation, restrict heterochromatin formation H4K20 SUV4-20H1, SUV4- Transcriptional activation, 20H2, SET8/PR- heterochromatin formation, SET7, NSD1 response to DNA dam- age, cell cycle dependent silencing and mitosis H3R2 PRMT4/CARM1 Transcriptional activation H3R8 PRMT5 Transcriptional repression, mitosis H3R17 PRMT4/CARM1 Transcriptional activation H3R26 PRMT4/CARM1 Transcriptional activation H4R3 PRMT1, PRMT5, Transcriptional repression PRMT7 (PRMT5). Transcriptional activation (PRMT1).

Table 1.5: Methylation of histone lysine and arginine residues by various methyl- transferase and their function. A specific lysine and arginine residue can be modified by several methyltransferases and the modification of each residue by methylation has different transcriptional outcome.

56 Demethylase Histone modifications PAD4/ PADI4 H2AR3, H3R2, H3R8, H3R17, H3R26, H2AR3(Me), H3R2(Me), H3R8(Me), H3R17(Me), H3R26(Me) LSD1 H3K4(Me), H3K4(Me2), H3K9(Me), H3K9(Me2) JHDM1 H3K36(Me), H3K36(Me2) JHDM2A/JMJD1A H3K9(Me2) JMJD2A/ JHDM3A H3K9(Me3), H3K9(Me2), H3K36(Me3), H3K36(Me2) JMJD2B H3K9(Me3) JMJD2C/GASC1 H3K9(Me3), H3K9(Me2), H3K36(Me3), H3K36(Me2) JMJD2D H3K9(Me3), H3K9(Me2), H3K9(Me)

Table 1.6: Known histone demethylases and their substrate specificity. PAD4/ PADI4 is not a true demethylase but is the only known enzyme that can antagonize arginine methylation. The lysine demethylases are represented by the amine oxidase, LSD1, and jumonji domain containing oxygenases.

57 CHAPTER 2

SIN3A/HDAC2 AND PRMT5 ARE ASSOCIATED WITH HUMAN SWI/SNF COMPLEXES AND ARE INVOLVED IN CAD TRANSCRIPTIONAL REPRESSION

2.1 Abstract

hSWI/SNF complexes have been implicated in transcriptional activation; but their

role in transcriptional repression is not well studied. Previous work in the laboratory has shown that the mSIN3A/HDAC corepressor complex copurifies with BRG1 and

BRM-based hSWI/SNF complexes. In this chapter interactions of mSIN3 isoforms

A and B have been further analyzed, and interaction of PRMT5 with hSWI/SNF complexes has been identified. In addition the methyltransferase activity of PRMT5 has been characterized. Protein-protein interaction studies indicate that c-MYC,

PRMT5, and mSIN3A interact with the same hSWI/SNF subunits, and c-MYC as-

sociated hSWI/SNF complexes lack PRMT5 and mSIN3A. Northern blot analysis

of the c-MYC target gene, CARBAMOYL-PHOSPHATE SYNTHASE-ASPARTATE

CARBAMOYLTRANSFERASE-DIHYDROOROTASE (CAD), showed that both BRG1

remodeling activity and histone deacetylation are important for transcriptional repres-

sion of CAD. Using chromatin immunoprecipitation assays, it has been demonstrated

58 that MAD1, BRG1, mSIN3A, HDAC2, and PRMT5 are associated with CAD pro- moter during repression, while c-MYC and BRG1 are recruited to the CAD promoter to mediate transcriptional activation. Since BRG1 can regulate MYC target gene, the role of BRG1 and BRM in MYC/RAS-mediated transformation was studied, which revealed that both BRG1 and BRM remodeling activity are required for transforma- tion.

2.2 Introduction

Expression of several genes changes during normal cell growth and differentia- tion, which correlate with alterations in chromatin structure. The BRG1 and BRM chromatin remodeling complexes have been implicated in transcriptional activation of many inducible genes, but their role in gene repression is not well known [87, 200].

The findings that the mSIN3/HDAC corepressor complex copurifies with BRG1 and

BRM chromatin remodelers, and that the NURD complex contains HDAC 1 and 2, suggests that ATP-dependent chromatin remodeling is involved in transcriptional re- pression [281, 321, 337, 260, 155]. Additionally, BRG1, BRM and BAF45/INI1 have been linked to transcriptional repression, but the molecular mechanism of hSWI/SNF mediated repression is not well understood [283, 284, 339, 341].

mSIN3A/HDAC corepressor complexes mediate gene repression by deacetylating histones at the promoter of genes targeted by retinoid acid and thyroid hormone re- ceptors, IKAROS, , and MYC/MAX/MAD proteins [2, 119, 158, 198, 150, 228].

The MYC/MAX/MAD network plays an important role in the control of normal cell growth and proliferation by regulating the expression of cell cycle regulators, for example CDC25A, CYCLIN A, and p53 [72, 67]. MYC has been implicated in

59 transcriptional activation, apoptosis, and oncogenic transformation. MYC is an im- mediate early gene that is expressed in early G1 and activates expression of its target genes by binding to the E box (CACGTG) as a MYC-MAX heterodimer [12].

Binding of MYC-MAX results in histone acetylation and gene activation, whereas

displacement of MYC-MAX by the MAD-MAX heterodimer, recruits HDAC com-

plexes like mSIN3/HDAC, N-CoR and represses transcription [6, 7, 246, 183, 24, 66].

Expression of both c-MYC and MAD proteins fluctuates as cells progress through the

cell cycle, while MAX levels are unaltered [103, 104]. Thus, the balance of MYC-MAX and MAD-MAX complexes governs the regulation of MYC target gene by recruiting histone modifying activities.

To extend our understanding of how hSWI/SNF complexes regulate transcrip-

tion, the interacting proteins of hSWI/SNF complexes were analyzed and PRMT5 was identified as a hSWI/SNF-associated protein. Both recombinant and hSWI/SNF- associated PRMT5 methylated histones H3 and H4 and methylation was efficient on

hypoacetylated histones. Furthermore when the role of BRG1, mSIN3A/HDAC2, and PRMT5 was analyzed in the regulation of CAD, it was found that BRG1 was recruited to the CAD promoter during transcriptional activation as well as repres-

sion and recruitment of mSIN3A/HDAC2 along with PRMT5 was associated with transcriptional repression of CAD. Since it was found that MYC directly interacted with BRG1- and BRM-based hSWI/SNF complexes and BRG1 regulated MYC target

gene, CAD, the role of BRG1 and BRM in MYC and RAS-mediated transformation

was tested. Soft agar growth assays indicated that expression of either inactive BRG1

or BRM interfered with the transforming activity of MYC and RAS.

60 2.3 Materials and Methods

2.3.1 Plasmid constructions, DNA digestion, PCR reaction, ligation, and transformation

Details of the vectors and inserts used for constructing various plasmids used for bacterial, insect, and mammalian expression are described in the Appendix A

(Plasmid construction). Vectors were digested with appropriate restriction enzymes and following electrophoresis, bands containing the specific DNA fragment was excised and DNA was eluted from the gel. DNA inserts for ligation reactions were obtained by either PCR amplification or by restriction enzyme digestion of plasmids carrying the desired insert DNAs.

To PCR amplify DNA inserts, 250 ng of template DNA was mixed with 50 pmol of

5’ and 3’ specific primers, 2 mM MgCl2, 1X Taq polymerase buffer, 250 µM dNTPs and 2.5 U Platinum Taq polymerase in a final volume of 50 µl. A hot start was performed by incubating the mix of DNA template, primers and MgCl2 in a volume of 25 µl at 99oC for 5 min, which was then cooled on ice for 2 min before adding the remaining components. The reactions were incubated successively at 95oC for

1.5 min, 50oC for 1.5 min; 72oC for x min (x= 1 min/1Kb of DNA). These three steps were repeated 29 times before reactions were incubated at 72oC for 10 min and maintained at 4oC. The amplification of the PCR product was verified by analyzing 5

µl of the above PCR reaction on 1% TBE-Agarose gel by electrophoresis and the rest

45 µl of the PCR reaction was purified by phenol:chloroform extraction and DNA was precipitated as described above for the preparation of vector. Next day, the PCR product was resuspended in 15 µl TE [pH 8.0], and digested with the appropriate

61 restriction enzyme(s) for 3 h. The digested PCR product was separated on 1% TAE-

Agarose gel and the desired band was eluted from the gel.

To generate a plasmid clone, vector and insert were ligated using 4 µl of eluted vector with either 7.5 µl or 15 µl of eluted insert DNA in the presence of 1X T4 DNA ligase buffer and 4 µl of T4 DNA ligase in a final volume of 40 µl. The reactions were incubated in a 16oC water-bath, which is kept in the cold room for 14-16 h.

Next day, the ligation reaction was transformed in CaCl2 competent DH5α cells by incubating the ligation reaction with 65 µl of DH5α cells, 170 µl of TCM (10 mM

Tris-HCl [pH 8.0], 10 mM CaCl2, 10 mM MgCl2) on ice for 1 h. The mixture was subjected to heat shock at 42oC for 2 min, and then 1 ml LB media was quickly added before cells were allowed to grow at 37oC for 1 h. Cells were collected by centrifugation at 1000 rpm for 4 min, resuspended in 100 µl of LB media and plated onto LB plates containing the appropriate antibiotic. Cells were grown at 37oC for

16 h to form colonies, which were then inoculated in 3 ml LB media and grown for another 14-16 h before plasmid DNA was harvested using DNA mini prep protocol, and clones containing the desired insert in the correct orientation were confirmed by restriction enzyme digestion analysis.

2.3.2 Coupled in vitro transcription and translation

Coupled in vitro transcription and translation was performed using the TNT

T3/T7 coupled reticulocyte lysate system (Promega Inc., catalog no. L5010). Briefly,

1 µg of plasmid DNA was mixed with 12.5 µl of reticulocyte lysate, 1.0 µl of TNT reaction buffer, 0.5 µl of 1 mM amino acid mix lacking cysteine and methionine, 0.5

µl of RNasin, 0.5 µl of either T3 or T7 polymerase, and 2 µl of Expre [35S]- protein

62 labeling mix (Perkin Elmer, catalog no. NEG072) in a volume of 25 µl, which was

incubated at 30oC for 2 h. To determine the efficiency of in vitro translation, 2 µl

of the above reaction was separated on 8% SDS-PAGE gel and processed as follows.

The gel was washed twice with MilliQ water for 10 min, treated with 1 M salicylic

acid for 20 min to enhance autoradiography, dried at 80oC for 50 min and finally

exposed to X-ray film overnight at −80oC. Next day, the film was developed and

the region of dried gel corresponding to the full length in vitro translated protein was

excised and [35S] methionine and cysteine incorporation was determined by soaking

the gel slice in 3 ml scintillation fluid and counting in a scintillation counter. The

cpm (counts per minute) represents the radiolabel incorporation in 2 µl of the sample.

2.3.3 GST pull-down assay

Expression of GST fusion proteins was carried out as described previously [81].

GST-PRMT5, GST-MYC, and GST-PAH1, 2 and 4 fusion proteins were induced with

2 mM IPTG for 4 h at 37oC, while 2 h induction worked well for GST-PAH3. To

immobilize GST fusion proteins, 30 µl of GST beads were pre-washed three times with

buffer STE-100 (20 mM Tris-HCl [pH 7.6], 5 mM MgCl2, 100 mM NaCl, 1 mM EDTA)

supplemented with 0.5% NP-40 and BSA (1mg/ml). Then, approximately 500-800

µg of bacterial extract was incubated with the prewashed GST beads on ice for 30

min. Following GST protein binding, bound beads were washed three times with 300

µl of buffer STE-100. Washed beads were then blocked in 250 µl of buffer STE-100

containing uninduced bacterial extract (1mg/ml), BSA (1mg/ml), carnation milk (5

mg/ml) and ethidium bromide (100 µg/ml) at 4oC for 5 to 6 h. Next, approximately

8 x 104 cpm of in vitro-translated and 35S -labeled hSWI/SNF subunits were added

63 to the above reactions and incubated at 4oC for 12 to 16 h. Beads were washed

three times with 300 µl of STE-100 containing BSA (1mg/ml), and carnation milk (5

mg/ml), followed by two washes with STE-150, which contains 150 mM NaCl, and

the retained proteins were released by boiling in 25 µl of 2X SDS-PAGE loading dye

for 3 min and analyzed by SDS-PAGE followed by autoradiography as detailed in

section 2.3.2.

2.3.4 Co-in vitro translation and immunoprecipitation

To co-translate and radiolabel two proteins simultaneously, the plasmids carrying

the corresponding cDNAs in the same orientation (T3 or T7) were mixed together in

equal amounts in a coupled in vitro transcription and translation reactions according

to section 2.3.2. PRMT5 was in vitro translated in combination with BRG1, BRM,

BAF155, BAF57, BAF45/INI1, or mSIN3A. Based on the initial experiment, if the two proteins did not translate well, the ratio of the two plasmids was readjusted

to achieve equivalent translation of each protein. To immunoprecipitate proteins,

roughly 6-7 x 104 cpm of combined in vitro translated proteins were mixed with 20 µl of either pre-immune or immune BRG1, BRM, BAF155, BAF57, BAF45/INI1 and mSIN3A (AK-11) (Santa Cruz Inc., catalog no. sc-767) antibodies and incubated on ice for 20 min. Next, the volume was increased to 200 µl with IP buffer (20 mM

Tris-HCl [pH 7.4], 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% aprotinin) and samples were incubated at 4oC on nutator for 5 to 6 h. To capture the antibody-

protein complexes, 75 µl of preblocked protein A sepharose beads (preblocked with

BSA (0.5 mg/ml) for 2 h) were added, and samples were incubated at 4oC on nutator

for additional 12 h. Next day, the beads were collected by centrifugation at 2000

64 rpm for 2 min, and washed three times with 300 µl of STE-150 (20 mM Tris-HCl [pH

7.6], 5 mM MgCl2, 150 mM NaCl, 1 mM EDTA) supplemented with 0.05% NP40, and once using 300 µl of STE-150. The beads were finally resuspended in 25 µl of

2X SDS-PAGE gel loading dye, boiled for 3 min, and 18 µl of the supernatant was separated on 8% SDS-PAGE and analyzed by autoradiography as in section 2.3.2.

2.3.5 Site directed mutagenesis

To generate catalytically inactive PRMT5, we mutated amino acid residues G367 and R368 to A367 and A368 in the catalytic domain of PRMT5 using the QuickChange site directed mutagenesis kit (Stratagene, Inc.), which has been replaced by the

QuickChange Multisite-directed mutagenesis kit (Stratagene, Inc., catalog no. 200514).

To generate the flag tagged PRMT5 R368A and G367A/R368A mutations, 5MHMT1 and 3MHMT1 or 5MHMT2 and 3MHMT2 mutagenic primer sets were used in a PCR reaction as described below. As template DNA, 500 ng of pBS(KS+)/FL-PRMT5 was mixed with 50 pmol of each 5’ and 3’ primer, 10% DMSO, 5% glycerol, and either 3.0, 4.0, 5.0 or 6.0 mM MgCl2 in a volume of 31 µl. The reaction was heat denatured at 99oC for 5 min and then cooled on ice for 2 min. Following the hot start, 0.5 mM dNTPs, 1X Pfu polymerase buffer, 2.5 U of native Pfu polymerase, and water to a final volume of 50 µl was added to each reaction and subjected to thermal cycling in the PCR machine according to the parameters as follows. Step 1:

94oC for 1.5 min, Step 2: 50oC for 1.5 min, Step 3: 68oC for 8 min, Step 4: Go to step 1 for 25 cycles, Step 5: 4oC. After the PCR amplification, the DNA template was digested by treating the reactions with 20 U DpnI at 37oC for 3 h. To verify the amplification of template DNA, 10 µl of the above PCR reactions were analyzed

65 on 1% TBE-agarose gel. The successful mutagenesis reactions were transformed in

Epicurian-XL1 blue cells as follows. 25 µl of the PCR reaction was mixed with 50 µl of cells and incubated on ice for 1h. Then the samples were subjected to heat shock at 42oC for 1 min and incubated on ice for 2 min. Next, 0.5 ml of NZY broth media that was preheated to 42 oC was added and cells were allowed to grow at 37oC for 1.5 h. The cells were then collected by centrifugation at 2000 rpm for 5 min, resuspended in 100 µl of media, and plated on LB-agar plates containing 100 µg/ml ampicillin, which were allowed to grow for 16 h at 37oC. Plasmid DNA was isolated from six different colonies and the mutants were identified by DNA sequencing.

2.3.6 Expression of wild type and mutant FL-PRMT5 in Sf9 cells

To express wild type (WT) and mutant (Mut) flag tagged PRMT5 protein in

Sf9 cells we used the Bac-To- Bac baculovirus expression system (Life technologies,

Inc.). As a first step, we transposed pFastbac/WT-Fl PRMT5 and pFastbac/Mut-Fl

PRMT5 individually in the bacmid DNA, which is present in DH10Bac cells. 250 ng of each plasmid DNA was mixed with 65 µl of CaCl2 competent DH10Bac cells and incubated on ice for 30 min, prior to the heat shock at 42oC for 45 sec. After the heat shock, the cells were incubated on ice for 2 min and then 900 µl of SOC media (Invitrogen Inc., catalog no. 15544034) was added, before they were allowed to grow for 4 h at 37oC. Finally, 100 µl of the transformation reaction was plated on LB plates containing kanamycin (50 µg/ml), gentamycin (7 µg/ml), tetracycline

(10 µg/ml) on which 5 µl of IPTG (1M) and 125 µl of X-gal (20 mg/ml) has been spread. Bacterial colonies were allowed to grow for 16-20 h and the white colonies, which carry the transposed bacmid DNA, were streaked on new plates and grown for

66 16 h at 37oC. The true white colonies (4-6) were then inoculated in triplicates in 4 ml

LB media containing kanamycin (50 µg/ml), gentamycin (7 µg/ml) and tetracycline

(10 µg/ml) for 24 h prior to harvesting the bacmid DNA. To prepare bacmid DNA, cells were pelleted and gently resuspended (gentle vortexing) in 300 µl of solution

I (15 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 µg/ ml RNaseA). Then, 300 µl of solution II (0.2 N NaOH, 1% SDS) was added, gently mixed by inversions and incubated for 5 min at room temperature. To remove protein and genomic DNA,

300 µl of 3 M potassiun acetate [pH 5.5] was added with gentle mixing, and samples were allowed to precipitate for 10 min on ice. The clear supernatant containing the bacmid DNA was collected by centrifugation at 14000 rpm for 10 min and mixed with

0.8 ml of isopropanol to precipitate the bacmid DNA. The samples were incubated

on ice for 10 min and the bacmid DNA was collected by centrifugation at 14000 rpm

for 15 min. The bacmid DNA was washed twice with 0.5 ml of 70% ethanol, air dried

for 10 min, and then resuspended in 30 µl of TE [pH 8.0]. To analyze the quality

of bacmid DNA, 4 µl of DNA was analyzed on 0.5% TAE agarose gel containing 0.5

µg/ml ethidium bromide at 23V for 14-16 h. The bacmid DNA is stored in styrofoam

boxes at −20oC.

As the final step, to express the wild type and mutant PRMT5 in Sf9 cells, 1 x

106 Sf9 cells were plated in 6 well plates for 1 h and transfected with 2, 6, and 17 µl of

respective bacmid DNA. For each protein, three different bacmid DNA clones were

tested. DNA was diluted in 100 µl of HyQ SFX-insect media, mixed with another

100 µl of media containing 7.5 µl of cellfectin (Invitrogen Inc., catalog no. 71362),

and incubated for 45 min at room temperature. Then, 0.8 ml of media was added

to the above tubes and the mixture was added to the cells in the 6 well plate that

67 had been washed once with 2 ml media. The cells were incubated at 27oC for 5 h, before they were washed once with 2 ml of 1X PBS, and incubated in 2 ml media for 72 h. The supernatant containing the baculovirus, was collected and clarified by

centrifugation at 2000 rpm for 4 min. This supernatant represents the virus master

stock, from which two 1 ml freezer stocks were prepared by supplementing the 2%

FBS and stored at −80oC. To amplify the viral titer, 100 µl of the master stock was

used to infect 16 x 106 cells in 150 mm plate for 72 h. The supernatant was collected

and clarified as above. This viral supernatant represents the 1st viral amplification,

which is used in subsequent experiments. To verify the expression of protein, 100 µl

of the 1st amplification virus was used to infect 8 x 106 cells in 100 mm plate for 48,

60 and 72 h. After each time point, nuclear and cytosolic extracts were prepared,

and expression of recombinant protein was analyzed by Western blotting on 20 µg

of extract using anti-flag antibodies. The clone, which shows maximum expression

was used for large-scale protein production according to the time point with highest

protein expression. To express the recombinant protein on a large scale, the 1st

amplification virus was amplified once more in twenty 150 mm plates with 16 x 106

cells using 100 µl of the virus. The 2nd amplification virus was collected and clarified

72 h after infection. To induce wild type and mutant PRMT5 expression, 300 ml of

cells at 2.6 x 106 cells/ml were infected with 200 ml of 2nd amplification virus for 60

h. After 60 h, nuclear extracts were prepared from the infected cells and expression

was verified by Western blotting with anti-flag antibodies.

68 2.3.7 Purification of flag-tagged recombinant PRMT5 and BRG1 and BRM based hSWI/SNF complexes and pro- tein identification by mass spectrometry

Wild type and mutant Fl-PRMT5 were purified by immunopurification using anti-

flag M2 beads (Sigma Inc., catalog no. A2220). To purify flag-tagged protein from 40

mg of Sf9 nuclear extract, 1 ml of packed M2 beads was used, which was successively

washed with 4 ml of 1 M glycine [pH 3.5] and 4 ml of TBS (20m M Tris-HCl [pH

7.4], 150 mM NaCl) three times. Then, the beads were equilibrated with 10 ml of

BC150 before incubation with the extract for 14 h at 4oC on the nutator, in 15 ml

tubes. Next day, the beads were repacked in a 3 ml syringe column, and the extract

was allowed to pass through twice to maximize binding. The unbound proteins were

removed by successive washing with 6 ml of BC 150, BC 300 and BC100. To elute

the bound flag tagged proteins, about 500 µl of flag peptide (resuspended in BC100

at 1 mg/ml) was added, and the void volume (40% column volume) was collected.

Next, the beads were incubated with flag peptide for 1 h, such that there is about 100

µl of flag peptide solution on top of the column. Then, approximately 15 fractions

of 100 µl were collected with constant addition of flag peptide on top of the column.

About 1.2 ml of flag peptide was used per column followed by elution with another

500 µl of BC100. To analyze the purity and identify the concentrated fractions, 5 µl

of each eluted fraction was analyzed by silver staining.

Flag tagged BRG1- and BRM-based hSWI/SNF complexes were purified as above, except that 2.5 ml of packed M2 bead volume was used with approximately 190 mg of nuclear extract from HeLa cells expressing either flag-tagged wild type BRG1 or

BRM. The peak fractions were pooled and concentrated by TCA precipitation. To

69 precipitate BRM complex, 400 µl of Fl-BRM complex was mixed with deoxycholic acid (DOC) to a final concentration 0.02% (4 µl of 2% DOC stock) and incubated at room temperature for 15 min. Next, TCA was added to a final concentration of

6% (136 µl of 24% TCA stock), mixed by inversion (five times), and incubated on ice for 8 h. Then the TCA precipitate was collected by centrifugation at 14,000 rpm for

10 min at 4oC and the supernatant was carefully removed. Then 200 µl of ice-cold acetone was added to the pellet and incubated on ice for 15 min before centrifugation at 14,000 rpm for 10 min at 4oC. Finally, supernatant was carefully removed, pellet was dried for 2 min at room temperature, resuspended in 15 µl of 2X SDS-gel loading dye and analyzed by SDS-PAGE followed by Coomassie blue staining. The visible bands were excised and sent to Dr. Paul Tempst laboratory for identification by mass spectrometry.

Anti-flag M2 bead purified flag-tagged BRG1 and BRM complex fractions (200

µl) were further fractionated on a linear 22-50% glycerol gradient (1.8 ml) for 16 h at 29,000 rpm, 4oC in table top ultra centrifuge. To pour the linear gradient,

FPLC system was used and the gradient was poured at a rate of 1 ml/min. After centrifugation, 100 µl fractions were collected from the bottom of the gradient using the 21G3/4 vacutainer safetylok blood collection needle set (Becton Dickenson Inc., catalog no. 367296) and 20 µl of each fraction was analyzed by silver staining to identify the peak fractions.

2.3.8 Mammalian-two hybrid experiment and transfection using Lipofectamine

HeLa S3 cells were transfected with 2.25 µg of total DNA (1 µg reporter plasmid, pG5Hsp70-CAT, 0.5 µg of either Gal4 alone or Gal4 fusion protein plasmids, 0.5

70 µg of either VP16 alone or VP16 fusion protein encoding plasmid, and 0.25 µg of pCMV-β-galactosidase) in 60 mm plates at 70% confluency using lipofectamine plus reagent as per manufacturer’s instructions (Invitrogen Inc., Lipofectamine catalog no.

18324-012; Plus reagent catalog no. 10964-021) for 5-6 h. A mock transfection with

1 µg of pBS KS(+) instead of Gal4 and VP16 expressing plasmid was included to measure the basal reporter activity. Briefly, 2.25 µg of DNA was diluted in 240 µl of

DMEM without FBS, mixed with 10 µl of plus reagent, and incubated for 10 min at room temperature. Next, 250 µl of DMEM media without FBS, but containing 12 µl of lipofectamine was added to each reaction, mixed and incubated for 45 min at room temperature. Then, 1 ml of media without FBS was added to each reaction, which was transferred to the plates, with HeLa S3 cells that had been washed twice with

2 ml of 1X PBS, and contained 1 ml DMEM without FBS. The plates were swirled

4-5 times and incubated for 6 h at 37oC. Next, the media was removed, cells were washed twice with 2 ml of 1X PBS, 3 ml of DMEM supplemented with 10% FBS was added to the plates, and cells were allowed to grow for about two days. The cells were harvested 42 h post transfection, washed twice with 1X PBS and whole cell lysate was prepared by repeated freeze-thaw cycles (5 times) in 150 µl of 25mM Tris-HCl

[pH 7.6]. The lysates were precleared by centrifugation at 14,000 rpm for 10 min at 4oC, and used to measure β-Galactosidase and chloramphenicol acetyl transferase activity.

2.3.9 β-Galactosidase and Chloramphenicol acetyl transferase (CAT) assays

To measure the efficiency of transfection, β-galactosidase assay was performed by incubating 15 µl of extract with 1.5 µl of 100X Mg Buffer (100 mM MgCl2, 4.4 M

71 β-mercaptoethanol), 117 µl of 0.1 M sodium phosphate buffer [pH 7.4] (8.2 ml of 0.5M

Na2HPO4, 1.8 ml of 0.5 M NaH2PO4, and water to 50 ml), and 16.5 µl of substrate o-nitrophenyl galactoside (ONPG, 8 mg/ml) at 37oC till yellow color developed. The reaction was immediately terminated by quick addition of 1 M sodium carbonate (250

µl). Next, the absorbance of the samples was taken at 415 nm.

To perform CAT assays, the extracts were first heat inactivated for 10 min at 65oC and clarified by centrifugation at 14,000 rpm for 10 min at 4oC. Then to set up CAT reactions, the normalized volume of extract based on the β-galactosidase activity was aliquoted in a final volume of 15 µl (diluted with 25mM Tris-HCl [pH 7.6]), and incubated with 2.5 µl of CAT mix (33.3 ng AcetylCoA (Sigma, catalog no. A2056) and 0.025 µCi [14C]-chloramphenicol (Perkin Elmer, catalog no. NEC408A)) for 1 h at

37oC. Reactions were terminated by adding 250 µl of ethyl acetate, and the acetylated and unacetylated forms of chloramphenicol were extracted by rigorous vortexing of the samples for 1 min followed by centrifugation at 14,000 rpm 4oC for 10 min. The upper organic phase containing chloramphenicol was dried, pellet was resuspended in 10 µl of ethylacetate, and spotted on Silica gel 1B TLC plate (J.T. Baker Inc., catalog no. 4462-04), 1.5 cm away from each other and 2 cm from the bottom of the plate. The samples were spotted by slowly applying the solution drop by drop as it dried on the plates. The TLC plate was allowed to dry at room temperature for additional 30 min, and then the acetylated forms of chloramphenicol were separated by capillary action in a saturated chamber containing 100 ml running buffer (9:1

Chloroform: Methanol). After the buffer reached almost to the top (1 to 1.5 cm from the top end), plates were taken out, dried, and exposed to phosphorImager screen overnight. To calculate the CAT activity, which is represented by the percentage of

72 chloramphenicol acetylation, the acetylated (upper spots) and unacetylated (lower most spot) forms of chloramphenicol were quantitated using ImageQuant program.

The signal of [acetylated]/ [acetylated + unacetylated] x 100 represents the percentage of acetylation.

2.3.10 Histone methyltransferase assay

Histone methylation was performed using either recombinant PRMT5 or flag- tagged BRG1 and BRM complexes with 2 µg of H1-depleted HeLa core histones as substrates. Each reaction contained 15 mM HEPES [pH 7.9], 5 mM MgCl2, 20% glycerol, 1 mM ETDA, 0.25 mM DTT, 0.5 mM PMSF, and 2.75 µCi of [3H]-S-

Adenosyl methionine (Perkin Elmer, Inc., catalog no. NET-155H) in a total volume of 25 µl. Samples were incubated at 30oC for 1.5 h, and then the reaction were terminated by adding 5 µl of 2X SDS sample loading dye, which was separated on an 18% SDS-PAGE. To inhibit the methyltransferase activity of PRMT5, general protein methylase inhibitor methyl-thio adenosine (MTA) was used. In case of MTA treatment, 250 ng of BRG1 complex was preincubated with either 150 or 750 mM

MTA for 15 min, prior to the addition of 2.75 µCi of [3H]-S-Adenosyl methionine and

BC100 (15 mM HEPES [pH 7.9], 5 mM MgCl2, 20% glycerol, 1 mM ETDA, 0.25 mM DTT, 0.5 mM PMSF) to a final volume of 25 µl. Histones were visualized by

Coomassie blue staining, and methylated histones were detected by autoradiography.

The gels were treated with 1 M salicylic acid [pH 6.0] for 30 min, dried at 80oC for

50 min, and exposed to X-ray film at −80oC for 2-4 days before developing the autoradiograph.

73 2.3.11 MBP-MORF expression, purification and HAT assay

MBP-MORF was expressed and purified according to the previously described protocol [221]. DH5α cells carrying pMBP-MORF were inoculated in 25 ml LB containing 0.2% glucose and 75 µg/ml ampicillin for about 14 h. Next day, 5 ml of overnight culture was inoculated in fresh 450 ml LB containing 0.2% glucose and 75

µg/ ml ampicillin such that the final O.D is 0.02. The cells were incubated at 37oC and grown till OD reaches 0.4 (about 3 h), and then they were induced with 1 mM

IPTG at 37oC for 2 h. The cells were collected by centrifugation at 4,000 rpm for 10 min, washed in 25 ml of 1X PBS and resuspended in 2.5x pellet volume of buffer B

(buffer B: 20 mM Tris-HCl [pH 8.0], 10% glycerol, 150 mM KCl, 5 mM MgCl2, 0.1%

NP-40, and protease inhibitors). Next, cells were lysed by sonication using a microtip

Branson sonifier 450, at 50% duty cycle and 5% output, 10 times for 20 sec each. The lysate is cleared by centrifugation at 10,000 rpm for 10 min and stored at −80oC. To verify the induction of MBP-MORF, 15 and 30 µg of extract was separated on 8%

SDS-PAGE and analyzed by coomassie staining.

To purify MBP-MORF, 10 mg of extract was bound to 500 µl of amylose resin

(NEB Inc., catalog no. E8021S) that was pre-washed with 7.5 ml of buffer B. The extract was incubated with the amylose resin for 2.5 h at 4oC on a nutator. The resin was then packed in a column and washed successively with 6 column volumes of buffer B-150 (contains 150 mM KCl), 10 column volumes of buffer B-500 (contains

500 mM KCl) and 5 column volumes of buffer B-150. The bound proteins were eluted by incubating the beads with elution buffer (0.5% maltose in buffer B-150) for 15 min, and 50 µl fractions were collected. After collecting every 3 fraction, the column was incubated with 150 µl of elution buffer for 10 min before resuming fraction collection,

74 and total of 15 fractions were collected. To analyze the purification and estimate concentration of MBP-MORF, 5 µl of each fraction was separated on 8% SDS-PAGE and visualized by silver staining.

To test the activity of the purified MBP MORF, which catalyzes the acetylation of histones H3 and H4, HAT assay was performed on N-terminal histone H3 and H4 peptide (aa 1-20). N-terminal peptides (4 µg) were incubated with approximately

150 ng of MBP-MORF in the presence of 0.125 µCi [3H]- acetylCoA, and 4 µl of 5X bufferA ( 250 mM Tris-HCl [pH8.0], 50% glycerol, 5 mM DTT, 0.5 mM EDTA, 5 mM PMSF and 50 mM sodium butyrate) in a 20 µl reaction at 30oC for 30 min.

Next, the reactions were spotted on P-81 Whatman paper (Whatman Inc., catalog no. 3698325) and dried for 30 min. The unbound free [3H]- acetyl CoA is removed

by washing the P-81 paper 5 times with 10 ml of 0.1 M sodium carbonate buffer

[pH 9.0] for 7 min each. The papers are then dried for 30 min, immersed in 3ml of scintillation fluid and acetylation measured by scintillation counting.

2.3.12 Acetylation of N-terminal histone peptides

H2A and H2B histone peptides were chemically labeled using the HDAC assay kit as specified by the manufacturer (Upstate Biotech.,Inc., product discontinued and replaced by catalog no. 17-320). To acetylate histone H3 and H4 N-terminal tails, purified MBP-MORF was used. 20 µg of either H3 or H4 peptide was incubated with approximately 1 mg of MBP-MORF in the presence of 1X buffer A (50 mM Tris-HCl

[pH 8.0], 10% glycerol, 1mM DTT, 0.1 mM EDTA, 1 mM PMSFand 10 mM sodium butyrate), and 0.75 µCi [3H]- acetylCoA in a final volume of 35 µl at 37oC for 30 min.

75 To purify the acetylated peptides the reaction was loaded on Isolute SCX-column

(Argonaut Inc., catalog no. 5300002-A) that was prewashed once successively with

500 µl of solution I (10 mM HCl in 100% methanol) and solution II (10mM HCl,

0.04% methanol). After 10 min incubation of the acetylated peptide reaction on the column, the unbound sample was eluted by centrifugation at 1,000 rpm for 1 min.

The sample was reloaded, spun, and the column was washed twice with 500 µl of solution III (10 mM HCl in 10% methanol). The bound peptides were eluted twice successively by incubating the column with 75 µl of elution buffer (3 N HCl in 50% isopropanol) for 15 min before collection by centrifugation at 2,000 rpm for 1 min.

The two eluants were combined, dried using the speed vacuum pump (about 1 h), and resuspended in 50 µl of autoclaved Milli Q water. The labeling of the peptide was determined by measuring the counts of 1 µl sample in 3 ml of scintillation fluid.

The acetylated peptides had a specific activity of approximately 6700 cpm/ µg and

79% of the labeled peptide was recovered after purification as described above.

2.3.13 Histone deacetylation assay

To measure the histone deacetylase activity of BRG1 and BRM complexes, im- munopurified hSWI/SNF complexes were incubated with equal amounts of each pu- rified acetylated histone peptide. HDAC reaction was set up by mixing 0.25 µg of immunopurified hSWI/SNF complexes with acetylated histone peptide (1 x 104 cpm), and BC100 to a final volume of 20 µl, and reactions were incubated at 37oC for 5 h.

To control for the specificity of HDAC activity, reactions containing HDAC inhibitor, sodium butyrate (50 mM) were also included. The reactions were terminated by adding 50 µl of freshly prepared quenching solution (259 µl of 12.1 N HCl, 28 µl of

76 17.4 N acetic acid and 2.713 ml MilliQ water). The released [3H]-acetate was ex- tracted by adding 600 µl of ethyl acetate and rigorously vortexing the reactions for

3 min. The extracted [3H]-acetate was quantitated by measuring in duplicate 200 µl aliquots of the upper ethylacetate phase in 3 ml of scintillation fluid.

2.3.14 Northern blot analysis

Total RNA was extracted from cells using the guanidium thiocyanate-phenol- chloroform protocol as described [315]. Currently, trizol reagent (Invitrogen Inc., catalog no.15506-018) is used for isolation of total RNA as described in section 3.3.4.

To measure the mRNA levels, approximately 30 µg of total RNA was loaded on a

1% formaldehyde agarose gel (3.5 g of agarose melted in 217.5 ml DEPC-treated water, cooled to about 60oC, and then add 70 ml of 5X gel running buffer (0.1 M

MOPS [pH 7.0], 40 mM sodium acetate, 5 mM EDTA) and 62.5 ml formaldehyde) that was pre-run at 5V/cm for 5 min immediately before the samples were loaded.

To prepare the samples, 4.5 µl of total RNA (equal to 30 µg), was mixed with 2 µl of 5X gel running buffer, 3.5 µl of formaldehyde and 10 µl of formamide, denatured at 65oC for 15 min, cooled on ice for 2 min, and then 2 µl of gel loading buffer

(0.25% bromophenol blue, 0.25% xylene cyanol, 50% glycerol, 1 mM EDTA) was added before loading. The samples were separated by electrophoresis at 4 V/cm in

1X gel running buffer until the bromophenol dye has migrated about 9 cm. The gel was gently removed, rinsed twice with DEPC-water for 15 min, and then soaked in

0.05 N NaOH for 30 min to allow in gel alkali hydrolysis of RNA. Following this, the gel was washed once with DEPC water for 5 min, and soaked in 20X SSPE-NaOH

[pH 7.4] (3.6 M NaCl, 0.2 M NaH2PO4, 0.02 M EDTA, and adjust pH to 7.4 with 10N

77 NaOH) for 45 min. Next, the gel was transferred onto Hybond-XL nylon membrane

(Amersham Pharmacia Biotech. Inc., catalog no. RPN1510S) as described [242] in

20 X SSPE overnight by capillary action. Next day, the membrane was removed after marking the wells with a pencil, and rinsed in 6X SSPE for 5 min. Then the

membrane was dried for 30 min, and RNA was crosslinked to the membrane using

the Stratalinker 1800 in the auto-crosslink mode. Next, the membrane was soaked in

6X SSPE for 5 min, prior to blocking with 5 ml ExpressHyb solution (Clontech, Inc.,

catalog no. 636831) for 2 h at 42oC. Following prehybridization, the membranes were

hybridized to the desired DNA probes in 5 ml of ExpressHyb containing 1.5 to 2.0

x106 cpm/ml of [32P] radiolabeled DNA probe for 2 h at 42oC. To remove unbound

radiolabeled probes, the membrane was washed successively twice with 2X SSPE

containing 0.05% SDS at room temperature for 20 min, once with 1X SSPE containing

0.1% SDS at room temperature for 30 min, and once with 1X SSPE containing 0.1%

SDS at 50oC for 30 min. The membrane was finally rinsed in 6X SSPE and exposed

to a phosphorImager screen overnight. Probes used to detect human CAD (+172

to +467), human NUC LEOLIN (+348 to + 670), and human GAPDH (+177 to

+578) were synthesized by RT-PCR using specific primers (sequences are provided

in Appendix B) and 10 µg of total RNA from HeLa cells.

To generate the cDNA for each probe, individual RT reaction was set up with

10 µg of HeLa S3 total RNA, 20 pmol of the reverse primer, 3.5 mM MgCl2 and 1X

Taq polymerase buffer, in a volume of 16.5 µl. The reactions were heat denatured

successively at 80oC for 4 min, cooled on ice and again incubated at 94oC for 5 min,

followed by cooling on ice for 2 min. Then 0.5 µl of RNasin (RNase inhibitor, Promega

Inc., catalog no. N2115) was added, and the reactions were incubated at 42oC for 30

78 min prior to adding 3.0 µl of RT cocktail (1.5 µl AMV Reverse Transcriptase (10U/

µl; Promega Inc., catalog no. M5101), 0.8 µl 25 mM dNTP mix and 0.7 µl DEPC treated water), and incubating at 42oC for 1 h. The samples were heat inactivated at 99oC for 5 min and stored at −20oC. Next, 2 µl of the above RT reaction was amplified in a PCR reaction as described in section 2.3.1.

2.3.15 Radiolabeling of DNA probes

To generate high specificity radiolabeled DNA probes, the DNA fragments were labeled throughout with [α-32P]-dCTP. A labeling reaction was set up with 200 ng of DNA, which was denatured in a volume of 29.8 µl at 100oC for 3 min, and then incubated on ice for 2 min. To the denatured DNA, 0.4 µl of each dATP, dGTP, dTTP

(25 mM stock solution) was added followed by 5 µl of BSA (1mg/ ml), 5 µl of 10X

Klenow buffer (Eco pol buffer), 3 µl of random hexamers (IDT Inc., resuspended as

0.1 O.D/ µl), 1 µl of Klenow polymerase (5 U/ µl, NEB Inc., catalog no. M0210S),

5 µl of [α-32P]-dCTP, and reaction was incubated at 37oC for 45 min. Then, the reaction was heat inactivated at 65oC for 20 min, volume was increased to 100 µl with water, and purified by gel exclusion over sephadex G-50 column. To prepare the column, 1 ml of swelled G-50 beads were packed in 1 ml syringe by consecutively adding the bead slurry, and spinning the column at 2,000 rpm for 30 sec. Once the column is packed, spin at 2,000 rpm for 4 min to remove any buffer that might be left within the packed beads. To remove the unincorporated free dNTPs, the sample was loaded slowly at the center of the column, and spun at 2,000 rpm for 4 min. The eluant contains the labeled probe and the specific activity (cpm/µg) of the probe was

79 determined by scintillation counting of 1 µl eluant. A successful labeling reaction

will yield probe at > 1x 107 cpm/µg specific activity.

2.3.16 Metabolic labeling and double immunoprecipitation assay

Cellular proteins were labeled in vivo by incubation 1.2 x 107 cells in media lacking methionine and cysteine (Gibco BRL Inc., catalog no.21013024) at 37oC for 2 h. Next,

760 µCi of [35S]- labeled methionine and cysteine was added and cells were allowed to grow for an additional 4 h. Then, nuclear extracts were prepared according to the protocol previously described [168]. Approximately, 500 µg of [35S]- labeled nuclear extracts were incubated with either preimmune or immune antibodies in a 250 µl reaction containing IP buffer (20 mM Tris-HCl [pH 7.4], 100 mM NaCl, 5 mM MgCl2,

1 mM EDTA, 0.1% NP-40, 1% aprotinin). After 1 h incubation on ice, during which the samples were mixed by gentle flicking, 75 µl of protein A sepharose beads were

added to bind the immune complexes, and the samples were incubated at 4oC for

another 14 h. The beads were collected by centrifugation at 2,000 rpm for 2 min, and

washed 5 times with 500 µl of IP buffer containing 150 mM NaCl. Bound protein

complexes were released by heat denaturation at 75oC for 5 min in the presence of 100

µl IP buffer supplemented with 0.5% SDS, and the supernatant was used to perform a

second immunoprecipitation using specific antibodies as described above. Following

the second overnight binding, the beads were washed 5 times with 500 µl of IP buffer

containing 150 mM NaCl, boiled in 25 µl of 2X SDS-PAGE loading dye, spun at

14,000 rpm for 3 min, and 18 µl of the supernatant was loaded onto a 8% SDS-PAGE

gel. After the electrophoresis, the gel was treated with 1 M salicylic acid for 30 min,

80 dried at 80oC for 50 min, and then exposed to X-ray film overnight at −80oC before

developing the autoradiogram.

2.3.17 Chromatin immunoprecipitation assay

Cross-linked chromatin was prepared as described previously [82]. Approximately,

1x 107 cells were cross-linked with 1% formaldehyde on shaker at room temperature

for 10 min, and the reaction was terminated by adding glycine [pH 3.5] to a final concentration of 0.125 M. The cells were washed twice with 5 ml of 1X TBS (20 mM Tris-HCl [pH 8.0], 150 mM NaCl), and harvested in 1 ml of lysis buffer (50 mM Tris-HCl [pH 8.1], 100 mM NaCl, 5 mM EDTA, 0.5% SDS, protease inhibitors).

Cells were collected by centrifugation and resuspended in 250 µl of IP buffer (100 mM

Tris-HCl [pH 8.6], 5 mM EDTA, 1.7% Triton X-100, protease inhibitors). Chromatin was solubilized to a bulk size of 0.5-2 Kbp by sonication using a microtip Branson sonifier 450 (Output control at 5 and duty cycle of 30%), clarified by centrifugation at 14,000 rpm at 4oC for 10 min and stored at −80oC. The solubilized chromatin is stable for about 2 months. To immunoprecipitate specific chromatin fragments,

250 µl of solubilized chromatin was precleared with 30 µl of pre-blocked protein A beads (0.2 mg/ ml salmon sperm DNA (stock 10 mg/ml; Boehringer Mannheim

GmbH Inc., catalog no. 223646), 0.5 mg/ ml BSA) at 4oC on nutator for 2 h.

The supernatant was moved to a new tube after centrifugation at 14,000 rpm for 3 min, and the specific antibodies were added (anti- BRG1- 25 µl; anti-BRM- 40 µl; anti-flag- 35 µl; anti-PRMT5 full length (skb)- 45 µl; anti-MYC (N-262) (Santa Cruz

Inc., catalog no. sc-764)-5 µg; anti-MAD (Santa Cruz Inc., catalog no. sc-222)- 5 µg; anti-HDAC2 (Zymed Inc., catalog no. 515100)- 5 µg; anti-mSIN3A (K-20) (Santa

81 Cruz Inc., catalog no. sc-994)- 5 µg; anti-H3(Me2)R8-35 µl; anti- H4(Me2)R3- 40

µl), and tubes were incubated on nutator at 4oC for 5-6 h . To capture the immune complexes, 40 µl of pre-blocked protein A beads were added, and the reactions were left on the nutator at 4oC for 14 h. Bound nucleoprotein complexes were collected by centrifugation at 2,000 rpm for 2 min, and washed once successively with 300 µl of mixed micelle buffer (20 mM Tris-HCl [pH 8.1], 150 mM NaCl, 5 mM EDTA, 5%

[w/v] sucrose, 0.2% Triton X-100 and 0.2% SDS), buffer-250 (50 mM HEPES [pH 7.5],

250 mM NaCl, 1 mM EDTA, 0.1% deoxycholine, 0.2% Triton X-100), LiCl detergent buffer (10 mM Tris-HCl [pH 8.0], 250 mM LiCl, 1 mM EDTA, 0.5% deoxycholine,

0.25% NP-40) and TE [pH 7.6]. To elute the immune complexes, the washed beads were rigorously vortexed in 100 µl of elution buffer (50 mM Tris-Hcl [pH 8.0], 10 mM EDTA, 0.1% SDS) and incubated at 65oC for 10 min. The samples were spun at 14,000 rpm for 3 min, the supernatant was transferred to new tubes, and the beads re-eluted with 100 µl TE [pH 8.0] supplemented with 0.067 % SDS. Both the eluants were combined and DNA-protein crosslink was reversed by incubation at

65oC overnight. The immunoprecipitated DNA was purified by phenol: chloroform extraction following proteinase K treatment (250 µl proteinase K solution in TE [pH

8.0] containing 100 µg of proteinase K, 15 µg of tRNA) at 37oC for 2 h. The DNA was precipitated in the presence of 1/10th volume of 4 M LiCl and 2 volumes of 100% ethanol at −20oC overnight, resuspended in 35 µl TE [pH 8.0] supplemented with

RNase (0.2 µg/ µl), and tRNA removed by incubating the samples at 37oC for 30 min. Eluted DNA was PCR-amplified using 10 µl of the eluted DNA along with 100 pmol each of 5’ primer and 3’ primers in a PCR reaction (50 µl) supplemented with

2 µCi of [α-32P] dCTP for 35 cycles as described in section 2.3.1. Sequence of the

82 specific primers used to amplify human CAD (-157 to +161) and ODC (-5 to +377)

promoter sequences are provided in Appendix B.

2.3.18 Cell culture and transformation assay

Wild type and HeLa S3 cell lines that express Fl-BRG1, Fl-BRG1 (K798R) and

Fl-BRM cell lines and NIH3T3 cells were grown in Dulbecco’s modified Eagle’s media

(DMEM) supplemented with 10% fetal bovine serum (FBS). For the growth of flag

tagged BRG1 and BRM cell line, 2.5 µg/ml of puromycin was included to prevent

the loss of plasmid expressing flag tagged protein. HeLa S3 cells were synchronized

in G1 by plating 1.2 x 107 cells per 100 mm plate in the presence of serum. After 4 h,

cells were washed twice with 1X PBS, starved for 48 h in media without serum, and

released from the block by adding serum to a final concentration of 40%. To establish

NIH3T3 cell lines that express c-MYC, Ha-RAS and either flag-tagged mutant BRG1

or mutant BRM, 0.5 µg of each retroviral vector was transfected in 70% confluent

NIH3T3 cells either individually or in combination using lipofectamine-puls reagent

as described in section 2.3.8. Cells were then selected in the presence of 2.5 µg/ml

puromycin (for flag tagged Mut BRG1 and BRM) and 480 U/ml hygromycin (for

MYC and Ha-RAS) for two to three weeks, and used to measure anchorage indepen-

dent growth and growth rates as described [92]. To compare the proliferation rate

of NIH3T3 based cell lines, 2 x 105 cells were plated in duplicates and viable cells

were counted by adding trypan blue dye which only stains dead cell, after 2, 4, and

6 days. This experiment is repeated four times as above.

Transformation assay was performed with 2 x 102 cells of either NIH3T3 cells

expressing empty pBabe vector or cells expressing MYC and RAS with or without

83 flag tagged mutant BRG1 or BRM in triplicates. 3% stock agar solution was prepared by adding 1 g of agar to 30 ml of milliQ water, which was autoclaved for 20 min and incubated at 56oC. To grow cells in anchorage independent manner, working

solution of agar (0.3%) was prepared by 1:10 dilution of the 3% agar stock in DMEM

supplemented with 10% FBS and equilibrated at 56oC until needed. Next, 0.5 ml

media containing 200 cells was transferred to 15 ml tubes, and then 0.3% agar solution

was moved to 40oC for 10 min. Finally, 4 ml of 0.3% agar stock, which is at 39-40oC

as assessed by sensitivity on skin, is added to the cell, mixed, and quickly transferred

to bacterial non-coated 60 mm petridishes (Tritech Research Inc., catalog no. 3308).

The agar is allowed to cool for 10 min, and then the plates are transferred to 37oC

tissue culture incubator for 10-14 days to form colonies, which are then counted and

photographed.

2.3.19 Antibodies and Western blot analysis

Proteins were electrophoresed on an 8-10% SDS-polyacrylamide gel, transferred

onto nitrocellullose membrane (Osmonics, Inc., catalog no. EP4HY00010) overnight

at 200 mA, and Western blotting was performed as described below using the anti-

bodies listed in Appendix C. The membrane was blocked for 2 h in 20 ml of blocking

solution [1X TBS (10 mM Tris-Hcl pH7.4, 127 mM NaCl) containing 0.05% Tween-20

and 5% carnation milk], and the blocking solution was changed every 30 min. Next,

the membrane was rinsed three times with 10 ml of wash buffer (1X TBS supple-

mented with 0.05% Tween-20) for 10 min before incubation for 2 h with the desired

primary antibody in 10 ml of antibody solution (diluted in wash buffer containing 2%

84 milk carnation; Anti-skb-1:200, Anti-N-HMT-1:500, Anti-BRG1-1:3,000; Anti-BRM-

1:200, Anti-BAF155-1:200; Anti-BAF60-1:200; Anti-BAF57-1:500; Anti-MYC-1:500,

Anti-MAD1-1:500, Anti-SIN3A-1:500; Anti-HDAC2-1:2,500, Anti-β ACTIN-1:3,000,

Anti–β TUBULIN-1:100, Anti-Flag-1:500; Anti-ST7-1:400). Then the membrane was

washed three times in 10 ml of wash buffer for 10 min each and incubated with the required secondary antibody for 1 h in 10 ml of secondary antibody solution (horse raddish-peroxidase linked anti-rabbit or anti-mouse antibody diluted at 1:3,000 in wash buffer). Finally, the membrane was washed extensively in 10 ml of wash buffer

four times for 5 min each, before specific antibody signal was detected using a equi-

volume mixture of ECL reagent 1 and 2 (1 ml each) (Amersham Pharmacia Biotech.,

Inc., catalog no. RPN2106).

2.4 Results

2.4.1 mSIN3A and mSIN3B interact directly with BRG1, BRM, BAF57 and BAF45/INI1 hSWI/SNF subunits

Affinity purification of BRG1 and BRM complexes from HeLa cell lines expressing

either flag tagged BRG1 or BRM indicated that mSIN3A/HDAC is associated with

hSWI/SNF complexes [260]. To identify the subunits of hSWI/SNF complexes that

interact with mSIN3/HDAC complex, GST pull down experiments were performed

using mSIN3A and B proteins as they are core components of the mSIN3/HDAC

complex. Both SIN3 proteins contain four conserved PAH (paired amphipathic he-

lix), and one HID (histone deacetylase interaction) domains (Figure 2.1A). To map

the interactions, GST fusion protein of each individual PAH domain was incubated

with in vitro translated and 35S-labeled hSWI/SNF subunits, and analyzed by au-

toradiography (Figure 2.1B). While, both BRG1 and BRM interacted with PAH3

85 and 4 domains of mSIN3A and B, BAF57 and BAF45/INI1 were able to interact with only PAH4 domain of mSIN3A and B. These interactions were specific, because

PAH1 and 2 domains did not interact with any of the above proteins, and also no

interaction was seen between PAH3/4 and hSWI/SNF subunits, BAF170, BAF155,

and BAF53.

To further define the regions of hSWI/SNF subunits that interact with SIN3

proteins, different regions of BRG1, BRM, and BAF57 were tested for their ability

to interact with PAH3 and 4 domains of mSIN3A and B. Deletion of the C-terminal

region of BRG1 and BRM, which contain the bromodomain known to be involved

in protein-protein interaction, did not affect the interactions with PAH3/4. When

the N-terminal region of BAF57 containing the HMG domain, which mediates DNA

binding was tested in GST pull down experiments it was unable to interact with

GST-PAH4 fusion protein (Figures 2.2A and B). Thus the kinensin like coiled-coil

region of BAF57 is crucial for mSIN3A and B interactions. These results suggest that

mSIN3/HDAC complex associates with hSWI/SNF complexes through interactions

with multiple subunits.

2.4.2 Protein arginine methyltransferase, PRMT5 is associ- ated with flag-tagged BRG1-and BRM-based hSWI/SNF complexes

Purification of flag-tagged BRG1 and BRM complexes from the respective HeLa

cell lines have indicated that there are additional proteins that are associated with the

complexes. To identify these proteins, both flag-tagged BRG1 and BRM complexes

were affinity purified and concentrated by TCA precipitation. The purified complexes

were visualized by Coomassie blue staining, and the unknown band migrating around

86 66KDa as well as the other prominent bands were excised and sent to Dr. P.Tempst laboratory for identification by mass spectrometry (Figure 2.3A and B) [306].

Mass spectrometry analysis confirmed the presence of known hSWI/SNF sub- units and indicated that BRM complex contains both BAF60a and b isoforms. Also, the previously unidentified 66 KDa subunit was found to be PRMT5, which is a type II protein arginine methyltransferase (Figure 2.3B). To verify this result, poly-

clonal antibodies were generated against GST-PRMT5 fusion protein and Western blot analysis of both flag-tagged BRG1 and BRM complexes show that PRMT5 is

associated with the immunopurified complexes (Figure 2.4A). When nuclear ex-

tracts from HeLa cells that do not express any flag-tagged proteins was purified on

anti-flag beads, and analyzed by Western blotting using the anti-PRMT5 antibod-

ies, no signal was detected, indicating that PRMT5 is not a contaminant of anti-flag

purification (Figure 2.4A, lane 1). To further confirm the association of mSIN3A

and PRMT5 with hSWI/SNF complexes in vivo, HeLa nuclear extracts that have

been fractionated on BioRex-70 and contain hSWI/SNF complexes were immuno-

precipitated using either preimmune or immune anti-BRG1 and BRM antibodies.

Both mSIN3A and PRMT5 were detected in the anti-BRG1 and BRM immunopre-

cipitates, suggesting that both proteins are associated with endogenous hSWI/SNF

complexes (Figure 2.4B). Previous work by Sif et. al [260], have shown that BRG1-

based hSWI/SNF complex can be further fractionated into two distinct complexes

which differ in their remodeling activity. Fractionation of affinity purified BRG1- and

BRM-based hSWI/SNF complexes on glycerol gradients reveal that as previously de-

scribed, BRM co-sediments with mSIN3A and PRMT5 proteins, while the BRG1

87 complex I contains both mSIN3A and PRMT5, which are lacking in complex II (Fig- ure 2.4C). Altogether, mSIN3A and PRMT5 coexist in both BRG1- and BRM-based hSWI/SNF complexes.

2.4.3 PRMT5 interacts directly with components of hSWI/SNF complexes and mSIN3A

To identify the hSWI/SNF subunits that interact with PRMT5, GST pull-down assays were performed using full-length PRMT5. Similar to the results presented in Figure 2.1B both hSWI/SNF ATPases, BRG1 and BRM, along with BAF57 and

BAF45/INI1 interacted with GST-PRMT5 (Figure 2.5A). Since mSIN3A coexists with PRMT5 in hSWI/SNF complexes, the ability of PRMT5 to directly interact with mSIN3A was tested and it was found that mSIN3A directly associate with

GST-PRMT5. To further confirm the interaction results above, PRMT5 was co- translated and 35S-labeled with either BRG1, BRM, BAF155, BAF57, BAF45/INI1, or mSIN3A and then immunoprecipitated in the presence of specific hSWI/SNF and mSIN3A antibodies (Figure 2.5B). As expected from the above results, PRMT5 was co-immunoprecipitated with BRG1, BRM, BAF57, BAF45/INI1, and mSIN3A, however, anti-BAF155 antibodies did not precipitate PRMT5, indicating that the interactions were specific. The specificity of the antibodies was tested by incubat- ing each antibody with in vitro translated PRMT5 protein, which shows that the antibodies used above do not cross react with PRMT5 (Figure 2.5C).

To demonstrate that hSWI/SNF subunits interact with PRMT5 in vivo, mammalian- two hybrid experiments were performed. BRG1, BRM, BAF57 and BAF45/INI1 cDNAs were cloned in frame with the activation domain of herpes simplex virus pro- tein, VP16; while PRMT5 cDNA was fused in frame with GAL4 (aa1-147) DNA

88 binding domain. Both Gal4 fusion and one of the VP16 fusion-expressing plasmids were co-transfected in HeLa cells along with a GAL4 driven CAT reporter plasmid

that contains five GAL4 binding site (pG5-Hsp70-40 CAT) (Figure 2.6 A). Analysis

of CAT expression in transfected cells, indicated that only in the presence of either

BRG1, BRM, BAF57, or BAF45/INI1 fused VP16 protein, CAT gene was activated 2-

to 5-fold, suggesting that all the tested hSWI/SNF components interact with PRMT5

in vivo. In addition, it was found that GAL4-PRMT5 fusion protein could repress the

basal expression of CAT reporter gene, raising the possibility that PRMT5 functions

as a transcriptional repressor. These results demonstrate that like mSIN3 proteins,

PRMT5 interaction with BRG1 and BRM based hSWI/SNF complexes is mediated

through multiple contacts.

2.4.4 PRMT5 interacts with mSIN3 and hSWI/SNF com- plex components through both N and C- terminal amino acid sequences

To map the region of PRMT5 that mediates protein-protein interaction with

mSIN3 and hSWI/SNF, PRMT5 protein was divided into two regions and expressed

as GST fusion proteins. GST-N-PRMT5 represents the unique N-terminal region (aa

5-280) that lacks the catalytic domain, while GST-C-PRMT5 (aa 281-637) contains

the conserved catalytic domain of PRMT family. GST pull down experiments were

conducted using immobilized GST-N-PRMT5 and GST-C-PRMT5 along with GST

and GST-PAH2 as negative controls, and full length GST-PRMT5 as the positive

control (Figure 2.6 B). In vitro translated and 35S-labeled BRG1, BRM, BAF170,

BAF57, BAF45/INI1, mSIN3A, mSIN3B, and PRMT5 were tested for their ability

to interact with N and C-terminal regions of PRMT5. Similar to full length PRMT5,

89 both N and C-terminal GST-PRMT5 fusions were able to interact with hSWI/SNF component and mSIN3A and B proteins. As seen in figure 2.5A, no interaction was observed with in vitro translated BAF170 subunit. Since, it has been reported that PRMT5 could oligomerize in vivo, the interaction of PRMT5 with itself was tested, which show that PRMT5 binds itself through both N and C regions [235].

These results were verified by performing mammalian two hybrid experiments using

GAL4-N-PRMT5 and GAL4-C-PRMT5 fusion proteins (Figure 2.6 C). Similar to the GST pull down experiments, both N- and C- PRMT5 could mediate interactions with hSWI/SNF subunits, though BRG1, BRM, BAF57, and BAF45/INI1. In addi- tion, like full length PRMT5, both N- and C-PRMT5 could repress CAT expression.

These results suggest that the interaction of PRMT5 with the specific hSWI/SNF subunits and mSIN3 corepressor is not limited to a certain region, and it occurs through multiple contacts in PRMT5 protein.

2.4.5 PRMT5 methylates histones H3 and H4

Since PRMT5 belongs to the family of arginine methyltransferases, which have been shown to methylate histones and regulate transcription, the ability of PRMT5 to acts as a histone methyltransferase (HMTase) was tested in vitro. When recombinant

GST-PRMT5 was used in HMTase assay, it was unable to methylate any histones.

Since some proteins require posttranslational modification to be active, flag tagged

PRMT5 was expressed in Sf9 cells using the Bac- to Bac- baculovirus expression system. Using anti-flag column, recombinant PRMT5 was purified, and incubated with HeLa core histones in the presence of [3H]-S-adenosyl methionine (SAM), which shows that PRMT5 methylates histones, H3 and H4 (Figure 2.7 A). Similarly, when

90 either BRG1- or BRM- based hSWI/SNF complexes that contain PRMT5, were tested in HMTase experiments, H3 and H4 were methylated, and this methylation was aug- mented when the reactions were supplemented with recombinant PRMT5 (Figure

2.7 A). To verify that the methylation activity was catalyzed by PRMT5, catalyti- cally inactive PRMT5 was generated by mutating conserved glycine 367 and arginine

368 to alanine in the catalytic domain (Fl-Mut PRMT5). When Fl-Mut PRMT5 was incubated with histones, there was no detectable methylation, attributing the methylase activity to PRMT5 (Figure 2.7A). Hence, both recombinant PRMT5 and hSWI/SNF complexes target the same histones for methylation.

The results presented in figure 2.4 show that both mSIN3A and PRMT5 coexist

in hSWI/SNF complexes, and work by various groups have established cross-talk be-

tween histone acetylation and methylation. Thus, the ability of PRMT5 to methylate

acetylated histones was tested. Acetylated histones were isolated from sodium bu-

tyrate treated HeLa cells, while HeLa core histones purified from untreated cells are

hypoacetylated. Comparison of PRMT5 methylase activity on these histones show

that hypoacetylated histones are better substrates for PRMT5 containing BRG1 and

BRM complexes, suggesting that histone deacetylation is a prerequisite for PRMT5

to methylate histones, H3 and H4 (Figure 2.7). Since most post translational modi-

fications including acetylation occur on N-terminal tails, N-terminal tails of histones

H2A, H2B, H3, and H4 were acetylated. H2A and H2B were acetylated chemically

using the Upstate HDAC assay kit, and H3 and H4 were acetylated using bacteri-

ally expressed and purified MBP-MORF in the presence of [3H]-AcetylCoA (Figures

2.8 A and B). Acetylated peptides were individually purified, and incubated with

mSIN3A/HDAC and PRMT5 containing-BRG1 and BRM complexes in the presence

91 and absence of HDAC inhibitor, sodium butyrate to show that the release of acetate was due to HDAC activity (Figure 2.8 C). In agreement with the above results, both hSWI/SNF complexes deacetylated only H3 and H4 tails. Taken together, these re- sults suggest that hSWI/SNF associated PRMT5 relies on the activity of associated mSIN3/HDAC complex for efficient histone methylation.

2.4.6 CAD expression is upregulated in the presence of cat- alytically inactive BRG1 or HDAC inhibitor

Since both mSIN3A/HDAC2 and PRMT5 are associated with hSWI/SNF com- plexes, the role of BRG1 remodeling activity in the transcriptional regulation of mSIN3A target genes was evaluated. mSIN3A/HDAC corepressor complex is in- volved in the transcriptional repression of MYC/MAX/MAD target genes, that are activated by the binding of MYC-MAX heterodimer, and repressed when MAD-MAX is bound to the E- box [6, 7]. The MYC target genes, CAD and NUCLEOLIN

(NUC ), which are repressed in serum starved cells and are activated on induction of

MYC expression by serum stimulation, were selected for further analysis [33]. The expression of both genes was measured in HeLa cells that were synchronized by serum starvation, and then allowed to enter cell cycle by serum stimulation. Northern blot analyses show that the expression of CAD in wild type HeLa cells is induced 3 to

5-fold in serum stimulated cells after 2-6 h and the basal expression is re-established by 14-16 h post serum stimulation, consistent with previous findings in NIH3T3 and

Rat1a cells [33, 175]. In contrast, when CAD expression was analyzed in HeLa cells expressing catalytically inactive BRG1 (K798R, Mut BRG1), it was found that CAD gene is derepressed 3-fold, suggesting that BRG1 remodeling activity is essential for proper regulation of CAD expression (Figure 2.9A and B, compare HeLa and Mut

92 BRG1 CAD mRNA levels at T= 0, 14 and 16 h). Since, CAD is a known MYC and

MAD target gene, the role of histone deacetylation in CAD repression was verified by treating HeLa cells that have been synchronized and serum stimulated with HDAC inhibitor, depsipeptide for 4 h (HeLa + Depsi). Northern blot results show that

CAD is derepressed 6-7 fold in the presence of depsipeptide (Figure 2.9A and B).

However, the contribution of PRMT5 to the regulation of CAD expression could not be evaluated, because high dose of methyltransferase inhibitor, methylthioadenosine

(MTA) that repressed hSWI/SNF-associated PRMT5 activity in vitro was toxic to cells (Figure 2.9C). To determine if BRG1 was involved in the regulation of other

MYC/MAX/MAD target genes, we analyzed the expression of NUC on the same blot, and found that unlike CAD, NUC induction was delayed and the mRNA levels were lower in Mut BRG1 cells (Figure 2.9A and B). However, as expected inhibi- tion of histone deacetylation resulted in 4-fold derepression of NUC , suggesting that

BRG1 is not involved in the transcriptional repression of NUC , which is dependent on histone deacetylation. Furthermore, analysis of GAPDH expression in the presence of Mut BRG1 and depsipeptide, reveal that neither had any affects on its expression

(Figure 2.9A and B). Altogether, these results show that chromatin remodeling and histone deacetylation are involved in transcriptional repression of CAD.

2.4.7 BRG1, mSIN3A/HDAC2, and PRMT5 are recruited to the CAD promoter

Finding above have shown that BRG1 mediated chromatin remodeling and histone deacetylation are required for normal regulation of CAD expression, however, it is not clear if these activities are required at the CAD promoter, and are directly involved

93 in the transcriptional repression. To address this issue, chromatin immunoprecipi-

tation (ChIP) experiments were performed using anti-BRG1 as well as anti-HDAC2 antibodies, and recruitment on the CAD promoter was measured by PCR using the

indicated primers (Figure 2.10A). First, the recruitment of c-MYC and MAD pro- teins, which are linked with transcriptional activation and repression, respectively, were analyzed in HeLa cells that had been synchronized and then serum stimulated for different time points. The recruitment of c-MYC at 2 and 6 h, and MAD1 at 0 and 16 h correlated with CAD expression (compare Figures 2.9A and 2.10A). How-

ever, when BRG1 recruitment was analyzed, it was found that BRG1 was associated with CAD promoter during both transcriptional activation and repression (2, 6, 14,

and 16 h), suggesting that BRG1 is involved in both processes (Figure 2.10 A). In addition, the kinetics of Mut BRG1 recruitment to the CAD promoter in HeLa cells expressing catalytically inactive BRG1 was same (compare Figure 2.10A, left and right panels). When BRG1 recruitment to another MYC/MAX/MAD target gene,

ODC , was analyzed, it was found that though c-MYC is associated with the ODC promoter, there was no BRG1 recruited to the promoter, showing specificity of BRG1 binding to the CAD promoter (Figure 2.10B).

Further analysis of the recruitment of BRG1 complex associated proteins, mSIN3A and HDAC2, revealed that these proteins are associated with CAD promoter at T= 0 h, when the gene is repressed and this association is lost at 2 h post serum stimulation upon CAD induction. Association of these proteins was re-established by 6 h, which is

followed by transcriptional repression of CAD (Figure 2.10A). Taken together, these

results indicate that mSIN3A and HDAC2 are recruited to CAD promoter through

their interactions with various subunits of hSWI/SNF complex to repress CAD.

94 Though the role of histone methylation in CAD expression has not been tested, and PRMT5, which associates with BRG1 complex as well as mSIN3A, has been implicated in transcriptional repression of CYCLINE1 , the recruitment of PRMT5 to the promoter of CAD was tested [71]. ChIP assay results show that PRMT5 is recruited to the CAD promoter concomitantly with HDAC2 and remains associated with the CAD promoter even after 16 h of serum stimulation, suggesting that it is involved in CAD repression. To evaluate if the differential recruitment of the above proteins was due to changes in their expression, Western blot analysis was performed on nuclear extracts from HeLa cells that had been synchronized and serum stimulated as before (Figure 2.10C). BRG1, PRMT5 and HDAC2 protein levels did not fluctuate at different time points, indicating that chromatin-modifying activities are recruited to CAD promoter in a cell cycle dependent manner.

2.4.8 c-MYC directly interacts with hSWI/SNF complexes that lack mSIN3A and PRMT5

Having found that BRG1 is recruited to CAD promoter during activation along with c-MYC, and since BAF45/INI1 has been shown to interact with c-MYC to ac- tivate MYC driven reporter gene in a BRG1 dependent manner [42], association of c-MYC with hSWI/SNF complexes was tested. HeLa nuclear extracts were incubated with GST-MYC, and the bound proteins were analyzed by Western blotting for the presence of BRG1- and BRM- based hSWI/SNF complex components (Figure 2.11A, lanes 1-4). The GST pull down experiment shows that both BRG1 and BRM com- plexes can specifically interact with c-MYC, since no interactions were observed with either GST or GST-PAH2A proteins. To exclude the possibility that other c-MYC interacting nuclear proteins like TRRAP mediated the interactions, immunopurified

95 hSWI/SNF complexes that lack TRRAP were used in pull down assays (Figure 2.11 lane 5-8) [182, 183]. Similar to the results with nuclear extract pull downs, BRG1,

BRM, BAF155, and BAF45/INI1 were detected in the GST-MYC reaction. Since

mSIN3A/HDAC and PRMT5 are associated with hSWI/SNF complexes, their pres-

ence in the GST-MYC pull down reactions were analyzed, and Western blotting

revealed that hSWI/SNF complex that interacts with GST-MYC lacks PRMT5 and mSIN3A/HDAC, indicating that different pools of hSWI/SNF complexes exist in vivo that are associated with either activator or repressor proteins.

To verify this interaction in vivo, immunoprecipitation experiments were con- ducted using metabolically 35S-labeled nuclear extracts from asynchronous HeLa cells

(Figure 2.11B). Nuclear extracts were incubated with either preimmune or immune

BAF155, BAF57, or BAF45/INI1 antibodies. After extensive washing, the retained

protein complexes were released by heat denaturation and immunoprecipitated again

using anti-MYC antibodies. When the first immunoprecipitation was performed us-

ing any of the hSWI/SNF subunit antibodies, c-MYC was immunoprecipitated unlike

the control preimmune antibody, indicating that hSWI/SNF associate with c-MYC

in vivo.

To identify the hSWI/SNF subunits that mediate interaction with c-MYC, GST

pull down experiments were performed using immobilized GST-MYC and 35S-labeled

hSWI/SNF proteins. Similar to the results in figure 2.1 and 2.3, BRG1, BRM,

BAF57, and BAF45/INI1 were able to interact specifically with c-MYC (Figure 2.11C,

lane 4). To map the c-MYC regions that can interact with hSWI/SNF subunits, GST

fusion proteins that include either the transcriptional activation domain (TAD), the

96 hinge region, or the DNA binding domain (DBD) were generated, and used in GST- pull down assays (Figure 2.11D). Unlike BRG1 and BAF45/INI1, which appeared to

interact equally well with all three c-MYC regions, BRM showed preference towards

the hinge region, while BAF57 interacted with the TAD and hinge region. Collec-

tively, these results suggest that c-MYC associates with BRG1 and BRM complexes

by physically interacting with the same hSWI/SNF subunits that are targeted by

mSIN3 and PRMT5 corepressors.

2.4.9 Expression of catalytically inactive BRG1 or BRM in- hibits NIH3T3 co-transformation by c-MYC and Ha- RAS

c-MYC is an oncogene whose overexpression has been linked to human can- cers [203]. Previous work has reported that overexpression of c-MYC along with

Ha-RAS can induce transformation in embryonic fibroblast since they are involved in the regulation of genes that participate in cell growth, cytoskeleton structure and chro-

matin modification [48, 159, 264, 257, 345]. Since c-MYC interacts with hSWI/SNF

complexes and BRG1 complex can regulate MYC target genes like CAD, the role of

BRG1 and BRM in c-MYC mediated transformation was evaluated. For this purpose,

stable polyclonal NIH3T3 cell lines were established that expressed either c-MYC and

active Ha-RAS or catalytically inactive flag-tagged BRG1 (Mut BRG1) or BRM (Mut

BRM) in addition to c-MYC and Ha-RAS. (Figure 2.12A). To assess transforma-

tion, these cells were grown in soft agar for two weeks that will allow transformed

cells to form colonies, and this experiment revealed that the transformation potential

of MYC and Ha-RAS was severely inhibited by both Mut BRG1 and Mut BRM (Fig- ure 2.12B). Despite low expression of Mut BRM, it was as efficient as Mut BRG1

97 in suppressing growth of MYC and RAS overexpressing cells in soft agar. Further

analysis of proliferation in these cells indicated that expression of both Mut BRG1 and BRM decreased the rate of MYC/RAS transformed cell proliferation by 1.5 to

4.0- fold, while expression of empty retroviral vector that contains the Mut BRG1

and BRM had no effect on cell growth and proliferation (Figure 2.12C). Transfec-

tion of c-MYC/Ha-RAS cells with retroviral vector alone had no effect on cell growth

(Figures 2.12C). These results suggest that the chromatin remodeling activities of

BRG1 and BRM are important for c-MYC/Ha-RAS mediated transformation.

2.5 Discussion

The work in this chapter shows that PRMT5, a type II arginine methyltransferase

that catalyzes symmetric methylation, is associated with BRG1- and BRM- based

hSWI/SNF complexes that also contain mSIN3A, and methylates hypoacetylated hi-

stone H3 and H4. In addition, the association of corepressors, PRMT5 and mSIN3

A and B isoforms, is mediated through interactions with BRG1, BRM, BAF57, and

BAF45/INI1, which also interact with transcriptional activator c-MYC. The inter-

actions of c-MYC and PRMT5/mSIN3A have been found to be mutually exclusive.

Further in vivo analysis of MYC target gene, CAD, by Northern blot and ChIP exper-

iments have indicated that BRG1 remodeling activity as well as histone deacetylation

by mSIN3A/HDAC2 is required for efficient repression of CAD, and that PRMT5 is

also recruited to the CAD promoter during transcriptional repression. Furthermore,

soft agar growth assays indicate that hSWI/SNF remodeling activity is crucial for

MYC and RAS-mediated transformation.

98 2.5.1 Role of BRG1 based hSWI/SNF complex in repression

In vitro interaction studies suggest that recruitment of PRMT5 and mSIN3A/HDAC2

to the CAD promoter is mediated through their interactions with specific hSWI/SNF subunits, which can also form a complex with c-MYC. Though, the association of

BRG1 with the CAD promoter varied when cells were arrested in G1 (T=0 h), or

stimulated to enter S phase (T=2 h), BRG1 was recruited to the CAD promoter when

the gene was repressed by serum starvation (T=0 h), activated by serum induction

(T=2 and 6 h), and re-repressed as cells transversed through cell cycle (T=14 and

16 h). It is known that MYC recruitment results in histone acetylation and acti- vation of target genes, including CAD, and ChIP experiments in this chapter show that c-MYC binding correlates with enhanced BRG1 recruitment, and loss of PRMT5 and mSIN3A/HDAC2 at the CAD promoter, raising the possibility that BRG1-based hSWI/SNF complexes participate in both transcriptional activation and repression of

CAD [82, 183]. Though we have linked histone deacetylation directly with transcrip-

tional repression of CAD, no direct evidence has been provided in this chapter that

links PRMT5 with CAD repression, because high doses of protein methyltransferase

inhibitor, MTA, that were effective in vitro was toxic to the cells. Taken together,

the findings that BRG1 and c-MYC are associated with CAD promoter during acti-

vation, whereas CAD repression correlates with the recruitment of BRG1 along with

associated mSIN3A/HDAC2 and PRMT5 to the CAD promoter, and that c-MYC

associated hSWI/SNF complexes lack PRMT5/mSIN3A, argue that when c-MYC

protein expression is increased, interaction of hSWISNF complexes with repressors

PRMT5 and mSIN3/HDAC would be minimized. As a consequence, c-MYC would

recruit repressor-free hSWI/SNF complexes to activate gene expression, and promote

99 cell growth and proliferation. Since c-MYC protein has a short half life, its levels de-

crease as cell progress through cell cycle, which will restore hSWI/SNF’s association

with repressor proteins and the repression of activated genes will be re-established.

2.5.2 hSWI/SNF complexes and histone deacetylation and methylation

Histones are modified in various ways as described in chapter 1, which influence the

chromatin structure and transcription. In addition, different modifications cross-talk

to either promote or inhibit other modifications at a locus (Chapter 1), for example,

histone H3K9 methylation and H3S10 phosphorylation are mutually exclusive [229].

The findings that both histone deacetylase proteins and arginine methyltransferase,

PRMT5, co-exist in hSWI/SNF complexes, suggest that there might be a cross-talk between them. The data presented in this chapter shows that hSWI/SNF complexes can deacetylate histone H3 and H4 tails, and that hSWI/SNF-associated PRMT5 methylates hypoacetylated histones better. Additionally, both mSIN3A/HDAC2 and

PRMT5 are recruited to the CAD promoter during repression. These results sug- gest that histone deacetylation is a prerequisite for PRMT5 to methylate histones, which in combination with deacetylated histones might provide a stronger repressive signal. It is known that mSIN3/HDAC co-repressor complexes cannot deacetylate nucleosomal histones, and currently it is not clear if PRMT5 can methylate nucle-

osomal histones; however, association of both activities with hSWI/SNF chromatin

remodeling complexes, indicates that they might require remodeling of nucleosome structure before they can modify histones in vivo.

100 2.5.3 Role of hSWI/SNF complexes in cell growth

As discussed in chapter 1, the hSWI/SNF chromatin remodeling complexes are

either BRG1- or BRM-based, which are highly similar ATPases. Though the two

proteins show some redundancy, in vivo studies have indicated that there are differ-

ences in their functions [274, 234, 259, 31]. BRG1 is an essential gene, while BRM

is dispensable for viability since deletion of both alleles is not lethal, suggesting that

they perform distinct functions to maintain normal cell growth and proliferation.

The findings that mutations of BRG1, BRM, and BAF45/INI1 are associated with

human cancers, indicate that their remodeling activity is essential to maintain normal

cell growth. To address the importance of BRG1 and BRM activity in controlling

expression of genes targeted by well known oncoproteins, c-MYC and RAS, the ef-

fect of catalytically inactive BRG1 and BRM in MYC and RAS transformation was

studied. The findings indicate that the oncogenic potential of MYC/RAS was re-

duced in the presence of inactive BRG1 and BRM, with BRM being more critical for MYC/RAS-mediated transformation because low expression of mutant BRM was sufficient to interfere with colony formation in soft agar. A large number of genes are known to be regulated by MYC and RAS and these results suggest that both

BRG1 and BRM are involved in their regulation directly or indirectly [48, 345, 257].

Since the data presented in this chapter shows that BRG1 and BRM complexes play an essential role in c-MYC/Ha-RAS-induced transformation, it would be interesting to identify the genes whose expression is altered by BRG1 and BRM such that they promote tumorigenesis.

101 Figure 2.1: Both isoforms of mSIN3 can interact with hSWI/SNF subunits. (A) Schematic representation of mSIN3A and B showing the conserved PAH and HID domains.

(B) BRG1, BRM, BAF57 and BAF45/INI1 can interact with the C-terminal PAH3 and PAH4 domains of mSIN3A and 3B. Approximately 1-2 µg of either GST (lanes 2 and 8), GST-mSIN3A PAH1 through 4 (lanes 3-6), or GST-mSIN3B PAH1 through 4 (lanes 9-12) were immobilized on GST beads and tested for their ability to interact with the indicated 35S -labeled hSWI/SNF subunits. The upper panels show Coomassie blue stained gels of the GST and GST-PAH fusion proteins.

102 Figure 2.1

103 Figure 2.2: Interaction of mSIN3 isoforms with hSWI/SNF components do not require BRG1 and BRM bromodomain or BAF57 HMG domain.

(A) Schematic diagram shows the functional domains of the BRG1 and BRM AT- Pases of the hSWI/SNF complex (Upper panel). C-terminally truncated BRG1 and BRM were in vitro translated and incubated with either GST (lane 2) or GST-PAH3 and 4 fusion proteins (Lower panel; lane 3-6).

(B) Upper panel depicts the domain structure of BAF57. The BAF57 C-terminal kinesin-like coiled coil region is required for interaction with mSIN3A and B. GST pull down experiments were performed using in vitro translated full length or C-terminal truncated BAF57 with immobilized GST or GST-PAH4 A and B. Input lanes represent 25% of the total in vitro translated protein used in each reaction.

104 Figure 2.2

105 Figure 2.3: Flag-tagged BRG1 and BRM complexes contain PRMT5. (A) Scheme for purification of flag-tagged BRG1 and BRM complexes.

(B) Coomassie blue stained gels showing affinity purified hSWI/SNF complexes. Arrows indicate bands that were excised and identified by mass spectrometry. Amino acid sequences of four of the thirteen peptides analyzed, which identify the 66 KDa polypeptide as PRMT5, are shown.

106 Figure 2.4: PRMT5 is associated with mSIN3A containing BRG1 and BRM com- plexes.

(A) Antibodies raised against cloned PRMT5 recognize the hSWI/SNF-associated 66 KDa polypeptide. Approximately 250 ng of affinity purified flag-tagged BRG1 and BRM complexes were analyzed by Western blotting using the indicated antibodies.

(B) PRMT5 and mSIN3A co-exist in the same BRG1 and BRM complexes. Partially purified hSWI/SNF complexes were incubated with either PI, anti-BRG1 or anti-BRM antibodies, and protein complexes were analyzed by Western blotting using the indicated antibodies.

(C) Affinity and glycerol gradient purified BRG1 and BRM complexes were analyzed by Western blotting using 20 µl of either BRG1 complex I or II, and BRM complex. Input lanes 1 and 4 show affinity purified Fl-BRG1 and Fl-BRM complexes.

107 Figure 2.4

108 Figure 2.5: PRMT5 can specifically interact with hSWI/SNF subunits and mSIN3A.

(A) In vitro translated hSWI/SNF subunits and mSIN3A were synthesized in the presence of [35S]-methionine and cysteine using the promega TNT-coupled retic- ulocyte lysate, and incubated with either GST (lane 2), GST-PAH2A (lane 3) or GST-PRMT5 (lane 4). For reference, 25% of the input is shown in lane 1.

(B) PRMT5 cDNA was co-translated with either BRG1, BRM, mSIN3A, BAF57, BAF45/INI1, or BAF155 cDNA in the presence of [35S]-methionine and cys- teine, and the protein mixtures were subjected to immunoprecipitation using preimmune (PI) and immune (I) antibodies as indicated.

(C) Anti-hSWI/SNF and anti-mSIN3A antibodies do not cross-react with PRMT5. In vitro translated and 35S -labeled PRMT5 was incubated with the indicated antibodies and the reactions were treated as in B. As a control, anti-PRMT5 immunoprecipitation is shown.

109 Figure 2.5

110 Figure 2.6: PRMT5 interacts with hSWI/SNF components in vivo through both N and C-terminal regions.

(A) Mammalian two hybrid experiment was performed using GAL4- PRMT5 as the bait and the indicated VP16 fusion proteins as prey in HeLa S3 cells. The in- teraction in vivo was assessed by the expression of CAT, which was quantitated by the acetylation of chloramphenicol. As controls, a mock transfection with pBluescript DNA and another transfection with GAL4 alone in the presence or absence of VP16 alone was included. The CAT activity has been normalized using β-Galactosidase expression.

(B) GST pull down assays were performed using [35S] labeled in vitro translated hSWI/SNF subunits, PRMT5 and mSIN3A and B with immobilized GST-N- PRMT5 (lane 4), GST C-PRMT5 (lane 5) and GST-PRMT5 (lane 6) proteins. As controls, GST alone and GST-PAH2A are included. Input lane represents 10% of the total protein used in the reactions.

(C) Mammalian two hybrid experiment was performed using GAL4 fusion protein containing either N-PRMT5 (aa 5-280) or C-PRMT5 (aa 281-637) as the bait to map the PRMT5 domains that mediate interaction with hSWI/SNF subunits.

111 Figure 2.6

112 Figure 2.7: Recombinant and hSWI/SNF-associated PRMT5 can methylate histones H3 and H4.

(A) HeLa core histones were incubated with increasing amounts (75 and 150 ng) of individually expressed and affinity purified wild type (WT) Fl-PRMT5, or mutant Fl-PRMT5 (G367A/R368A) as indicated. Similar amounts of either wild type or mutant Fl-PRMT5 were added to reactions containing 250 ng of affinity purified BRG1 or BRM complexes.

(B) BRG1 and BRM-associated PRMT5 can efficiently methylate hypoacetylated H3 and H4. The left panel shows separation of the different isoforms of HeLa core histones isolated from either asynchronous cells, or cells treated with 10 mM sodium butyrate (NaB) on a Triton/Acetic Acid/Urea (TAU) gel. The right panel shows methylation of hypoacetylated (H) and hyperacetylated (Ac-H) HeLa core histones using 250 ng of affinity purified BRG1 and BRM-associated PRMT5.

113 Figure 2.7

114 Figure 2.8: MBP-MORF acetylates histones H3 and H4 N-terminal peptides, which are deacetylated by hSWI/SNF complex.

(A) Purification of MBP-MORF. 10mg of bacterial extract containing MBP-MORF was bound to amylose resin and eluted with maltose. 5 µl of the purified fraction was analyzed by silver staining.

(B) MBP-MORF is able to methylate histones H3 and H4 N-terminal peptides. 4 µg of N-terminal peptides were incubated with 150 ng of purified MBP-MORF in the presence of [3H]-acetylCoA and the incorporation of [3H]-acetate was measured by filter binding.

(C) mSIN3A/HDAC2 and PRMT5-containing BRG1 and BRM complexes can deacetylate H3 and H4 peptides. Equal amounts of acetylated H2A, H2B, H3 or H4 peptides were incubated with 250 ng of affinity purified BRG1 and BRM-based hSWI/SNF complexes in the presence or absence of 10 mM NaB as indicated.

115 Figure 2.8

116 Figure 2.9: BRG1 chromatin remodeling and histone deacetylation are essential for efficient repression of the MYC target gene, CAD.

(A) Cells were serum starved and total RNA was isolated from either wild type cells (WT BRG1), cells that express catalytically inactive BRG1 (Mut BRG1), or cells treated with 60 nM depsipeptide (depsi) at the indicated times. Northern blot analysis was performed using equal amounts of total RNA, and mRNA was detected using 32P -labeled cDNA probes. To control for sample loading 18S and 28S rRNAs were visualized by ethidium bromide staining.

(B) Bar graphs show quantitation of bands shown in (A). Fold induction for each gene is reported as the ratio of the total number of counts for each sample to the wild type uninduced sample (T= 0 h). Quantitation of mRNA was performed using a Molecular Dynamics PhosphorImager. To control for sample loading 18S and 28S rRNAs were visualized by ethidium bromide staining.

(C) Methyl-thio adenosine (MTA) is unable to inhibit hSWI/SNF associated histone methyltransferase activity efficiently. hSWI/SNF complex was preincubated for 15 min with increasing dose of MTA ( 150 and 750 mM) before the addition of methyl donor, [3H]-SAM (lane 3, 4). Ctrl, represents a reaction without flag tagged BRG1 complex and lane 2 shows the HMTase activity of the FL-BRG1 complex used in the assay. Lower panel shows the Coomassie blue staining of the gel before autoradiography.

117 Figure 2.9

118 Figure 2.10: Cell cycle dependent recruitment of wild type and mutant BRG1, mSin3A/HDAC2 and PRMT5 to the CAD promoter.

(A) Schematic representation of the CAD promoter region showing the position of the primer pair used to PCR-amplify the sequences from -157 to +161. Black boxes (E boxes) depict MYC-MAX and MAD-MAX DNA binding sites. ChIP assays were performed using chromatin from either HeLa cells (anti-MYC, anti- MAD1, anti-WTBRG1, anti-mSIN3A, anti-HDAC2 and anti-PRMT5), or HeLa cells that express flag-tagged mutant BRG1 (anti-Fl-Mut BRG1, anti-HDAC2 and anti-PRMT5). Chromatin was prepared either after 48 h serum starva- tion (T= 0 h), or after serum stimulation for the indicated times. Input lanes represent 1% of the total chromatin used in each reaction. As controls, Mock, which represents samples without chromatin, no antibody (No Ab) and preim- mune (PI) reactions are shown. Total DNA used in the input reactions was diluted 300-fold, and because there was no variation in input lanes only two representative gels are shown.

(B) Schematic representation of the ODC promoter region showing the primers used to PCR-amplify the sequences from -5 to +377. ChIP assays were performed using the indicated antibodies.

(C) Nuclear extracts were prepared from either cells blocked in G1 (T= 0 h), or cells blocked and then released for the indicated times. Approximately 20 µg of nuclear extracts was analyzed by SDS-PAGE, and proteins were detected using anti-BRG1, anti-HDAC2 and anti-PRMT5 antibodies.

119 Figure 2.10

120 Figure 2.11: c-MYC can directly interact with BRG1 and BRM complexes. (A) Approximately 1-2 µg of either GST, GST-PAH2A or GST-MYC was incubated with 2 mg of HeLa nuclear extracts (lanes 2-4), or 500 ng of immunopurified hSWI/SNF complexes (lanes 6-8), and the indicated subunits were detected by Western blotting. Input lanes 1 and 5 show 1% and 50% of the total amount of proteins used in each experiment, respectively.

(B) Endogenous c-MYC can be found in association with hSWI/SNF subunits. HeLa S3 cells were metabolically labeled, and nuclear extracts were first immunopre- cipitated (1oAb) using either anti-BAF45/INI1, anti-BAF155 or anti-BAF57. Retained complexes were released by heat denaturation, and immunoprecipi- tated (2oAb) using anti-MYC antibodies. As, controls, anti-MYC and preim- mune (PI) immunoprecipitations are shown.

(C) c-MYC can associate with hSWI/SNF complexes by directly interacting with BRG1, BRM, BAF57 and BAF45/INI1. In vitro 35S -labeled hSWI/SNF sub- units were incubated with either GST (lane 2), GST-PAH2A (lane 3), or GST- MYC (lane 4), and bound proteins were detected by autoradiography.

(D) hSWI/SNF subunits interact differently with c-MYC. The upper panel shows the conserved domains of c-MYC. GST pull down assays were performed using the indicated GST fusion proteins. As controls, GST and GST-PAH2A are shown. The input lanes in (C) and (D) represent 10% of the total amount of protein used in each reaction.

121 Figure 2.11

122 Figure 2.12: Expression of catalytically inactive BRG1 and BRM reduces the trans- forming activity of c-MYC and Ha-RAS.

(A) Western blot analysis shows that both c-MYC and Ha-RAS are equally expressed in stably transfected NIH3T3 cell lines. Anti-Flag antibodies were used to detect epitope-tagged mutant BRG1 and BRM. Anti-HDAC2 was included as a control to show equal loading.

(B) Anchorage-independent growth of stably transfected NIH3T3 cell lines. Sta- ble cell lines that express either MYC+RAS, MYC+RAS+MutBRG1, or MYC+RAS+MutBRM were placed in soft agar medium and cultured for two weeks. Soft agar colony assays were performed with triplicate plates and re- peated four times using 2 x 102 cells. Representative pictures showing the morphology and sizes of transformed cells are shown (magnification 40X).

(C) Growth rates of stably transfected NIH3T3 cell lines. Approximately 2 x 105 cells were seeded into two separate 60 mm tissue culture dishes and counted every two days. This experiment was repeated four times, and the data points represent the average counts from duplicate plates. Standard deviations are included, but are too small for the error bars to appear on the graph.

123 Figure 2.12

124 CHAPTER 3

HUMAN SWI/SNF-ASSOCIATED PRMT5 METHYLATES HISTONES H3R8 AS WELL AS H4R3 AND REPRESS TRANSCRIPTION OF ST7 AND NM23 TUMOR SUPPRESSOR GENES

3.1 Abstract

PRMTs are involved in transcriptional regulation, but their role in controlling cell growth and proliferation is unclear. In chapter 2, it was demonstrated that PRMT5 can interact with flag-tagged BRG1 and BRM-based hSWI/SNF complexes, and that both recombinant and hSWI/SNF-associated PRMT5 methylate hypoacetylated hi- stones H3 and H4. In this chapter, the association of PRMT5 with endogenous hSWI/SNF complexes has been confirmed and the methylation sites of PRMT5 on

N-terminal histone tails have been identified as H3R8 and H4R3. Moreover, al- though H3K9 and H3K14 acetylation inhibited PRMT5 mediated methylation, sym- metric methylation of H3R8 and H4R3 slightly decreased acetylation of H3 and H4

N-terminal peptide by MORF. To study the role of PRMT5 in gene expression as

well as cell growth and proliferation, a PRMT5 anti-sense (AS-PRMT5) cell line

was established. Comparative microarray analysis revealed that when PRMT5 lev-

els were reduced, 227 genes were derepressed while only 43 genes were repressed.

125 Further studies on two tumor suppressor genes identified by the microarray analy- sis, SUPPRESSOROFTUMORIGENICITY7 (ST7 ) and NON − METASTATIC23

(NM23 ), indicate that PRMT5 directly regulates their transcriptional expression.

Repression of ST7 and NM23 correlates with increased PRMT5 recruitment and

H3R8 methylation along with reduced H3K9 acetylation at the promoters. Further-

more, overexpression of PRMT5 in NIH3T3 cells stimulates proliferation and induces

transformation. These findings suggest that the BRG1 and BRM-associated PRMT5

regulates cell growth and proliferation by controlling expression of genes involved in

tumor suppression.

3.2 Introduction

Gene expression is a tightly regulated process, and deficiency in the activities in-

volved in this regulatory pathway hinder normal cell growth and can lead to cancer.

hSWI/SNF complexes can be purified either alone or in combination with mSIN3A/HDAC

and PRMT5, indicating that there are different pools of BRG1 and BRM-based

hSWI/SNF complexes [214, 260]. In the previous chapter, it was demonstrated that

flag-tagged BRG1 and BRM complexes contain PRMT5, and that the PRMT5 and

mSIN3A/HDAC2 containing BRG1-based hSWI/SNF complex represses the

MYC/ MAX/ MAD target gene, CAD [214]. These studies and work by various

groups indicate that chromatin remodeling complexes can act in concert with var-

ious histone-modifying enzymes to modulate chromatin structure and gene expres-

sion [87, 200].

126 Histone methylation has been linked to transcriptional activation as well as repression.

While methylation of histone H3R17 by PRMT4/CARM1 leads to transcriptional ac- tivation, methylation of H3 and H4 by PRMT5 has been associated with repression,

although the residues modified by PRMT5 are unknown [40, 71, 214]. PRMT5 is

a type II arginine methyltransferase, which is involved in diverse cellular functions

including RNA processing, signal transduction, and transcription [17]. Studies in

S.pombe show that PRMT5 positively regulates Shk1 kinase, which is involved in

RAS signaling, and is required for normal cell growth [98]. Deletion of PRMT5 re-

sults in slow growth that is rescued by re-expression of either S.pombe or human

PRMT5, indicating that PRMT5 is functionally conserved [99]. Moreover experi-

ments in X .laevis, show that depletion of PRMT5 delays entry into mitosis whereas

overexpression of PRMT5 overrides replication checkpoint and accelerates mitotic

entry [323]. Although PRMT5 has been associated with transcriptional repression

of CYCLINE1 and CAD, it is not clear whether PRMT5 is involved in regulating a

broader spectrum of genes and can affect cell growth and proliferation in mammals.

In this chapter, the role of PRMT5 in global gene expression as well as cell growth

and proliferation has been evaluated. The association of PRMT5 with endogenous

BRG1 and BRM complexes has been verified and it has been demonstrated that

immunopurified recombinant and hSWI/SNF-associated PRMT5 target H3R8 and

H4R3 residues. PRMT5 knockdown in NIH3T3 cells shows that PRMT5 regulates

expression of cell cycle regulators and tumor suppressor genes. Analysis of tumor sup-

pressor genes, ST7 and NM23 , revealed that enhanced PRMT5 recruitment results

in increased methylation of H3R8 with concomitant decrease in H3K9 acetylation

and transcriptional repression. Furthermore, PRMT5 stimulated cell proliferation

127 and anchorage independent colony formation in NIH3T3 cell, which correlated with reduced expression of ST7 and NM23 tumor suppressor genes. This indicates that the BRG1 and BRM-associated PRMT5 is an important regulator of cell growth and proliferation.

3.3 Materials and Methods

3.3.1 Purification of flag-tagged INI1 complex

To purify hSWI/SNF complexes that contain both BRG1 and BRM ATPases, flag tagged- INI1 complex was purified on anti-flag M2 beads using nuclear extracts from

HeLa S3 cell line expressing flag tagged INI1 (Fl-INI1-11) according to the procedure described in section 2.3.7

3.3.2 Histone methyltransferase assay using HeLa core his- tone and histone N-terminal peptides as substrates

Methylation assays were performed using either 2.5 µg HeLa core histones or 4 µg histone N-terminal peptides (synthesized at KECK facility http://keck.med.yale.edu) and immunopurified Sf9 expressed flag-tagged PRMT5 (250 ng) or hSWI/SNF- asso- ciated PRMT5 (400 ng) in a 25 µl reaction as described in section 2.3.10. After a 1.5 h

incubation at 30oC, reactions that used HeLa core histones were treated as described previously in section 2.3.10, while reactions containing histone N-terminal peptides

were spotted on Whatman P-81 filter paper and processed to remove unincorporated

[3H]-SAM as described in section 2.3.11.

128 3.3.3 Cell culture and establishment of NIH3T3/WT-PRMT5 and NIH3T3/AS-PRMT5 cell lines.

Both HeLa S3 and NIH3T3 cell lines were cultured in DMEM containing 10% fetal bovine serum. HeLa S3 and flag-tagged INI1-11 cell lines were grown at the

National Cell Culture Center (Biovest International, Inc.). To generate cell lines that express flag-tagged PRMT5 or anti-sense PRMT5, 5 x 105 NIH3T3 cells in 60 mm plates were transfected with 2 µg of pBabe/Fl-PRMT5 or pBabe/AS-PRMT5, using

Lipofectamine plus reagent as described in section 2.3.8. After 48 h of transfection drug resistant cells expressing either flag-tagged PRMT5 or AS-PRMT5 were selected for two weeks in the presence of 3 µg/ml puromycin. After 2 weeks cells resistant

to puromycin survived and this represents the polyclonal culture. Cells were further

split and freezer stocks were prepared for long-term storage of the cells. To generate

clonal NIH3T3 cells expressing AS-PRMT5, 1 x 102 cells were plated in 100 mm tissue

culture plate and allowed to form colonies. Several individual colonies were isolated,

expanded into cell lines, and analyzed by RT-PCR to assess endogenous PRMT5

levels. After screening several clones, NIH3T3/AS-PRMT5 clone 15 was used in

subsequent experiments because expression of endogenous PRMT5 was reduced by

more than 90% as quantitated by RT-PCR (section 3.3.4).

3.3.4 Reverse transcriptase-polymerase chain reaction (RT- PCR) and microarray analysis

RNA was prepared from NIH3T3, NIH3T3/AS-PRMT5, and NIH3T3/Fl-PRMT5

cell lines using Trizol reagent (Invitrogen, Inc., catalog no. 15506-018). Cells from

approximately 90% confluent plate was collected, washed twice with 5 ml of 1X PBS,

and resuspended in 1 ml of trizol reagent by pipeting up and down. Next, 200 µl

129 of chloroform was added, mixed by vigorously shaking of the tube (fifteen times)

and incubated at room temperature for 5 min. Then the sample was spun at 11,000

rpm for 15 min at 4oC and the upper aqueous layer was transferred to a new tube.

To precipitate total RNA, 500 µl of isopropanol was added, mixed by inversion (five

times), and incubated at room temperature for 10 min. To collect total RNA, sample

was spun at 11,000 rpm for 15 min at 4oC, pellet was washed once with 1 ml of 75% ethanol, and the RNA pellet was air dried for 5 min at room temperature. Finally,

RNA was resuspended in 30 µl of DEPC-treated water and stored at -80oC until

needed. To prepare cDNA template for PCR, reverse transcription reaction was set

up with approximately 10-20 µg of total RNA in a 20 µl reaction containing 20 pmol

of specific 3’ primer, 3.5 mM MgCl2, 1 mM dNTPs, 1X Taq Pol buffer, 15 U AMV-RT

(Promega Inc., catalog no. M5101), and 2.5 U RNasin as described in section 2.3.14.

Either 0.2 µl or 2.0 µl of reverse transcriptase reaction was PCR- amplified using

specific primers in a 50 µl reaction containing 2.5 U of Taq polymerase (Invitrogen,

Inc., catalog no. 10966-034) and 2 µCi of [α-32P]dCTP as described in section 2.3.1.

Amplified fragments were separated from non-specific products by electrophoresis in

1X TBE on a 5% native polyacrylamide gel (18 ml of 40:1 acrylamide:bisacrylamide

solution containing 1X TBE, 12 ml water, 200 µl 10% APS, and 50µl TEMED) at

180 V that had been pre-run for 1 h at 160 V. After electrophoresis, gel was washed

twice with water for 10 min, stained with ethidium bromide (0.25 µg/ml) for 10 min

to visualize the pBR322 DNA-MspI digest marker (NEB Inc., catalog no. N3032S),

and then dried at 80oC for 1.5 h before exposure to a phosphorImager screen overnight

and quantitation using ImageQuantv5.0. Specific mouse primer pairs were used to

amplify the following genes from the NIH3T3 fibroblast cell lines: NM23 (+103 to

130 +313), ST7 (+ 448 to + 717), RRM1 (+181 to + 591), CYCLIN B2 (+172 to +

489), CYCLIN E2 (+69 to + 409), NF-κB (+ 393 to + 762), GAS2 (+ 275 to +595),

CDC20 (+161 to + 540), CDK4 (+ 151 to + 542), MYT1L (+ 796 to + 1112), ALL

(+ 186 to + 432), SERPIN (+368 to + 798), PRMT5 (+ 1093 to + 1306), GAPDH

(+178 to + 578) and β- ACTIN (+140 to + 585). The specific primer sequences are provided in Appendix B.

Microarray analysis was performed using total RNA isolated from either puromycin- resistant NIH3T3 or NIH3T3/AS-PRMT5 cells. Affymetrix MG-U74Av2 high den-

sity expression array chips, which include 6,000 cDNA clones and 8,000 expressed se- quence tags, were used to identify genes whose expression was altered when PRMT5 levels were reduced. The analysis was performed by the Ohio State University Com- prehensive Cancer Center microarray facility (http://www.osuccc.osu.edu/microarray).

Gene expression levels were estimated from GeneChip(r) PM probe intensities by means of an enhanced two-array version of the Li-Wong PM-only algorithm [43].

The enhanced algorithm: 1) scales all PM and MM probe intensities so as to min- imize between-array differences in the scaled MM probe intensity distributions; 2)

applies between-array variance analysis to the scaled PM probe intensities in order

to estimate PM-specific sensitivities; 3) estimates gene expression levels by regressing

scaled PM probe intensities on estimated PM probe sensitivities within each probe

set; 4) tests a probe-level GLM (General Linear Model) within each probe set in

order to estimate the p-values for between-array differential gene expression. The

estimated p-values can be several orders of magnitude lower than 0.05, as required

by the Bonferroni correction, which applies when simultaneously testing thousands

of genes for significant differential expression [128].

131 3.3.5 BrdU incorporation assay and cell cycle profile analysis

To assess the percentage of cells in S phase in a particular time interval, which indicates the proliferation status of the cell, BrdU incorporation was measured using the BrdU flow kit (Becton Dickenson Inc., catalog no. 559619). To label cells in S phase, 1 x 106 cells were incubated in media containing 10 µM BrdU for either 4.5 or 9 h. Next, cells were harvested, washed once with 1 ml of 1X PBS, resuspended

in 100 µl of BD cytofix/cytoperm buffer and incubated on ice for 30 min to fix and

permeabilize the cells. Then, cells were washed with 1 ml of 1X BD wash/perm buffer

and incubated with 100 µl of BD cytoperm plus buffer for 10 min before washing and

refixing with 100 µl of BD cytofix/cytoperm buffer for 5 min on ice. To expose

incorporated BrdU, washed cells were treated with 100 µl of DNase solution (30 µg

of DNase diluted in 1X PBS) for 1 h at 37oC, followed by washing with 1 ml of 1X

BD wash/perm buffer and staining with 1 µl of FITC-conjugated anti-BrdU antibody

(diluted 1:50 in 1X BD Perm/Wash buffer) for 20 min at room temperature. Samples

were then washed twice with 1 ml of 1X BD Perm/wash buffer, resuspended in 1 ml of

staining buffer (1X PBS containing 3% FBS) containing 20 µl of 7-amino-actinomycin

D (7-AAD) before cells were analyzed by FACS analysis.

To determine if NIH3T3 cells expressing AS-PRMT5 were undergoing apoptosis,

2 x 105 cells were plated and allowed to grow for 4 days before the DNA content was

examined by FACS analysis using a FACScalibur flow cytometer (Becton Dickenson).

To stain DNA, cells were harvested, washed twice in 10 ml of 1X PBS, and resus-

pended in 200 µl of 1X PBS. To fix and permeabilize the cells, 800 µl of 100% ethanol

was added drop by drop along with slow vortex to achieve a final concentration of

80% and fixed cells were stored at 4oC overnight. Next day, the cells were washed

132 with 10 ml of 1X PBS and resuspended in 1 ml 1X PBS supplemented with 18 µl of

RNase A (10 mg/ml). To digest cellular RNA, samples were incubated at 37oC for

5 min and then, DNA was stained by incubating the cells with 0.5 µl of propidium iodide (10 mg/ml, Sigma Inc., catalog no. P4170) at room temperature for 45 min before measuring DNA content.

3.3.6 Proliferation, and anchorage independent and depen- dent growth assays

To measure the proliferation rate of NIH3T3/Fl-PRMT5 and NIH3T3/AS-PRMT5,

2 x 105 cells were seeded into 60 mm plates and allowed to grow for 6 days. Pro- liferation of each cell line was repeated three times using duplicate plates, and cells were counted every two days. To assess the anchorage independent growth potential of NIH3T3/Fl-PRMT5 and NIH3T3/AS-PRMT5, 2 x 102 cells were seeded into 60 mm plates in DMEM medium containing 0.3% agar as described previously in section

2.3.18. Each cell line was tested for its ability to grow in soft agar using triplicate plates. Colonies were counted and the average from three different experiments is shown. For anchorage dependent growth, 4 x 103 cells were plated in 100 mm plates.

After 7 days, plates were gently washed three times with 10 ml of prewarmed 1X

PBS (37oC) and fixed with 5 ml of 10% buffered formalin (Sigma Inc., catalog no.

HT5O-12) at room temperature for 15 min. The plates were then washed three times with 10 ml of MilliQ water to remove formalin and stained with 5 ml of 0.1% crystal violet (Sigma Inc., catalog no. C-0775) at room temperature for 10 min. Excess free stain was removed by repeated washing with MilliQ water and the plates were allowed to dry before scanning.

133 3.3.7 Dot blot analysis and chromatin immunoprecipitation (ChIP) assay

To test anti-H3(Me2)R8 antibodies, 1 and 2 µg of methylated and unmethylated

H3 peptides were spotted on nitrocellulose membrane using the minifold slot blot sys-

tem (Schleicher and Schuell Inc., catalog no. 77480)as follows before Western blotting.

Two pieces of 3 mm watman paper (8 cm x 8 cm) with one piece of nitrocellulose mem-

brane (8 cm x 8 cm) on top that were pre-wet in 1X TBS (10 mM Tris-HCl [pH 7.4],

127 mM NaCl) was assembled on the minifold blot system platform, and then the slot

blot plate was attached on the top tightly. Next vacuum was applied and adjusted such that there was slow suction of liquid and once the excess fluid was removed,

the samples were loaded in a volume of 150 µl of 1X TBS and wells were washed

with 250 µl of 1X TBS. Finally the membrane was removed and Western blot was performed as described previously in section 2.3.19. ChIP assays were performed on chromatin prepared from 90% confluent (roughly 5 x 106) NIH3T3, NIH3T3/Fl-WT

PRMT5, and NIH3T3/AS-PRMT5 cells as described in section 2.3.17. When ChIP experiments were performed using anti-PRMT5 antibody, the N-terminal specific (aa

5-200) anti-N-HMT(30 µl) antibody was used instead of the full length PRMT5 anti- body (anti-skb). The eluted DNA was resuspended in 35 µl of TE [pH 8.0] containing

RNase (0.2 µg/µl) and for Mock, No Ab, PI, and I reactions 10 µl of eluted DNA was amplified and 15 µl of each PCR reaction was analyzed. For the input lane,

0.6 µl of eluted DNA was PCR-amplified and 10 µl was loaded on the gel. Specific mouse primer pairs were used to amplify mouse ST7 (-228 to +209), NM23 (-211 to

+254), and MYT1l (-258 to +214) sequences. Sequence of the specific primers used

134 to amplify the promoter regions of mouse ST7 , NM23 , and MYT1L are provided in

Appendix B.

3.4 Results

3.4.1 Recombinant and hSWI/SNF-associated PRMT5 methy- lates H3R8 and H4R3

Using flag tagged BRG1 and BRM cell lines it has been demonstrated in chap- ter 2 that PRMT5 is associated with epitope-tagged hSWI/SNF complexes and

that PRMT5 methylates histones H3 and H4, but the arginine residues targeted

by PRMT5 are unknown [214]. Since the amounts of PRMT5-associated BRG1 and

BRM specific hSWI/SNF complexes purified by affinity chromatography were lim- ited, hSWI/SNF complexes (Fl-hSWI/SNF) containing both BRG1 and BRM were purified by anti-flag affinity purification from Fl-INI1-11 nuclear extracts. The Fl-

INI1-11 is a HeLa S3 based cell line expressing flag tagged BAF45/INI1, which is

a subunit of both BRG1- and BRM-based hSWI/SNF complexes [259]. As control,

HeLa S3 nuclear extracts were also purified over an anti-flag column, and the col-

lected fractions were analyzed by sliver staining as well as Western blotting (Figures

3.1A and B). As previously observed in chapter 2, PRMT5 was enriched in fractions containing both BRG1 and BRM ATPases but not in control fractions purified using

HeLa nuclear extract (Figure 3.1B).

The histone methyltransferase activity of PRMT5 containing hSWI/SNF com-

plexes (Fl-hSWI/SNF) was verified using H1-depleted HeLa core histones as the sub-

strate (Figure 3.1C). As expected, the Fl-hSWI/SNF complex as well as wild type

(WT) recombinant Sf9 expressed PRMT5 methylated histones H3 and H4. When

the levels of PRMT5 protein in Fl-hSWI/SNF complex was estimated by Western

135 blotting, it was found that 7 to 9-fold more recombinant Fl-PRMT5 was used in the histone methyltransferase assay to detect H3 and H4 methylation (Figures 3.1C

and D). This suggests that association of PRMT5 with BRG1 and BRM complexes

stimulates its methyltransferase activity.

Since most histone modifications that affect gene transcription occur in the N-

terminal tail of histones, the ability of recombinant and hSWI/SNF-associated PRMT5

to methylate histone N-terminal tails was tested. Purified N-terminal histone H3

and H4 peptides (>95% purity) that include the first 20 amino acids were incubated

with either recombinant Sf9 expressed PRMT5 or PRMT5-containing Fl-hSWI/SNF

complex in the presence of [3H]-SAM. Both recombinant and hSWI/SNF-associated

PRMT5 specifically methylated N-terminal H3 and H4 peptides, while no methyla-

tion was observed when BSA or a control H3 peptide (aa 60-84), which contains four

arginine residues were used as substrates (Figure 3.1E). In contrast, catalytically

inactive Fl-PRMT5 (G367A/R368A) was unable to methylate H3 and H4 N-terminal

tails. These results show that PRMT5 targets arginine residues in the N-terminal

peptides of H3 and H4.

To identify the specific arginine residue(s) methylated by PRMT5, H3 and H4

peptides carrying specific arginine to alanine substitutions were used as substrates

in methylation assays (Figure 3.2). When Fl-hSWI/SNF complex was incubated

with unlabeled wild type and mutant peptides, only H3R8A and H3R2A/R8A/R17A

were not methylated (Figure 3.2A). Mutation of either H3R2A or H3R17A had no

significant impact on H3 peptide methylation suggesting that H3R2 and H3R17 are

not required for PRMT5-mediated H3 methylation. To confirm the specificity of

PRMT5 toward H3R8, recombinant Fl-PRMT5 was used in the methylation assay

136 (Figure 3.2B). Similar to the result using hSWI/SNF complex, Fl-PRMT5 was able to methylate wild type H3, H3R2A, and H3R17A, but not peptides carrying H3R8A mutation indicating that arginine 8 on H3 is the primary site of PRMT5 mediated methylation. Using a similar strategy H4 arginine residues methylated by PRMT5 were identified (Figure 3.2C and D). When either recombinant or Fl-hSWI/SNF complex was incubated with H4 N-terminal peptides containing H4R3A mutation,

H4 peptide methylation was lost suggesting that arginine 3 on H4 is preferentially methylated by PRMT5. These results reveal that recombinant and hSWI/SNF- associated PRMT5 target the same arginine residues in the H3 and H4 N-terminal tails.

3.4.2 H3K9 and K14 acetylation interferes with PRMT5- mediated H3 methylation while H3R8 and H4R3 methy- lation do not inhibit acetylation

Histone methyltransferase assay using hyperacetylated HeLa core histones show that BRG1- and BRM-associated PRMT5 do not efficiently methylate highly acety- lated histones (Figure 2.7) [214]. Hence the cross-talk between histone H3 N-terminal peptide acetylation and H3R8 methylation was tested. When H3 peptides that were acetylated at lysine 9 or lysine 14 (Upstate Inc.) were used as substrate in methyla- tion assays, hSWI/SNF-associated PRMT5 was unable to methylate either acetylated peptides, indicating that H3 acetylation inhibits H3R8 methylation (Figure 3.3A).

To investigate if the reverse was true, the ability of MBP-MORF to acetylate sym- metrically dimethylated H3R8 and H4R3 peptides was analyzed. It has been shown previously in chapter 2 that MBP-MORF can efficiently methylate non-modified hi- stones H3 and H4 N-terminal tails (Figure 2.8B). When either unmethylated or

137 dimethylated H3 and H4 peptides (aa 1-20), were incubated with MBP-MORF in the presence of [3H]- acetylCoA, both unmethylated and methylated peptides were efficiently acetylated (Figure 3.3B). These results indicate that though deacetylation

is crucial for H3R8 methylation, demethylation of H3R8 and H4R3 is not required to

initiate histone tail acetylation.

3.4.3 Identification of genes regulated by PRMT5

PRMT5 has been implicated in the control of yeast cell morphology by interacting

with the Shk1 kinase, but its role in the control of cell cycle progression and prolif-

eration in mammalian cells is poorly understood. To evaluate the effect of H3R8

and H4R3 methylation on gene expression, PRMT5 target genes were identified by

reducing its level in NIH3T3 cells. To knockdown PRMT5 expression, an anti-sense

NIH3T3 cell line (AS-PRMT5) was established where PRMT5 transcript levels were

reduced by more than 90% as detected by RT-PCR while GAPDH and β-ACTIN

mRNA levels were unaltered (Figure 3.4A). To verify the knockdown of PRMT5

protein expression, Western blot analysis was performed on nuclear extracts from

either puromycin-resistant NIH3T3 (NIH3T3-Vector) or AS-PRMT5 cells. In agree-

ment with the RT-PCR results, endogenous PRMT5 levels were reduced 2.6-fold in

the anti-sense cell line, while MAD levels were unaffected (Figure 3.4B). These results

demonstrated that the anti-sense construct specifically reduced PRMT5 expression.

To identify genes regulated by PRMT5, a comparative microarray analysis was

performed using RNA from NIH3T3-Vector and AS-PRMT5 cell lines, which revealed

that 227 genes were up-regulated, while only 43 genes were down-regulated in the AS-

PRMT5 cells (Appendix D Tables 1 and 2). This result suggested that more genes

138 were repressed by PRMT5 either directly or indirectly, and that genes affected by

PRMT5 included proteins involved in cell adhesion and signalling, cell cycle progres- sion, tumor suppression, metabolic pathways, protein degradation, and chromatin remodeling. Among the genes with tumor suppressor activity, suppressor of tumori- genecity 7 (ST7 ), non − metastatic23 (NM23 ), growtharrestspecific1 and 2 (GAS1 and GAS2 ), lysyl oxidase-like (LOXl), and retinoblastomalike − 1 (p107 ) were dere- pressed 2.5 to 5-fold. Similarly, cell cycle regulators such as CDK4, CYCLINB2 ,

CYCLINE2 , and CDC20 were also induced 2 to 4-fold in the anti-sense cell line

(Appendix D Tables1). To confirm the microarray analysis data, semiquantitative

RT-PCR analysis was performed on few of the genes that were either up or down- regulated (Figure 3.4C and D). All tumor suppressor and cell cycle inducer genes were derepressed to the same extent as determined by microarray analysis. Simi-

larly, expression of down- regulated genes such as myelin transcription factor 1-like

(MYT1l), ALL1-fused gene from chromosome 1q (ALL1), and serine proteinase in- hibitor E1 (SERPIN E1) was also confirmed by RT-PCR analysis (Appendix D Table

2).

3.4.4 PRMT5 stimulates cell proliferation and induces an- chorage independent growth

Since PRMT5 negatively regulates expression of cell cycle inducers as well as

tumor suppressor genes, the effect of PRMT5 on cell growth and proliferation was

investigated. A cell line was established in NIH3T3 cells that overexpress flag-tagged

PRMT5 (Fl-PRMT5) and its growth characteristics were compared to the NIH3T3-

Vector and AS-PRMT5 cell lines (Figure 3.5A and B). When the proliferation of

AS-PRMT5 cells was compared to parental NIH3T3 as well as puromycin resistant

139 NIH3T3 cells, it was observed that AS-PRMT5 cells grew 2 to 3-fold slower. In contrast, Fl-PRMT5 cells grew 3 to 4-fold faster than NIH3T3 cells similar to the effect observed when NIH3T3 cells were transformed by overexpressing MYC and Ha-

RAS. These results indicate that the level of PRMT5 protein is critical to maintain normal cell growth and proliferation. To understand the growth characteristic of

AS-PRMT5 and Fl-PRMT5 cells, the cell cycle profile as well as the rate of DNA synthesis was measured in AS-PRMT5 and Fl-PRMT5 cells (Figure 3.5C and D).

BrdU incorporation, which is a measure of DNA synthesis, was reduced in AS-PRMT5 cells compared to NIH3T3 and Fl-PRMT5 cells, and the cell cycle profile measured by the DNA content showed that no cell death was induced by altered PRMT5 expression. These results suggest that reduced proliferation of AS-PRMT5 cells is caused by a slow transition from G1 to S phase.

Since Fl-PRMT5 cells exhibited a proliferation rate that was similar to MYC/RAS transformed NIH3T3 cells, the transforming ability of PRMT5 protein was analyzed by monitoring colony formation by FL-PRMT5 cells (Figure 3.6). When equal num- bers of sense and anti-sense PRMT5 cells were cultured in an anchorage dependent manner, and the colonies were stained using crystal violet dye, only cells that overex- pressed Fl-PRMT5 formed colonies at a rate comparable to MYC/RAS-transformed cells (Figure 3.6A). Similarly, when Fl-PRMT5 and AS-PRMT5 cells were grown in soft agar, both AS-PRMT5 and NIH3T3-Vector cells failed to grow while Fl-PRMT5 and MYC/RAS-transformed cells were able to form colonies in an anchorage inde- pendent manner (Figure 3.6B). Moreover, overexpression of Fl-PRMT5 increased the number of colonies by 3-fold in comparison to MYC/RAS-transformed NIH3T3

140 cells (Figure 3.6B). These results show that PRMT5 stimulates cell growth and proliferation, and induces transformation.

3.4.5 PRMT5 directly regulates expression of ST7 and NM23

Recruitment of PRMT5 to the promoter region of CAD and CYCLINE1 genes cor- relates with their transcriptional repression [71, 214]. To understand the mechanism by which PRMT5 regulates cell proliferation and induces transformation, expression of two tumor suppressor genes (ST7 and NM23 ) that were identified to be upregu- lated upon PRMT5 knockdown were further analyzed (Figure 3.4C and Appendix D

Tables1). ST7 and NM23 genes were chosen because reduced expression of both genes has been associated with a variety of cancers including breast, prostate, ovarian, colon, head and neck, gastric, pancreatic, and renal cell carcinomas [117, 165, 166, 298, 335].

As a control, expression of myelin transcription factor 1-like (MYT1l) that was defi- cient in AS-PRMT5 cells was also examined. When the mRNA levels of ST7 , NM23 , and MYT1l were measured in NIH3T3 and AS-PRMT5 cells, ST7 and NM23 were up-regulated 2.5 and 3.8-fold in the AS-PRMT5 cell line, respectively, while MYT1l was down-regulated 5-fold (Figure 3.7A). In stark contrast, the steady state levels of ST7 and NM23 were repressed 2 and 4-fold, respectively, in cells overexpressing

Fl-PRMT5. When MYT1l expression was measured in the Fl-PRMT5 cell line, its levels were increased 5 to 6-fold. These results suggest that PRMT5 can repress as well as induce gene expression.

To distinguish between direct and indirect affects of PRMT5 on the expression of genes that were altered by the knockdown and overexpression of PRMT5, ChIP assays were performed. Recruitment of PRMT5 to the promoter regions of ST7 ,

141 NM23 , and MYT1l was analyzed in NIH3T3 and AS-PRMT5 cells (Figure 3.7B).

Endogenous PRMT5 was associated with the promoter regions of ST7 and NM23 in

NIH3T3 cells while PRMT5 recruitment was lost in the AS-PRMT5 cells. Also, the recruitment of PRMT5 to the promoter regions of ST7 and NM23 correlated with their transcriptional repression (Compare figures 3.7A and B). In Fl-PRMT5 cells that overexpress PRMT5, association of exogenously expressed flag tagged- PRMT5 with ST7 and NM23 promoter was accompanied by increased ST7 and NM23 tran- scriptional repression (Compare figures 3.7A and B). ChIP experiments also show

that unlike ST7 and NM23 , endogenous and flag tagged-PRMT5 were not associated

with MYT1l promoter, indicating that MYT1l is not a direct target of PRMT5.

3.4.6 PRMT5 methylates H3R8 at the ST7 and NM23 pro- moter, and opposes H3K9 acetylation

Although PRMT5 was recruited at the ST7 and NM23 promoter region, it is not clear if PRMT5 mediated transcriptional repression involves histone methylation at

H3R8 and H4R3. Since no antibodies were available against symmetrically methy-

lated H3R8, a rabbit polyclonal antibody was raised to recognize this modification.

The specificity of anti-H3(Me2)R8 antibody was tested on immobilized unmethy-

lated H3 peptides (aa 1-20), internal H3 peptide (aa 60-84), symmetrically dimethy-

lated H3R8, and non-histone protein (BSA) that contains arginine residues (Figure

3.8A). Western blot analysis indicates that the antibody is specific for symmetri-

cally dimethylated H3R8. To detect H3R8 symmetric methylation at the promoters

of ST7 and NM23 that are associated with PRMT5, ChIP assays were conducted

on cross-linked chromatin from FL-PRMT5, NIH3T3, and AS-PRMT5 using the

anti-H3(Me2)R8 antibody. Symmetric methylation of H3R8 was detected at both

142 ST7 and NM23 but not MYT1l promoter regions in NIH3T3 and Fl-PRMT5 cells

(Figure 3.8B). However, when ChIP assays were performed using chromatin from

AS-PRMT5 cells, anti-H3(Me2)R8 failed to immunoprecipitate the ST7 and NM23 promoter regions. These results along with the microarray and RT-PCR data suggest that methylation of H3R8 plays an essential role in transcriptional repression of ST7

and NM23 tumor suppressor genes.

Since histone acetylation has been linked with transcriptional activation, and us-

ing in vitro methyltransferase assays it was found that H3K9 acetylation inhibits

H3R8 methylation, the acetylation status of H3K9 at the promoter regions of ST7 ,

NM23 , and MYT1l was analyzed (Figures 3.3A and 3.8C). ChIP assays were per-

formed using anti-H3acetylK9 (H3AcK9) antibody in NIH3T3 cells, as well as NIH3T3

cells that express Fl-PRMT5 and AS-PRMT5 (Figure 3.8C). In accord with the in

vitro methylation assays, H3K9 acetylation was inhibited in cells that overexpress

Fl-PRMT5, while H3K9 was acetylated at the ST7 and NM23 promoters in NIH3T3

and AS-PRMT5 cells. Thus, these findings show that there is negative cross-talk

between H3K9 acetylation and H3R8 methylation in vivo.

3.4.7 ST7 and NM23 tumor suppressors are differentially tar- geted by BRG1 and BRM- based hSWI/SNF com- plexes

Since PRMT5 associates with BRG1 and BRM-based hSWI/SNF complexes in

vivo the recruitment of both BRG1 and BRM remodeling proteins was also tested

(Figure 3.1 and chapter 2). When ChIP assays were performed using cross-linked

chromatin from either Fl-PRMT5, NIH3T3, or AS-PRMT5 cells, it was found that

BRG1 was associated with ST7 and MYT1l, but not NM23 , while BRM was recruited

143 to the promoter sequences of NM23 and MYT1l and not ST7 (Figures 3.9A and B).

Also, BRG1 recruitment to the ST7 promoter region was independent of PRMT5

recruitment, whereas BRM association with the NM23 promoter depends on the

presence of PRMT5 (compare Fl-PRMT5, NIH3T3 and AS-PRMT5 panels in figures

3.9A and B). These results indicated that different genes require distinct PRMT5-

containing chromatin remodeling complexes for their regulation.

To verify whether the remodeling activity of BRG1- and BRM-based hSWI/SNF

complexes were required for ST7 , NM23 , and MYT1l expression, the mRNA levels

of these genes were analyzed in HeLa S3 cells that express either catalytically inactive

flag tagged BRG1 or BRM (Figure 3.9C). In the presence of catalytically inactive

BRG1, ST7 was derepressed 2 to 3-fold while NM23 expression was unaltered in the mutant BRM cells (Figure 3.9D). MYT1l whose promoter is bound by both BRG1 and BRM was derepressed 3 and 2.5-fold in the presence of mutant BRG1 and BRM,

respectively (Figure 3.9D). These results suggest that not all PRMT5 target genes

are directly affected by BRG1 and BRM chromatin remodeling activity.

3.5 Discussion

In this chapter, the association of PRMT5 with hSWI/SNF complexes has been

further validated by immunopurification of Fl-hSWI/SNF complexes from flag tagged-

INI1-11 cell line. It has been shown that recombinant and hSWI/SNF-associated

PRMT5 preferentially methylate histones H3R8 and H4R3. Identification of PRMT5

regulated genes by microarray analysis show that PRMT5 controls cell growth and

proliferation by modulating expression of both cell cycle inducers as well as tumor

suppressor genes. Using sense and anti-sense PRMT5 cell lines, it has been found that

144 BRG1 and BRM-associated PRMT5 is directly involved in transcriptional repression of ST7 and NM23 tumor suppressor genes. These findings suggest that PRMT5

controls cell growth and proliferation by maintaining appropriate expression of tumor

suppressor genes.

3.5.1 PRMT5 methylates specific arginine residues in H3 and H4 N-terminal tails

H3 and H4 N-terminal tails contain highly conserved lysine and arginine residues,

which are modified by acetylation, methylation, phosphorylation, ubiquitination as

well as sumoylation that impact transcription and these modifications can either syn-

ergize or antagonize to specify the transcriptional outcome (Chapter 1). The role of

lysine acetylation and methylation in modulating chromatin structure and transcrip-

tion has been well studied, and the mechanisms by which arginine methyltransferases

influence gene expression are now emerging.

Using N-terminal histone tail peptides, it has been shown that H3R8 and H4R3

are specifically methylated by both recombinant and hSWI/SNF-associated PRMT5.

However, it is possible that there are other sites within H3 and H4 that are methylated

by PRMT5, and attempts to identify additional H3 and H4 methylation sites by mass

spectrometry have failed due to low recovery of the modified peptides.

It was observed that hyperacetylated H3 and H4 are not efficiently methylated

by hSWI/SNF-associated PRMT5, suggesting that lysine acetylation might interfere

with PRMT5 mediated arginine methylation (Chapter 2, [214]). In agreement, it

was found that acetylation of H3K9 or K14 blocks H3R8 methylation on histone

H3 N-terminal peptide, however H3R8 and H4R3 methylation did not inhibit H3

and H4 N-terminal tail acetylation by MORF (Figure 3.3). The 25% decrease in

145 H3(Me2)R8 peptide acetylation could be due to loss of H3K9 acetylation caused by stearic hinderance from H3R8 methyl groups. Moreover, a certain level of cross-talk between H3K9 acetylation and H3R8 methylation has been observed at the ST7 and

NM23 promoters in vivo (Figures 3.8B and C). Although, PRMT5 recruitment cor-

relates with H3R8 methylation it is not clear how PRMT5 repress transcription.

It is possible that methylation of H3R8 sterically hinders H3K9 acetylation, which

might be an important step in gene activation. PRMT5 also targets H4R3, which is

asymmetrically dimethylated by PRMT1 and is associated with transcriptional acti-

vation [297]. In this case, when H4R3 is either unmethylated or mono-methylated,

PRMT5 could catalyze its symmetric dimethylation and prevent transcriptional acti-

vation by PRMT1. However, the mechanism(s) underlying transcriptional repression

upon H3R8 and H4R3 symmetric dimethylation is still unknown.

Attempts to detect H4R3 methylation using commercially available anti-H4(Me2)R3

antibodies (Upstate Inc.) in ChIP assays failed, but the possibility that H4R3 is

methylated at the ST7 and NM23 promoters can not be excluded since these anti-

bodies have not been tested in ChIP assays. Therefore, to address this issue anti-

H4(Me2)R3 antibodies that specifically recognize symmetrically methylated H4R3 will

be generated and tested in ChIP assays. It will be important to determine whether

methylation of H3R8 and H4R3 occurs simultaneously in order to repress ST7 and

NM23 gene expression, or that methylation of either H3R8 or H4R3 will be sufficient

to induce transcriptional repression.

146 3.5.2 PRMT5 regulates cell growth and proliferation by mod- ulating expression of ST7 and NM23 tumor suppressor genes

To investigate the role of PRMT5 in transcriptional regulation, global gene ex-

pression in AS-PRMT5 cells was measured by microarray analysis and the differential expression of some up and down-regulated genes was verified by RT-PCR (Figures

3.4C and D, Appendix D Tables1 and 2). It was found that more genes were dere- pressed when PRMT5 levels were reduced, consistent with its role in transcriptional repression. Based on the microarray results, it was expected that AS-PRMT5 cell lines would grow faster in comparison to control NIH3T3, because many cell cycle reg- ulators including CYCLINE2 , CYCLINB2 , and CDK4 , whose expression is enhanced in many human tumors, were up-regulated [218, 245, 310]. However, results from pro- liferation assays indicated that AS-PRMT5 cells grow 2-fold slower than NIH3T3 cells, while cells overexpressing PRMT5 proliferate like MYC/RAS-transformed NIH3T3 cells. Since PRMT5 has been linked to transcriptional repression and several tumor suppressor genes were found to be up-regulated in the AS-PRMT5 cell, it is possi- ble that PRMT5 promotes growth by repressing tumor suppressor genes. In accord, when expression of ST7 and NM23 was analyzed in cell lines that express either sense or anti-sense PRMT5, it was found that both genes were derepressed when PRMT5 levels were reduced and repressed when PRMT5 levels were increased (Figure 3.7A).

ST7 is a tumor suppressor genes on human chromosome 7q31.1, a region frequently associated with loss of heterozygosity in different human cancers [334]. Re-expression of ST7 in the PC3 prostate cancer cell line inhibits cell growth and tumorigenecity

147 in nude mice [124, 335]. Recent studies have reported that ST7 expression is re- pressed in 44% of breast cancer tissue compared to their normal counterpart, and F-

MuLV virus induced tumorigenesis is accompanied with transcriptional repression of

ST7 [124, 327]. Similarly, NM23 expression is reduced in highly metastatic melanoma cells [268]. Furthermore, re-introduction of NM23 into K-1735TK melanoma cells de- creased their ability to form colonies in soft agar and reduced incidence of primary tumor formation in nude mice [165]. These studies demonstrate that ST7 and NM23 are involved in tumor suppression and confirm the results that when ST7 and NM23 levels are reduced in cells that overexpress PRMT5, their ability to hyperproliferate and form colonies in an anchorage independent manner is enhanced. There are other genes whose expression is affected when PRMT5 levels are reduced, and they might also be involved in regulating cell growth and proliferation. Therefore, it is impor- tant to study the expression and evaluate the role of various tumor suppressor genes like LOXL, GAS1 , GAS2 , and p107 , which are affected by PRMT5 knockdown to understand the mechanism of PRMT5 induced transformation.

PRMT5 directly represses ST7 and NM23 transcription and BRG1 was specifi- cally recruited to the ST7 promoter, while BRM was targeted to NM23 . Using cell lines that express catalytically inactive BRM, it was found that MYT1l was dere- pressed, while NM23 was unaffected though BRM was recruited to NM23 promoter.

It is possible that in the presence of mutant BRM a regulator of NM23 , which inhibits

NM23 expression is upregulated, or alternatively another remodeling protein might compensates for BRM function on NM23 gene, thereby maintaining transcriptional repression of NM23 . When expression of ST7 and MYT1l was analyzed in mutant

BRG1 expressing cells, both genes were derepressed, confirming the role of BRG1 in

148 transcriptional repression of direct target genes. The observations that BRG1 and

BRM-associated PRMT5 can induce transformation in immortalized cell and is in- volved in transcriptional repression of anti-cancer genes suggest that PRMT5 is an attractive molecule to target in cancer therapy.

149 Figure 3.1: PRMT5 co-elutes with flag-tagged BAF45/INI1 complexes. (A and B) Either HeLa S3 or Fl-INI1 nuclear extract (180 mg) was incubated with anti-flag M2 affinity gel, and hSWI/SNF complexes were eluted with 20-fold molar excess of flag peptide. Eluted complexes were analyzed by silver staining (A) or by Western blotting (B) using specific antibodies. Western blots were performed using 20 µg of Fl-INI1 nuclear extract (Input, lane 1), 15 µl of eluted fraction using HeLa nuclear extract (Ctrl, lane 2), and 15 µl of eluted flag-tagged hSWI/SNF (Fl-hSWI/SNF) complexes (lane 3).

(C) Recombinant and hSWI/SNF-associated PRMT5 can methylate histones H3 and H4. H1-depleted HeLa core histones were incubated with either Fl-hSWI/SNF complexes, affinity purified wild type (WT) Fl-PRMT5, or mutant (Mut) Fl- PRMT5/G367A-R368A in the presence of 3H-SAM. Histones were visualized by Coomassie blue staining, and methylated products were detected by autora- diography.

(D) Levels of PRMT5 in Fl-hSWI/SNF complexes were quantitated by Western blot analysis using increasing amounts of either immunopurified Fl-hSWI/SNF fractions (100, 200 and 400 ng) or Sf9 expressed and affinity purified Fl-PRMT5 (6.25, 12.5 and 25 ng). Asterisk (*) indicates a long exposure of anti-PRMT5 Western blot.

(E) Methylation of histone H3 and H4 N-terminal tails was carried out as described in (C). Reactions were spotted onto Whatman P-81 filter paper, and methy- lated peptides were quantitated by liquid scintillation counting as described in Materials and Methods. As controls, methylation of BSA and H3 peptide (aa 60-84) are shown.

150 Figure 3.1

151 Figure 3.2: Recombinant and hSWI/SNF-associated PRMT5 can specifically methy- late H3R8 and H4R3. Wild type and mutant H3 peptides containing an arginine to alanine substitution at a single position (2, 8 or 17) or at all three positions were incubated with either flag-tagged hSWI/SNF complexes (A), or flag-tagged WT-PRMT5 (B) in the pres- ence of 3H -SAM. Similarly, wild type and mutant H4 peptides with either a single point mutation (R3A, R17A, R19A), or a triple point mutation (R3A/R17A/R19A) were incubated with either flag-tagged hSWI/SNF complexes (C) or flag-tagged WT- PRMT5 (D).

152 Figure 3.3: Interplay of H3R8 and H4R3 methylation with histone N-terminal tail lysine acetylation.

(A) Either WT or H3K9 and H3K14 acetylated peptides were methylated with PRMT5 containing-hSWI/SNF complexes as described for Figure 3.1E.

(B) Histone N-terminal peptides that were either unmodified or symmetrically methylated at H3R8 and H4R3 were subjected to acetylation with MBP-MORF in the presence of [3H]-AcetyleCoA and the acetylation was quantitated by liq- uid scintillation counting.

153 Figure 3.4: Characterization of sense and anti-sense (AS) PRMT5 cell lines. (A) Expression of endogenous PRMT5 was assessed in NIH3T3 and anti-sense cell lines. RT-PCR was performed using total RNA from either NIH3T3 cells (lanes 1-3) or AS-PRMT5 cells (lanes 4-6) using primers specific for either PRMT5, GAPDH or β-ACTIN. PCR reactions were performed using 2 µl (lanes 1, 3, 4 and 6) or 0.2 µl (lanes 2 and 5) of the RT reactions. Control (Ctrl) represents PCR reactions lacking the 5’ primer (lanes 1 and 4).

(B) Approximately 20 µg of nuclear extracts from NIH3T3 and AS-PRMT5 cells were analyzed by Western blotting using anti-PRMT5 and anti-BRG1 antibodies. RT-PCR was conducted as described in panel A using primers specific for the indicated up-regulated genes (C) and down-regulated genes (D). As a control, GAPDH levels were also analyzed.

154 Figure 3.5: PRMT5 induces cell growth and proliferation. (A) Proliferation of NIH3T3, stable cell lines that express AS-PRMT5, Fl-PRMT5, or MYC/RAS-transformed NIH3T3 cells was measured using 2 x 105 cells. The experiment was repeated three times in duplicates. Standard deviations are included but are too small for the error bars to appear on the graph.

(B) Approximately 40 µg of whole cell extract from the indicated cell lines were analyzed by Western blotting using anti-flag antibodies to detect expression of Fl-PRMT5 in the flag-tagged PRMT5 cell line. The same blot was stripped and probed with either anti-PRMT5 or anti-MAD antibodies.

(C) BrdU incorporation in NIH3T3, AS-PRMT5, and Fl-PRMT5 cells was deter- mined after 4.5 or 9 h incubation with BrdU. The percentage of BrdU positive cells was determined by FACS analysis.

(D) NIH3T3, AS-PRMT5, Fl-PRMT5 cells were grown for 4 days, stained with propidium iodide, and the DNA content of each cell line was analyzed by FACS analysis. The percentage of cells in each stage of the cell cycle including cells undergoing apoptosis (A) is shown.

155 Figure 3.5

156 Figure 3.6: Overexpression of PRMT5 stimulates anchorage dependent and indepen- dent growth.

(A) Approximately 4 x 103 of either NIH3T3, AS-PRMT5, Fl-PRMT5 or MYC/RAS-transformed cells were grown for 7 days and colonies were stained with crystal violet.

(B) Equal number (2 x 102) of drug-resistant cells containing either vector alone, AS-PRMT5, Fl-PRMT5 or MYC and RAS were grown in soft agar for ten days. Representative pictures showing the morphology and size of transformed cells are shown at 40X magnification. Colony formation assays were performed in triplicates and repeated three times. The number shown below each figure represents the average number of colonies from nine plates.

157 Figure 3.7: BRG1 and BRM-associated PRMT5 is directly involved in transcriptional repression of ST7 and NM23.

(A) RT-PCR was performed on total RNA from either NIH3T3, AS-PRMT5 or PRMT5 cell lines for ST7, NM23, MYT1L and GAPDH. PCR reactions for each gene was carried out using either 2 µl (lanes 1, 3, 4, 6, 7 and 9) or 0.2 µl (lanes 2, 5 and 8) of the RT reaction. Ctrl, represents PCR reactions without 5’ primer (lanes 1, 4 and 7).

(B) ChIP assays were conducted using cross-linked chromatin from either NIH3T3, AS-PRMT5 or Fl-PRMT5 cells using either preimmune (PI) or immune (I) anti-PRMT5 or anti-flag antibodies.

158 Figure 3.8: Symmetric H3R8 methylation and H3K9 acetylation at the ST7, NM23 and MYT1L promoter are inversely related.

(A) Specificity of anti-H3(Me2)R8 was determined by Western blot analysis using 1 and 2 µg of symmetrically methylated H3R8 peptide, unmethylated N-terminal and internal H3 peptides, or BSA.

(B and C) ChIP assays were conducted using cross-linked chromatin from either Fl-PRMT5, NIH3T3 or AS-PRMT5 cells either preimmune (PI) or immune (I) anti-H3(Me2)R8 antibodies (B), or anti-H3AcK9 antibodies (C). As controls, Mock (reaction without chromatin) and No antibody (Ab) (reaction with chro- matin but without antibody) reactions are shown.

159 Figure 3.9: BRG1 and BRM are differentially recruited to methylated ST7 and NM23 promoters.

(A and B) Cross-linked chromatin was prepared from asynchronous Fl-PRMT5, NIH3T3 and AS-PRMT5 cell lines, and ChIP assays were conducted using either preimmune (PI) or immune (I) anti-BRG1 (A) and anti-BRM (B) antibodies.

(C) Levels of catalytically inactive BRG1 and BRM were measured by Western blotting using 30 µg of nuclear extract from either mutant (Mut) BRG1 or BRM cell lines. HeLa S3 nuclear extract was used as a control, and proteins were detected using the indicated antibodies.

(D) MYT1L, NM23, ST7 and GAPDH transcript levels were analyzed by RT-PCR using RNA from either HeLa cells or HeLa cells that express either mutant BRG1 or BRM. PCR reactions were carried out using either 2 µl (lanes 1, 3, 5 and 7) or 0.2 µl (lanes 2, 4 and 6) of the RT reaction. Ctrl, represents PCR reactions without 5’ primer (lane 1).

160 Figure 3.9

161 CHAPTER 4

PRMT5 OVEREXPRESSION IS ASSOCIATED WITH GLOBAL AS WELL AS ST7 PROMOTER SPECIFIC H3R8/H4R3 SYMMETRIC METHYLATION IN MANTLE CELL LYMPHOMA

4.1 Abstract

In chapters 2 and 3 it has been shown that PRMT5 interacts with h SWI/SNF complexes, and methylates histones H3R8 and H4R3. In addition, overexpression of PRMT5 induces hyperproliferation and anchorage independent growth of NIH3T3 cells. In this chapter, the role of PRMT5 in human cancer is being studied. PRMT5 expression in normal B lymphocytes and a panel of lymphoid cancer cell lines as well as mantle cell lymphoma (MCL) clinical samples was analyzed. PRMT5 protein levels were found to be elevated in all cancer cell lines and clinical samples examined despite its low rate of transcription and mRNA stability. Remarkably, when the association of PRMT5 mRNA with polysomes was compared between normal and transformed B cells, it was found that PRMT5 mRNA is translated more efficiently in Mino and JeKo

MCL cells. Furthermore, micro-RNA analysis shows that miR-96 is down-regulated

2.5 to 3-fold in MCL cell lines, and is involved in inhibiting PRMT5 translation.

Additionally, PRMT5 recruitment and H3R8 and H4R3 methylation were found to

162 be enhanced at the ST7 promoter, and that is accompanied by decreased expression of ST7 in Mino and JeKo cells as well as MCL clinical samples. Moreover, knock down of PRMT5 expression in the MCL cell line JeKo, and Burkitt’s lymphoma cell line Raji, reduces cell proliferation. The work reported in this chapter indicates that

aberrant expression of PRMT5 leads to altered epigenetic modification of chromatin,

which impacts transcription of anti-cancer genes in mantle cell lymphoma.

4.2 Introduction

Cancer is caused by aberrant expression of genes that regulate cell growth and

proliferation, and understanding the molecular mechanisms that alter their gene ex-

pression is important for designing therapies for cancer. The chromatin-modifying

enzymes, which can either activate or repress gene expression, are involved in can-

cer etiology [70, 243]. It has been reported that interplay and cross-talk between

chromatin-modifying enzymes is necessary for efficient regulation of gene expression,

and that changes affecting the activity or targeting of chromatin modifiers can trigger

cancer [76, 114].

PRMT5 can impact transcription in either methylase dependent or independent

manner. As a component of the androgen receptor cofactor complex, PRMT5 pos-

itively modulates androgen receptor-driven transcription independent of its methyl-

transferase activity [126]. In contrast, PRMT5 methyltransferase activity is impor-

tant for its ability to methylate histones H3 and H4, and regulate transcription of its

target genes [55, 215]. Furthermore, methylation by PRMT5 can also have either a

163 positive or negative effect on its substrates. For example, PRMT5-mediated methyla- tion of SmD1 and SmD3 is crucial for their incorporation into snRNPs, which are in- volved in RNA splicing; whereas, methylation of the MBD2 subunit of NURD reduces its ability to associate with methylated DNA and repress transcription [85, 107, 278].

The findings in chapter 3 show that reduced levels of PRMT5 in NIH3T3 cells inhibit cell growth and proliferation, while enhanced expression of PRMT5 induces hyperproliferation and anchorage independent growth [215]. Recently, it was de- termined that suppression of ER-negative breast cancer cell proliferation by 17-β

estradiol treatment is accompanied by suppression of PRMT5 expression [189]. More-

over, PRMT5 overexpression has been implicated in gastric cancer, highlighting the

significance of normal expression of PRMT5 in the control of cell growth and prolif-

eration [108].

To assess the role of PRMT5 in human lymphoid cancers, its expression in a

panel of transformed lymphoid cells including mantle cell lymphoma (MCL) cell

lines and MCL patient samples was analyzed. MCL is a subtype of non-Hodgkin’s

B cell lymphoma characterized by the presence of t(11;14)(q13;q32) chromosomal

translocation. Patients afflicted by MCL show a very poor prognosis and survival

rate of 3-4 years. MCL is incurable due to the lack of effective treatments, and identi-

fication of novel therapeutic targets remains the focus of ongoing MCL research [309].

Using MCL as a model system, when the levels of PRMT5 and global methylation of

histones H3R8 and H4R3 were examined, and it was found that PRMT5 is highly ex-

pressed in a wide variety of lymphoid cancer cell lines including MCL clinical samples,

and consequently global symmetric methylation of H3R8 and H4R3 is also enhanced

in these cells. In addition, PRMT5 expression is regulated at the translational level

164 through miR-96, and enhanced PRMT5 expression correlates with transcriptional si-

lencing of ST7 in all lymphoid cancer cell lines and MCL clinical samples examined.

Furthermore, knockdown of PRMT5 expression inhibited growth and proliferation of the JeKo MCL cell line, as well as the Raji Burkitt’s lymphoma cell line. This

work identifies PRMT5 as a key global chromatin modifier whose aberrant expres-

sion contributes to silencing of the ST7 tumor suppressor, and is associated with

lymphomagenesis.

4.3 Materials and Methods

4.3.1 Cell culture and B cell isolation

Normal and transformed lymphoid cells were cultured in RPMI-1640 supple-

mented with 10-20% FBS. Normal B cells were isolated from tonsils obtained from

Children’s Hospital through the Cooperative Human Tissue Network (CHTN). Ton-

silar tissues were minced extensively in RPMI-1640 containing 10% FBS and strained through a collector sieve (Bellco Glass, Inc.) to remove tissue debris. Next, mono- cytes were removed by adhesion to tissue culture plates overnight, and B cells were isolated by depletion of T lymphocytes. To deplete T cells, the tonsilar cells were collected after overnight incubation in 150 mm tissue culture plates, mixed with 8-

fold excess of sheep red blood cells (Colorado Serum Company, catalog no. CS1115),

spun at 1,200 rpm for 10 min, and incubated on ice for 1 h. Next, cells were gently

resuspended by inversion and allowed to equilibrate to room temperature for 1 h. To separate B cells from rosetted T lymphocytes, 10 ml of ficoll-paque (Amersham, Inc.,

catalog no. 17-1440-03) was added, and samples were spun at room temperature for

30 min before collecting the ”buffy coat” layer containing B cells. B lymphocytes

165 were washed with media and purity of the isolated cells was determined by FACS analysis using anti-CD19-PE antibody. Two aliquots of purified 5 x 105 cells were transferred to FACS analysis tube (BD falcon polystyrene tubes, Fisher Inc., catalog no. 14959-5), washed once with 1 ml of 1X PBS and cells were collected by centrifu- gation at 1,500 rpm for 10 min. The cells were resuspended in 10 µl of IgG from murine serum (Sigma Inc., catalog no. I8765), and one sample, which was the control, was labeled with 20 µl of PE labeled mouse IgG1κ (BD Pharmingen Inc., catalog no.

555749), while the other sample was labeled using 20 µl of anti-PE-human CD19 antibody (BD Pharmingen Inc., catalog no. 555413). The samples were incubated on ice for 10 min and washed once using 1 ml of 1X PBS to remove the access PE stained antibody. The cells were finally fixed by resuspending in 500 µl of 0.1% for- malin and stored protected from light at 4oC until analyzed by flow cytometry. B lymphocytes isolated by this procedure were 90-95% pure. Normal and transformed human B lymphocytes were collected under an IRB-approved and HIPPA-compliant protocol.

4.3.2 B cell activation

To activate tonsilar B cells, 2.5 x 106 cells were seeded into 12 well plates in 0.5 ml RPMI-1640 supplemented with 10%FBS, 15 ng/ml IL-4 (BioSource International

Inc., catalog no. PHC0044), and 15 µg/ml goat anti-human IgM+G (Jackson Im- munoResearch Laboratories Inc., catalog no. 109-005-044), and grown for either 4 or

6 days. To assess the proliferation of B cells upon activation, cells were treated with

10 µM BrdU for 72 h prior to fixing and staining as detailed in section 3.3.5. To analyze the expression of PRMT5 upon B cell activation, 1 x 108 cells were activated

166 as above and nuclear as well as cytosolic extracts were prepared for Western blot

analysis.

4.3.3 RIPA cell lysate preparation and Western blot analysis

To prepare whole cell extracts using RIPA buffer lysis, 1 x 107 cells were lysed in

150 µl of RIPA lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1%

SDS and 0.5% sodium deoxycholate) and incubated on ice for 20 min. Cell lysates

were clarified by centrifugation at 14,000 rpm, 4oC for 10 min and Western blots

were performed using nuclear, cytosolic, or whole cell extracts. Nuclear and cytosolic

extracts were prepared as described previously [168]. Proteins were separated on

8-10% SDS-PAGE, transferred to nitrocellulose membrane and detected by enhanced chemiluminescence.

4.3.4 Immunofluorescence analysis

For immunofluorescence experiments, PRMT5, ST7, H3(Me2)R8, and H4(Me2)R3

were visualized by plating 1 x 104 cells on glass coverslips, which were pretreated with

poly-L-lysine (Sigma Inc., catalog no. P1524). To coat coverslips with poly-L-lysine,

they were immersed in 2 ml of 0.06% poly-L-lysine solution in 6 well plate for 1 h and

then rinsed twice with 2 ml of 1X PBS before 2 ml media containing 1 x 104 cells was

added. The next day, cells were washed with 1X PBS, fixed in 4% paraformaldehyde

at room temperature for 15 min, washed three times with 1X PBS and treated with

0.1% Triton X-100 at room temperature for 10 min. Cells were then blocked in

10% goat serum (diluted in 1X PBS; Gibco BRL Inc., catalog no. 16210064) for 2

h, washed twice with 1X PBS, and incubated with 40 µl of either preimmune (PI),

anti-PRMT5, anti-ST7, affinity purified H3(Me2)R8, or anti-H4(Me2)R3 antibody

167 (all antibodies were used at 1:25 dilution in 2% goat serum) on parafilm at 37oC for

2 h. The serum antibodies were initially depleted of non-specific binding proteins by incubating 500 µl of the antibody containing serum with 50 µl of GST-agarose

beads on nutator at 4oC for 2 h. The supernatant was used as the antibody stock for

the immunoflourescence experiments. To remove excess primary antibody, cells were

washed extensively with 1X PBS (three times) for 10 min before incubation with 40

µl of FITC-labeled goat anti-rabbit antibody (Sigma Inc., catalog no. F0382) at 37oC

for 1 h. Cells were washed again with 2 ml of 1X PBS (three times) for 5 min, nuclei

were stained with 2 ml of DAPI (0.2 µg/ml, Sigma Inc., catalog no. D9542) for 5 min

at room temperature before coverslips were washed three times in 2 ml of 1X PBS

for 5 min, quickly rinsed in water and mounted on glass slides using 5 µl of prolong

antifade reagent (Molecular probes Inc., catalog no. P7481). PRMT5, ST7, and

symmetrically methylated H3R8 and H4R3 proteins were visualized by fluorescence

microscopy using a Zeiss axioscope at 100X magnification in the campus microscopy

and imaging facility (http://cmif.osu.edu).

4.3.5 5Azacytidine, depsipeptide and DRB treatment

To study the effects of DNA methylation and histone deacetylation, 1 x 107 cells

were treated with 8 µM 5Azacytidine (5AzaC; Sigma Inc., catalog no. A2385) or

60 nM depsispeptide (Depsi) for 24 h, respectively. After treatment cells were col-

leceted, washed once with 10 ml 1X PBS before RNA was harvested using Trizol

reagent (Invitrogen, Inc., catalog no. 15596-018). To study mRNA stability, 1 x 107

cells were treated with 50 µM DRB (5,6-Dichloro-1-b-D-ribofuranosylbenzimidazole;

Sigma Inc., catalog no. D1916) for 0.5, 1, or 2 h before RNA was isolated as above.

168 4.3.6 Real time RT-PCR

Reverse transcription (RT) was performed on 2 µg of total RNA using a reverse

transcription kit (Applied Biosystems, Inc. catalog no. N8080234). Briefly, 20 µl re- action containing 2 µg RNA, 1X TaqMan RT buffer, 5.5 mM MgCl2, 0.5 mM dNTP’s,

2.5 µM random hexamer, 0.4 U/µl RNase inhibitor, and 1.25 U/µl multiscribe reverse

transcriptase was first incubated at 25oC for 10 min, followed by incubation at 42oC

for 1 h, and then finally inactivated at 95oC for 5 min. Real time PCR was performed

using the TaqMan system (Applied Biosystems, Inc., catalog no. 4304437) in a 10 µl

reaction containing 1 µl of RT reaction, 1X TaqMan universal master mix, 0.8 µM

of each primer, and 0.2 µM MGB probe (custom made at Applied Biosystems Inc.).

For the internal control, expression of GAPDH was measured using 1X pre-mixed

GAPDH primer/probe set (Applied Biosystems, Inc., Assay ID HS99999905-M1).

ST7 (+341 to +407) and PRMT5 (+1712 to +1828) gene specific primers and probes

used for real time RT-PCR reactions are listed in Appendix B. To normalize RT-PCR results for DRB treated samples, 18S was used as an internal control (Applied Biosys- tems, Inc., catalog no. 4319413E). Normalized fold expression of ST7 and PRMT5 was calculated relative to normal B lymphocytes using the 2−∆∆Ct method [∆Ct =

Ct(gene)-Ct(control), ∆∆Ct = ∆Ct- ∆Ct(B cell) and fold expression= 2−∆∆Ct].

4.3.7 Nuclear run on assay

To measure the rate of PRMT5 transcription, nuclei were prepared from 4 x 107

Mino and JeKo MCL cell lines, or 1 x 108 normal B lymphocytes. Cells were washed

with 1X PBS, resuspended in 500 µl of lysis buffer (10 mM Tris-HCl [pH 7.4], 10

mM NaCl, 3 mM MgCl2, 0.5% NP-40), and incubated on ice for 5 min. Nuclei were

169 collected by centrifugation and resuspended in an equal volume of storage buffer (50

mM Tris-HCl [pH 8.5], 5 mM MgCl2, 0.1 mM EDTA, 40% glycerol). To initiate run on transcription, 50 µl of nuclei were incubated with 50 µl of 2X reaction cocktail

containing 25 µl of 4X reaction buffer (100 mM HEPES-KOH [pH 7.5], 10 mM MgCl2,

300 mM KCl, 20% glycerol, 10 mM DTT), 12.5 µl of 8X nucleotide mix (2.6 mM ATP,

CTP, GTP and 3 µM UTP), 5 µl of [α-[32P] UTP, and 7.5 µl DEPC-treated water

at 30oC for 30 min. Nuclear run on transcription was terminated by adding 2 µl of

DNaseI at 37oC for 10 min. Next, reactions were incubated with 300 µl of stop buffer

(10 mM Tris-HCl [pH 8.0], 350 mM NaCl, 7 M urea, 1 mM EDTA, 2% SDS), 300

µg proteinase K, and 100 µg tRNA at 50oC for 2 h. Total RNA was trichloroacetic

acid (TCA)-precipitated by adding TCA to a final concentration of 10% (45 µl),

incubated on ice for 20 min, and collected by centrifugation at 4oC for 15 min.

Radiolabeled RNA was resuspended in 50 µl TE [pH 8.0] containing 0.5% SDS. The

radioactive counts were determined by measuring 1 µl of the resuspended radiolabelled

RNA by liquid scintillation counting and 1.5 to 2 x 106 cpm of labeled RNA were

mixed with 1.5 to 2 ml of prehybridization buffer (50% formamide, 10X Denhardt’s

solution, 6X SSPE, 0.2% SDS) before hybridization to Hybond-XL nylon membrane,

which contained 5 µg of immobilized DNA fragments from either pBluescript digested

with PvuII (nt 526 to 975), β-ACTIN (nt +140 to + 585), or PRMT5 (nt +101

to +590), at 42oC for 96 h. The β-ACTIN fragment was generated by RT-PCR

using primers 5b-ACT and 3b-ACT, while PRMT5 DNA fragment was amplified

by PCR from pBS(KS+)/FL-PRMT5 using primers 5HMT3 and 3HMT2 (primer

sequences are provided in the Appendix B). The nylon membrane containing DNA

fragments was pre-blocked in 4 ml of prehybridization buffer at 42oC for 24 h.

170 Following hybridization at 42oC for 96 h, the blot was washed twice in 20 ml of 6X

SSPE containing 0.2% SDS at 42oC for 30 min and analyzed by phosphorImager.

4.3.8 Genomic DNA isolation, construction of PRMT5 pro- moter driven luciferase plasmids and transfection of HeLa and JeKo cells

To amplify the PRMT5 promoter region, genomic DNA isolated from HeLa cells

was used as the template DNA. HeLa cells from a 90% confluent plate were harvested,

washed once with 1X PBS, and lysed in 10 ml of ice cold buffer L (5 mM PIPES [pH

8.0], 85 mM KCl, 1 mM CaCl2, 5% sucrose, and 0.5% NP 40) by pipeting twice. The nuclei were collected by centrifugation at 1500 rpm for 2 min and washed with 1 ml of buffer L lacking NP 40. The nuclei pellet was resuspended in 100 µl of buffer M (15 mM Tris-HCl [pH 7.5], 15 mM NaCl, 60 mM KCl, 5 mM β-mercaptoethanol, 1 mM

CaCl2, 3 mM MgCl2, and 0.34 M sucrose) and lysed by adding 100 µl of buffer O (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 15 mM EDTA, and 0.3% SDS). Next, samples were deproteinated by proteinase K treatment (400 µl of 1X NEB buffer 2, 200 µl buffer O, 600 µg proteinase K) at 60oC for 14-16 h, and then DNA was purified by phenol: chloroform extraction followed by ethanol precipitation. To amplify human

PRMT5 promoter sequences, 100 pmol of promoter specific primers (-1000 to +190) were used in a PCR reaction along with 1 µg of HeLa genomic DNA as described in section 2.3.1. The various PRMT5 promoter constructs were generated as described in Appendix A.

To assess the activity of PRMT5 promoter sequences, the promoter containing pGL2 plasmids were transfected in HeLa and JeKo cells by electroporation. In case of HeLa cells, 5 x 106 cells were electroporated with 4.5 µg of pGL2 plasmid and

171 0.5 µg of pCMV-β Gal (β-galactosidase expressing plasmid) in 100 µl of nucleofector

reagent V using program V001, while transfection of JeKo cells was performed using

5 x 106 cells with 12 µg of pGL2 plasmid and 3 µg of β-galactosidase plasmid in 100

µl of nucleofector reagent T using program T-27. Currently, JeKo cells are being

electroporated in reagent L using program X001, which provides a better transfection efficiency (50 to 60%). To electroporate, 5 x 106 cells were transferred to a 15 ml tube

and collected by centrifugation at 1,000 rpm for 4 min. Media was completely removed

and the DNA was added followed by 100 µl of the required nucleofector reagent. The mixture was transferred to the supplied cuvettes, which was electroporated in the

amaxa electroporator according to the above specific program. Next, 0.5 ml of media

was quickly added to the cells, which were transferred using the supplied plastic

pasteur pipets to 3 ml of media in 6 well plate. Cells were harvested 40 h after

transfection and lysed in 100 µl of 1X passive lysis buffer provided with the Dual

luciferase assay kit (Promega, Inc., catalog no. E1910). Luciferase assay was carried

out using 40 µl of cell lysate and normalized with β-galactosidase activity, which was

measured as described in section 2.3.9. To measure luciferase assay, 100 µl of the

LAR II reagent (supplied in the kit) was added to the cuvette (BD Biosciences Inc.) and then quickly mixed with 40 µl of lysate before reading the luciferase activity using protocol no. 6 in the luminometer. To stop the reaction, 100 µl of Stop-glow reagent

(provided with the kit) was added and re-read in the luminometer. In case renilla luciferase was used in the experiments, the reading after the addition of stop-glow

provides the measure of renilla luciferase activity.

172 4.3.9 Polyribosome profiling

Approximately 4 x 107 normal B or 2 x 107 MCL cells were washed twice with

1X PBS and lysed in 250 µl of lysis buffer (20 mM HEPES [pH 7.5], 100 mM KCl,

10 mM MgCl2, 0.25% NP-40, 100 µg/ml cycloheximide (Sigma Inc., catalog C4859),

100 U/ml RNasin, 1 mM DTT, and 0.5 mM PMSF) by passing through a 27.5 gauge

needle five times. Lysates were cleared by centrifugation at 14,000 rpm, 4oC for

5 min and loaded immediately onto a linear 15% to 40% sucrose gradient (4.8 ml)

poured using a FPLC system with freshly prepared 15% and 40 % sucrose gradient

solution. Gradients were then centrifuged in a SW55 rotor at 43,000 rpm, 4oC for 2.5

h. Fractions were collected and absorbance of each fraction (200 µl) was measured at

254 nm before total RNA was extracted using Trizol reagent as described in section

3.3.5 and RNA was resuspended in 7.5 µl of DEPC-treated water. To synthesize

cDNA, reverse transcription reaction was performed with 3.5 µl of the above isolated

total RNA in a 10 µl reaction as described in section 4.3.6. PRMT5 mRNA levels were

measured by real time RT-PCR using specific primers and probe as described above in

section 4.3.6, and as an internal control β-ACTIN mRNA levels were also determined

using the 1X premixed human β-ACTIN primer/probe set (Applied Biosystems Inc.,

Assay ID no. HS99899903-M1).

4.3.10 RNase protection assay (RPA)

To detect expression of micro RNAs (miR), RPA was performed on 20 µg of total

RNA isolated from either normal B lymphocytes or transformed MCL cell lines.

However, when miR expression was detected in MCL clinical samples, 5 µg of total

RNA was used. Labeled probes were constructed using the mirVana miRNA probe

173 construction kit (Ambion, Inc., catalog no. 1550). Essentially, a single stranded DNA oligonucleotide corresponding to the desired miR sequence (sense strand sequence where all Us have been replaced with Ts) and containing the T7 promoter sequence

(CCTGTCTC) at the 3’end was hybridized to the T7 promoter primer (provided with the kit). Briefly, 1 µl of T7 promoter primer was mixed with 3 µl of DNA hybridization buffer, 1 µl of above DNA oligonucleotide (100 pmol/µl), heat denatured

at 70oC for 5 min, and then cooled at room temperature for another 5 min. Next,

to synthesize double stranded DNA T7 promoter primer was extended using Klenow

DNA polymerase by adding 1 µl of 10X klenow reaction buffer, 1 µl of 10X dNTP mix,

2 µl of water, 1 µl of Exo-Klenow and incubating the reaction at 37oC for 30 min.

The resulting double stranded (ds) DNA template can be stored indefinitely at -20 oC. To generate the miR probe the ds DNA template was in vitro transcribed using

T7 RNA polymerase in the presence of [α-[32P]CTP at 37oC for 30 min by adding

0.5 µl dsDNA template, 1 µl of 10X transcription buffer, 0.5 µl of 10 mM ATP, 0.5

µl of 10 mM GTP, 0.5 µl of 10 mM UTP, 2.5 µl of [α-32P]CTP (10 µCi/µl), 1 µl of T7 RNA polymerase and 4.5 µl water. Transcription was terminated by adding

DNase I to the reaction, and labeled RNA was purified by loading samples onto a

0.8 mm thick 12% acrylamide-urea gel (7.2 g Urea, 1.5 ml 10X TBE, 4.5 ml of 40% acrylamide (38:2), and water to a final volume of 15 ml). Labeled full-length RNA probe was excised out and eluted from the gel by adding 150 µl of probe elution buffer

(provided with the kit) and incubating the gel slice at 37oC for 30 min. The sample was centrifuged at 14,000 rpm for 2 min and the supernatant was transferred to a new tube. The labeling of the probe was determined by measuring the counts of 1 µl of the eluted probe by liquid scintillation counting. To measure the level of each miR,

174 RPA was performed using the mirVana miRNA detection kit (Ambion, Inc., catalog no. 1552). Briefly, 5 x 104 cpm of each miR-specific probe was mixed with 20 µg of total RNA in the presence of 10 µl mirVana hybridization buffer in a final volume of

20 µl, heat denatured at 95oC for 3 min, and then incubated at 42oC for 16 h. As a control, miR-specific probes were also incubated with 20 µg of yeast tRNA (provided with the kit). Next, reactions were digested with 1:100 dilution of RNase A+T1 in

150 µl of RNase digestion buffer at 37oC for 45 min. To inactivate RNase A+T1 and to precipitate protected dsRNA, 225 µl of RNase Inactivation/PPT solution and 225

µl of 100% ethanol were added to each reaction, mixed, and incubated at −80oC for 2 h. Protected dsRNA was harvested by centrifugation at 14,000 rpm, 4oC for 15 min and pellets were resuspended in 10 µl of gel loading buffer II. Samples were heated at 95oC for 3 min and loaded immediately after flushing the wells with running buffer onto a 15% acrylamide-urea gel that was pre-run at 15 A for 1 h. Samples were separated at 15 A till the bromophenol dye migrated about 7 cm from the bottom of the well. Protected RNAs were visualized by phosphorImaging, and quantitated using ImageQuant v5.0.

4.3.11 Construction of double stranded (ds) RNA and dsRNA transfection in JeKo and Raji lymphoma cells.

To evaluate the effects of miR-96 on PRMT5 protein expression, either wild type or To evaluate the effects of miR-96 on PRMT5 protein expression, either wild type or mutant miR-96 specific dsRNA was electroporated into JeKo and Raji cells using the Amaxa Biosystems electroporator. Approximately 5 x 106 JeKo or Raji cells were electroporated with 2.5 µg of miR-96 dsRNA in 100 µl of the reagent L (pro- gram X001) and V (program M013), respectively. Cells were then plated in 3 ml of

175 RPMI-1640 containing 10% FBS, and lysed 30 h later in 100 µl of RIPA lysis buffer

(50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% SDS and 0.5% sodium

deoxycholate) before proteins were detected by Western blotting. To generate wild

type and mutant miR-96 dsRNAs, T7-driven single stranded DNA oligonucleotides

corresponding to the underlined wild type miR-96 sense:

(50-AATTTGGCACTAGCACATTTTTGCCCTGTCTC-30) and anti-sense:

(50- AAGCAAAAATGTGCTAGTGCCAAACCTGTCTC-30), or mutant miR-96 sense:

(50- AAGGCTCACATAGCACATTTTTGCCTCGTCTC-30) and anti-sense:

(50-AAGCAAAAATGTGCTATGTGAGCCCTCGTCTC-30) were transcribed indi- vidually using the Ambion silencer siRNA construction kit (Ambion, Inc., catalog no. 1620). To transcribe sense and antisense oligonucleotides, 100 pmol of each

primer was mixed with 1 µl of T7 promoter primer, 3 µl of DNA hybridization buffer,

heat denatured at 70oC for 5 min, cooled to room temperature for 5 min, and then

converted to double stranded (ds) DNA using Klenow by supplementing the reactions

with 1 µl of 10X Klenow reaction buffer, 1 µl of 10X dNTP mix, 1 µl of Exo-Klenow,

and 2 µl water and incubating the reactions at 37oC for 30 min. Next, the above ds

DNA template was used in transcription reaction by mixing 2 µl of ds template DNA,

10 µl of 2X NTP mix, 2 µl of 10X T7 reaction buffer, 2 µl of T7 enzyme mix, and 4

µl of water and the reactions were incubated at 37oC for 2 h. Then, the sense and

anti-sense wild type or mutant miR-96 transcription reactions were annealed at 37oC

for 14 h, and dsDNA templates along with RNA leader sequences were removed by

DNase and RNase treatment in a volume of 100 µl in the presence of 6 µl digestion

buffer, 3 µl of RNase, and 2.5 µl of DNase at 37oC for 2 h. Next, wild type or mutant

miR-96 dsRNAs were purified through an Ambion filter cartridge (provided with the

176 kit) as described below before electroporation into JeKo and Raji cells. The reactions were incubated with 400 µl of siRNA binding buffer for 5 min at room temperature

and then applied to the filter cartridge that has been pre-wet with 100 µl of siRNA wash buffer. The cartridge was centrifuged at 10,000 rpm for 1 min, and washed

twice with 500 µl of siRNA wash buffer. Finally, the siRNA was eluted with 100 µl

of DEPC-treated water that has been pre-heated to 75oC by centrifugation at 12,000

rpm for 2 min and stored at -80oC.

4.3.12 Transfection of normal and transformed B lympho- cytes, and luciferase assays

To study the effect of wild type and mutant 3’UTR of PRMT5 on gene expres-

sion, normal B cells, JeKo and Raji cell lines were transfected by electroporation.

Approximately 2.5 x 107 B cells, or 5 x 106 JeKo and Raji cells were electroporated

with 15 µg of total DNA (6 µg of pCMV-Luc construct, 2 µg of pRenilla-Luc control

plasmid, and 7 µg of pBS(KS+)) using B cell specific nucleofector (program U15),

L nucleofector (program X001), and V nucleofector (program M013) reagent, respec-

tively as described in section 4.3.8. After electroporation cells were plated in 3 ml of

RPMI-1640 containing 10% FBS for 24 h, and then collected, washed once with 1X

PBS, and lysed in 100 µl of 1X passive lysis buffer provided with the Dual luciferase

assay kit (Promega, Inc., catalog no. E1910). Dual luciferase reporter analysis was

carried out using 40 µl of cell lysate as described in section 4.3.8.

177 4.3.13 In vitro transcription, capping, polyadenylation, and translation

PRMT5 with or without wild type 3’UTR, PRMT5 with mutant 3’UTR, and

BAF45 cDNAs were in vitro transcribed and capped simultaneously using EcoRV- linearized pBS(KS+)-based constructs with T7 RNA polymerase by incubating 1 µg of linearized template with 10 µl of 2X NTP/CAP buffer, 2 µl of 10X reaction buffer, and 2 µl of T7 enzyme mix in a final volume of 20 µl at 37oC for 2 h according to the message machine kit (Ambion, Inc., catalog no. 1344). Next, polyadenylation was carried out in a 100 µl reaction containing 20 µl transcription reaction, 1X E-PAP

o buffer, 2.5 mM MnCl2, 1 mM ATP, and 1 U of E-PAP at 37 C for 20 min (Poly(A) tailing kit, Ambion Inc., catalog no. 1350). When 1, 2, or 8 U of E-PAP was used for 1 h in polyadenylation reaction, the resulting poly-A tail RNA was not trans- lated in vitro and further titrations were performed to determine the polyadenylation reaction conditions that generate poly-A RNA that could be translated. RNA was purified by phenol: chloroform extraction and then precipitated for 14-16 h at −20oC by adding 1/10th volume of ammonium acetate stop buffer (provided with message machine kit) and equal volume of isopropanol. Next, the RNA was collected by cen- trifugation at 14,000 rpm, 4oC for 15 min, washed once with 500 µl of 75% ethanol,

resuspended in 20 µl of DEPC-water and then in vitro translation was performed

using 0.25 µg of the in vitro transcribed RNA in a 10.4 µl reaction. To assess the

effect of wild type and mutant miR-96 on PRMT5 translation, in vitro transcribed

RNA was mixed with 2.1 µl of 2X Oligohyb buffer (20 mM Tris-HCl [pH 8.0], 20 mM

NaCl) and 2.5 ng (1:1 molar ratio) or 5.0 ng (1:2 molar ratio) of either wild type modified miR-96 (UUUGGCACGCAGGGAUUUUUGC) or mutant modified miR-96

178 (UGGUCACCGCAGGGAUUUUUGC) RNA in a final reaction volume of 4.2 µl.

Since PRMT5 mRNA is about 2,200 bp and miR-96 is 22 bp, to achieve equimolar ra- tio of the two RNA, the amount of miR-96 RNA that should be used will be 1/100th of

PRMT5 mRNA. Both wild type and mutant modified miR-96 RNA oligonucleotides

were synthesized as 5’-phosphorylated RNAs (Integrated DNA Technologies, Inc.).

To anneal wild type or mutant modified oligonucleotides to PRMT5 RNA, reactions were heated at 75oC for 5 min and allowed to incubate at room temperature for 10 min. In vitro translation was initiated by adding 5 µl of nuclease treated reticulocyte lysate (Promega Inc., catalog no. L4960), 0.2 µl of 1 mM aa mix without methionine and cysteine, 0.2 µl of RNase inhibitor, and 8.8 µCi of [35S]methionine and cysteine

(Perkin Elmer, catalog no. NEG072), and reactions were incubated at 30oC for 30

min. Next, translation was terminated by adding 10.2 µl of 2X SDS sample loading

buffer, and in vitro synthesized proteins were separated on 8% SDS-PAGE. Gels were

treated with 1 M salicylic acid for 20 min, dried, and labeled products were visualized

by autoradiography.

4.3.14 ChIP assay

ChIP experiments were performed using soluble cross-linked chromatin from ap-

proximately 1 x 107 normal or transformed B cells as described previously in section

2.3.16, except that washing with the mixed micelle buffer was carried out twice. To

assess recruitment to the ST7 promoter, real time PCR was performed as described

in section 4.3.6 in triplicates using 3 µl of the ChIP eluted DNA (total 35 µl) from

179 preimmune and immune antibody reactions in a 10 µl PCR reaction using ST7 pro-

moter specific primers (-125 to -15) and probe (-74 to -54 ) that are listed in Appendix

B.

4.3.15 Generation of lentiviral particles and infection of JeKo cells.

To prepare lentiviral particles containing either empty vector or anti-sense (AS)

PRMT5, 40% confluent 293T cells (100 mm plates) were transfected with 5 µg of pVSV-G, 15 µg of pCMV∆8.2, and 20 µg of either pRRL-puro or pRRL-AS-PRMT5

using the calcium phosphate precipitation protocol [242]. Briefly, 40 µg of DNA was

mixed with 20 µl of CaCl2 (Stock 2.5 M) and 0.1X TE [pH 8.0] in a final volume of

500 µl. Next, 500 µl of 2X HBS (0.28 M NaCl, 0.05 M HEPES, 1.5 mM Na2HPO4,

pH to 7.0 with NaOH, and filtered through 0.45 µm filter) was added slowly with

constant mixing by bubbling in air. The samples were incubated at room temperature

for 20 min to allow calcium-DNA precipitates to form. Then, the media from the

cells was removed and replaced with fresh media supplemented with 10% FBS and

0.25 µl/ml chloroquine (100 mM). After the 20 min incubation, the calcium-DNA

precipitate mixture was added, and cells were incubated for 8 h before removing the

transfection mix. The cells were finally washed twice with 3 ml of 1X PBS and

grown in 4 ml of DMEM containing 10% FBS for 48 h. Supernatants containing

replication-deficient lentivirus were harvested 48 h post-transfection and clarified by

centrifugation at 2,000 rpm for 4 min. To generate enough lentivirus containing

supernatant, ten 100 mm plates were transfected as above and supernatant was pooled

and used as the virus stock. To determine the amount of virus that was required

to achieve knockdown of PRMT5, 7.5 x 105 JeKo cells were infected with either

180 125, 250, or 500 µl of control empty vector or AS-PRMT5 lentivirus supernatant in a final volume of 1 ml RPMI-1640 supplemented with 10% FBS and 8 µg of polybrene for 48 h. PRMT5 expression was analyzed by Western blotting on 20

µg of the RIPA extract prepared as described in section 4.3.3 from the above cells.

Knockdown of PRMT5 expression was maximal when 250 µl of lentivirus supernatant was used and the following experiments were performed under these conditions.

To monitor proliferation, infected cells were counted every 2 days for 6 days post- infection from four different infections and media was changed every two days. To assess cell proliferation by BrdU incorporation, control empty vector and AS-PRMT5 virus infected cells from day 2, and 4 were treated with 10 µM BrdU for 4 h before staining with anti-FITC labeled BrdU antibody as described in section 3.3.8.

4.3.16 Statistical analysis

To statistically validate data generated using multiple samples within different groups, analysis of variance (Anova) was used to calculate the P-value. To identify differentially expressed genes or recruitment of various chromatin remodelers between two groups, paired t-tests were used to calculate the P-value. In all cases GraphPad

Prism4 software was used to generate P-values.

181 4.4 Results

4.4.1 PRMT5 is overexpressed and symmetric methylation of histones H3R8 and H4R3 is increased in lymphoid cancer cell lines

In previous chapters it has been shown that PRMT5 interacts with BRG1 and

BRM-based hSWI/SNF complexes, and that overexpression of PRMT5 in immortal-

ized but not transformed, NIH3T3 cells induces hyperproliferation and transforma-

tion [215]. Hence, the role of PRMT5 in human malignancies of lymphoid origin for

which normal controls are readily available was investigated. Nuclear and cytosolic

extracts from normal CD19+ B lymphocytes, patient-derived lymphoma (Burkitt’s

lymphoma: Daudi, Jijoye, BL30, P3HRI, and Raji; mantle cell lymphoma: Mino,

JeKo and SP53), in vitro EBV-transformed lymphoma (LCL-147), and leukemia cell

lines (EOL1, NB4, Mv411) were analyzed by Western blotting (Figure 4.1A). PRMT5

protein levels were increased in both nuclear and cytosolic fractions of transformed

lymphoid cells compared to normal B cells. Since PRMT5 associates with BRG1 and

BRM chromatin remodeling complexes, and mutations of hSWI/SNF components

have been implicated in various human cancers including leukemia and lymphoma,

the levels of BRG1, BRM, and BAF57 in normal and transformed lymphoid cells was also analyzed. hSWI/SNF subunits were expressed in all samples except for

the Burkitt’s lymphoma P3HRI cell line where BRM expression was undetectable.

Because PRMT5 was found in both nuclear and cytosolic fractions of transformed

lymphoid cells, while its localization was restricted to the cytosol of normal B lym-

phocytes, expression of a cytosolic protein, α-TUBULIN, was measured in the same

extracts. Western blot analysis revealed that α-TUBULIN is expressed exclusively

182 in the cytosol, indicating that the presence of PRMT5 in the nucleus of transformed

cancer cell lines is not due to cytosolic contamination.

To further confirm the Western blot data, PRMT5 protein was detected in nor-

mal B, and Mino and JeKo MCL cells by immunoflourescence staining using either

pre-immune (PI) or immune anti-PRMT5 antibody (Figure 4.1C). Only when anti-

PRMT5 antibody was used, a signal was detected that was significantly higher in both

Mino and JeKo MCL cells, confirming that the level of PRMT5 protein is elevated

in transformed cells. In chapter 3, it was demonstrated that PRMT5 methylates his-

tones H3R8 and H4R3 in vitro, and PRMT5 recruitment to ST7 and NM23-H1 pro-

moters has been linked to symmetric methylation of H3R8 at these promoters [215].

Therefore, global H3R8 and H4R3 methylation in normal B and MCL cells was ana-

lyzed using antibodies that can specifically recognize either symmetrically methylated

H3R8 or H4R3 (Figure 4.1B). Both anti-H3(Me2)R8 and anti-H4(Me2)R3 antibod-

ies failed to detect these modifications in normal B lymphocytes. In stark contrast,

Mino and JeKo MCL cell lines exhibited high levels of symmetrically methylated

H3R8 and H4R3. Collectively, these studies suggest that increased levels of PRMT5

induce symmetric methylation of H3R8 and H4R3, and might be associated with

MCL pathology.

4.4.2 PRMT5 expression is not induced by cell proliferation

The results above show that PRMT5 expression is significantly high in lymphoid

cancer cell lines compared to non-transformed B cells that are isolated from either peripheral blood or tonsils. Unlike the transformed cells, normal B cells are resting

and non-proliferating. To determine if enhanced expression of PRMT5 is a result of

183 cell proliferation rather than transformation, B cells were activated to induce pro- liferation using a combination of IL-4 treatment and IgG receptor crosslinking with anti-IgG+M, and then PRMT5 expression was measured (Figure 4.2). Upon activa- tion, resting B cells enter the cell cycle and proliferate. To verify B cell proliferation, resting and activated B cells were grown in the presence of BrdU, which is incorpo- rated in DNA during S phase. It was found that B cells incorporated BrdU upon activation, and the percentage of BrdU positive cells increased from 28% to 46% as cells were maintained in the activation media (Figure 4.2A). Having confirmed that activated B cells were proliferating, PRMT5 expression in nuclear and cytosolic ex- tracts prepared from resting and activated B cells was measured by Western blotting

(Figure 4.2B). It was found that proliferation of normal B cells do not result in in- creased PRMT5 expression. Similarly, when B cells were activated using a different protocol that includes IL-4 treatment in combination with receptor cross-linking and activation by anti-CD40 and anti-IgG, there was no increase in PRMT5 expression

(Figure 4.2C). Taken together, these results indicate that enhanced expression of

PRMT5 is associated with cell transformation rather than cell proliferation.

4.4.3 PRMT5 mRNA level and stability are reduced in Mino and JeKo MCL cells

Having found that expression of PRMT5 protein is enhanced in various trans- formed lymphoid cell lines, PRMT5 expression was measured to determine if in- creased transcript levels resulted in the observed high levels of PRMT5 protein (Fig- ure 4.3A). Real time RT-PCR revealed that despite elevated levels of PRMT5 protein in cancer cells, the steady state levels of PRMT5 mRNA were 2 to 5-fold lower in

184 transformed lymphoid cells (p< 10−4), suggesting that there are other mechanisms in- volved in up-regulation of PRMT5 protein expression. To understand the underlying

cause for decreased PRMT5 gene expression in transformed lymphoid cells, we ex- amined the rate of PRMT5 transcription in normal B cells as well as patient-derived

Mino and JeKo MCL cell lines (Figure 4.3B). Consistent with the real time RT-PCR

results, nuclear run on analysis indicated that the rate of PRMT5 mRNA transcrip-

tion was 2.9 and 2.4-fold lower in Mino (p=0.028) and JeKo (p=0.011) MCL cell lines, respectively. These results show that PRMT5 mRNA transcription is more efficient in normal B lymphocytes and suggest that PRMT5 is transcriptionally repressed in

MCL cell lines.

To understand the mechanisms involved in PRMT5 repression, the role of DNA

methylation and histone deacetylation was evaluated. Normal B lymphocytes and

MCL cells, Mino and JeKo, were treated with the DNA methylase inhibitor, 5-

AzaCytidine (5AzaC), and the histone deacetylase inhibitor, depsipeptide (Depsi).

Following drug treatment, the levels of PRMT5 mRNA were measured by real time

RT-PCR (Figure 4.3C). It was found that in the presence of 5AzaC PRMT5 ex- pression was derepressed by 2.8-fold in normal B (p= 0.001), 1.8-fold in Mino (p=

0.0067), and 1.5-fold in JeKo (p= 0.0261) cells. Similarly, when the cells were treated with depsipeptide PRMT5 mRNA levels were increased by 4.7-fold in normal B (p=

0.0001), 4.5-fold in Mino (p= 0.0016), and 2.83-fold in JeKo (p= 0.0183) cells. These results suggest that both DNA methylation and histone deacetylation impact PRMT5 gene transcription in both normal and transformed MCL cells.

To gain further insight in the regulation of PRMT5 expression, the region of

PRMT5 promoter that is important for its transcription was mapped. A search was

185 performed to identify the binding sites for the various transcription factors in the 1

Kb region of PRMT5 promoter sequence, which showed that there were three AML1 and nine GATA binding sites ( http://www.cbrc.jp/research/db/TFSEARCH.html)

(Figure 4.4A). To identify the region that is important for transcriptional activ-

ity, four plasmid constructs that contained either the 1.2 Kb region (-1000 to +

190, pGL2-PRMT5 ), or one of the three shorter regions (−274 to + 190, pGL2-

PRMT5 ∆(−274)), (-203 to + 190, pGL2-PRMT5 ∆(−203)), and (-100 to + 190, pGL2-PRMT5 ∆(−100)) upstream of a promoterless luciferase gene were generated

(Figure 4.4A). It was desired to transfect normal B and MCL cells, Mino and JeKo, with the above promoter constructs, but due to the low efficiency of transfection these

experiments failed in normal B and Mino cells. However, JeKo cells were successfully

transfected, and when the activity of full length and truncated PRMT5 promoter se-

quences were compared, it was found that luciferase expression was greatly diminished

by all three promoter truncations (Figure 4.4B). The above study indicated that the

region between −1000 to −274 is essential for promoter activity (Figure 4.4B and

C). To verify these finding, the above plasmids were also transfected in HeLa cells

and similar results were observed (Figure 4.4C). Having found that all three AML1

sites were present in the −1000 to −274 region, these sites were mutated individually

and in combination in the full length promoter construct to evaluate the significance

of AML1 binding site in PRMT5 transcription (Figure 4.4D). Mutations of all three

AML1 sites abolished luciferase expression suggesting that AML1 binding sites are

important for PRMT5 promoter activity. Furthermore, to evaluate the contribution

of each AML1 site, individual AML1 site mutants were transfected, which show that

luciferase expression is lost upon mutation of sites A and C, whereas mutation of

186 AML1 B site had no effect. Similar results were obtained when the mutant promoter

constructs were transfected in HeLa cells (Figure 4.4E). These results suggest that

AML1 is a key regulator of PRMT5 promoter activity.

Though some knowledge has been gained about the transcriptional regulation of

PRMT5 , it is unclear how PRMT5 protein expression is enhanced in Mino and JeKo

cells. To address this question, I assessed if differences in PRMT5 mRNA stability

between normal and transformed B cells can account for PRMT5 translation in these cells (Figure 4.5). Normal B lymphocytes as well as Mino and JeKo MCL cells

were treated with DRB to inhibit mRNA synthesis, and PRMT5 mRNA levels were measured at various time intervals by real time RT-PCR. 18S rRNA was used as an internal control to normalize PRMT5 mRNA levels in these experiments, because low dose and short time treatment with DRB does not affect RNA polymerase I transcription. It was found that in comparison to normal B cells (t1/2=226 min), the half-life of PRMT5 mRNA was reduced 2.7-fold in Mino (t1/2=84 min) and 2.3-fold in

JeKo (t1/2=96 min) MCL cell lines (p=0.009). Taken together, these findings along with the observed decrease in PRMT5 mRNA transcription suggest that increased expression of PRMT5 protein in transformed MCL cell lines is due to changes in

PRMT5 mRNA translation.

4.4.4 Aberrant expression of miR-96 is associated with en- hanced PRMT5 translation

To compare the translation of PRMT5 mRNA in normal and transformed B cells, cell lysates from normal B lymphocytes and MCL cell lines, Mino and JeKo, were separated by sucrose gradient sedimentation in the presence of cycloheximide (Figure

4.6). Gradient fractions were collected, and total RNA was extracted to measure the

187 levels of PRMT5 mRNA by real time RT-PCR (Figure 4.6). The amount of β-ACTIN mRNA in each fraction was used as an internal control to normalize the amount of

PRMT5 mRNA present in each fraction. Sucrose gradient analysis demonstrated that

although PRMT5 mRNA was detected in polyribosomal fractions 10 to18 in normal

and transformed B cells, only 27% of total PRMT5 mRNA was polyribosomal in normal B lymphocytes. However, the fraction of PRMT5 mRNA associated with polyribosomes was enriched 2 and 2.7-fold in Mino (p=0.0003) and JeKo (p=0.002) cells, respectively. This change in the polyribosome profile of PRMT5 mRNA and the Western blot results suggest that PRMT5 translation is enhanced in transformed

Mino and JeKo MCL cells.

Recently, it has been shown that miRs can inhibit mRNA translation by binding

through their seed sequence to partially complementary sites in the 3’UTR of target mRNAs. Hence, the presence of potential miR binding site in the 3’UTR of PRMT5

was determined by searching the Wellcome Trust Sanger Institute miRNA registry.

At that time, 42 potential miRs that can bind to the 3’UTR of PRMT5 were found,

but only 9 conformed to the seed sequence binding requirements [19] (Figure 4.7A).

Therefore, the expression of all 9 miRs in normal and transformed B cells was mea-

sured using RNase protection assays (Figure 4.7B). Of all the tested PRMT5 -specific

miRs including miR-607, only miR-96 was expressed in normal and transformed B

lymphocytes; however, its expression was 2 to 2.5-fold lower in Mino (p=0.012) and

JeKo (p=0.005) MCL cell lines (Figure 4.7B). When the expression of an unrelated

control miR, miR-197, was analyzed it was found that its expression was unaltered in normal and transformed B cells (Figure 4.7B).

188 4.4.5 miR-96 can inhibit PRMT5 translation in vivo, and its binding site is critical for translational regulation

To verify if miR-96 can effect PRMT5 translational regulation in vivo, double stranded (ds) miR-96 RNA was introduced in the patient-derived JeKo MCL cell line. When JeKo cells were electroporated with either wild type or mutant miR-96 and the level of PRMT5 protein was measured, it was found that PRMT5 expression decreased by 37%, whereas the levels of β-ACTIN were unaffected (Figure 4.8A).

Similarly, when the same experiment was performed in Burkitt’s lymphoma cell line,

Raji, PRMT5 protein level was reduceded by 50%, highlighting the importance of miR-96 expression in translational regulation of PRMT5. To determine if reduced expression of PRMT5 is the result of translational inhibition, but not PRMT5 mRNA degradation, the steady state level of PRMT5 mRNA in mock transfected as well as cells transfected with wild type and mutant miR-96 were measured (Figure 4.8B).

As expected, PRMT5 mRNA levels were unaffected in the presence of exogenously transfected miR-96 in JeKo and Raji cells.

Next, the ability of miR-96 to inhibit PRMT5 translation was studied in vitro.

Recent work has shown that translational gene silencing by miRs in vitro requires the presence of a 7-methyl G cap and a poly(A) tail [301]. Therefore, in vitro translation experiments were conducted using 7-methyl G capped and poly(A)-tailed PRMT5 mRNA with and without wild type 3’UTR, or with mutant 3’UTR where the miR-96 seed sequence binding site was mutated. Both wild type and mutant miR-96 failed to inhibit PRMT5 translation in vitro, suggesting that there are other components miss- ing in the rabbit reticulocyte lysate, which might stabilize miR-96 binding to its target

189 site. Because miR-96 anneals to the PRMT5 3’UTR primarily through its seed se- quence (+2 to +8), it was assumed that the PRMT5 mRNA:miR-96 hybrid molecule would not be stable thermodynamically at 30oC (∆G =-13.76 kcal/mol, Tm=22oC).

To make up for the lack of stability and to strengthen the PRMT5 mRNA:miR-96 interaction, six consecutive ribonucletotides (+9 to +14) were changed in miR-96

(∆G =-27.5 kcal/mol, Tm=44oC), and its ability to inhibit PRMT5 translation in vitro was tested (Figure 4.9A). Modified wild type miR-96 inhibited PRMT5 transla- tion efficiently at a 1:1 molar ratio only when PRMT5 mRNA with wild type 3’UTR was used. Translation of PRMT5 mRNA without 3’UTR or with mutated 3’UTR, and the unrelated BAF45 mRNA was unaffected. Similarly, when modified mutant miR-96, where the seed sequence (+2 to +8) has been mutated, was used in these in vitro translation experiments, there was no noticeable change in translation of

PRMT5 mRNA or control BAF45 mRNA.

Since exogenous expression of miR-96 can reduces PRMT5 expression in vivo, the importance of miR-96 binding site for translational inhibition of PRMT5 was evaluated (Figure 4.9B). Wild type and mutant PRMT5 3’UTRs were subcloned downstream of a CMV-driven luciferase reporter, and electroporated into either nor-

mal B lymphocytes, or transformed JeKo and Raji lymphoma cells. In the presence

of wild type PRMT5 3’UTR, luciferase expression was unchanged in B cells, whereas in JeKo and Raji cells, it was enhanced 2.4 (p=0.004) and 1.8-fold (p=0.026), respec- tively, indicating that reduced expression of miR-96 in transformed lymphoma cell lines permits enhanced luciferase expression. When the miR-96 seed sequence bind- ing site was mutated, luciferase expression was increased 1.3-fold in B cells (p=0.035)

190 and 1.2-fold in JeKo (p=0.006) and Raji cells (p=0.037). These results show that miR-96 contributes to proper regulation of PRMT5 translation.

4.4.6 ST7 is repressed in lymphoid cancer cell lines that over- express PRMT5

In chapter 3, ST7 was identified as a direct target genes of PRMT5, where it

was demonstrated that recruitment of PRMT5 to the ST7 promoter induce sym-

metric methylation of histone H3R8 and is associated with their transcriptional re-

pression [215]. Since PRMT5 protein levels are abnormally high in the analyzed

transformed lymphoid cell lines, the impact of elevated PRMT5 levels on ST7 ex-

pression was determined. Real time RT-PCR analysis showed that ST7 mRNA level

was reduced 2 to 12-fold (p< 10−4) in cancer cell lines (Figure 4.10). Remarkably,

when ST7 protein levels were measured by Western blotting, all transformed lym-

phoid cell lines lacked ST7 expression, suggesting that there is an inverse relationship

between PRMT5 and ST7 protein levels (Figure 4.11A).

To further confirm the expression of ST7, ST7 protein was detected by immunoflu-

orescence in normal B cells as well as Mino and JeKo MCL cell lines. In accord with

the Western blot data, ST7 was detected in normal B cells but not in transformed

MCL cell lines (Figure 4.11B). It is worth noting that expression of ST7 was not de-

tected when either nuclear or cytosolic extracts were used; however, ST7 was readily

detectable when whole cell lysates were prepared using RIPA buffer. These results

and the immunofluorescence staining pattern in normal B lymphocytes suggest that

ST7 protein is insoluble and probably membrane-bound.

Expression of ST7 mRNA was inhibited in all transformed lymphoid cell lines

examined, suggesting that ST7 promoter activity is altered in these cancer cell lines.

191 To verify if PRMT5 is directly involved in inducing ST7 repression, ChIP experi- ments using anti-PRMT5 specific antibodies were conducted (Figure 4.12A). When cross-linked chromatin was immunoprecipitated from either normal or transformed B lymphocytes, PRMT5 recruitment to the ST7 promoter was enriched 3 and 5.5-fold

in transformed Mino (p=10−3) and JeKo (p=0.006) MCL cell lines, respectively.

Consistent with the findings in NIH3T3 cells (chapter 3), BRG1 recruitment to the

ST7 promoter was enhanced 1.6 and 3-fold in Mino (p=10−2) and JeKo (p=0.0007),

respectively, while BRM recruitment was unaffected. These results show that the

BRG1-associated PRMT5 is involved in ST7 transcriptional repression in transformed

B cells. Since PRMT5 can symmetrically methylate H3R8 and H4R3, the methyla-

tion status of these residues at the ST7 promoter was evaluated (Figure 4.12B). ChIP

analysis showed that methylation of H3R8 is augmented 1.7 (p=0.013) and 2.7-fold

(p<10−4) in Mino and JeKo cells, respectively. Similarly, methylation of H4R3 is

enriched 3.7 (p=0.002) and 2.5-fold (p<10−4) in these MCL cell lines. We have also

analyzed BRG1, BRM, and PRMT5 recruitment as well as H3R8 and H4R3 methyla-

tion at the ST7 promoter in two other Burkitt’s lymphoma cell lines, Daudi and Raji,

and found similar results (Figure 4.12C and D). These findings show that increased

expression of PRMT5 is directly involved in ST7 gene repression, and that PRMT5

recruitment as well as H3R8 and H4R3 methylation are altered in transformed lym-

phoid cell lines.

4.4.7 PRMT5 is overexpressed and its target gene, ST7 is repressed in MCL clinical samples

Results presented above show that PRMT5 protein levels are aberrantly increased

in a variety of transformed lymphoid cells including Mino and JeKo MCL cell lines,

192 and as a consequence ST7 expression is inhibited. To confirm these findings and to assess whether the inverse relationship between PRMT5 and ST7 exists in tumors isolated from MCL patients, the above experiments were repeated in MCL clinical samples. When PRMT5 mRNA levels were measured in several MCL clinical sam- ples, it was found that similar to MCL cell lines; clinical samples exhibit reduced levels

of PRMT5 mRNA (Figure 4.13). Furthermore, when PRMT5 protein expression was

measured by Western blotting and immunoflourescence in MCL clinical samples 6 and

7, which were not limiting, it was found that PRMT5 levels were elevated in both

nuclear and cytosolic fractions (Figure 4.14A). Similarly, immunofluorescence analy-

sis revealed that PRMT5 overexpression correlates with enhanced global symmetric

methylation of H3R8 and H4R3 in these MCL patient samples (Figure 4.14B). Since,

miR-96 expression is reduced in JeKo cells and it has been shown to inhibit PRMT5

translation in JeKo and Raji cells, miR-96 expression was measured in MCL clinical

samples. RNase protection assay results show that miR-96 expression is decreased in

MCL clinical samples 6 and 7 indicating that reduced expression of miR-96 is one of

the aberrations associated with PRMT5 translation in MCL (Figure 4.15).

Since PRMT5 can directly inhibit ST7 transcription in MCL cell lines, ST7

mRNA and protein expression was analyzed in multiple MCL patient samples. Real

time RT-PCR show that ST7 mRNA levels are reduced 2 to 30-fold (p<10−4), and

Western blot analysis indicated that ST7 protein expression was severely inhibited in

all MCL clinical samples examined (Figures 4.16A and B). In addition, immunoflu-

orescence staining of ST7 protein in MCL samples from patients 6 and 7 confirmed

the Western blot results (Figure 4.16C). Next, the recruitment of BRG1, BRM, and

PRMT5 to the ST7 promoter in MCL patient samples 6 and 7 was measured by ChIP

193 experiments (Figure 4.17A). BRG1 recruitment was enriched 3.6 (p=0.006) and 2- fold (p=0.002), while PRMT5 recruitment was enhanced 14 (p=0.0002) and 8-fold

(p=0.003) in MCL clinical samples 6 and 7. As a result of increased BRG1-associated

PRMT5 recruitment to the ST7 promoter, symmetric methylation of H3R8 was aug- mented 7 (p=0.016) and 3-fold (p=0.003) in clinical samples 6 and 7, while analysis

of H4R3 methylation in MCL patient 6 and 7 show that methylation was elevated 4.6

(p=0.001) to 5-fold (p=0.008) (Figure 4.17B). Collectively, these results demonstrate that altered expression of PRMT5 directly affects H3R8 and H4R3 global methyla- tion and triggers changes in target gene expression which may in turn contribute to

MCL.

4.4.8 Knocking down PRMT5 alters the growth characteris- tics of transformed lymphoid cell lines

It was observed in chapter 3 that overexpression of PRMT5 in NIH3T3 cells

induces hyperproliferation, while knockdown of PRMT5 reduces cell growth and

proliferation. Since PRMT5 levels are high in patient-derived MCL cell lines and

MCL clinical samples, the affect of reducing PRMT5 protein levels on MCL cell

growth was investigated. To lower PRMT5 expression, JeKo cells were infected with

either recombinant control or antisense (AS)-PRMT5 lentivirus, and PRMT5 protein

expression was evaluated at different time points after infection. PRMT5 protein lev-

els were reduced by day 4, while β-ACTIN expression was unaltered (Figure 4.18A).

When the proliferation of control and AS-PRMT5 expressing JeKo cells was mea-

sured, there was a noticeable 1.8-fold decrease in growth by day 6 (p<10−4) (Figure

4.18B). To evaluate if this decrease in cell growth and proliferation was a direct result

194 of cell death and/or slow cell cycle progression, the DNA content and BrdU incorpo- ration of control and AS-PRMT5 infected cells was determined. It was found that

AS-PRMT5 cells incorporated BrdU 25% less efficiently than parental cells infected with control vector, and did not display apoptotic cell death (Figure 4.19A and B).

These results suggest that reducing the expression of PRMT5 interferes with growth

of transformed B cells.

Since PRMT5 can directly regulate ST7 transcription, ST7 mRNA levels were

measured in control and AS-PRMT5 expressing JeKo cells (Figure 4.18C). Real time

RT-PCR results show that as PRMT5 protein levels decrease by day 6, transcription

of ST7 is induced 1.7-fold (p=10−3). Despite this transcriptional derepression, ST7

protein levels did not increase, suggesting that there might be other mechanisms in-

volved in regulating ST7 protein expression. Similar effects on cell proliferation and

ST7 mRNA expression were observed when PRMT5 was knocked down in Burkitt’s

lymphoma cell line, Raji (Figure 4.20). These findings provide more evidence that

knocking down PRMT5 expression in cancerous B cell impacts their growth charac-

teristics, and that ST7 expression is regulated posttranscriptionally.

4.5 Discussion

The findings that epigenetic modifications of chromatin are associated with can-

cer makes it important that how post-translational changes of histones impact gene

expression, and how abberations of this process contributes to disease are investi-

gated and understood to provide better therapies for cancer. In this chapter, it has

been found that PRMT5 protein expression is induced in a wide variety of human

lymphoid cancer cells including patient-derived cell lines and MCL clinical samples.

195 Overexpression of PRMT5 has been linked to aberrant expression of miR-96, and

as a consequence of these changes global symmetric methylation of H3R8 and H4R3

is increased. More specifically, expression of the PRMT5 target gene, ST7, is in- hibited in lymphoid cancer cell lines with altered PRMT5 and miR-96 expression.

Furthermore, knock down of PRMT5 expression reduces proliferation of transformed

lymphoid cell lines, suggesting that reduced levels of PRMT5 are critical for normal

cell growth.

4.5.1 Reduced miR-96 expression is associated with enhanced PRMT5 translation in MCL

Analysis of B cell nuclear and cytosolic extracts showed that PRMT5 is barely

detectable in the nucleus, whereas transformed lymphoid cell lines and MCL clinical

samples contained significantly higher levels of PRMT5 protein in both compartments

(Figures 4.1 and 4.14). In addition, PRMT5 expression is not influenced by the pro-

liferation status of the cell, indicating that enhanced PRMT5 level is associated with

cell transformation (Figure 4.2). This is further supported by the observation that

global expression of PRMT5 protein is increased in both nucleus and cytosol of various

solid tumor cell lines including glioma (U251, Gli3605), adenocarcinoma (HeLa S3,

SW13), breast and lung carcinoma (BT549, A549), and hepatoma (HepG2) in com-

parison to immortalized and non-transformed NIH3T3, Rat1a, and PC12 cell lines

(Figure 4.21). Nuclear PRMT5 is associated with chromatin remodelers including

BRG1/BRM-based hSWI/SNF and NURD complexes, and is involved in transcrip- tional repression of cell cycle regulators and tumor suppressor genes [107, 214, 215].

Therefore, when PRMT5 levels are elevated either by overexpression or as it is the

196 case in transformed cancer cells, it might repress transcription of key target genes and promote tumorigenesis.

PRMT5 expression is regulated at both transcriptional and translation stages and

that increased expression of PRMT5 in transformed Mino and JeKo MCL cell lines

is a direct result of enhanced PRMT5 mRNA translation (Figures 4.3, 4.5, and 4.6).

What is more striking is the finding that expression of one of the miRs predicted to bind to the PRMT5 3’UTR was reduced in patient-derived Mino and JeKo MCL cells as well as MCL clinical samples, and re-expression of miR-96 in two distinct lymphoma cell lines reduced PRMT5 translation in vivo (Figures 4.7, 4.8, and 4.15).

Furthermore, mutation of the miR-96 seed sequence abolished PRMT5 translational inhibition in vivo, indicating that the observed repressive effect of miR-96 is specific.

Further support for the involvement of miR-96 in regulating PRMT5 translation comes from the observation that the PRMT5 3’UTR can enhance luciferase transla- tion in transformed JeKo cells with low miR-96 expression (Figure 4.9B). However, attempts to recapitulate translational inhibition of PRMT5 in vitro were not success- ful when wild type miR-96 was used. The fact that transfection of wild type miR-96 in JeKo or Raji cells resulted in decreased expression of PRMT5, and the findings that mutation of the miR-96 binding site slightly improved luciferase translation ar- gue that there might be other cellular factors and miRs involved in stabilizing the

PRMT5 mRNA:miR-96 interaction as well as inhibiting PRMT5 translation (Figures

4.8A, 4.9B, and 4.15). Recently, more PRMT5-specific miRs have been discovered, and it is unknown if their levels are altered in MCL cells. More work is required to determine if there are additional aberrantly expressed PRMT5-specific miRs, and

197 whether restoring their expression either individually or in combination would re- sult in efficient PRMT5 translational inhibition. The lack of stability of PRMT5 mRNA:miR-96 hybrid molecules was compensated by increasing the complementar- ity beyond the seed sequence, and this modified miR-96 could specifically inhibit

PRMT5 translation in vitro (Figure 4.9A). Thus normal expression of miR-96 is essential for proper PRMT5 translation.

4.5.2 PRMT5 overexpression enhances global H3R8 and H4R3 methylation, and is accompanied by ST7 suppression

It was shown in chapter 2 and 3 that association of PRMT5 with hSWI/SNF complexes enhances its histone methyltransferase activity, and endows it with the ability to influence target gene expression so that the net outcome is to promote cell growth and transformation [215]. Since PRMT5 preferentially targets histones

H3R8 and H4R3, their methylation status was analyzed in transformed MCL cells, which show that these sites are highly methylated in MCL cell lines as well as clinical samples (Figures 4.1C and 4.14B). More specifically, when histone methylation was analyzed at the ST7 promoter in both MCL cell lines and MCL clinical samples, it was found that H3R8 and H4R3 were hypermethylated (Figures 4.12 and 4.17). While recruitment of PRMT5 to the ST7 promoter enhances H3R8 and H4R3 methylation, there is no correlation between the levels of histone H3R8/H4R3 methylation and

ST7 transcriptional suppression (Figures 4.10, 4.12, 4.16, and 4.17). To exclude the possibility that ST7 repression in MCL cells is not due to DNA methylation, ST7 mRNA level was measured in MCL cells in the presence of DNA methylase inhibitor,

5Azacytidine (5AzaC) (Figure 4.22). RT-PCR results indicated that inhibition of

DNA methylation does not restore normal ST7 expression in MCL cells.

198 As discussed in chapter 1, it is well established that different posttranslational

modifications of histones can act either synergistically or antagonistically to specify

transcriptional outcome [76]. For instance, histone H3K9 and H3K27 trimethylation

marks colocalize at the repressed UBX promoter [236]. Therefore, it is possible that in the case of ST7 transcription there are additional epigenetic marks involved in its

regulation, which may vary between cell lines and clinical samples. Recent studies

have clearly shown that methylated histones can be recognized and bound by specific

methyl binding protein, and that this is important for transcriptional regulation.

Good examples of how epigenetic marks are critical for determining transcriptional

outcome are provided by the WDR5 protein, which can bind to dimethylated H3K4

and support transcriptional activation of HOXC8, and by HP1, which can recognize

tri-methylated H3K9 and induce silencing [115, 313]. Thus, it is going to be important

to determine if additional silencing epigenetic marks are present at the ST7 promoter,

and if there are specific factors that can bind symmetrically methylated H3R8 and

H4R3.

The results in this chapter suggest that expression of ST7 is regulated both at

the transcriptional and translational levels. Knocking down PRMT5 expression in

transformed JeKo and Raji cells induces ST7 transcription, but does not affect ST7

protein expression (Figures 4.18C and 4.20). Similarly, transformed lymphoid cells

that express low levels of ST7 mRNA do not exhibit any detectable levels of ST7

protein (Figures 4.11 and 4.16). Like, decreased expression of miR-96 leads to en-

hanced PRMT5 translation it is possible that aberrant expression of ST7-specific

miR(s) might be inhibiting ST7 translation. More experiments are required to ex-

amine expression of ST7-specific miRs in normal and transformed B cells.

199 4.5.3 Role of PRMT5 in mantle cell lymphomagenesis

There is increasing evidence that associates misexpression and/or mutation of

histone modifying enzymes with cancer etiology. For instance, the mixed lineage

leukemia gene, MLL1 which methylates H3K4 and activates transcription, is fre-

quently translocated in acute leukemias and as a consequence more than 50 different

leukemogenic MLL fusion proteins are generated [35]. In contrast, overexpression

of EZH2, which has been tightly linked to gene silencing, has been documented in

prostate, breast, and gastric cancers and appears to correlate with the high degree of

invasiveness of tumors [148, 180, 290]. However, the role of histone arginine methy-

lation in oncogenesis is not well understood. Evidence in support of the role played

by PRMT5 in cancer came from studies, which showed that PRMT5 is frequently upregulated in gastric carcinoma [108]. Furthermore, when the MDA-MB-231 breast cancer cell line is transfected with ER-α and treated with 17β-estradiol, there is a clear decrease in cell proliferation, which is accompanied by reduced PRMT5 ex- pression [189]. The results presented in this chapter show that PRMT5 undergoes translational upregulation in cancer cells of lymphoid origin including MCL clinical samples. As a result PRMT5 protein expression is elevated and symmetric methy- lation of H3R8 and H4R3 is augmented. The impact of enhanced histone arginine methylation is misregulated gene expression, and in the case of the PRMT5 target gene, ST7 , there is a clear inhibition of transcription, which appears to be associated with cancer cell growth. We also show that knocking down PRMT5 expression in

MCL and Burkitt’s lymphoma cell lines reduces cell proliferation (Figures 4.18 and

200 4.20). Thus, it appears that PRMT5 is a key histone-modifying enzyme that con- trols cell growth by modulating expression of target genes through histone arginine methylation.

Elevated expression of PRMT5 can impact other pathways besides methylation of histones H3R8 and H4R3. Recently, PRMT5 was shown to interact with and methylate MBD2 that is associated with the NURD complex [107]. The consequence of this methylation is reduced MBD2 binding to methylated DNA and inability to repress transcription [278]. Thus, it is possible that in addition to histone modi-

fication, PRMT5 promotes tumorigenesis by negatively modulating the activity of

MBD2-based NURD complex. Therefore, it is going to be interesting to examine the methylation status of MBD2 in cancer cells that overexpress PRMT5, and to verify if expression of NURD target genes is altered in these cancer cells. Furthermore our

findings, which show that reducing expression of PRMT5 can inhibit cell prolifera- tion, provide a good starting point to devise new strategies aimed at addressing the therapeutic relevance of targeting PRMT5.

201 Figure 4.1: PRMT5 is overexpressed in lymphoma and leukemia cell lines. (A) Expression of PRMT5 and hSWI/SNF subunits were assessed by Western blot- ting using either nuclear (N) and cytosolic (C) extracts from normal CD19+ B cells (40 µg), or the indicated transformed cell lines (20 µg). To discern between N and C fractions, we measured α-TUBULIN expression.

(B) Anti-H3(Me2)R8 and anti-H4(Me2)R3 antibodies do not cross-react and are highly specific. 1 and 2 µg of BSA or the indicated peptides were spotted on ni- trocellulose membrane, and detected using anti-H4(Me2)R3 or anti-H3(Me2)R8.

(C) Immunofluorescence of PRMT5, H4(Me2)R3, and H3(Me2)R8 in normal B cells, Mino and JeKo cells. Normal B and MCL cells were fixed and incubated with either pre-immune, or immune anti-PRMT5, anti-H3(Me2)R8, and H4(Me2)R3 antibodies. FITC-labeled goat anti-rabbit antibody was used to detect PRMT5 and modified histones, and DAPI was used to stain nuclei. Pictures were taken at 100X magnification.

202 Figure 4.1

203 Figure 4.2: B cell proliferation does not induce PRMT5 expression. (A) Resting B cells were activated by the addition of IL4 and anti-IgG and M for either 0, 4 or 6 days, and newly synthesized DNA was labeled by BrdU for 72 h and detected using anti-FITC labeled BrdU antibody.

(B) Nuclear (N) and cytosolic (C) extracts (20 µg) were prepared from 0, 4 and 6 day activated B cells and PRMT5 and β-ACTIN expression was measured by Western blotting.

(C) Western blot analysis was performed using anti-PRMT5 and anti-β-ACTIN antibodies on nuclear (N) and cytosolic (C) extracts prepared from B cells that had been activated for 72 h with a combination of IL4, anti-CD40 and anti-IgG. For comparison of PRMT5 expression, nuclear and cytosolic extracts from Mino and JeKo cells is included.

204 Figure 4.3: Expression of PRMT5 is regulated at transcriptional level. (A) PRMT5 mRNA level was measured by real time RT-PCR in normal as well as transformed B cells. The bar graph shows normalized fold expression of PRMT5 mRNA in various cell lines relative to normal B cells using GAPDH as internal control. RT-PCR analysis was performed three times in triplicates and the graph shows the average standard deviation (SD).

(B) The rate of PRMT5 transcription is altered in MCL cell lines. Nuclear run on assays were conducted as described in materials and methods. Control (Ctrl) PvuII-PvuII DNA fragment of pBluescript KS(+), and β-ACTIN and PRMT5 cDNA PCR fragments were immobilized on Hybond XL membrane and detected with radiolabeled RNA isolated from the indicated cells. Signals were quantitated and expression of PRMT5 was reported relative to β-ACTIN.

(C) MCL cells were treated with 5Azacytidine (5AzaC) and depsipeptide (Depsi) for 24 h before total RNA was isolated and PRMT5 expression measured by real time RT-PCR analysis using 18S as the internal control.

205 Figure 4.3

206 Figure 4.4: AML1 sites in the promoter region of PRMT5 are critical for its activity.

(A) Schematic representation of PRMT5 promoter sequence. Some of the restriction sites and transcription factors, AML1 (A, B and C) and GATA (a-i) binding sites are depicted with respect to the start of transcription (+1). The schematics of the truncated promoter are shown below the full-length promoter construct.

(B and C) Full length and truncated PRMT5 promoter constructs were transfected in JeKo (B) and HeLaS3 (C) cells in duplicates and the luciferase activity was measured.

(D and E) Full length PRMT5 promoter construct containing either individually mutated AML1 A, B and C binding sites or all three mutated AML1 binding sites were transfected in JeKo (D) and HeLaS3 (E) cells and promoter activity was measured by the expression of luciferase gene.

207 Figure 4.4

208 Figure 4.5: PRMT5 mRNA is more stable in B cells. Normal and transformed B cells were treated with DRB for the indicated times, and total RNA was isolated and quantitated by real time RT-PCR. PRMT5 mRNA ex- pression was normalized using 18S as internal control. To calculate the half-life (t1/2) of PRMT5 mRNA, data points in each graph were generated from four distinct ex- periments conducted in triplicates and plotted in a log-linear scale using XMGRACE v5.2.14.

209 Figure 4.6: PRMT5 mRNA is translated more efficiently in MCL cell lines. (A) Polyribosome profiles of normal and transformed B cells. Whole cell lysate were fractionated by 15-40% sucrose gradient sedimentation, and polyribosome pro- files were determined by measuring the absorbance of each fraction at 254nm. Fractions representing 40S, 60S, 80S and polyribosomes are indicated.

(B) RNA isolated from each fraction was used to measure the level of PRMT5 mRNA by real time RT-PCR, and β-ACTIN was used as an internal control to normalize PRMT5 mRNA levels in each fraction. The amount of PRMT5 mRNA present in each fraction is reported relative to the fraction containing the lowest copy number of PRMT5 mRNA. The data points in each graph represent the average from triplicate RT-PCR reactions SD.

210 Figure 4.7: Differential expression of PRMT5-specific miRNAs in normal and trans- formed B cells.

(A) Schematic representation of PRMT5 mRNA depicting the position of potential miRNA binding sites within the 3’UTR.

(B) RPA was performed on 20 µg of total RNA isolated from the indicated cells using either miR-96, miR-197 or miR-607 probe. Probe represents 1/10th of the total amount of labeled probe used in each reaction, and control shows digestion of the probe in presence of yeast tRNA. Ethidium bromide stained gels are included to show equal loading.

211 Figure 4.8: PRMT5 expression is downregulated by miR-96 in transformed B cells. (A) JeKo and Raji cells were electroporated with 2.5 µg of either wild type or mutant miR-96 double stranded RNA, and 20 µg of RIPA extracts were analyzed by Western blotting.

(B) Transfection of miR-96 reduces PRMT5 protein expression and does not alter PRMT5 mRNA levels. JeKo and Raji cells (5 x 106) were electroporated with either wild type or mutant miR-96, and PRMT5 mRNA was measured by real time RT-PCR. Expression of PRMT5 mRNA was normalized using GAPDH as internal control.

212 Figure 4.9: Effect of modified miR-96 and PRMT5 3’UTR on translation in vitro and in vivo.

(A) Modified miR-96 inhibits PRMT5 translation. In vitro translation was carried out in the absence or presence of increasing amounts of modified wild type or mutant miR-96 using 0.25 µg of PRMT5 mRNA without 3’UTR (PRMT5), with wild type 3’UTR (PRMT5-WT 3’UTR), or with mutant miR-96 binding site 3’UTR (PRMT5-Mut 3’UTR). BAF45 mRNA was used as a control.

(B) Normal B (25 x 106) or transformed JeKo and Raji (5 x 106) cells were elec- troporated in the presence of pRL-TK with either pCMV-LUC, pCMV-LUC fused to wild type PRMT5 3’UTR, or pCMV-LUC fused to mutated miR-96 binding site PRMT5 3’UTR construct, and luciferase expression was measured using dual luciferase reporter assay. Luciferase activity is represented relative to pCMV-LUC for each cell line, and has been normalized using Renilla luciferase.

213 Figure 4.10: ST7 is repressed in transformed lymphoid cell lines. ST7 transcription is repressed in lymphoid cancer cell lines as determined by real time RT-PCR. This experiment has been repeated three times in triplicates and normalized using GAPDH as an internal control.

214 Figure 4.11: ST7 is silenced in lymphoid cell lines. (A) ST7 protein expression was analyzed by Western blotting using 20 µg of RIPA extracts from either normal B cells, or the indicated transformed lymphoid cell lines. β-ACTIN levels were detected to ensure equal loading.

(B) Fixed normal or transformed B cells were incubated with either pre-immune or immune anti-ST7 antibody. ST7 protein was visualized using goat FITC labeled anti-rabbit antibody, while nuclei were stained with DAPI. Pictures were taken at 100X magnification.

215 Figure 4.12: Recruitment of BRG1 associated PRMT5 correlates with enhanced H3R8 and H4R3 methylation at ST7 promoter. ChIP assays were performed on cross-linked chromatin from normal and transformed MCL cell lines, Mino and JeKo (A and B) or Burkitt’s lymphoma cell lines, Raji and Daudi (C and D) using either preimmune (PI) or the indicated immune antibodies, and the retained DNA was amplified by real time PCR using ST7 -specific primers and probe. Fold enrichment with each antibody was calculated relative to the PI sample. Each ChIP experiment was repeated twice in triplicates.

216 Figure 4.13: PRMT5 mRNA expression in MCL clinical samples. Steady state levels of PRMT5 mRNA were determinied by real time RT-PCR analysis using total RNA from the indicated cells. PRMT5 mRNA expression was normalized to GAPDH.

217 Figure 4.14: MCL patients overexpress PRMT5 protein. (A) Nuclear and cytosolic extracts from normal CD19+ B cells or MCL clinical samples 6 and 7 (20 µg) were analyzed by Western blotting using either anti- PRMT5 or control β-ACTIN antibody.

(B) Immunofluorescence of normal B and transformed MCL cells from clinical sam- ples 6 and 7 after staining with DAPI, PI, or antibodies to PRMT5, H3(Me2)R8, and H4(Me2)R3.

218 Figure 4.14

219 Figure 4.15: Expression of miR-96 is reduced in MCL clinical samples 6 and 7. To measure miRNA expression, RPA was performed on 5 µg of total RNA isolated from normal B cells and MCL clinical samples 6 and 7 using specific radiolabeled probe to detect miR-96, miR-197, and miR-607 expression. Probe represents 1/20th of the total amount of labeled probe used in each reaction, and control shows digestion of the probe in the presence of yeast tRNA. Ethidium bromide stained gels are included to show equal loading.

220 Figure 4.16: Overexpression of PRMT5 correlates with ST7 silencing in MCL clinical samples.

(A) Real time RT-PCR analysis of ST7 mRNA level in MCL patient samples 1-10. Expression of ST7 was normalized using GAPDH as an internal control.

(B) Western blot analysis was performed on 20 µg of RIPA extracts from normal B cells, Mino, JeKo, and MCL clinical samples 1-10 using the indicated antibodies.

(C) Immunofluorescence of normal B cells and MCL clinical samples 6 and 7 cells after staining with DAPI, PI, or immune anti-ST7 antibody. Pictures were taken at 100X magnification.

221 Figure 4.17: Recruitment of BRG1, PRMT5, and methylation of H3R8 and H4R3 at the ST7 promoter in MCL clinical samples. ChIP experiments were performed on cross-linked chromatin from normal B cells and MCL clinical samples 6 and 7 using either PI or the indicated immune antibodies. Immunoprecipitated DNA was amplified by real time PCR, and the fold enrichment of ST7 promoter sequences with each antibody was calculated relative to the PI sample.

222 Figure 4.18: Knocking down PRMT5 expression affects growth of transformed MCL JeKo cell.

(A) Western blot analysis was performed on 20 µg of RIPA extract from JeKo cells after infection with either vector or AS-PRMT5 lentivirus for 0, 2, and 4 days using anti- PRMT5 and control anti-β-ACTIN antibodies.

(B) Proliferation of Jeko cells infected with either control vector or AS-PRMT5 lentivirus. Cells were counted every two days for 6 days, and the experiment was repeated four times in duplicates.

(C) ST7 mRNA expression in lentivirus infected JeKo cells was evaluated by real time RT-PCR at day 0, 2, 4, and 6. ST7 mRNA expression is represented relative to control vector infected JeKo cells, and is normalized to GAPDH .

223 Figure 4.19: Comparison of cell cycle profile and BrdU incorporation between JeKo cells infected with either mock or AS-PRMT5 lentivirus. JeKo cells were infected with lentivirus containing either empty vector or AS-PRMT5 for 0, 2, and 4 days and their cell cycle profile was analyzed by 7-AAD staining (A) and BrdU incorporation was measured after 4 h of growth in the presence of 10 µM BrdU by anti-FITC labeled BrdU antibody (B). Ctrl represents the background binding of anti-FITC labeled BrdU antibody to 2 day infected cells to which BrdU was not added.

224 Figure 4.20: PRMT5 knockdown alters growth of Raji cells. (A) Western blot analysis was performed on 20 µg of RIPA extract from Raji cells infected with either control vector or AS-PRMT5 lentivirus at the indicated times using anti-PRMT5 and control anti-β-ACTIN antibodies.

(B) Proliferation of Raji cells was evaluated after infection with either control vector or AS-PRMT5 lentivirus. Viable cells were counted every two days for 6 days, and the proliferation assay was repeated twice in triplicates.

(C) ST7 mRNA expression was analyzed by real time RT-PCR in Raji cells infected with either control vector or AS-PRMT5 lentivirus after 0, 2, 4 and 6 days. ST7 mRNA level was normalized to GAPDH .

225 Figure 4.21: PRMT5 is overexpressed in adherent cancer cell lines compared to im- mortalized cells. Western blot analysis was performed on nuclear (N) and cytosolic (C) extracts from various cell lines using the indicated antibodies.

226 Figure 4.22: Effect of 5-Azacytidine on ST7 expression. Normal B lymphocytes and transformed MCL cell lines and MCL clinical samples 4 and 6 were treated with 8 µM of 5-Azacytidine (5AzaC), which is a DNA methylase inhibitor for 24 h before total RNA was isolated and ST7 expression was measured by real time RT-PCR analysis.

227 CHAPTER 5

SYNOPSIS AND FUTURE WORK

The work in this thesis has showed that protein arginine methyltransferase 5

(PRMT5) is associated with both BRG1 and BRM- based hSWI/SNF complexes, and

is involved in mediating transcriptional repression. PRMT5 directly interacts with

hSWI/SNF complex through interactions with BRG1, BRM, BAF57, and BAF45/INI1

subunits as well as the corepressor protein mSIN3A, which also associates directly

with hSWI/SNF complexes. It has been shown that the BRG1-based hSWI/SNF

complex is involved in both transcriptional activation and repression of the MYC

target gene, CAD; and that transcriptional repression is mediated through the re-

cruitment of mSIN3A/HDAC2 and PRMT5 corepressor proteins. Further work fo-

cused on characterizing the activity and understanding the in vivo role of PRMT5,

leading to the conclusion that PRMT5 methylates histones H3R8 and H4R3, and reg-

ulates cell growth and proliferation. It has been demonstrated that overexpression

of PRMT5 promotes hyperproliferation, while reducing PRMT5 expression decreases

cell proliferation. Using a NIH3T3 cell line, where expression of PRMT5 has been

knocked down, 227 genes were upregulated while 43 genes were downregulated. Fur-

thermore, overexpression of PRMT5 and global hypermethylation of H3R8 and H4R3

have been associated with mantle cell lymphoma (MCL). It is demonstrated that

228 PRMT5 is involved in directly repressing transcription of ST7 by methylating histone

H3R8 and H4R3. Protein expression studies indicated that all cancer cells that were

analyzed, overexpress PRMT5, which correlates with the lack of ST7 expression.

Moreover, reducing PRMT5 expression in lymphoma cells decreases proliferation of

transformed JeKo and Raji cells.

Furthermore, the regulation of PRMT5 expression in normal and transformed B cells was investigated, which show that PRMT5 expression is regulated both tran- scriptionally and translationally. PRMT5 overexpression in MCL cells is attributed to the loss of translational inhibition of PRMT5 mRNA in transformed cells, caused partially by low expression of miR-96. In addition, AML1 binding sites in the PRMT5 promoter have been found to be crucial for transcription, and both histone acetylation as well as DNA methylation is involved in the transcriptional regulation of PRMT5 .

Though, this work has provided some insight in the regulation of PRMT5 expression and function, there are several unanswered questions that are discussed below and require further work.

It has been demonstrated that PRMT5 methylates H3 and H4 in the N-terminal tails of histones and that its association with hSWI/SNF complexes significantly augments the methylase activity of PRMT5. To detect H3 and H4 methylation by recombinant flag tagged PRMT5, 7-9 fold more PRMT5 protein was used in histone methyltransferase (HMTase) assay as compared to hSWI/SNF associated PRMT5, and it was found that the activity of PRMT5 was still 2 to 2.3 and 8 to 10 fold lower for H3 and H4 peptides respectively (Figure 3.2, Chapter 3). These results indicated that hSWI/SNF association with PRMT5 stimulated its activity 14 to 18 and 56 to

72- fold for H3 and H4 peptides, respectively. However, currently it is unknown if

229 the intact hSWI/SNF complex or the subunits that directly interact with PRMT5, namely BRG1, BRM, BAF57, and BAF45/INI1, are sufficient to stimulate histone

methyltransferase activity of PRMT5. To address this issue, HMTase assays will

be conducted in the presence of individual hSWI/SNF subunits to determine their

stimulatory effect on PRMT5 activity. Furthermore, work by other groups indicates

that PRMT5 can also methylate histone H2A when PRMT5 is associated with either

MEP50 or BLIMP1, indicating that associated proteins can alter PRMT5 substrate

specificity [4, 90]. The substrate specificity of hSWI/SNF-associated PRMT5 in the

presence of MEP50 and BLIMP1 proteins will be tested.

Studies aimed at understanding the regulation of PRMT5 expression show that

PRMT5 is under transcriptional as well as translational control (Chapter 4). It

has been found that AML1 binding sites are important for the promoter activity

of PRMT5 and that PRMT5 mRNA expression is regulated by DNA methylation

and histone acetylation (Figure 4.3C and 4.4). However, the mechanisms that are

involved in the transcriptional downregulation of PRMT5 mRNA in transformed

cells are unknown, and further work is required to address this issue. In addition the

effect of AML1 on PRMT5 expression needs to be analyzed by comparing PRMT5

expression in normal and AML1 deficient cell lines, and examining the recruitment of

AML1 and its associated factors to the PRMT5 promoter. Also, ChIP assays will be

performed using antibodies that recognize the various transcriptional repressive marks

including H3R8, H3K9, and H3K27 methylation to gain insight in the mechanism that

control PRMT5 transcription.

Though miR-96 is involved in translational repression of PRMT5, the under-

standing of translational control mechanisms is still incomplete. The observation

230 that mutation of miR-96 binding site has no significant affect on the expression of

the luciferase in B cells, which show higher expression of miR-96 compared to MCL cells suggest that there are other miRs involved in the translational regulation of

PRMT5. More miRs have been identified that can regulate PRMT5 translation. It will be important to examine the expression of other potential PRMT5 binding miRs in normal and transformed cells and analyze their affects on PRMT5 translation.

It has been observed that PRMT5 is overexpressed in a variety of cancer cells and that increased expression is accompanied by enhanced nuclear localization of

PRMT5. As a consequence, global levels of H3R8 and H4R3 methylation are in- creased, which correlates with repression of the PRMT5 target gene, ST7. Another

aspect of PRMT5 overexpression that needs to be addressed is the importance of

nuclear translocation of PRMT5 in tumorigenesis, and to identify the mechanism

involved in this process. Preliminary experiments have indicated that PRMT5 is

phosphorylated by the MAP kinase ERK2 in vitro, and that mutation of proba-

ble serine phosphorylation site in PRMT5 causes a loss in the expression pattern of

GFP fused PRMT5 (Figure 5.1). Experiments designed to study the nuclear level of

PRMT5 in the presence of various MAP kinase and nuclear import inhibitors will help

to understand the underlying nuclear translocation process and its role in oncogenic

transformation.

During the course of this study, it has been shown that ST7 expression is regulated

by PRMT5 mediated H3R8 and H4R3 methylation, and that ST7 is repressed in all

cancer cells overexpressing PRMT5. However, the transcriptional repression of ST7

does not correlate with the lack of ST7 protein in transformed cell, which suggest

that ST7 mRNA is also translationally regulated, like PRMT5 mRNA. Protein

231 data also shows that ST7 is localized in cytosol and is probably membrane bound.

Currently, the function of ST7 and the mechanism of tumor suppression by ST7 are

unknown. Experiments will be designed to identify proteins that interact with ST7,

for example yeast-two hybrid assay with ST7 as the bait or immunoprecipitation

experiments using immobilized ST7. These experiments can provide important clues

to the biological pathways where ST7 might function.

Microarray data has identified that 227 genes are repressed by PRMT5 in NIH3T3

cells, which contains a number of tumor suppressor genes including ST7, NM23-H1,

GAS1, GAS2, LOXL and p107. Both ST7 and NM23 − H1 have been identified

as direct targets of PRMT5 and work has focused on the expression of ST7 in MCL

cancer model system. We did not pursue our studies on NM23 − H1 because it does

not seem to act as a tumor suppressor gene for hematological malignancies. Though

decreased expression of NM23-H1 has been associated with the tumorigenecity and

invasiveness of melanoma, gastric, and breast carcinoma, analysis of NM23-H1 expres-

sion in lymphoma cells has indicated that NM23-H1 levels are elevated in tumorigenic cells, suggesting a difference in the function of NM23-H1 protein in adherent and sus- pension cells. The expression of LOXl, GAS1, GAS2, and p107 will be analyzed in various lymphoma and leukemia cell lines and ChIP experiments will be performed to identify if any of these genes are direct targets of PRMT5. The tumor suppres- sive function of these genes will be evaluated in lymphoma cells by establishing cell

lines that overexpress individual tumor suppressor gene and growth characteristics

of the cell lines will be studied in vitro and in vivo. These experiments will provide

important insight in various pathways that are affected by overexpression of PRMT5

in tumorigenic cells.

232 Figure 5.1: PRMT5 localization is altered by the mutation of potential ERK2 phos- phorylation sites and ERK2 phosphorylates PRMT5 in vitro.

(A) HeLa cells were transfected using lipofectamine with 2 µg of plasmids expressing either GFP alone or GFP-PRMT5 fusion proteins (wild type (WT) PRMT5, or PRMT5 carrying S402A, S529A, or S266A/S402A/S529A mutations). Cells were fixed 24 h after transfection and DNA was stained with DAPI. Pictures were taken at 100X using Zeiss axioscope. Overlay shows the merged picture of GFP and DAPI images.

(B) Recombinant flag-tagged PRMT5 (62.5, 125, and 250 ng) was incubated with 1U of activated ERK2 in the presence of [γ-P32] ATP for 30 min at 30oC. Lane 1 shows a reaction without ERK2 using 125 ng of Fl-PRMT5. The reactions were separated on 8% SDS-PAGE gel and analyzed by phosphoImager.

233 Figure 5.1

234 APPENDIX A

PLASMID CONSTRUCTIONS

Name Construction Description pBS(KS+)/ Primers, 5N1met and 3FG- Contains C-terminal flag 5N1-3 Flag SKB1 MET amplified PCR product tagged PRMT5 cDNA (71 (PRMT5) from pGex2TK/Fl SKB1 was to 1871) in T7 orientation. digested with BamHI-EcoRI This clone contains only 21 and ligated to BamHI-EcoRI bp of 5’UTR sequence, which linearized pBS(KS+). was introduced through the 5N1Met primer. pBS(KS+)/ Primers, 5N2met and 3FG- Contains C-terminal flag 5N2-3 Flag SKB1 MET amplified PCR product tagged PRMT5 cDNA (39 (PRMT5) from pBS(KS+)/5N1-3 Flag to 1871) in T7 orientation. SKB1(PRMT5) was digested This clone contains only 53 with BamHI-EcoRI and lig- bp of 5’UTR sequence, which ated to BamHI-EcoRI lin- was introduced through the earized pBS(KS+). 5N2Met primer.

Continued

Table A.1: pBluescript (KS+)-Ampr based plasmids

235 Table A.1 continued pBS(KS+)/ KpnI digested 1855bp DNA Contains PRMT5 cDNA (39 PRMT5 + 3’UTR fragment from pEGFP C1/ to 2254) in T7 orientation. WT PRMT5 + 3’UTR was The initial CAG to TAG mu- ligated to KpnI linearized tation has been corrected by pBS(KS+) 5N2-3 Flag SKB1. site directed mutatgenesis us- ing primer PRMT5 SDM1. This clone contains the com- plele 3’UTR sequence and has no flag tag. pBS(KS+)/ Site directed mutagenesis was Contains mutations in five PRMT5 + 3’UTR performed on pBS(KS+)/ bases at the miR-96 seed se- with mutant PRMT5 + 3’UTR with quence binding site to abolish miR-96 site Mir96SDM primer using miR-96 annealing to PRMT5 Quick change multisite 3’UTR. directed mutagenesis kit. pBS(KS+)/Flag Site directed mutagenesis was Contains C-terminal flag SKB1(PRMT5) performed on pBS(KS+)/ tagged PRMT5 cDNA where mutant R368A 5N2-3 Flag SKB1 with the Arg 324 has been mutated primers 5MHMT1 and to Ala. This clone is in T7 3MHMT1 using Quick change orientation. site directed mutagenesis kit. pBS(KS+)/Flag Site directed mutagenesis was Contains C-terminal flag SKB1(PRMT5) performed on pBS(KS+)/ tagged PRMT5 cDNA where double mutant Flag SKB1 mutant R368A the Gly 323 and Arg 324 have G367A/R368A with primers 5MHMT2 and been mutated to Ala. This 3MHMT2 using Quick change clone is in T7 orientation. site directed mutagenesis kit. pBS(KS+)/ Primers, 5N2met and 3HMT3 Contains PRMT5 cDNA (39 PRMT5 aa 1-280 amplified PCR product from to 708) in T7 orientation, pBS(KS+) 5N2-3 Flag which includes aa 1-236. Can SKB1(PRMT5) was digested be used for in vitro transla- with BamHI and ligated to tion of N-terminal PRMT5. BamHI linearized pBS(KS+).

Continued

236 Table A.1 continued pBS(KS+)/ Primers, 5HMT1 and 3HMT3 Contains PRMT5 cDNA cor- PRMT5 aa 5-280 amplified PCR product from responding to aa 5-236 in T3 pBS(KS+) 5N2-3 Flag orientation. Does not work SKB1(PRMT5) was digested for in vitro translation of N- with EcoRI-BamHI and terminal PRMT5. ligated to EcoRI-BamHI linearized pBS(KS+). pBS(KS+)/ Primers, 5HMT5 and 3HMT3 Contains PRMT5 cDNA cor- PRMT5 aa 281- amplified PCR product from responding to aa 236-593 in 637 pBS(KS+) 5N2-3 Flag T3 orientation. Can be used SKB1(PRMT5) was digested for in vitro translation of C- with EcoRI-BamHI and terminal PRMT5. ligated to EcoRI- BamHI linearized pBS(KS+). pBS(KS+)/ST7 Primers, 5hST7 and 3hST7 Contains full length ST7 amplified 1837bp PCR prod- cDNA cloned in T7 orienta- uct from pSuperscript cDNA tion. Can be used as template library was blunt ended by for in vitro transcription and klenow fill in reaction and lig- translation of ST7. ated to EcoRV linearized and CIP treated pBS(KS+). pBS(KS+)/ BamHI digested 481bp Contains the 481bp NM23-H1 hNM23-H1 DNA fragment from cDNA cloned in T7 orienta- pCDNA3.1/hNM23-H1 was tion. Can be used as template ligated to BamHI linearized for in vitro transcription and and CIP treated pBS(KS+). translation of NM23-H1. pBS(KS+)/c-Myc EcoRI-SalI digested 1.6Kbp Contains c-MYC cDNA in T7 DNA fragment from pBabe- orientation. Can be used as Hyg/c-MYC was ligated template for in vitro tran- to EcoRI-SalI linearized scription and translation of c- pBS(KS+). MYC.

Continued

237 Table A.1 continued pBS(KS+)/CMV- CMV promoter was excised Contains CMV promoter Luc as EcoRI- HindIII fragment driven luciferase gene in T7 from pPINCO- GFP and lig- orientation. ated to EcoRI- HindIII di- gested pBS(KS+) to generate pBS(KS+)/ CMV. To intro- duce the Luc gene, HindIII digested PCR product of primers 5Luc and 3Luc from pGL2 basic was ligated to HindIII linearized and CIP treated pBS(KS+)/ CMV. pBS(KS+)/Fl Site directed mutagenesis was Contains C-terminal flag PRMT5 S266A performed on pBS(KS+)/ tagged PRMT5 cDNA where 5N2-3 Flag SKB1 with primer the Ser 222 has been mutated 5PRMT5S266A using Quick to Ala. This clone is in T7 change multi-site directed orientation. mutagenesis kit. pBS(KS+)/Fl Site directed mutagenesis was Contains C-terminal flag PRMT5 S402A performed on pBS(KS+)/ tagged PRMT5 cDNA where 5N2-3 Flag SKB1 with primer the Ser 358 has been mutated 5PRMT5S402A using Quick to Ala. This clone is in T7 change multi-site directed orientation. mutagenesis kit. pBS(KS+)/Fl Site directed mutagenesis was Contains C-terminal flag PRMT5 S529A performed on pBS(KS+)/ tagged PRMT5 cDNA where 5N2-3 Flag SKB1 with primer the Ser 485 has been mutated 5PRMT5S529A using Quick to Ala. This clone is in T7 change Multi-site directed orientation. kit.

Continued

238 Table A.1 continued pBS(KS+)/Fl Site directed mutagenesis was Contains C-terminal flag PRMT5 S266A/ performed on pBS(KS+)/ tagged PRMT5 cDNA where S402A 5N2-3 Flag SKB1 with theSer 222 and 358 has been primers 5PRMT5S266A and mutated to Ala. This clone is 5PRMT5S402A using Quick in T7 orientation. change multi-site directed mutagenesis kit. pBS(KS+)/Fl Site directed mutagenesis was Contains C-terminal flag PRMT5 S402A/ performed on pBS(KS+)/ tagged PRMT5 cDNA where S529A 5N2-3 Flag SKB1 with the Ser 358 and 485 has been primers 5PRMT5402A and mutated to Ala. This clone is 5PRMT5S529A using Quick in T7 orientation. change multi-site directed mutagenesis kit. pBS(KS+)/Fl Site directed mutagenesis was Contains C-terminal flag PRMT5 S266A/ performed on pBS(KS+)/ tagged PRMT5 cDNA where S402A/ S529A 5N2-3 Flag SKB1 with the Ser 222, 358 and 485 has primers 5PRMT5S266A, been mutated to Ala. This 5PRMT5S402A and clone is in T7 orientation. 5PRMT5S529A using Quick change multi-site directed mutagenesis kit. pBS(KS+)/ NotI digested BRG1 fragment Contains aa 1-1443 of BRG1. BRG1(1-1443) from pBS(KS+)/FL BRG1 Can be used for in vitro trans- was ligated to NotI linearized lation of BRG1 lacking AT- and CIP treated pBS(KS+). Pase domain. pBS(KS+)/ XmnI digested BRM frag- Contains aa 1-1437 of BRM. BRM(1-1437) ment from pBS(KS+)/FL Can be used for in vitro trans- BRM was ligated to XmnI lation of BRM lacking AT- linearized and CIP treated Pase domain. pBS(KS+).

Continued

239 Table A.1 continued pBS(KS+)/ SalI digested BAF57 N- Contains aa 1-157 of BAF57. BAF57(1-157) terminal fragment from Can be used for in vitro trans- pBS(KS+)/FL BAF57 was lation of BAF57 lacking ki- ligated to SalI linearized and nensin like coiled coil domain. CIP treated pBS(KS+). pBS(KS+)/SUZ12 EcoRI digested 2.2 kbp Contains the full length DNA fragment from pfast- SUZ12 cDNA in T7 orienta- bac/SUZ12 was ligated to tion. Can be used for in vitro EcoRI linearized and CIP translation of SUZ12. treated pBS(KS+). pBS(KS+)/EED BamHI-EcoRI digested 1.4 Contains the full length EED kbp DNA fragment from cDNA with a His tag at pFastbac/His-EED was lig- the 5’ end in T7 orientation. ated to BamHI-EcoRI lin- This clone translates poorly earized pBS(KS+). in vitro. pBS(KS+)/EZH2 BamHI digested 2.25 kbp Contains the full length EZH2 DNA fragment from pfast- cDNA in T7 orientation. Can bac/EZH2 was ligated to be used for in vitro transla- BamHI linearized and CIP tion of EZH2. treated pBS(KS+). pBS(KS+)/ EcoRI-KpnI digested 1.4 Contains the full length RbAp48 kbp DNA fragment from RbAp48 cDNA in T7 orienta- pfastbac/RbAp48 was ligated tion. Can be used for in vitro to EcoRI-KpnI linearized translation of RbAp48. pBS(KS+).

240 Name Construction Description pGex2TK/PAH1A Primers, 5PAHI and 3PAHI Expresses N-terminal GST and or 5BPAH1 and 3BPAH1 am- fused to the PAH1 domain pGex2TK/PAH1B plified PCR product from of mSIN3A or B in bacteria. pVZmSIN3A or B was di- Used for GST-pull down ex- gested with BamHI-EcoRI periments. and ligated to BamHI-EcoRI linearized pGex2TK. pGex2TK/PAH2A Primers, 5PAHII and 3PAHII Expresses N-terminal GST and or 5BPAH2 and 3BPAH2 am- fused to the PAH2 domain pGex2TK/PAH2B plified PCR product from of mSIN3A or B in bacteria. pVZmSIN3A or B was di- Used for GST-pull down ex- gested with BamHI-EcoRI periments. and ligated to BamHI-EcoRI linearized pGex2TK. pGex2TK/PAH3A Primers, 5PAHIII and Expresses N-terminal GST and 3PAHIII or 5BPAH3 and fused to the PAH3 domain pGex2TK/PAH3b 3BPAH3 amplified PCR of mSIN3A or B in bacteria. product from pVZmSIN3A Used for GST-pull down ex- or B was digested with periments. BamHI-EcoRI and ligated to BamHI-EcoRI linearized pGex2TK. pGex2TK/PAH4A Primers, 5PAHIV and Expresses N-terminal GST and 3PAHIV or 5BPAH4 and fused to the PAH4 domain pGex2TK/PAH4B 3BPAH4 amplified PCR of mSIN3A or B in bacteria. product from pVZmSIN3A Used for GST-pull down ex- or B was digested with periments. BamHI-EcoRI and ligated to BamHI-EcoRI linearized pGex2TK. pGex-Myc A kind gift from Dr. M.Fuchs Expresses N-terminal GST- and Dr. D. Livingston MYC fusion protein in bacte- ria.

Continued

Table A.2: pGex2TK-Ampr (Pharmacia) based plasmids for GST fusion protein ex- pression

241 Table A.2 continued pGex2TK /c-Myc Primers, 5NMYC and Expresses N-terminal GST TAD 3NMYC amplified PCR fused to TAD of MYC (aa1- product from pBabe-Hyg/ 149) in bacteria. c-MYC was digested with BamHI-EcoRI and ligated to BamHI-EcoRI linearized pGex2TK. pGex2TK /c-Myc Primers, 5MMYC and Expresses N-terminal GST Hinge 3MMYC amplified PCR fused to middle hinge frag- product from pBabe-Hyg/ ment of MYC (aa150-354) in c-MYC was digested with bacteria. BamHI-EcoRI and ligated to BamHI-EcoRI linearized pGex2TK. pGex2TK /c-Myc Primers, 5CMYC and Expresses N-terminal GST DBD 3CMYC amplified PCR fused to DBD of MYC (aa355- product from pBabe-Hyg/ 439) in bacteria. c-MYC was digested with BamHI-EcoRI and ligated to BamHI-EcoRI linearized pGex2TK. pGex2TK/Fl Primers, 5FGMET and 3FG- Expresses N-terminal GST- SKB1(PRMT5) MET amplified PCR product fused to full length PRMT5 from HBP-ALL cDNA library (aa 4-593) fusion protein in was digested with BamHI- bacteria. EcoRI and ligated to BamHI- EcoRI linearized pGex2TK. pGex2TK/N-term Primers, 5FGSKB6 and Expresses N-terminal GST PRMT5 3SKB208 amplified PCR fused to PRMT5 (aa 6-164) product from pBS(KS+) protein in bacteria. 5N2-3 Flag SKB1(PRMT5) was digested with BamHI- EcoRI and ligated to BamHI- EcoRI linearized pGex2TK.

Continued

242 Table A.2 continued pGex2TK/C-term Primers, 5HMT6 and 3FG- Expresses C-terminal GST- PRMT5 Met amplified PCR product PRMT5 (aa 237-593) fusion from pBS(KS+) 5N2-3 Flag protein in bacteria. Used for SKB1(PRMT5) was digested GST pull down experiments with BamHI-EcoRI and lig- and N-PRMT5 antibody pro- ated to BamHI-EcoRI lin- duction. earized pGex2TK. pGex2TK/ST7 Primers, 5GST-ST7N and Expresses N-terminal GST Nterm 3GST-ST7N amplified PCR fused to ST7 (aa 102-278) product from pBS(KS+)/ protein in bacteria. ST7 was digested with BamHI-EcoRI and ligated to BamHI-EcoRI linearized pGex2TK. pGex2TK/ST7 Primers, 5GST-ST7C and Expresses N-terminal GST Cterm 3GST-ST7C amplified PCR fused to ST7 (aa 325-558) fu- product from pBS(KS+)/ sion protein in bacteria. ST7 was digested with BamHI-EcoRI and ligated to BamHI-EcoRI linearized pGex2TK. pGex2TK/hNM23- Primers, 5GST-NM23H1 Expresses N-terminal GST H1 and 3GST-NM23H1 am- fused to full length human plified PCR product from NM23H1 (aa 2-132) fusion pBS(KS+)/ hNM23H1 was protein in bacteria. digested with BamHI-EcoRI and ligated to BamHI-EcoRI linearized pGex2TK.

243 Name Construction Description pFastbac/ 5N1-3 BamHI-EcoRI digested Expresses C-terminal flag Flag 1800bp DNA fragment from tagged wild type PRMT5 SKB1(PRMT5) pBS(KS+)/ 5N1-3 Flag protein in Sf9 cells. Needs to SKB1 was ligated to BamHI- be transformed in DH10bac EcoRI linearized pFastbac cells to prepare bacmid DNA for Sf9 transfection. Expression has not been tested. pFastbac/ 5N2-3 BamHI-EcoRI digested Expresses C-terminal flag Flag 1832bp DNA fragment from tagged wild type PRMT5 SKB1(PRMT5) pBS(KS+)/ 5N1-3 Flag protein in Sf9 cells, which SKB1 was ligated to BamHI- is active and can methylate EcoRI linearized pFastbac. histones and peptides. pFastbac/Flag BamHI-EcoRI digested Expresses C-terminal flag SKB1(PRMT5) 1832bp DNA fragment from tagged mutant PRMT5 mutant R368A pBS(KS+)/ Flag SKB1 protein in Sf9 cells, which mutant R368A was ligated contains a mutation of cat- to BamHI-EcoRI linearized alytic site Arg 324 residue. pFastbac Activity has not been tested. pFastbac/Flag BamHI-EcoRI digested Expresses C-terminal flag SKB1(PRMT5) 1832bp DNA fragment tagged mutant PRMT5 pro- double mutant from pBS(KS+)/ Flag tein in Sf9 cells, which is G367A/R368A SKB1(PRMT5) double mu- catalytically inactive due to tant G367A/R368A was mutation of catalytic site ligated to BamHI-EcoRI residues Gly 323 and Arg linearized pFastbac. 324. pFastbac/Flag Primers 5Gcn5 and 3Gcn5 Expresses C-terminal flag GCN5 amplified 1386bp PCR prod- tagged yeast GCN5 HAT pro- uct from yeast genomic tein in Sf9 cells. Expression DNA was PstI-KpnI digested has been confirmed but the and ligated to PstI-KpnI activity of purified protein linearized pFastbac. has not been tested.

Continued

Table A.3: pFastbac - Ampr (Gibco BRL) based plasmids for protein expression in Sf9 cells

244 Table A.3 continued pFastbac/Flag Primers, 5Esa and 3Esa am- Expresses C-terminal flag Esa1 plified 1400bp PCR product tagged yeast ESA1 HAT pro- from yeast genomic DNA was tein in Sf9 cells. Expression BamHI-EcoRI digested and and activity activity has not ligated to BamHI-EcoRI lin- been tested. earized pFastbac. pFastbac/Flag A kind gift from Dr. X. J. Expresses C-terminal flag MORF Yang. tagged HAT protein, MORF in Sf9 cells. Expression and activity activity has not been tested. pFastbac/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S266A 1832bp DNA fragment from tagged mutant PRMT5 pro- pBS(KS+)/ Fl PRMT5 tein in Sf9 cells, where Ser S266A was ligated to BamHI- 266 has been mutated to EcoRI linearized pFastbac. Ala. To be used to study the role of phosphorylation by MAP kinase on PRMT5 localization and activity. Has been expressed. pFastbac/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S402A 1832bp DNA fragment from tagged mutant PRMT5 pro- pBS(KS+)/ Fl PRMT5 tein in Sf9 cells, where Ser S402A was ligated to BamHI- 358 has been mutated to EcoRI linearized pFastbac. Ala. To be used to study the role of phosphorylation by MAP kinase on PRMT5 localization and activity. Has been expressed.

Continued

245 Table A.3 continued pFastbac/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S529A 1832bp DNA fragment from tagged mutant PRMT5 pro- pBS(KS+)/ Fl PRMT5 tein in Sf9 cells, where Ser S529A was ligated to BamHI- 222 has been mutated to EcoRI linearized pFastbac. Ala. To be used to study the role of phosphorylation by MAP kinase on PRMT5 localization and activity. Has been expressed. pFastbac/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S266A/ 1832bp DNA fragment from tagged mutant PRMT5 pro- S402A pBS(KS+)/ Fl PRMT5 tein in Sf9 cells, whereSer 222 S266A/ S402A was ligated and 358 has been mutated to BamHI-EcoRI linearized to Ala. To be used to study pFastbac. the role of phosphorylation by MAP kinase on PRMT5 localization and activity. Has been expressed. pFastbac/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S402A/ 1832bp DNA fragment from tagged mutant PRMT5 pro- S529A pBS(KS+)/ Fl PRMT5 tein in Sf9 cells, whereSer 358 S402A/ S529A was ligated and 485 has been mutated to BamHI-EcoRI linearized to Ala. To be used to study pFastbac. the role of phosphorylation by MAP kinase on PRMT5 localization and activity. Has been expressed. pFastbac/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S266A/ 1832bp DNA fragment from tagged mutant PRMT5 pro- S402A/ S529A pBS(KS+)/ Fl PRMT5 tein in Sf9 cells, whereSer S266A/ S402A/S529A was 222, 358 and 485 has been ligated to BamHI-EcoRI mutated to Ala. To be used linearized pFastbac. to study the role of phos- phorylation by MAP kinase on PRMT5 localization and activity. Has been expressed.

Continued

246 Table A.3 continued pFastbac/FL-ST7 BamHI-EcoRI digested Expresses C-terminal flag 1815bp DNA fragment from tagged ST7 (596 aa) pro- pBabe-Hyg /Flag ST7 was tein in Sf9 cells. Has been ligated to BamHI-EcoRI expressed. linearized pFastbac. pFastbac/ FL- Primers, 5PAD4 and 3FL- Expresses C-terminal flag PAD4 PAD4 amplified 2 kbp PCR tagged PAD4/PADI4 (634 fragment from pCMV6- aa) protein in Sf9 cells. XL4/PAD4 (Origene Inc.) Expression has not been was digested with BamHI- tested. EcoRI and ligated to BamHI- EcoRI linearized pFastbac.

247 Name Construction Description pBabe-Hyg/Ha A kind gift from Dr. Expresses Ha-Ras protein Ras L.Gustavo. and confers hygromycin resistance. pBabe-Hyg/E1A A kind gift from Dr. Expresses viral E1A protein L.Gustavo. and confers hygromycin resis- tance. pBabe-Hyg/c- A kind gift from Dr. Expresses c-MYC protein Myc L.Gustavo. and confers hygromycin resistance. pBabe- BamHI-EcoRI digested Expresses C-terminal flag Puro/5N2-3 Flag 1832bp DNA fragment from tagged full length wild type SKB1(PRMT5) pBS(KS+)/ 5N2-3 Flag PRMT5 protein. Confers SKB1 was ligated to BamHI- puromycin resistance. EcoRI linearized pBabe-puro. pBabe-Puro/Flag BamHI-EcoRI digested Expresses C-terminal flag SKB1(PRMT5) 1832bp DNA fragment from tagged full length mutant mutant R368A pBS(KS+)/ Flag SKB1 PRMT5 protein carrying Arg mutant R368A was ligated 324 to Ala mutation. Confers to BamHI-EcoRI linearized puromycin resistance. pBabe-puro. pBabe-Puro/Flag BamHI-EcoRI digested Expresses C-terminal flag SKB1(PRMT5) 1832bp DNA fragment tagged full length mutant double mutant from pBS(KS+)/ Flag PRMT5 protein that is G367A/R368A SKB1(PRMT5) double mu- catalytically inactive due to tant G367A/R368A was Gly 323 and Arg 324 to Ala ligated to BamHI-EcoRI mutation. Confers puromycin linearized pBabe-puro. resistance. pBabe- Primers 5AHMT1 and Expresses 992bp PRMT5 Puro/antisense 3AHMT2 amplified 992bp cDNA (39 to 1031) in an- SKB1(PRMT5) PCR fragment from tisense orientation. Confers pBS(KS+)/ 5N2-3 Flag puromycin resistance. SKB1was digested with BamHI-SalI and ligated to BamHI-SalI linearized pBabe-puro.

Continued

Table A.4: Retroviral expression plasmids (all plasmids carry - Ampr) for mammalian expression.

248 Table A.4 continued pBabe-Puro/N- BamHI fragment from Expresses N-terminal PRMT5 sense pBS(KS+)/ PRMT5 aa PRMT5 (aa1-236) pro- 1-280 was ligated to BamHI tein and confers puromycin linearized and CIP treated resistance. pBabe-Puro. pBabe-Puro/N- BamHI fragment from Expresses antisense to N- PRMT5 antisense pBS(KS+)/ PRMT5 aa terminal PRMT5 (aa1-236) 1-280 was ligated to BamHI and confers puromycin resis- linearized and CIP treated tance. pBabe-Puro. pBabe- BamHI- EcoRI fragment from Mammalian expression of Puro/PRMT5 pCMV-Luc/ PRMT5 3’UTR the RNA corresponding 3’UTR was ligated to BamHI- EcoRI to 3’UTR of PRMT5 and linearized pBabe-Puro. confers puromycin resistance.. pBabe-Hyg/Flag Primers 5FLST7 and 3FLST7 Mammalian Expresses C- ST7 amplified 1815bp PCR frag- terminal flag tagged full ment from pBS(KS+)/ length human ST7 (596aa) ST7was digested with SalI isoformB protein and confers and ligated to SalI linearized hygromycin resistance. and CIP treated pBabe-Hyg. pBabe-Hyg/AS-Fl Primers 5FLST7 and 3FLST7 Expresses 1815bp flag tagged ST7 amplified 1815bp PCR frag- ST7 cDNA in antisense ment from pBS(KS+)/ ST7 orientation and confers hy- was digested with SalI and gromycin resistance. Can ligated to SalI linearized and be used to knockdown ST7 CIP treated pBabe-Hyg. expression. pBabe-Bsd/Fl Primers 5FLNM23H1 and Expresses C-terminal flag NM23-H1 3FLNM23H1 amplified tagged 481bp 132aa hu- 481bp PCR fragment from man NM23-H1 and confers pBS(KS+)/ hNM23-H1was blastacidin resistance. digested with BamHI- EcoRI and ligated to BamHI- EcoRI linearized pBabe-Bsd.

Continued

249 Table A.4 continued pBabe/ EGFP fragment from Expresses catalytically mu- EGFP/Mut pPINCO-GFP (NotI-Klenow- tant BRG1 protein. The cells BRG1 HindIII )was inserted into containing the construct can linearized pBabe/ Puro/ Mut be selected by GFP expres- BRG1 (ClaI-Klenow-HindIII) sion. pRev- Primers 5AHMT1 and Expresses 992bp of PRMT5 TRE/antisense 3AHMT2 amplified 992bp cDNA in antisense orientation SKB1(PRMT5) PCR fragment from in the presence of inducible pBS(KS+)/ 5N2-3 Flag rTTA protein and confers hy- SKB1was digested with gromycin resistance. BamHI-SalI and ligated to BamHI-SalI linearized pRev-TRE. pMSCV- BamHI-SalI digested CMV- Expresses luciferase gene un- Neo/CMV-Luc Luc DNA fragment from der CMV promoter and con- pBS(KS+)/CMV-Luc was fers neomycin resistance. ligated to BamHI-SalI lin- earized pMSCV-Neo. pMSCV- BamHI-ClaI digested and Expresses luciferase gene un- Hyg/CMV-Luc blunt ended (Klenow der CMV promoter and con- fill in reaction) CMV- fers hygromucin resistance. Luc DNA fragment from pBS(KS+)/CMV-Luc was ligated to blunt ended ClaI linearized and CIP treated pMSCV-Hyg. pMSCV- BamHI-ClaI digested and Expresses luciferase gene un- Puro/CMV-Luc blunt ended (Klenow der CMV promoter and con- fill in reaction) CMV- fers puromycin resistance. Luc DNA fragment from pBS(KS+)/CMV-Luc was ligated to blunt ended ClaI linearized and CIP treated pMSCV-puro

Continued

250 Table A.4 continued pMSCV-Neo/AS- Primers 5ASXhoI and Expresses 992bp of PRMT5 PRMT5 3ASEcoRI amplified 992bp cDNA(39 to 1031) in anti- PCR fragment from sense orientation and confers pBS(KS+)/ 5N2-3 Flag neomycin resistance. SKB1was digested with XhoI-EcoRI and ligated to XhoI-EcoRI linearized pMSCV-Neo. pMSCV-Neo/AS- BamHI-SalI digested CMV- Expresses both, 992bp of PRMT5/CMV- Luc DNA fragment from PRMT5 cDNA (39 to 1031) Luc pBS(KS+)/CMV-Luc was in antisense orientation and ligated to BamHI-SalI luciferase gene from a CMV linearized pMSCV-Neo/ promoter. Confers neomycin AS-PRMT5 resistance. pMSCV- BamHI digested hNM23H1 Expresses both, 132 aa human Neo/hNM23-H1 from pBS(KS+)/ hNM23-H1 NM23-H1 and luciferase gene CMV-Luc was ligated to BglII linearized from a CMV promoter. Con- and CIP treated pMSCV- fers neomycin resistance. Neo/ CMV-Luc. pMSCV- BamHI-EcoRI digested FL- Expresses C-terminal flag Puro/hNM23-H1 NM23H1 from pBabe-Bsd tagged full length human /Fl NM23-H1 was ligated NM23-H1(132 aa) and con- to BglII- EcoRI linearized fers puromycin resistance. pMSCV-Puro. pMSCV-Hyg/Fl SalI digested FL-ST7 DNA Expresses full length hu- ST7 fragment from pBabe-Hyg man ST7 isoformB (596 aa) /Flag ST7 was ligated to XhoI and confers hygromycin resis- linearized and CIP treated tance. pMSCV-Puro. pMSCV-Hyg/AS- SalI digested FL-ST7 DNA Expresses flag tagged 1815bp Fl ST7 fragment from pBabe-Hyg human ST7 isoformB cDNA /Flag ST7 was ligated to XhoI in antisense orientation and linearized and CIP treated confers hygromycin. Resis- pMSCV-Puro. tance.

251 Name Construction Description pVSV-G A kind gift from Encodes for the envelope pro- Dr. L. Comai tein of the lentivirus. It is cotransfected for generating lentivirus particles. pCMV∆8.2 A kind gift from Encodes for the Pol and Gag Dr. L. Comai protein of the lentivirus. It is cotransfected with pRRL- based vector for generating lentivirus particles. pRRL/SV40 Puro BamHI-ClaI sticky-blunt Lentiviral expression vec- 1055 bp DNA fragment tor encoding puromycin from pBabe-puro was resistance gene. ligated to BamHI-SalI sticky-blunt linearized pRRLsin.hCMVIRESGFP. pRRL/SV40 BamHI-ClaI sticky-blunt Expresses 992 bp of PRMT5 Puro- AS PRMT5 2047 bp DNA fragment cDNA in antisense orienta- from pBabe-puro/AS-SKB1 tion. To be used for gen- was ligated to BamHI- erating AS-PRMT5 express- SalI sticky-blunt linearized ing lentivirus. Also confers pRRLsin.hCMVIRESGFP. puromycin resistance. pRRL/SV40 BamHI-ClaI sticky-blunt Expresses C terminal flag Puro- Fl WT 2937 bp DNA fragment from tagged full length PRMT5 PRMT5 pBabe-Puro/5N2-3 Flag cDNA. To be used for gener- SKB1 was ligated to BamHI- ating wild type PRMT5 ex- SalI sticky-blunt linearized pressing lentivirus. Also con- pRRLsin.hCMVIRESGFP. fers puromycin resistance. pRRL/SV40 Hyg- BamHI-NheI sticky-blunt Expresses full length MYC MYC 2915 bp DNA fragment cDNA. To be used for gen- from pBabe-hyg/MYC erating MYC expressing was ligated to BamHI- lentivirus. Also confers SalI sticky-blunt linearized hygromycin resistance. pRRLsin.hCMVIRESGFP.

Continued

Table A.5: Lentiviral, pRRLsin IRES GFP based expression plasmids.

252 Table A.5 continued pRRL/GFP NheI-SalI blunt-sticky 2547 Expresses full length PRMT5 PRMT5 bp DNA fragment from in fusion with N-terminal w/o 3UTR pEGFP C1/ Fl WT PRMT5 EGFP. The EGFP- PRMT5 was ligated to BamHI- protein is also flag tagged at SalI blunt-sticky linearized the C-terminal of PRMT5. pRRLsin.hCMVIRESGFP. Does not have any selectable marker. pRRL/GFP NheI-SalI blunt-sticky 2923 To expresses full length PRMT5+3UTR bp DNA fragment from PRMT5 in fusion with N- pEGFP C1/ WT PRMT5 + terminal EGFP. The EGFP 3’UTR was ligated to BamHI- fusion will be expressed in SalI blunt-sticky linearized the presence of PRMT5 pRRLsin.hCMVIRESGFP. 3’UTR. Does not have any selectable marker. NOTE: This clone will expresses truncated EGFP-PRMT5 due to a CAG to TAG muta- tion at position 1719 in the pEGFPC1/ WT PRMT5+ 3’UTR. pRRLsin IRES XbaI-BamHI digested RT- To express miR-96 in cells GFP/miR-96 PCR product obtained us- through lentivirus infection. ing primers 5mir96cDNA and 3mir96cDNA on B cell RNA was ligated to XbaI-BamHI digested pRRLsin IRES GFP. pRRLsin IRES XbaI-BamHI digested RT- To express miR-19a in cells GFP/miR-19a PCR product obtained using through lentivirus infection. primers 5mir19acDNA and 3mir19acDNA on B cell RNA was ligated to XbaI-BamHI digested pRRLsin IRES GFP.

Continued

253 Table A.5 continued pRRLsin IRES XbaI-BamHI digested RT- To express miR-19b in cells GFP/miR-19b PCR product obtained using through lentivirus infection. primers 5mir19bcDNA and 3mir19bcDNA on B cell RNA was ligated to XbaI-BamHI digested pRRLsin IRES GFP. pRRLsin IRES XbaI-BamHI digested RT- To express miR-25 in cells GFP/miR-25 PCR product obtained us- through lentivirus infection. ing primers 5mir25cDNA and 3mir25cDNA on B cell RNA was ligated to XbaI-BamHI digested pRRLsin IRES GFP. pRRLsin IRES XbaI-BamHI digested RT- To express miR-32 in cells GFP/miR-32 PCR product obtained us- through lentivirus infection. ing primers 5mir32cDNA and 3mir32cDNA on B cell RNA was ligated to XbaI-BamHI digested pRRLsin IRES GFP. pRRLsin IRES XbaI-BamHI digested RT- To express miR-92 in cells GFP/miR-92 PCR product obtained us- through lentivirus infection. ing primers 5mir92cDNA and 3mir92cDNA on B cell RNA was ligated to XbaI-BamHI digested pRRLsin IRES GFP.

254 Name Construction Description pBXG1/PAH1A Primers 5BXAH1 and Expresses Gal4 DNA bind- and 3BXAH1 or 5BXH1 and ing domain (aa1-147) fused pBXG1/PAH1B 3BXH1 amplified PCR frag- in frame with PAH1 domain ment from pVZmSIN3A or B of mSIN3A or B. Used in was digested with SpeI- XbaI mammalian two-hybrid ex- and ligated to SpeI- XbaI periments as the bait to test linearized pBXG1. interactions with hSWI/SNF subunits. pBXG1/PAH2A Primers 5BXAH2 and Expresses Gal4 DNA bind- and 3BXAH2 or 5BXH2 and ing domain (aa1-147) fused pBXG1/PAH2B 3BXH2 amplified PCR frag- in frame with PAH2 domain ment from pVZmSIN3A or B of mSIN3A or B. Used in was digested with SpeI- XbaI mammalian two-hybrid ex- and ligated to SpeI- XbaI periments as the bait to test linearized pBXG1. interactions with hSWI/SNF subunits. pBXG1/PAH3A Primers 5BXAH3 and Expresses Gal4 DNA bind- and 3BXAH3 or 5BXH3 and ing domain (aa1-147) fused pBXG1/PAH3B 3BXH3 amplified PCR frag- in frame with PAH3 domain ment from pVZmSIN3A or B of mSIN3A or B. Used in was digested with SpeI- XbaI mammalian two-hybrid ex- and ligated to SpeI- XbaI periments as the bait to test linearized pBXG1. interactions with hSWI/SNF subunits. pBXG1/PAH4A Primers 5BXAH4 and Expresses Gal4 DNA bind- and 3BXAH4 or 5BXH4 and ing domain (aa1-147) fused pBXG1/PAH4B 3BXH4 amplified PCR frag- in frame with PAH4 domain ment from pVZmSIN3 A or B of mSIN3A or B. Used in was digested with SpeI- XbaI mammalian two-hybrid ex- and ligated to SpeI- XbaI periments as the bait to test linearized pBXG1. interactions with hSWI/SNF subunits.

Continued

Table A.6: pBXG1– Ampr based plasmids for Gal4 fusion protein expression.

255 Table A.6 continued pBXG1/WT Primers 5HMT1 and 3HMT1 Expresses Gal4 DNA bind- SKB1(PRMT5) amplified 1767bp PCR frag- ing domain (aa1-147) fused in ment from pBS(KS+)/ 5N2-3 frame with wild type PRMT5 Flag SKB1was digested with aa 5-593. Used in mammalian EcoRI- BamHI and ligated two- hybrid experiments as to EcoRI- BamHI linearized the bait to test the PRMT5 pBXG1. interacting partners. pBXG1/Double Primers 5HMT1 and 3HMT1 Expresses Gal4 DNA bind- mutant amplified 1767bp PCR ing domain (aa1-147) fused in SKB1(PRMT5) fragment from pBS(KS+)/ frame with catalytically inac- Flag SKB1 double mutant tive PRMT5 aa 5-593. Used G367A/R368A was digested in mammalian two- hybrid ex- with EcoRI- BamHI and periments as the bait to test ligated to EcoRI- BamHI the PRMT5 interacting part- linearized pBXG1. ners. pBXG1/SKB1 Primers 5HMT1 and 3HMT3 Expresses Gal4 DNA bind- (PRMT5) aa amplified 693bp PCR frag- ing domain (aa1-147) fused 5-280 ment from pBS(KS+)/ 5N2-3 in frame with PRMT5 aa 5- Flag SKB1was digested with 236. Used in mammalian EcoRI- BamHI and ligated two- hybrid experiments as to EcoRI- BamHI linearized the bait to map the domains pBXG1. of PRMT5 interacting part- ners. pBXG1/SKB1 Primers 5HMT3 and 3HMT1 Expresses Gal4 DNA bind- (PRMT5) aa amplified 1074bp PCR frag- ing domain (aa1-147) fused in 281-637 ment from pBS(KS+)/ 5N2-3 frame with PRMT5 aa 237- Flag SKB1was digested with 593. Used in mammalian EcoRI- BamHI and ligated two- hybrid experiments as to EcoRI- BamHI linearized the bait to map the domains pBXG1. of PRMT5 interactions.

Continued

256 Table A.6 continued pBXG1/SKB1 Primers 5HMT3 and 3HMT4 Expresses Gal4 DNA bind- (PRMT5) aa amplified 600bp PCR frag- ing domain (aa1-147) fused in 281-480 ment from pBS(KS+)/5N2-3 frame with PRMT5 aa 237- Flag SKB1was digested with 436. Used in mammalian EcoRI- BamHI and ligated two- hybrid experiments as to EcoRI- BamHI linearized the bait to map the domains pBXG1. of PRMT5 interactions. pBXG1/SKB1 Primers 5HMT4 and 3HMT1 Expresses Gal4 DNA bind- (PRMT5) aa amplified 571bp PCR frag- ing domain (aa1-147) fused in 481-637 ment from pBS(KS+)/ 5N2-3 frame with PRMT5 aa 437- Flag SKB1was digested with 593. Used in mammalian EcoRI- BamHI and ligated two- hybrid experiments as to EcoRI- BamHI linearized the bait to map the domains pBXG1. of PRMT5 interactions.

257 Name Construction Description pACT/VP16 NarI-SalI digested and blunt Expresses VP16 activation Fl-Brg1 ended by klenow fill in, 5kbp domain (aa411-456) fused to Fl- BRG1 fragment from C-terminal flag tagged BRG1. pBS(KS+)/FL-BRG1 (T7) Used in mammalian two- hy- was ligated to AccI linearized, brid experiments to test inter- blunt ended and CIP treated action with Gal4 fusion pro- pACT. teins. pACT/VP16 HpaI-Klenow-KpnI ,blunt- Expresses VP16 activation hBrm sticky 4kbp Fl- hBRM frag- domain (aa411-456) fused ment from pBS(KS+)/FL- to C-terminal flag tagged BRM (T7) was ligated to hBRM. The first 253 aa NotI-Klenow- KpnI lin- are not included. Used in earized, pACT. mammalian two- hybrid ex- periments to test interaction with Gal4 fusion proteins. pACT/VP16 Primers, 5ACT-575FL and Expresses VP16 activation Fl-BAF57 3-BAF57-1 PCR amplified domain (aa411-456) fused 1227bp Fl-BAF57 fragment to C-terminal flag tagged from pBS(KS+)/ BAF57 was BAF57 (aa 2-411). Used in BamHI digested and ligated mammalian two- hybrid ex- to BamHI linearized and CIP periments to test interaction treated pACT. with Gal4 fusion proteins. pACT/VP16 Primers, 5BAF60 and Expresses VP16 activation BAF60 3BAF60 PCR amplified domain (aa411-456) fused to 1.3kbp BAF60 fragment BAF60 (aa 2- 435). Used in from pBS(KS+)/ BAF60 mammalian two- hybrid ex- was BamHI- SalI digested periments to test interaction and ligated to BamHI- SalI with Gal4 fusion proteins. linearized pACT. pACT/VP16 5INI1 and 3INI1 PCR ampli- Expresses VP16 activation Ini1(BAF45) fied 1125 bp Fl-BAF45 from domain (aa411-456) fused to pBS(KS+)/ Ini1 was digested BAF45 (aa 2-376). Used in with BamHI- SalI and lig- mammalian two- hybrid ex- ated to BamHI- SalI lin- periments to test interaction earized pACT. with Gal4 fusion proteins.

Table A.7: pACT– Ampr (Promega) based plasmids for VP16 fusion protein expres- sion.

258 Name Construction Description pGL2basic Primers 5PRMT5 P1 and Contains the human PRMT5 /hPRMT5 pro 3PRMT5 P1 PCR amplified promoter sequence from - 932 [BD] 1056 bp PRMT5 promoter to + 124 upstream of a pro- fragment from HeLa genomic moterless luciferase gene. To DNA was digested with KpnI study the activity of PRMT5 and ligated to KpnI linearized promoter. and CIP treated pGL2basic. pGL2basic Primers 5PRMT5 P1 and Contains the human PRMT5 /hPRMT5 pro 3PRMT5 P2 PCR amplified promoter sequence from - 932 [BE] opp. Ori 1136 bp PRMT5 promoter to + 204 in reverse orienta- fragment from HeLa genomic tion upstream of a promoter- DNA was digested with KpnI less luciferase gene. and ligated to KpnI linearized and CIP treated pGL2basic. pGL2basic KpnI digested PRMT5 pro- Contains the human PRMT5 /hPRMT5 moter fragment from pGL2 promoter sequence from - 932 promoter BE basic/ hPRMT5 pro [BE] to + 204 upstream of a pro- fragment opp. ori was ligated to KpnI moterless luciferase gene. To linearized and CIP treated study the activity of PRMT5 pGL2basic. promoter. pGL2 basic/Hyg BamHI- ClaI digested sticky- Contains the hygromycin re- blunt SV40 Hyg fragment sistance gene for selection. from pBabe/Hyg was lig- ated to BamHI-SalI linearized sticky- blunt pGL2 basic. pGL2basic KpnI digested PRMT5 pro- Contains the human PRMT5 /Hyg/hPRMT5 moter fragment from pGL2 promoter sequence from - 932 promoter BE basic/ hPRMT5 pro [BE] to + 204 upstream of a fragment opp. ori was ligated to KpnI promoterless luciferase gene linearized and CIP treated and confers hygromycin resis- pGL2basic/ Hyg. tance. Used to study the ac- tivity of PRMT5 promoter.

Continued

Table A.8: pGL2basic- Ampr (Promega) based plasmids for scoring promoter activity.

259 Table A.8 continued pGL2basic NdeI -KpnI digested and Contains the human PRMT5 /Hyg/hPRMT5 blunt ended 699bp fragment promoter sequence from - 495 promoter NdeI from pGL2 basic/ hPRMT5 to + 204 upstream of a -KpnI fragment pro [BE] opp. Ori was promoterless luciferase gene ligated to KpnI linearized, and confers hygromycin resis- blunt ended and CIP treated tance. This construct lacks pGL2basic/ Hyg. two AML1a sites. pGL2basic MluI -KpnI digested and Contains the human PRMT5 /Hyg/hPRMT5 blunt ended 458 bp fragment promoter sequence from - 254 promoter MluI from pGL2 basic/hPRMT5 to + 204 upstream of a -KpnI fragment pro [BE] opp. Ori was promoterless luciferase gene ligated to KpnI linearized, and confers hygromycin resis- blunt ended and CIP treated tance. This construct lacks all pGL2basic/Hyg. three AML1a sites. pGL2basic PstI -KpnI digested and Contains the human PRMT5 /Hyg/hPRMT5 blunt ended 387 bp fragment promoter sequence from - 183 promoter PstI from pGL2 basic/ hPRMT5 to + 204 upstream of a -KpnI fragment pro [BE] opp. Ori was promoterless luciferase gene ligated to KpnI linearized, and confers hygromycin resis- blunt ended and CIP treated tance. This construct lacks pGL2basic/Hyg. all three AML1a and three GATA sites. pGL2basic BamHI -KpnI digested and Contains the human PRMT5 /Hyg/hPRMT5 blunt ended 284 bp fragment promoter sequence from - 80 promoter BamHI- from pGL2 basic/ hPRMT5 to + 204 upstream of a KpnI fragment pro [BE] opp. Ori was promoterless luciferase gene ligated to KpnI linearized, and confers hygromycin resis- blunt ended and CIP treated tance. This construct lacks pGL2basic/Hyg. all three AML1a, SP1and five GATA sites.

Continued

260 Table A.8 continued pGL2basic Site directed mutagenesis Contains the human PRMT5 /Hyg/hPRMT5 was performed on pGL2 ba- promoter sequence from - promoter AML1a sic/Hyg/ hPRMT5 promoter 932 to +204 with mutated mutant BE fragment with primer AML1a site at -907 posi- 5’AML1a using Quickchange tion. Confers hygromycin re- Multi-site directed mutagene- sistance. Used to access the sis kit. contribution of AML1a site to the promoter activity. pGL2basic KpnI digested mutant Contains the human PRMT5 /hPRMT5 pro- PRMT5 promoter frag- promoter sequence from - moter AML1a ment from pGL2 ba- 932 to +204 with mutated mutant sic/Hyg/hPRMT5 promoter AML1a site at -907 position. AML1a mutant was ligated Used to access the contribu- to KpnI linearized and CIP tion of AML1a site to the pro- treated pGL2basic/Hyg. moter activity. pGL2basic Site directed mutagenesis Contains the human PRMT5 /Hyg/hPRMT5 was performed on pGL2 ba- promoter sequence from - promoter AML1b sic/Hyg/ hPRMT5 promoter 932 to +204 with mutated mutant BE fragment with primer AML1a site at -675 posi- 5’AML1b using Quickchange tion. Confers hygromycin re- Multi-site directed mutagene- sistance. Used to access the sis kit. contribution of AML1a site to the promoter activity. pGL2basic KpnI digested mutant Contains the human PRMT5 /hPRMT5 pro- PRMT5 promoter fragment promoter sequence from - moter AML1b from pGL2 basic/Hyg/ 932 to +204 with mutated mutant hPRMT5 promoter AML1b AML1a site at -675 position. mutant was ligated to KpnI Used to access the contribu- linearized and CIP treated tion of AML1a site to the pro- pGL2basic/ Hyg. moter activity.

Continued

261 Table A.8 continued pGL2basic Site directed mutagenesis Contains the human PRMT5 /Hyg/hPRMT5 was performed on pGL2 ba- promoter sequence from - promoter AML1c sic/Hyg/ hPRMT5 promoter 932 to +204 with mutated mutant BE fragment with primer AML1a site at -307 posi- 5’AML1c using Quickchange tion. Confers hygromycin re- Multi-site directed mutagene- sistance. Used to access the sis kit. contribution of AML1a site to the promoter activity. pGL2basic KpnI digested mutant Contains the human PRMT5 /hPRMT5 pro- PRMT5 promoter fragment promoter sequence from - moter AML1c from pGL2 basic/Hyg/ 932 to +204 with mutated mutant hPRMT5 promoter AML1c AML1a site at -307 position. mutant was ligated to KpnI Used to access the contribu- linearized and CIP treated tion of AML1a site to the pro- pGL2basic/ Hyg. moter activity. pGL2basic Site directed mutagenesis Contains the human PRMT5 /Hyg/hPRMT5 was performed on pGL2 ba- promoter sequence from - promoter sic/Hyg/ hPRMT5 promoter 932 to +204 with mutated AML1a,b mu- BE fragment with primers AML1a site at -907 and - tant 5’AML1 a and b using Quick 675 positions. Confers hy- change multi-site directed gromycin resistance. Used to mutagenesis kit. access the contribution of the two AML1a sites to the pro- moter activity. pGL2basic Site directed mutagenesis Contains the human PRMT5 /Hyg/hPRMT5 was performed on pGL2 ba- promoter sequence from - promoter sic/Hyg/ hPRMT5 promoter 932 to +204 with mutated AML1a,c mu- BE fragment with primers AML1a site at -907 and - tant 5’AML1 a and c using Quick 307 positions. Confers hy- change multi-site directed gromycin resistance. Used to mutagenesis kit. access the contribution of the two AML1a sites to the pro- moter activity.

Continued

262 Table A.8 continued pGL2basic /Hyg/ Site directed mutagenesis Contains the human PRMT5 hPRMT5 pro- was performed on pGL2 ba- promoter sequence from - moter AML1b,c sic/Hyg/ hPRMT5 promoter 932 to +204 with mutated mutant BE fragment with primers AML1a site at -675 and - 5’AML1 b and c using Quick 307 positions. Confers hy- change multi-site directed gromycin resistance. Used to mutagenesis kit. access the contribution of the two AML1a sites to the pro- moter activity. pGL2basic Site directed mutagenesis Contains the human PRMT5 /Hyg/hPRMT5 was performed on pGL2 promoter sequence from - promoter AML1 basic/Hyg/ hPRMT5 pro- 932 to +204 with mutated a,b,c mutant moter BE fragment with AML1a site at -907, -675 and primers 5’AML1 a, b and c -307 positions. Confers hy- using Quick change multi-site gromycin resistance. Used to directed mutagenesis kit. access the contribution of all three AML1a sites to the pro- moter activity. pGL2basic KpnI digested mutant Contains the human PRMT5 /hPRMT5 pro- PRMT5 promoter frag- promoter sequence from - moter AML1 a,b,c ment from pGL2 ba- 932 to +204 with mutated mutant sic/Hyg/hPRMT5 promoter AML1a site at -907, -675 and AML1 a,b,c mutant was lig- -307 positions. Used to access ated to KpnI linearized and the contribution of all three CIP treated pGL2basic/Hyg. AML1a sites to the promoter activity.

263 Name Construction Description pCMV-Luc Primers 5downUTRPRMT Contains the complete /PRMT5 3’UTR and 3MET2 PCR amplified PRMT5 3’UTR (1871 to 377bp fragment from HBP- 2248) sequence downstream ALL cDNA library was blunt of the firefly luciferase. Used ended and ligated to SalI to study the effects of 3’UTR linearized, blunt ended and on luciferase expression. CIP treated pCMV-Luc. pCMV-Luc Site directed mutagenesis was Contains the complete /PRMT5 3’UTR performed on pCMV-Luc/ PRMT5 3’UTR (1871 to Mut PRMT5 3’UTR with primer 2248) with mutated miR-96 MIR96SDM2 using Quick binding site downstream of change multi-site directed the firefly luciferase. Used to mutagenesis kit. study the effects of miR-96 binding site in 3’UTR on luciferase expression. pCMV-Luc Primers 5UTRLUC and Contains the complete /PRMT5 3’UTR 3UTRLUC PCR ampli- PRMT5 3’UTR along with 3’ (1800-2248) WT fied 448 bp fragment from end of the coding sequence pBS(KS+)/PRMT5+3’UTR (1800 to 2248) downstream was digested with SalI and of the firefly luciferase. Used ligated to SalI linearized and to study the effects of 3’UTR CIP treated pCMV-Luc. on luciferase expression. pCMV-Luc Primers 5UTRLUC and Contains the complete /PRMT5 3’UTR 3UTRLUC PCR ampli- PRMT5 3’UTR along with 3’ (1800-2248) Mut fied 448 bp fragment from end of the coding sequence pBS(KS+)/PRMT5+3’UTR (1800 to 2248) with mu- with mutant miR-96 site was tated miR-96 binding site digested with SalI and ligated downstream of the firefly to SalI linearized and CIP luciferase. Used to study treated pCMV-Luc. the effects of miR-96 binding site in 3’UTR on luciferase expression.

Continued

Table A.9: pCMV-Luc- Ampr based plasmids for monitoring 3’UTR activity.

264 Table A.9 continued pCMV-Luc/ Primers, 5actinB and 3act- Contains the complete β- β - ACTIN 3’UTR inB, PCR amplified 592 bp ACTIN 3’UTR sequence fragment from B cell random downstream of the firefly hexamer based RT reaction luciferase. Used to study the was digested with SalI and effects of 3’UTR on luciferase ligated to SalI linearized and expression. CIP treated pCMV-Luc. pCMV-Luc/ Primers, 5b-globin and 3b- Contains the complete β- β- GLOBIN globin, PCR amplified 132 bp Globin 3’UTR sequence 3’UTR fragment from B cell random downstream of the firefly hexamer based RT reaction luciferase. Used to study the was digested with SalI and effects of 3’UTR on luciferase ligated to SalI linearized and expression. CIP treated pCMV-Luc.

265 Name Construction Description pEGFP-C1/Fl BamHI-EcoRI digested Expresses C-terminal flag WT PRMT5 1832bp DNA fragment from tagged wild type PRMT5 pBS(KS+)/ 5N2-3Fl SKB1 protein fused to EGFP at the was ligated to BglII-EcoRI N-terminus. linearized pEGFP-C1. pEGFP-C1/WT Primers, 3MET1 and 3MET2 This clone expresses trun- PRMT5 + 3’UTR amplified 659bp PCR product cated GFP-PRMT5 due to a from HBP-ALL library was CAG to TAG mutation at po- digested with ScaI- EcoR1 sition 1719 that occurred dur- and ligated to ScaI- EcoRI ing the PCR reaction. The digested pEGFP-C1/ Fl WT clone contains the full length PRMT5. 3’UTR and EGFP is fused at the N-terminus. pEGFP-C1/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S266A 1832bp DNA fragment tagged mutant PRMT5 pro- from pBS(KS+)/Fl PRMT5 tein with EGFP fused at the S266A was ligated to BglII- N-terminus, where Ser 222 EcoRI linearized pEGFP-C1. has been mutated to Ala. pEGFP-C1/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S402A 1832bp DNA fragment tagged mutant PRMT5 pro- from pBS(KS+)/Fl PRMT5 tein with EGFP fused at the S402A was ligated to BglII- N-terminus, where Ser 358 EcoRI linearized pEGFP-C1. has been mutated to Ala. pEGFP-C1/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S529A 1832bp DNA fragment from tagged mutant PRMT5 pro- pBS(KS+)/ Fl PRMT5 tein with EGFP fused at the S529A was ligated to BglII- N-terminus, where Ser 485 EcoRI linearized pEGFP-C1. has been mutated to Ala. pEGFP-C1/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S266A/ 1832bp DNA fragment tagged mutant PRMT5 pro- S402A from pBS(KS+)/Fl PRMT5 tein with EGFP fused at the S266A/ S402A was lig- N-terminus, where Ser 222 ated BglII-EcoRI linearized and 358 has been mutated to pEGFP-C1. Ala.

Continued

Table A.10: pEGFP C1- Kanr (Clontech) based plasmids for GFP fusion protein expression in mammalian cells.

266 Table A.10 continued pEGFP-C1/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S402A/ 1832bp DNA fragment from tagged mutant PRMT5 pro- S529A pBS(KS+)/ Fl PRMT5 tein with EGFP fused at the S402A/S529A was ligated N-terminus, where Ser 358 to BglII-EcoRI linearized and 485 has been mutated to pEGFP-C1. Ala. pEGFP-C1/Fl BamHI-EcoRI digested Expresses C-terminal flag PRMT5 S266A/ 1832bp DNA fragment tagged mutant PRMT5 pro- S402A/ S529A from pBS(KS+)/Fl PRMT5 tein with EGFP fused at S266A/S402A/S529A was the N-terminus, where Ser ligated to BglII-EcoRI lin- 222, 358 and 458 has been earized pEGFP-C1. mutated to Ala.

267 Name Construction Description pG5-Hsp70-40 A king gift from Reporter plasmid for Gal4 fu- CAT Dr. T. Gilmore sion proteins. Contains five Gal4 binding sites upstream of a minimal Hsp70 promoter driven CAT gene pCMV-β Gal A kind gift from Dr. S. Jacob Expresses β-Galactosidase under CMV promoter. Used for normalization of transient transfections. pRL-TK Promega Expresses Renilla luciferase under the HSV TK promoter. Used for normalization of transient transfections.

Table A.11: Reporter and normalization plasmids.

Name Construction Description pMBP-MORF A king gift from Expresses MBP- MORF fu- Dr. X.J. Yang sion protein in bacteria. pCDNA3.1 A kind gift from Expresses human NM23-H1 /hNM23-H1 Dr. P.S. Steeg protein in mammalian cells and confers neomycin resis- tance. Continued

Table A.12: Miscelleneous plasmids.

268 Table A.12 continued pPINCO- GFP A kind gift from A retroviral vector containing Dr. M. Caliguiri as selectable marker EGFP under a CMV promoter. pRRLsin.h A kind gift from Lentiviral expression system CMVIRESGFP Dr. L. Comai. vector to package any gene. Does not have any selectable marker. Has an internal GFP. pCMV-Luc A kind gift from Dr. D. Expresses luciferase gene from Schoenberg a CMV promoter. Can be used to clone 3’UTR down- stream of the Luc gene. pET15b/ST7 Primers, 5ST7Histag and To express N-terminal His 3ST7Histag amplified 1756 tagged full length ST7 protein bp PCR product from in BL21 cells. Expression has pBS(KS+)/ST7 was digested not been verified. with XhoI and ligated to XhoI digested and CIP treated pET15b.

269 APPENDIX B

PRIMER SEQUENCES

Primer Sequence Use 5GDH CTCAACTACATGGTTTACATGTTC GAPDH RT-PCR (+178 to +202) 3GDH CCTTCCACGATACCAAAGTTGTCATG GAPDH RT-PCR (+552 to +578) 5BACT CATCGTGGGGCGCCCCAGGCACCA β-ACTIN RT-PCR (+140 to +166) 3BACT CAGCCAGGTCCAGACGCAGGATGGCA β-ACTIN RT-PCR (+557 to +585) 5CAD CTTAGTGCTCACCTATCCTCTGATCG CAD RT-PCR (+308 to +334) 3CAD GGGATGAAGGTTCTGTTCCATTCTG CAD RT-PCR (+578 to +603) 5NL GAAAGCAGCTGTCACTCCAGGCAAA NUCLEOLIN RT- (+378 to +402) PCR 3NL TCATCGTCCTCATCCTCTGAGGCAG NUCLEOLIN RT- (+662 to +686) PCR

Continued

Table B.1: Primers for RT-PCR and ChIP assays The primers that have been used to amplify either cDNA region for RT-PCR or promoter sequences for ChIP assays are listed with the primer sequence. All primers were designed for human sequences except where indicated under the “use” column.

270 Table B.1 continued

5CF2 AGTCTCTGCTGCTGCCGCCAA To amplify CAD pro- (-157 to -188) moter region by PCR for ChIP assay 3CR1 GAGAGGCGCATCACAGAGTGGGATAA To amplify CAD pro- (+135 to +161) moter region by PCR for ChIP assay 5ODF2 GCTTTGTCAGTCCCTCCTGTAGCCG To amplify ODC pro- (-5 to +20) moter region by PCR for ChIP assay 3ODR2 ATCACCCTTATCCAGCCGCGGGAGAA To amplify ODC pro- (+351 to +377) moter region by PCR for ChIP assay 5St7 CACTTCCTCGCTAATATCGGGAC Mouse ST7 RT-PCR (+448 to +425) 3St7 CTCGTCCTGGTACCCAAGTGTAC Mouse ST7 RT-PCR (+694 to +717) 5Nm23 GTGAGCGTACCTTCATTGCCA Mouse NM23 RT- (+103 to +124) PCR 3Nm23 CCACTGGTCCTGAGTGCATGT Mouse NM23 RT- (+282 to +303) PCR 5Myt GCCATTACAAGAGCTGTTCAGC Mouse MYT1L RT- (+796 to +818) PCR 3Myt CCGATCCACCTCATCTTCGTC Mouse MYT1L RT- (+1091 to +1112) PCR 5CB2 CCAAGAGCCATGTGACTATCC Mouse CYCLINB2 (+172 to +194) RT-PCR 3CB2 CACTGCAGAGCTGAGGGTTCTC Mouse CYCLINB2 (+466 to +489) RT-PCR 5CE2 CCAGCCAGACTCTCCGCAAG Mouse CYCLINE2 (+69 to +90) RT-PCR 3CE2 CAGGTCAGAATGCAGAACTTG Mouse CYCLINE2 (+387 to +409) RT-PCR 5Cdc20 CACTGCTTCAACTGGACGCACC Mouse CDC20 RT- (+161 to +183) PCR 3Cdc20 CTGAGGTTTGCCGCTGAGCC Mouse CDC20 RT- (+520 to +540) PCR

Continued

271 Table B.1 continued

5CDK4 GTCAGCACAGTTCGTGAGGTG Mouse CDK4 RT- (+151 to +173) PCR 3CDK4 CGGTACCAGAGCGTAACCACC Mouse CDK4 RT- (+521 to +542) PCR 5Rrm1 GTTGATCCTGCTCAGATCACC Mouse RRM1 RT- (+181 to +201) PCR 3Rrm1 CATATGCTGTGGTCTTTCAGCC Mouse RRM1 RT- (+570 to +591) PCR 5NF-kb CTGTCAACAGATGGCCCATACC Mouse NF-κB RT- (+393 to +415) PCR 3NF-kb CTCTGTCATCCGTGCTTCCAG Mouse NF-κB RT- (+741 to +762) PCR 5Gas2 GCAGCGGACCTGGCCTGTCTG Mouse GAS2 RT- (+275 to +296) PCR 3Gas2 GGAAATTGGCTGTGTTGTCTC Mouse GAS2 RT- (+575 to +595) PCR 5All GTAAGTAGCCAGTACAGCTCC Mouse ALL RT- (+186 to +207) PCR 3All GTCGACAGAGTGGATGCTGG Mouse ALL RT- (+412 to +432) PCR 5Serp GCAGATCCAAGATGCTATGGG Mouse SERPIN (+368 to +389) RT-PCR 3Serp GCACAGAGACGGTGCTGCCATC Mouse SERPIN (+776 to +798) RT-PCR 5HMT2 CTCTCAGTACCAGCAGGCCATCTA Mouse and human (+1093 to +1117) PRMT5 RT-PCR 3HMT2 CTGCCAGTTCTCTAGCGTCACCAC Mouse and human (+1282 to +1306) PRMT5 RT-PCR 5ST7 CTGAGTGGCGTCACAGGATGAAGAG To amplify mouse (-228 to -203) ST7 promoter re- gion by PCR for ChIP assay 3ST7 CGAGCTATGTCCAGCTGCCAGATTC To amplify mouse (+186 to +209) ST7 promoter re- gion by PCR for ChIP assay

Continued

272 Table B.1 continued

5NM23 AAGTGAGTCACAGGCAGCCGTAGGG To amplify mouse (-211 to -186) NM23 promoter re- gion by PCR for ChIP assay 3NM23 GCGTATCAAGCTGACAGGTCTGTAC To amplify mouse (+229 to +254) NM23 promoter re- gion by PCR for ChIP assay 5MYT1 CAGAAGGTCATATGCCGGGGATTTG To amplify mouse (-258 to -233) MYT1L promoter region by PCR for ChIP assay 3MYT1 CCGCTCACAAGTGACGGCAAGTTCA To amplify mouse (+189 to +214) MYT1L promoter region by PCR for ChIP assay 5hST7rt TGAAAATCAACGACAACTTG ST7 real time (+278 to +297) RT-PCR with the hST7 probe; 5’- 6FAM- CAC CGAAGTTCTA CGTGGCCC TAA-MGBNFQ -3’ [Replaced by 5ST7RTRT] 3hST7rt ATATTAGTGAGGAAGTGCCT ST7 real time (+354 to +373) RT-PCR with the hST7probe; 5’- 6FAM- CAC CGAAGTTCTA CGTGGCCC TAA-MGBNFQ - 3’ [Replaced by 3ST7RTRT]

Continued

273 Table B.1 continued

5ST7RTRT CCCCTAATTGCTTCCTCTACC ST7 real time RT-PCR (+817 to +837) using the probe no. 14 from the human universal probe library set (Roche Applied Sci- ences) 3ST7RTRT CCAAGAGAATATAAGCAGTTGCAC ST7 real time RT-PCR (+866 to +889) using the probe no. 14 from the human universal probe library set (Roche Applied Sci- ences) 5hNM23rt CGCCTTGTGGGTCTGAAAT NM23-H1 real time (+208 to +226) RT-PCR with the hNM23probe; 5’- 6FAM- TGCAAG CTTCCGAAGATCT TCTCA-MGBNFQ-3’ 3hNM23rt TTCACCAGGCCGGCAAAGAA NM23-H1 real time (+286 to +305) RT-PCR with the hNM23probe; 5’- 6FAM- TGCAAG CTTCCGAAGATCT TCTCA-MGBNFQ-3’ 5LOXL RT GCAGAGAGCCCACCTGTAC LOXL real time RT- (+1491 to +1509) PCR with LOXlRT probe; 5’- 6FAM TGGCCA GACACTTCTCCTC -MGBNFQ-3’

Continued

274 Table B.1 continued

3LOXL RT CACCCGCACATCGTAGTC LOXL real time RT- (+1561 to +1579) PCR with LOXlRT probe; 5’- 6FAM TGGCCA GACACTTCTCCTC -MGBNFQ-3’ 5PRMT5 RTRT CCTGTGGAGGTGAACACAGT PRMT5 real time RT- (+1712 to +1731) PCR with PRMT5RT probe; 5’VIC- TCTCAAA GTAGCCGGCAAAG CCAT-MGBNFQ - 3’ 3PRMT5 RTRT AGAGGATGGGAAACCATGAG PRMT5 real time RT- (+1808 to +1828) PCR with PRMT5RT probe; 5’VIC- TCTCAAA GTAGCCGGCAAAG CCAT-MGBNFQ - 3’ 5ChipST7 CCACTTGGCCTTCTCTTTC To amplify ST7 pro- (-102 to -121) moter region by real time PCR with the probe, hST7Chip; 5’ 6FAM- CCCTCG CGTTCTGGGTCC ATT-MGBNFQ- 3’ for ChIP assay 3ChipST7 GGTCCCTACAAGTGGCTTT To amplify ST7 pro- (-28 to -8) moter region by real time PCR with the probe, hST7Chip; 5’ 6FAM- CCCTCG CGTTCTGGGTCC ATT-MGBNFQ- 3’ for ChIP assay

Continued

275 Table B.1 continued

5ChipNM23 AAGCAGCTGGAAGGGTAAGA To amplify NM23-H1 (-143 to -123) promoter region by real time PCR with the probe, hNM23Chip; 5’-6FAM CTAACC CACTCCCGCGACT ACA-MGBNFQ 3’ for ChIP assay 3ChipNM23 CCGCCTCTTAGAGCTGAACT To amplify NM23-H1 (+176 to +196) promoter region by real time PCR with the probe, hNM23Chip; 5’-6FAM CTAACC CACTCCCGCGACT ACA-MGBNFQ 3’ for ChIP assay 5PRMT5 CHIP AGACCCTGAGATTGGTGGAT To amplify PRMT5 (-120 to -97) promoter region by real time PCR with PRMT5CHIP probe; 5’-6FAM ATTGGC AGGAAAAGCCACT CCCC-MGBNFQ-3’ for ChIP assay 3PRMT5 CHIP GGACGAGTCGCCTTAACAAC To amplify PRMT5 (-9 to +11) promoter region by real time PCR with PRMT5CHIP probe; 5’-6FAM ATTGGC AGGAAAAGCCACT CCCC -MGBNFQ-3’ for ChIP assay

276 Primer name Restriction Site Sequence (50 → 30) 5N1met BamHI CGCGGATCCGTGGACAGCGCGA GGAGAAAGATGGCGGCGATGGCG GTCGGGGGTGCTGGTGGGAG 5N2met BamHI CGCGGATCCGTGATTGGCTACT AGTATCAAGGAATCCCGGCGTGG ACAGCGCGAGGAGAAAGATGG 5FGMET BamHI CGCGGATCCATGGCGGTCGGGG GTGCTGGTGGGAGCCGC 3FGMET EcoRI CCGGAATTCTTACTATTTGTCA TCGTCGTCCTTGTAGTCGAGGCC AATGGTATATGAGCGGCCTGTG 5HMT1 EcoRI CCGGAATTCGCGGTCGGGGGGG GTGCTGGTGGG 3HMT1 BamHI CGCGGATCCCTAGAGGCCAATGG TATATGAGCGG 5HMT3 EcoRI CCGGAATTCCTCCAATACCTGGA ATACTTAAGC 3HMT3 BamHI CGCGGATCCCTAGTAGGAGCAGA ACTCCTTCTCTG 5HMT4 EcoRI CCGGAATTCTACAATGAGGTCCG AGCCTGTAGG 3HMT4 BamHI CGCGGATCCCTACAGCTTGGAGG AAGAGATGGGAGC 5HMT5 EcoRI CCGGAATTCGCGGTCGGGGGTGC TGGTGGGAGC 5HMT6 BamHI CGCGGATCCCTCCAATACCTGGA ATACTTAAGC 3MET1 ScaI AAAAGTACTACATGGCTTTGCCGG CTACTTTGA 5MHMT1 None GGTGCTGGGAGCAGGAGCGGGA CCCCTGGTGAAC

Continued

Table B.2: Primers used for cloning and mutagenesis Sequence of the primers used for cloning and mutagenesis as indicated in Appendix A (plasmid construction). The restriction enzyme sites that were introduced at the 5’ end are indicated in bold and the flag tag sequence is italicized.

277 Table B.2 continued

3MHMT1 None GTTCACCAGGGGTCCCGCTCCTG CTCCCAGCACC 5MHMT2 None GGTGCTGGGAGCAGCAGCGGGAC CCCTGGTGAAC 3MHMT2 None GTTCACCAGGGGTCCCGCTGCTG CTCCCAGCACC 5FGSKB6 BamHI CGCGGATCCGTCGGGGGTGCTG GTGGGAGCCGCGTGTCC 3SKB208 EcoRI CCGGAATTCTCAAGCCCCAATT TCAAGAGCCACTGCAAT 5’PRMT5S266A None GAAGACTATCTGCAGGCCCCGC TTCAGCCACTG 5’PRMT5S402A None GCTGACAATGAATTGGCGCCTG AGTGCCTGGATGG 5’PRMT5S529A None CGTCCAGAGACTCACGCTCCTG AGATGTTCTCATGG 5AHMT1 SalI ACGCGTCGACGTGATTGGCTACT AGTATCAAGGAATC 3AHMT2 BamHI CGCGGATCCCATATGTCTGAGAT TCCAGATTGTC 5ASXhoI XhoI CCGCTCGAGGTGATTGGCTACTA GTATCAAGGAATC 3ASEcoRI EcoRI CCGGAATTCCATATGTCTGAGAT TCCAGATTGTC 5downUTR BssHII TTGGCGCGCAATTAACCCTCACT AAAGGGGAATGTAGATTGCCCTG CGTGCCAAGTGTCCAGAGCCT TGG PRMT 3MET2 EcoRI CCGGAATTCGGCAGTAAAGGGCT ATAACTTTAT Mir96SDM None TGGCCTCTAGCCCTGCGAAGATA GTGTCCAGAGCCTTGG MIR96SDM2 None CTAAAGGGGGCCCTGCGAAGATA GTGTCCAGAGCCTTGG

Continued

278 Table B.2 continued

5UTRLUC SalI ACGCGTCGACGACTCACTCTCCT GGGATGTTCTC 3UTRLUC SalI, EcoRI ACGCGTCGACGAATTCGGCAGT AAAGGGCTATAACTTTAT 5PRMT5 P1 KpnI CGGGGTACCCTAAGTTCTGCCAG CGGCGCCT 3PRMT5 P1 KpnI CGGGGTACCCATCGCCGCCATCT TTCTCCTCG 3PRMT5 P2 KpnI CGGGGTACCCCCCTAGTGTGTCA GCTATTTCGG 5’AML1a None CTGCCAGCGGCGCCTGCGTGAAA TCTCTTGTACCGTGTTCTCC 5’AML1b None GAATTCCTTTTCTCTATCTTGCT TTGTGTGTGTGTGTTTAGGCG 5’AML1c None GCAGCGAAGCTGCGAGGAATTTC ACCGAGCCAGCCAGGAGTCG 5ACT-575FL BamHI CGCGGATCCGTTCATCAAAAAGA CCATCTTATGCCCCACCT 3-BAF57-1 EcoRI, BamHI GGAATTCCGGATCCGTTATTTG TCATCGTCGTCCTTGTAGTCTTC TTTTTTCTCATCTTCTGGTATGG GATCTGTTGG 5BAF60 BamHI CGCGGATCCGCGGCCCGGCTCCG GGTCAAGGG 3BAF60 SalI ACGCGTCGACCTATGTATTCCGG ATTCCCAGGG 5INI1 BamHI CGCGGATCCGCGCGCTGAGCAAG ACCTTCGGGCAG 3INI1 SalI ACGCGTCGACTTACCAGGCCGGG CCCGTGTTGGC 5hST7 BamHI CGCGGATCCCCGGGCCGGTGAAT CATCCCGGCAGACA 3hST7 EcoRI CCGGAATTCGAAGAGGGGTGATG AGTTCAGTTTTG 5GST-ST7N BamHI CGCGGATCCTTGCGCCCCCTTCT GGGAGGGGTT

Continued

279 Table B.2 continued

3GST-ST7N EcoRI CCGGAATTCTCACTGCTGAGAGC GTCGGTAACAGCCA 5GST-ST7C BamHI CGCGGATCCTTAATCCTACCCCC AGAACATATC 3GST-ST7C EcoRI CCGGAATTCTCAGTTTTGGAAAT GTTGAGATTG 5FLST7 SalI ACGCGTCGACCCGGGCCGGTGAA TCATCCCGGCAGACA 3FLST7 SalI ACGCGTCGACTTATTTGTCATCG TCGTCCTTGTAGTCGTTTTGGAA ATGTTGAGATTGAGGTGCGG 5ST7Histag XhoI CCGCTCGAGGCTGAAGCGGCCAC GGGCTTTCTG 3ST7Histag XhoI CCGCTCGAGTCAGTTTTGGAAAT GTTGAGATTG 5GST-NM23H1 BamHI CGCGGATCCGCCAACTGTGAGCG TACCTTCATTGC 3GST-NM23H1 EcoRI CCGGAATTCCTATTCATAGATCC AGTTCTGAGCAC 5FLNM23H1 BamHI CGCGGATCCTGCTGCGAACCACG TGGGTCCCGG 3FLNM23H1 EcoRI CCGGAATTCTTATTTGTCATCGT CGTCCTTGTAGTCTTCATAGATC CAGTTCTGAGCACA 5NMYC BamHI CGCGGATCCATGCCCCTCAACGT TAGCTTCACC 3NMYC EcoRI CCGGAATTCTCACAGCTTCTCTG AGACGAGCTTGGC 5MMYC BamHI CGCGGATCCGCCTCCTACCAGGC TGCGCGCAAA 3MMYC EcoRI CCGGAATTCTCACAACTCCGGGA TCTGGTCACGCAG 5CMYC BamHI CGCGGATCCGAAAACAATGAAAA GGCCCCCAAG

Continued

280 Table B.2 continued

3CMYC EcoRI CCGGAATTCCGCACAAGAGTTCC GTAGCTGTTC 5Gcn5 PstI AAAACTGCAGGGATTGGTAAGGG AAGACCGTGAGC 3Gcn5 KpnI CGGGGTACCTTATTTGTCATCGT CGTCCTTGATGTCATCAATAAGG TGAGAATATTCAGGTATTTC 5Esa BamHI CGCGGATCCGAGCTTTACCATTC TTTAGACGCTTCCTG 3Esa EcoRI CCGGAATTCTTATTTGTCATCGT CGTCCTTGTAGTCCCAGGCAAAG CGTAACTGAGAGGCAGTA 5actinB SalI ACGCGTCGACGCGGACTATGACT TAGTTGCGTTA 3actinB SalI ACGCGTCGACTAAGGTGTGCACT TTTATTCAACT 5b-globin SalI ACGCGTCGACGCTCGCTTTCTTG CTGTCCAATTT 3b-globin SalI ACGCGTCGACGCAATGAAAATAA ATGTTTTTTAT 5Luc HindIII CCCAAGCTTGCATTCCGGTACTG TTGGTAAAAT 3Luc HindIII CCCAAGTTACAATTTGGACTTTC CGCCCTTC 5PAD4 BamHI CGCGGATCCAGCCAGAGGGACGA GCTAGCCCGACG 3FL-PAD4 EcoRI CCGGAATTCTTACTATTTGTCAT CGTCGTCCTTGTAGTCGGGCACC ATGTTCCACCACTTGAAG 5mir96cDNA XbaI GCTCTAGAAGTGCCATCTGCTTG GCCGATTT 3mir96cDNA BamHI CGCGGATCCACGCGTTGCGGGTC CTGCTTTTCCCATATT 5mir19acDNA XbaI GCTCTAGACCCGGGATAGTTTTT GTTTGCAGTCCTCTGTT

Continued

281 Table B.2 continued

3 mir19acDNA BamHI CGCGGATCCATGCATGAAGGAAA TAGCAGGCCACCATCAGT 5mir19bcDNA XbaI GCTCTAGACCCGGGTCCTGTTAC TGAACACTGTTCTATGG 3mir19bcDNA BamHI CGCGGATCCTCTACAGACTTTTC ACTACCACAG 5mir25cDNA XbaI GCTCTAGAATGCATGAACTCCGG GACTGGCCAGTGTTGAG 3mir25cDNA BamHI CGCGGATCCACTAGTATCCGCAG TGTTGGGCCGGCACTGTC 5mir32cDNA XbaI GCTCTAGAACTAGTTGCTTGCTC TGGTGGAGATATTGCAC 3mir32cDNA BamHI CGCGGATCCCTGCAGCATGCACT CATGTGAAAATATCACAC 5mir92cDNA XbaI GCTCTAGAACGCGTGAAACTCAA ACCCCTTCTACACAGGT 3mir92cDNA BamHI CGCGGATCCCCCGGGGTCACAAT CCCCACCAAACTCAACAG

282 APPENDIX C

ANTIBODIES

Protein Immunogen Catalog Application detected no./Source tested BRG1 Unique N-terminal pep- Sif et al., 1998 WB, ChIP, IP tide BRM A BRM specific peptide Sif et al., 1998 WB, ChIP, IP BAF57 GST-BAF57 (aa 2-126) Covance Inc. WB, ChIP, IP BAF45/INI1 Dr. A.N. Im- IP balzano Flag epitope Flag peptide Covance Inc. WB, ChIP, IP PRMT5 GST-PRMT5 (aa 4-637) Covance Inc., WB, ChIP, IP Pal et al., 2003 PRMT5 GST-PRMT5 (aa 6-163) Covance Inc. WB, ChIP, IP, Im- munofloures- cence ST7 GST-ST7 (aa 325-585) Covance Inc. WB, Im- munofloures- cence

Continued

Table C.1: List of antibodies and sources Antibodies were either purchased or raised in NZW rabbits using the described im- munogen at Covance, Inc. The relevant reference is provided for the antibodies that had been generated previously in the lab.

283 Table C.1 continued

H3(Me2)R8 KHL conjugated to Covance Inc., WB, peptide containing sym- Pal et al., 2004 ChIP, Im- metrically methylated munofloures- arginine in the sequence cence [NH2-QTA(Me2)RKST QTA(Me2)RKST-COOH] H4(Me2)R3 KHL conjugated to pep- Covance Inc. WB, tide containing symmetri- ChIP, Im- cally methylated arginine munofloures- in the sequence [NH2- cence SG(Me2)RGKSG(Me2)RGK SG(Me2)RGK- COOH] Acetyl- Purchased 07-352, Upstate ChIP histone Biotechnology, H3(Lys9) Inc. MYC Purchased sc-40 (9E10), WB Santa Cruz sc-764 (N-262), ChIP, IP Santa Cruz MAD Purchased sc-222, Santa WB, ChIP Cruz, Inc. HDAC2 Purchased 51-5100 now WB, ChIP discontinued and replaced by 34-6400, Zymed mSIN3A Purchased sc-994 (K-20), WB Santa Cruz sc-767 (AK-11), ChIP, IP Santa Cruz Ha-RAS Purchased sc-35, Santa WB Cruz β-ACTIN Purchased A2066, Sigma WB α- Purchased CP06, Cal- WB TUBULIN biochem

284 APPENDIX D

GENES AFFECTED BY KNOCKDOWN OF PRMT5 IN NIH3T3 CELLS

Symbol Title Upfold P Set Mki67 antigen identified by monoclonal 7.59 99457 at antibody Ki 67 Mki67 antigen identified by monoclonal 7.48 161931 r at antibody Ki 67 Ptn pleiotrophin 7.16 97474 r at Hmgn2 high mobility group nucleosomal 6.45 101589 at binding domain 2 Sfrs1 splicing factor, arginine/serine-rich 6.35 160539 at 1 (ASF/SF2) Ect2 ect2 oncogene 5.71 97411 at Cspg2 chondroitin sulfate proteoglycan 2 5.69 100019 at Stmn1 stathmin 1 5.11 97909 at Gas1 growth arrest specific 1 5.03 94813 at Trip13 interac- 4.96 101372 at tor 13 Kif11 kinesin family member 11 4.87 99541 at

Continued

Table D.1: Genes up-regulated in anti-sense PRMT5 NIH3T3 using affymetrix high density expression arrays

285 Table D.1 continued

Tacc3 transforming, acidic coiled-coil 4.73 97238 at containing protein 3 2810429C13Rik RIKEN cDNA 2810429C13 gene 4.32 103071 at Met met proto-oncogene 4.23 100309 at 2810408K05Rik RIKEN cDNA 2810408K05 gene 4.17 94346 at Stk12 serine/threonine kinase 12 4.06 98469 at edr erythroid differentiation regulator 4 98525 f at Osf2-pending osteoblast specific factor 2 (fasci- 3.97 92593 at clin I-like) 1210001E11Rik RIKEN cDNA 1210001E11 gene 3.9 160182 at Cdc20 cell division cycle 20 homolog (S. 3.75 96319 at cerevisiae) Foxg1 forkhead box G1 3.72 161049 at Prim1 DNA primase, p49 subunit 3.67 96772 at Ncbp2 nuclear cap binding protein sub- 3.66 94509 at unit 2 Rbm3 RNA binding motif protein 3 3.66 96041 at Fignl1 fidgetin-like 1 3.58 160648 at Kif20a kinesin family member 20A 3.48 161856 f at Casp8ap2 caspase 8 associated protein 2 3.47 104290 at Kif23 kinesin family member 23 3.4 96168 at Rrm1 ribonucleotide reductase M1 3.38 100612 at Cdc25c cell division cycle 25 homolog C (S. 3.37 102934 s at cerevisiae) Hells helicase, lymphoid specific 3.35 93228 at Marcks myristoylated alanine rich protein 3.31 96865 at kinase C substrate 2810417H13Rik RIKEN cDNA 2810417H13 gene 3.3 93952 r at Kpna2 karyopherin (importin) alpha 2 3.29 92790 at Nr2f1 nuclear receptor subfamily 2, 3.26 102715 at group F, member 1 Hmgb2 high mobility group box 2 3.23 93250 r at Nr2f2 nuclear receptor subfamily 2, 3.2 103052 r at group F, member 2

Continued

286 Table D.1 continued

Pcnt2 pericentrin 2 3.15 99662 at Nme1 expressed in non-metastatic cells 3.13 92794 f at 1, protein (NM23A) (nucleoside diphosphate kinase) Mcmd4 mini chromosome maintenance de- 3.09 93041 at ficient 4 homolog (S. cerevisiae) AI181838 EST AI181838 3.07 161741 r at Slbp stem-loop binding protein 3.06 160471 at C1r complement component 1, r sub- 3.06 95415 f at component 2810408K05Rik RIKEN cDNA 2810408K05 gene 3.06 162094 f at Fmr1 fragile X mental retardation syn- 3.02 98441 at drome 1 homolog Gas2 growth arrest specific 2 2.99 94338 g at Has2 hyaluronan synthase 2 2.97 98865 at 5031439A09Rik RIKEN cDNA 5031439A09 gene 2.96 101001 at 2310034K10Rik RIKEN cDNA 2310034K10 gene 2.96 94389 at Gmnn geminin 2.95 160069 at 1110002B05Rik RIKEN cDNA 1110002B05 gene 2.91 96862 at 1810028N16Rik RIKEN cDNA 1810028N16 gene 2.91 97199 at 1810035L17Rik RIKEN cDNA 1810035L17 gene 2.85 96743 at Slc25a5 solute carrier family 25 (mitochon- 2.83 100617 at drial carrier; adenine nucleotide translocator), member 5 Odc ornithine decarboxylase, structural 2.83 160084 at 0610027F08Rik RIKEN cDNA 0610027F08 gene 2.81 97903 at Rcn2 reticulocalbin 2 2.8 93281 at Grcb gene rich cluster, B gene 2.79 92263 at Col6a2 procollagen, type VI, alpha 2 2.78 93517 at Ccnb2 cyclin B2 2.78 94294 at Nude-pending nuclear distribution gene E ho- 2.78 94910 at molog (Aspergillus) Fkbp4 FK506 binding protein 4 2.73 92809 r at

Continued

287 Table D.1 continued

Ttk Ttk protein kinase 2.73 103201 at St7 suppression of tumorigenicity 7 2.73 160591 at Slc38a4 solute carrier family 38, member 4 2.72 104286 at Idb2 inhibitor of DNA binding 2 2.72 93013 at C1r complement component 1, r sub- 2.69 161571 f at component 6330514M23Rik RIKEN cDNA 6330514M23 gene 2.68 97496 f at Cdk4 cyclin-dependent kinase 4 2.67 101017 at Rpa2 replication protein A2 2.67 98943 at G7e-pending G7e protein 2.65 104333 at Smarce1 SWI/SNF related, matrix associ- 2.65 96651 at ated, actin dependent regulator of chromatin, subfamily e, member 1 Cxcl12 chemokine (C-X-C motif) ligand 2.64 160511 at 12 D17H6S56E-2 DNA segment, Chr 17, human 2.63 101083 s at D6S56E 2 1110038L14Rik RIKEN cDNA 1110038L14 gene 2.61 97527 at Gas2 growth arrest specific 2 2.6 94337 at Eif4g2 eukaryotic translation initiation 2.59 100535 at factor 4, gamma 2 Racgap1 Rac GTPase-activating protein 1 2.58 94953 at Ndn necdin 2.57 101059 at Anp32b acidic nuclear phosphoprotein 32 2.56 96891 at family, member B Clcn4-2 chloride channel 4-2 2.55 104704 at Np95 nuclear protein 95 2.54 99564 at Slc35a1 solute carrier family 35 (CMP- 2.53 104380 at sialic acid transporter), member 1 Cetn3 centrin 3 2.52 92789 r at Loxl lysyl oxidase-like 2.51 103850 at Incenp inner centromere protein 2.51 93758 at Hspa4 heat shock protein 4 2.51 96594 at Pnp purine-nucleoside phosphorylase 2.5 93290 at

Continued

288 Table D.1 continued

Erh enhancer of rudimentary homolog 2.5 94040 at (Drosophila) Zfp26 zinc finger protein 26 2.5 102277 at Abcg2 ATP-binding cassette, sub-family 2.5 93626 at G (WHITE), member 2 Tia1 cytotoxic granule-associated RNA 2.49 160696 at binding protein 1 Cks1 CDC28 protein kinase 1 2.48 97468 at Nfib /B 2.46 160859 s at D4Wsu53e DNA segment, Chr 4, Wayne State 2.46 92542 at University 53, expressed Fin15 fibroblast growth factor inducible 2.46 97124 at 15 Nnt nicotinamide nucleotide transhy- 2.46 99009 at drogenase Prim2 DNA primase, p58 subunit 2.46 95549 at Gus beta-glucuronidase 2.46 97538 at Pkd2 polycystic kidney disease 2 2.45 100951 at 1110011K10Rik RIKEN cDNA 1110011K10 gene 2.45 95760 at Cpd1-pending cerebellar postnatal development 2.44 97345 at protein 1 5730408K05Rik RIKEN cDNA 5730408K05 gene 2.42 104195 at Apex1 apurinic/apyrimidinic endonucle- 2.42 93559 at ase 1 Gtf2i general transcription factor II I 2.42 94296 s at Mrpl20 mitochondrial ribosomal protein 2.42 94875 at L20 Cxcl12 chemokine (C-X-C motif) ligand 2.41 162234 f at 12 1500034E06Rik RIKEN cDNA 1500034E06 gene 2.41 97271 at 3010002G01Rik RIKEN cDNA 3010002G01 gene 2.41 99179 at 2310006I24Rik RIKEN cDNA 2310006I24 gene 2.4 95075 at Pcdh7 protocadherin 7 2.39 102280 at

Continued

289 Table D.1 continued

Gcat glycine C-acetyltransferase (2- 2.35 160628 at amino-3-ketobutyrate-coenzyme A ligase) Tubb2 tubulin, beta 2 2.35 94835 f at 2310003F16Rik RIKEN cDNA 2310003F16 gene 2.34 95408 at D4Ertd786e DNA segment, Chr 4, ERATO Doi 2.34 96059 at 786, expressed LOC228410 similar to cleavage stimulation fac- 2.33 100968 at tor subunit 3 Emp3 epithelial membrane protein 3 2.33 93593 f at Cd34 CD34 antigen 2.32 97773 at LOC218397 similar to Ras GTPase-activating 2.32 99467 at protein 1 (GTPase-activating pro- tein) (GAP) (Ras p21 protein ac- tivator) (p120GAP) (RasGAP) Eppb9 endothelial precursor protein B9 2.32 98992 at Sfrp2 secreted frizzled-related sequence 2.31 93503 at protein 2 Ccne2 cyclin E2 2.31 104142 at C530002L11Rik RIKEN cDNA C530002L11 gene 2.3 96710 at Cpd1-pending cerebellar postnatal development 2.28 97556 at protein 1 AU015605 expressed sequence AU015605 2.28 97853 at 2410026K10Rik RIKEN cDNA 2410026K10 gene 2.27 101047 at Tardbp TAR DNA binding protein 2.27 160377 at Tmem5 transmembrane protein 5 2.27 160642 at 1010001J12Rik RIKEN cDNA 1010001J12 gene 2.27 94455 at Hnrpl heterogeneous nuclear ribonucleo- 2.27 95232 at protein L Igbp1 immunoglobulin (CD79A) binding 2.26 162460 f at protein 1 Ahcy S-adenosylhomocysteine hydrolase 2.26 96024 at Ptov1 prostate tumor over expressed gene 2.26 99600 at 1 Tmpo thymopoietin 2.25 98982 at

Continued

290 Table D.1 continued

2400006A19Rik RIKEN cDNA 2400006A19 gene 2.24 160239 at H2afx H2A histone family, member X 2.24 93019 at Mcmd2 mini chromosome maintenance de- 2.24 93112 at ficient 2 (S. cerevisiae) 1010001C05Rik RIKEN cDNA 1010001C05 gene 2.24 93786 i at Ahcy S-adenosylhomocysteine hydrolase 2.24 96025 g at Ptprk protein tyrosine phosphatase, re- 2.23 160760 at ceptor type, K 0610006H08Rik RIKEN cDNA 0610006H08 gene 2.23 97318 at Slc25a5 solute carrier family 25 (mitochon- 2.21 100618 f at drial carrier; adenine nucleotide translocator), member 5 Cetn2 centrin 2 2.21 104733 at 2610016F04Rik RIKEN cDNA 2610016F04 gene 2.21 98893 at Carm1-pending coactivator-associated arginine 2.21 99169 at methyltransferase 1 Psmc3 proteasome (prosome, macropain) 2.2 93735 f at 26S subunit, ATPase 3 Imp4a-pending importin 4a 2.2 95034 f at Mtap4 microtubule-associated protein 4 2.19 92795 at Ifitm3l interferon induced transmembrane 2.19 93018 at protein 3-like Bat8 HLA-B associated transcript 8 2.19 97809 at Nfib nuclear factor I/B 2.19 99440 at Tyms 2.18 93237 s at 1500010B24Rik RIKEN cDNA 1500010B24 gene 2.18 93358 at 2610524G07Rik RIKEN cDNA 2610524G07 gene 2.18 93774 at 4930512K19Rik RIKEN cDNA 4930512K19 gene 2.18 96029 at Cops3 COP9 (constitutive photomor- 2.18 99113 at phogenic) homolog, subunit 3 (Arabidopsis thaliana) Ilf3 interleukin enhancer binding factor 2.17 160657 at 3 Hn1 hematological and neurological ex- 2.17 93276 at pressed sequence 1

Continued

291 Table D.1 continued

Rbl1 retinoblastoma-like 1 (p107) 2.17 104476 at 0610009E20Rik RIKEN cDNA 0610009E20 gene 2.16 160267 at Pdcd6ip programmed cell death 6 interact- 2.16 96252 at ing protein Arhj ras homolog gene family, member 2.15 104697 at J Sart3 squamous cell carcinoma antigen 2.15 161432 f at recognized by T-cells 3 Ech1 enoyl coenzyme A hydratase 1, 2.14 93754 at peroxisomal Nono non-POU-domain-containing, oc- 2.14 93830 at tamer binding protein 1110019J04Rik RIKEN cDNA 1110019J04 gene 2.14 95409 at U2af2 U2 small nuclear ribonucleoprotein 2.14 98404 at auxiliary factor (U2AF) Irak1 interleukin-1 receptor-associated 2.14 98595 at kinase 1 Cfdp craniofacial development protein 1 2.13 93784 at 2300002G02Rik RIKEN cDNA 2300002G02 gene 2.13 96626 at 2410071B14Rik RIKEN cDNA 2410071B14 gene 2.13 97896 r at 5031422I09Rik RIKEN cDNA 5031422I09 gene 2.13 96187 at Fstl follistatin-like 2.12 94833 at Anapc5 anaphase-promoting complex sub- 2.12 95100 at unit 5 Ppp1r7 protein phosphatase 1, regulatory 2.12 97979 at (inhibitor) subunit 7 Ctps2 cytidine 5’-triphosphate synthase 2 2.07 160652 at Calm3 calmodulin 3 2.07 92632 at Cpt1a carnitine palmitoyltransferase 1, 2.07 93320 at liver Hnrpa1 heterogeneous nuclear ribonucleo- 2.07 97272 at protein A1 Nfkb1 nuclear factor of kappa light chain 2.07 98427 s at gene enhancer in B-cells 1, p105

Continued

292 Table D.1 continued

2610016F04Rik RIKEN cDNA 2610016F04 gene 2.07 98894 at Psmb5 proteasome (prosome, macropain) 2.06 101558 s at subunit, beta type 5 1500002I10Rik RIKEN cDNA 1500002I10 gene 2.06 104305 at Gstz1 glutathione transferase zeta 1 (ma- 2.06 160350 at leylacetoacetate isomerase) Cdk4 cyclin-dependent kinase 4 2.06 160538 at Nol5 nucleolar protein 5 2.06 92569 f at Strap serine/threonine kinase receptor 2.06 94292 at associated protein Mrps21 mitochondrial ribosomal protein 2.06 94912 at S21 6230416J20Rik RIKEN cDNA 6230416J20 gene 2.06 99777 s at Chrac1 chromatin accessibility complex 1 2.05 100126 at Plagl1 pleiomorphic adenoma gene-like 1 2.05 92502 at 1110013G13Rik RIKEN cDNA 1110013G13 gene 2.05 103881 at Pros1 protein S (alpha) 2.05 104728 at Snrpa small nuclear ribonucleoprotein 2.04 100101 at polypeptide A 1110049G11Rik RIKEN cDNA 1110049G11 gene 2.04 104077 at Ube2e3 ubiquitin-conjugating enzyme E2E 2.04 93033 at 3, UBC4/5 homolog (yeast) 0610033H09Rik RIKEN cDNA 0610033H09 gene 2.04 94518 at 9430010O03Rik RIKEN cDNA 9430010O03 gene 2.04 95021 at Amd1 S-adenosylmethionine decarboxy- 2.11 101489 at lase 1 6330544B05Rik RIKEN cDNA 6330544B05 gene 2.11 98084 at MGC36997 hypothetical protein MGC36997 2.11 161076 at Ivd isovaleryl coenzyme A dehydroge- 2.1 104153 at nase Evl Ena-vasodilator stimulated phos- 2.1 160667 at phoprotein 2900010I05Rik RIKEN cDNA 2900010I05 gene 2.1 93581 at

Continued

293 Table D.1 continued

C330027G06Rik RIKEN cDNA C330027G06 gene 2.09 100410 at Gja1 gap junction membrane channel 2.09 100065 r at protein alpha 1 5430409I18Rik RIKEN cDNA 5430409I18 gene 2.09 160959 at Capg capping protein (actin filament), 2.08 160106 at gelsolin-like Echs1 enoyl Coenzyme A hydratase, 2.08 95426 at short chain, 1, mitochondrial Nup88 nucleoporin 88kDa 2.08 98146 at Cyp1b1 cytochrome P450, 1b1, 2.08 99979 at benz[a]anthracene inducible 3300001P08Rik RIKEN cDNA 3300001P08 gene 2.08 160791 at Ubc-rs2 ubiquitin C, related sequence 2 2.07 160165 at Pole2 polymerase (DNA directed), ep- 2.03 101920 at silon 2 (p59 subunit) 9430065L19Rik RIKEN cDNA 9430065L19 gene 2.03 160368 at Ctbp1 C-terminal binding protein 1 2.02 101081 at Rfc5 replication factor C (activator 1) 5 2.02 95612 at (36.5 kDa) 5730406I15Rik RIKEN cDNA 5730406I15 gene 2.02 96855 at Ywhab tyrosine 3-monooxygenase /tryp- 2.02 98053 at tophan 5-monooxygenase activa- tion protein, beta polypeptide Ncam1 neural cell adhesion molecule 1 2.01 100153 at 3110056M06Rik RIKEN cDNA 3110056M06 gene 2.01 103356 at 5730507C05Rik RIKEN cDNA 5730507C05 gene 2.01 104586 at Grim19-pending genes associated with retinoid- 2.01 93764 at IFN-induced mortality 19 KIAA0678 hypothetical protein KIAA0678 2.01 94422 at Set SET translocation 2.01 94456 at Nfyc nuclear transcription factor-Y 2.01 94968 at gamma Tgfbi transforming growth factor, beta 2.01 92877 at induced, 68 kDa

294 Symbol Title Downfold P Set H2-T23 histocompatibility 2, T region lo- 22.27 97173 f at cus 23 Ppie peptidylprolyl isomerase E (cy- 14.81 102278 at clophilin E) Pgk2 phosphoglycerate kinase 2 12.18 101388 at H2-K histocompatibility 2, K region 6.58 99379 f at Krt1-17 keratin complex 1, acidic, gene 17 5.96 92861 i at Krt1-17 keratin complex 1, acidic, gene 17 5.71 92862 f at 3930401K13Rik RIKEN cDNA 3930401K13 gene 5.28 93969 at Ckb creatine kinase, brain 4.59 93126 at Af1q-pending ALL1-fused gene from chromo- 4.4 95503 at some 1q H2-Q1 histocompatibility 2, Q region lo- 4.33 99378 f at cus 1 Canx calnexin 4.26 160180 at Serpine1 serine (or cysteine) proteinase in- 3.87 94147 at hibitor, clade E, member 1 Fabp4 fatty acid binding protein 4, 3.72 100567 at adipocyte Pva parvalbumin 3.29 96720 f at Lcn2 lipocalin 2 3.01 160564 at Gzma granzyme A 2.97 102995 s at Myt1l myelin transcription factor 1-like 2.72 96495 at H2-D1 histocompatibility 2, D region lo- 2.7 101886 f at cus 1 Tuba7 tubulin, alpha 7 2.68 93924 f at V2r1 vomeronasal 2, receptor, 1 2.65 95797 f at Thbd thrombomodulin 2.47 162023 f at Tnfrsf9 tumor necrosis factor receptor su- 2.46 97683 at perfamily, member 9

Continued

Table D.2: Genes down-regulated in anti-sense PRMT5 NIH3T3 using affymetrix high density expression arrays

295 Table D.2 continued

Tcf15 transcription factor 15 2.46 97717 at P5-pending protein disulfide isomerase-related 2.35 94209 g at protein Ccrn4l CCR4 carbon catabolite repres- 2.35 162384 f at sion 4-like (S. cerevisiae) Gata4 GATA binding protein 4 2.32 102713 at Cyr61 cysteine rich protein 61 2.32 92777 at Klra7 killer cell lectin-like receptor, sub- 2.32 97762 f at family A, member 7 Sel1h Sel1 (suppressor of lin-12) 1 ho- 2.27 92870 at molog (C. elegans) Vcam1 vascular cell adhesion molecule 1 2.25 92559 at Klra7 killer cell lectin-like receptor, sub- 2.24 97761 f at family A, member 7 Gpr14 G protein-coupled receptor 14 2.21 95306 at AI447804 expressed sequence AI447804 2.2 161791 r at Nupr1 nuclear protein 1 2.16 160108 at Tob1 transducer of ErbB-2.1 2.14 99532 at Efnb3 ephrin B3 2.12 103692 at Pabpc2 poly A binding protein, cytoplas- 2.1 101938 at mic 2 Il1a interleukin 1 alpha 2.1 94755 at Tcrg-V4 T-cell receptor gamma, variable 4 2.05 102745 at Tieg TGFB inducible early growth re- 2.05 99602 at sponse Mettl3 methyltransferase-like 3 2.04 160929 at Cnr1 cannabinoid receptor 1 (brain) 2.03 99892 at Tctex1 t-complex testis expressed 1 2.01 93516 at

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