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R .. . , . , . . _ fmsuukaofihivfim. .5: . wins“ ' LIBRARY # J 0‘ Michigan .5!!!“ University This is to certify that the dissertation entitled REGULATION OF THE HUMAN U6 SMALL NUCLEAR RNA TRANSCRIPTION BY THE RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN presented by THARAKESWARI SELVAKUMAR has been accepted towards fulfillment of the requirements for the PhD. degree in Cell and Molecular Biology 5m- Major Profy’ésor’s Signature 007‘ 01 / L00 8 —/ Date MSU is an Affirmative Action/Equal Opportunity Employer PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K:IProj/Acc&PralelRC/DateDue.indd REGULATION OF THE HUMAN U6 SMALL NUCLEAR RNA TRANSCRIPTION BY THE RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN By Tharakeswari Selvakumar A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Cell and Molecular Biology 2008 ABSTRACT REGULATION OF THE HUMAN U6 SMALL NUCLEAR RNA TRANSCRIPTION BY THE RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN By Tharakeswari Selvakumar The Retinoblastoma tumor suppressor (RB) protein restricts unregulated cell division by repressing transcription of genes whose products are essential for cell growth and division. RB represses a subset of RNA Polymerase (Pol) II- transcribed genes that contain E2F binding sites and are important for progression into S-phase. RB also acts as a general repressor of P01 1 and Pol III transcription. Considering that Pol I and Pol III transcribed products are essential for cell growth and division, repression of transcription by Pol I and Pol 111 can be an important aspect of the growth-suppressive function of RB. The growth inhibitory fimction of RB is linked to its anti-tumorigenic potential, and it is likely that understanding RB repression of Pol I and Pol III transcription can elucidate some important aspects of the RB tumor suppression mechanism. My study examines the mechanism for RB repression of U6 transcription by Pol III. Sequence comparison among the nine U6 copies in humans revealed that all the functional U6 copies are enriched in CpG dinucleotides at the promoter regions compared to the non-functional copies, and as the CpG sequence is the primary target for methylation in humans, this indicated a potential involvement of DNA methylation in regulation of U6 transcription. Existing evidence indicating a repressive role for DNA methylation on transcription, led to the hypothesis that DNA methylation contributes to U6 repression. In support, in vitro transcription from pre-methylated U6 templates demonstrated that DNA methylation has an inhibitory effect on U6 transcription. Earlier studies indicated that RB interacts with DNA methyltransferase (DNMT) l and DNMTI activity contributes to RB repression of E2F- transactivated Pol II transcription. Consistently, RB was found to direct recruitment of DNA methylation and DNA methyltransferases 1 and 3A to the U6 promoter during repression of Pol III transcription. Also, siRNA-mediated depletion of DNMTI, 3A and 3B in RB positive cells resulted in enhanced U6 transcription, suggesting a repressive role for DNA methyltransferases in U6 transcription. The results presented here implicate RB-directed promoter DNA methylation as an important aspect of the mechanism for RB-mediated repression of human U6 transcription. ACKNOWLEDGEMENTS I want to say many thanks to my mentor Dr. R. William Henry for being very supportive throughout the course of my PhD. His guidance has been instrumental in the successful completion of my thesis project. I would like to thank my graduate committee members Dr. Susan Conrad, Dr. Jon Kaguni, Dr. Min-Hao Kuo and Dr. Steve Triezenberg for their guidance and support. I would also like to thank the CMB program at Michigan State University for giving me the opportunity to pursue my doctoral study. I am also grateful to Dr. R. William Henry, Dr. Steve Triezenberg, Dr. David Amosti and Dr. Zachary Burton for helping me in securing a post doctoral position. I wish to thank all the previous Henry lab members —- Gauri Jawdekar, Anastasia Gridasova, Xianzhou Song, Craig Hinkley, Zakir Ullah, Liping Gu, Heather Hirsch and the current Henry lab members — Stacy Hovde, Nitin Raj and Alison Gjidoda for their help and support. I wish to thank my parents Mr. S. Selvakumar and Mrs. Umarani Selvakumar for their limitless love and patience, whose support has held me strong always. I wish to thank my brother Prem, sister Pragathi, all my cousins and the rest of my family in India for their love and affection. I wish to thank my dear friends Priya Raman, Srividhya Kidambi and Dr. Pooma Viswanathan for their wonderful company during my years at Michigan State University and for their life-long friendship. iv TABLE OF CONTENTS LIST OF FIGURES ...................................................................... vii KEY TO SYMBOLS AND ABBREVIATIONS .................................... ix CHAPTER 1: Introduction .................................................................................. 1 1. The Retinoblastoma Protein ....................................................... 1 2. RB functional domains ............................................................ 2 3. RB-E2F pathway ................................................................... 5 4. RB repression of Pol I and Pol II transcription .................................. 6 5. RB repression of RNA Polymerase III transcription ........................... 7 6. Mechanism for RB repression of RNA Polymerase III transcription ....... 9 7. RB represses U6 snRNA gene transcription .................................. 15 8. RB co-repressor proteins .......................................................... 18 8.]. DNA Methyltransferases ............................................... 19 8.1.1. DNA methylation mechanism .................................. 25 8.1.2. DNA methylation and cancer .................................. 25 8.1.3. DNA methylation and transcriptional repression ........... 30 8.1.4. DNA demethylation ............................................. 31 8.1.5. Non-CpG methylation in mammalian DNA ................. 33 8.1.6. Methyl CpG binding proteins ................................. 35 8.2. Histone deacetylases and RB-mediated repression ................ 38 8.3. SWI/SNF and RB mediated repression ............................... 41 8.4. Other co-repressor proteins ............................................. 42 8.4.1. Histone methyltransferases ..................................... 42 8.4.2. Topoisomerases .................................................. 43 8.4.3. Polycomb (PcG) group proteins ............................... 43 8.5. Summary .................................................................. 44 References ................................................................................... 46 CHAPTER 2: The Retinoblastoma tumor suppressor protein represses U6 snRNA transcription by a mechanism involving promoter DNA methylation. . . ......76 Abstract ............................................................................ 76 Introduction ........................................................................ 77 Materials and Methods ............................................................ 81 Results .............................................................................. 91 Discussion ........................................................................ 1 3 7 References ......................................................................... 142 CHAPTER [11: Summary ............................................................................... 148 Summary... 148 References ........................................................................ 155 APPENDIX: AP-l. The Retinoblastoma tumor suppressor protein induces a double-stranded break in template DNA ............................ 158 AP-2. HDACs 1 and 2 associate with the U6 promoter in RB positive cells .............................................................. 171 AP-3. The SWI/SNF component BRGI associates with the U6 promoter in RB positive cells ......................................... 172 AP-4. GST-RB expressed in E.Coli co-purifies with two predominant RNA species ............................................. 178 AP-5. Stimulatory effect of S-adenosyl homocysteine on Msp I methyltransferase function ............................................. 181 Materials and Methods .......................................................... 185 References ........................................................................ 189 vi Figure 1-1. Figure 1-2. Figure 1-3. Figure 1-4. Figure 1-5. Figure 1-6. Figure 2-1. Figure 2-2. Figure 2-3. Figure 2-4. Figure 2-5. LIST OF FIGURES Schematic representation of the RB functional domains. . .4 Types of RNA Polymerase III promoters ................... 11 General transcription machinery for RNA Polymerase III transcription .................................................. 13 The U6 snRNA transcription machinery .................. 17 Schematic representation of the DNMT family proteins ............................................................ 21 Proposed mechanism for DNA methylation ................. 27 CpG plot of all the nine U6 copies ............................ 93 The U6 start site CpG is methylated in vivo in MCF7 but not HeLa cells ............................................... 96 The U6 start site CpG gets methylated in vivo in HeLa cells in response to transient RB expression ............. 100 The start site CpG of the U6 snRNA gene becomes methylated in response to nuclear extract and GST-RB addition in vitro ................................................. 105 CpG methylation leads to reduced U6 transcription....112 vii Figure 2-6. Figure 2-7. Figure 2-8. Figure 2-9. Figure AP-1.l. Figure AP-l.2. Figure AP-l.3. Figure AP-1.4. Figure AP-2. Figure AP-3. Figure AP-4. Figure AP-S. Start site proximal cytosine methylation adds to the repressive effect caused by promoter CpG methylation on U6 repression ............................................... 115 DNMTl association with the endogenous U6 promoter coincides with RB activity status ................ 122 RB recruits DNMTl and DNMT3A to the endogenous U6 promoter ..................................... 130 Depletion of DNMTI, 3A and 3B in RB positive cells causes upregulation of U6 transcription .................... 135 RB causes a double stranded break in the U6 template DNA in vitro .................................................... 160 RB-induced double strand break is dependent on ATP... 164 Topoisomerases 11a and 1113 occupy the endogenous U6 promoter .................................................... 167 MO; probing to detect open complex formation ....................................................... 170 DNMTs 1 and 3A and HDACs l and 2 occupy the endogenous U6 promoter in U2OS but not SAOS cells..174 The SWI/SNF ATPase BRGl occupies the endogenous U6 promoter ..................................... 177 GST-RB expressed in E. Coli co-purifies with two predominant RNA species .................................... 180 S-adenosyl homocysteine (SAH) has a stimulatory effect on methyltransferase activity of MspI ............... 184 viii ATP de1 Brf— l Brf—2 Cdk ChIP C-Terminal Da DHFR DNA DNMT DSE GAPDH HDAC ICR MeCP MBD NTP PcG PCR KEY TO SYMBOLS AND ABBREVIATIONS Adenosine triphosphate B double prime 1 protein TFIIB related factor 1 TFIIB related factor 2 Cyclin-dependent kinase Chromatin immunoprecipitation Carboxy-terminal Dalton Dihydrofolate reductase Deoxyribonucleic acid DNA methyltransferase Distal sequence element Glyceraldehyde 3- phosphate dehydrogenase Histone deacetylase Internal control region Methyl CpG binding protein Methyl binding domain Nucleotide triphosphate Polycomb group complex Polymerase chain reaction ix Pol PSE rRNA RT-PCR SAM SAH SNAPc snRNA TBP TFIIB TFIIIA TFIIIB TFIIIC Topo tRNA Polymerase Proximal sequence element Retinoblastoma tumor suppressor protein Ribonucleic acid ribosomal RNA Reverse transcriptase polymerase chain reaction S-adenosyl methionine S-adenosyl homocysteine Small nuclear RNA activating protein complex Small nuclear RNA TATA binding protein Transcription factor IIB Transcription factor IIIA Transcription factor IIIB Transcription factor IlIC Topoisomerase transfer RNA CHAPTER I INTRODUCTION 1. The Retinoblastoma Protein The human Retinoblastoma susceptibility gene (RBI) was identified at genetic locus 13q14 on account of homozygous deletions and tumor-specific expression abnormalities in cases of retinoblastomas and sarcomas (53, 56, 116). The product of the RBI gene, the Retinoblastoma protein (RB), controls the cell- cycle and facilitates Gl growth arrest (92, 155, 167, 221). As a result, RB exerts an anti-proliferative effect against unregulated cell growth and division and fimctions as a checkpoint against tumorigenesis (66). RB binds and inactivates E2F transactivator proteins (E2Fs) 1, 2 and 3 (117). Some of the E2F-activated genes that are important for cell cycle progression include c-myc, B-myb, cdc2, dihydrofolate reductase and thymidine kinase (18, 25, 41, 146, 210). These genes contain variant forms of the consensus nucleotide sequence TTTCGCGC for E2F binding in their promoters (112, 146). RB is thought to repress transcription of E2F target genes by binding and inactivating E2F (51, 68, 179). Evidence suggesting active repression of transcription by the RB-E2F complex at targeted gene promoters containing E2F binding sites has been reported (44, 113, 167, 191, 222). The hypophosphorylated form of RB is thought to be the active form whereas the hyperphosphorylated form is inactive with respect to transcriptional repression at E2F target genes (25, 27). Cyclins D1, D2 and D3 as complexes with CDK4/CDK6 (47, 102, 221) and the Cyclin E-CDK2 complex (78) phosphorylate RB and the resultant inactivation during mid to late G1 permits cell cycle progression into S phase (47, 78, 102, 221). 2. RB functional domains RB contains the A and B pocket domains that are common to the related proteins p107 and p130, and which are involved in regulating cell cycle progression (Figure 1-1) (30, 36, 46). The A (amino acids 379-572) and B pocket (amino acids 646-772) domains (85, 88, 97, 107) are conserved across species (31, 115) and are crucial for the tumor suppressor function of RB (166). The spacer region from amino acids 573-645 serves a structural role to facilitate folding of the NB pocket. The amino acid sequence of the spacer is not crucial for A/B pocket activity (85, 88, 107).Viral oncoproteins such as the adenoviral ElA, SV40 large T antigen and human papillomavirus E7 interact with RB at the A/B pocket domain to inhibit RB function in infected cells (30, 85, 86, 88, 97). The region encompassing amino acids 379-928 comprising the NB pocket and the C terminal regions was found to be the minimal region required for tumor suppression (241). Many naturally occurring mutations observed in the Rb locus in tumors disrupt the pocket domain (63, 83) indicating that the pocket domain plays a crucial role in tumor suppression by RB. Factors that interact with RB at the pocket domain such as viral oncoproteins (115, 129, 232) and co-repressor proteins such as HDACs land 2 and BRGl (20, 43, 130, 132), contain an LXCXE motif that mediates their binding to RB. Structural analysis of the pocket domain Figure 1-1: Schematic representation of the RB functional domains. The A (amino acids 379-572) and B pocket (amino acids 646-772) domains (85, 88, 97) and the C pocket (amino acids 772-870) (225) are represented. The A/B pocket domain is involved in binding to proteins containing an LXCXE motif, such as viral oncoproteins (115, 129, 232) and co-repressor proteins such as HDACs 1 and 2 and BRGl (20, 43, 130, 132). The spacer region is shown (amino acids 572-646) (85, 88). The Large Pocket (amino acids 379-870) comprising the NB pocket and the C pocket which is involved in E2F binding is shown (77, 166). C terminal binding site for c-abl and MDM2 (at amino acids 772-870) (225, 238) are indicated. £32m uEBmanEuom on... «gm 9.5% mum mm» «9.00m mans.— aha 23 @533 «20.2 .590 mum at. EOE 585 3:3 N2. 52:0“. “932. m< 2.” :oooll .0 High . < _ cum Ohm NE. o3 mum ohm «IZ showed that the B domain contains the LXCXE binding site (115). However, the A domain was found to be important for maintenance of the active conformation of the B domain (105, 115). E2Fs do not contain an LXCXE motif, and they bind to RB via its Large Pocket domain comprising the NE and C pocket (amino acids 379-870) regions (77, 87, 107, 115, 166, 225). The C Pocket domain (amino acids 772-870) binds c-abl tyrosine kinase and MDM2 (107, 225, 238). The tyrosine kinase function of c-abl is inhibited when it is bound to RB (225) and this interaction is observed to be important for grth suppression by RB (226). The significance of the interaction between RB and MDM2 is not well characterized (66). The amino terminal region of RB contains cdk phosphorylation sites (31) and is thought to contribute to the tumor suppressive effect of RB (66, 173). However, contradictory evidence indicating that the removal of the amino- terrninal region of RB led to enhanced tumor suppressive potential of RB has also been presented (166, 240). 3. RB-EZF pathway The role of the RB-E2F regulatory mechanism in cell cycle control has been studied extensively (92, 167, 255). Overexpression of dominant-negative DPl, which is an E2F binding partner, inhibits progression in S phase and highlights the importance of E2F function and interaction with DPl in S phase progression (237). In another study, E2F3 knockout mice were delayed in entering S phase (89). In tumors triggered by T antigen expression, which inactivates RB (and p53) leading to the release of E2F, backcrossing the mice in an E2Fl-/- background impaired tumor growth. This suggested that the release of E2F] is important for tumor growth when RB is inactivated (202). Also, crossing RB -/- mice into an E2Fl_-/- background caused a significant reduction in ectopic cell cycle entry in the CNS and lens compared to RB-/- only mice (212). These lines of evidence suggest that the RB-E2F genetic interaction is involved in regulating cell cycle progression and tumor growth. 4. RB repression of Pol I and Pol II transcription RB represses transcription by RNA Polymerases I, II and III (227, 230). This section will focus on RB repression of Pol I and E2F-mediated Pol II transcription. Studies done by Cavanaugh et al., (23) have found that RB can inhibit Pol I transcriptional activation by UBF by binding to UBF through the RB pocket domain. RB can repress E2F-mediated Pol II transcription by binding to the E2F activation domain directly and blocking the transactivation function of E2F (51, 68). A second mechanism where RB recruits co-repressor complexes to E2F-target promoters has also been proposed (66). HDAC activity is required for RB repression of two E2F target genes, thymidine kinase and DHFR (130). hBRM (part of the hSWI/SNF chromatin remodeling complex) can cooperate in RB-mediated repression of E2F transcriptional activation (211). RB-mediated growth arrest can also occur in an E2F-independent manner. Overexpression of RB continues to cause growth arrest in the presence of a dominant negative E2F, suggesting that there are other additional RB targets for growth arrest (66, 248). Possibly, RB repression of general Pol I and Pol III transcription may be another important aspect of its growth arrest and tumor suppression mechanism. 5. RB repression of RNA polymerase III transcription RB represses general RNA polymerase III transcription (230). On comparing two osteosarcoma cell lines and the RB negative SAOSZ cells had elevated levels of Pol III transcription relative to the RB positive U208 cells (230). Moreover, primary fibroblasts from RB knockout mice had higher Pol III activity when compared to equivalent cells from wild type mice, suggesting that RB causes repression of Pol III transcription (230). RB repression of Pol III transcription can contribute to its anti-growth effect. Pol III-transcribed products such as the U6 snRNA, tRNA, and SSrRNA are required for key cellular processes such as protein synthesis and splicing which are important for normal cellular growth rate and division. During rapid growth and cell division in tumors, elevated levels of biosynthesis of cellular products are needed, thereby raising the demand for higher cellular levels of the protein synthesis (227) and possibly splicing machinery. When quiescent cells were subject to mitogenic stimulation to grow and divide, production of rRNA- and tRNA increased along with a corresponding increase in protein synthetic functions (178). The rate of growth is directly proportional to the rate of protein accumulation which in turn is dependent on the rate of protein synthesis (6). These results suggest a clear correlation between the cellular levels of the protein synthesis to the cellular growth rate (227). Therefore it is possible that by repressing transcription of Pol 111 products, RB exerts its anti-growth effect. As RB executes its tumor suppressor firnction by acting as a checkpoint against unwarranted cellular biosynthesis, growth and division (66, 221, 227), RB mediated repression of Pol III transcription could be an integral aspect of its tumor suppression function. One study has demonstrated that the activities of Pol III (and also P01 1) are elevated in murine tumors, whereas Pol II activity remained unaffected (189). The RB domains required for tumor suppression and those required for repressing Pol III transcription largely coincide (23, 230), indicating a potential link between the Pol III repression and tumor suppression functions of RB. The region encompassing amino acids 379-928 in the RB protein was the minimal region required for tumor suppression. This region was also the minimal region required for efficient repression of Pol III transcription. On the contrary, the region between amino acids 379-792 was the minimal region for repression of Pol II transcription. This suggests that tumor suppression by RB requires other additional functions apart from Pol II transcriptional repression, possibly, repression activity targeting Pol III transcription. Also many transforming agents such as oncoproteins from the simian virus 40, papovavirus, hepatitis B virus and chemical carcinogens cause elevated Pol III transcription (49, 133, 190, 194, 219, 229). Naturally-occurring mutations that inactivate RB as a ttunor suppressor were found to render RB inactive for repression of Pol III transcription also (103, 221, 231). Also, adenoviral ElA oncoprotein that binds and inactivates RB for tumor suppression inactivates RB as a repressor of Pol III transcription (230). These evidence lend support to the idea that the tumor suppressor ftmction and Pol III repressor functions of RB are linked. Detailed studies focused on understanding the mechanism for RB repression of Pol III transcription can help us gain insights into its tumor suppression mechanisms. 6. Mechanism for RB repression of Pol III transcription Pol III promoters have been classified as types 1 (SS rRNA), 2 (AdVAI and tRNA) and 3 (U6 snRNA) (Figure 1-2) (186). The type I Pol III promoters contain an A box, C Box and the Intermediate element (IE) together comprising the Internal Control Region (ICR) (19, 158-160). The type 2 promoters in AdVAI and tRNA genes consist of an A box and a B box (1, 57, 81, 192). The type III promoters found in U6 snRNA, 78K and H1 RNA genes are extragenic and consist a TATA Box, Proximal Sequence Element (PSE) and Distal Sequence Element (DSE) (76, 111, 126, 135). The TFIIIA, TFIIIB and TFIIIC factors are required for transcription of type I Pol III promoters, whereas only TFIIIB and TFIIIC are required in the case of type 2 promoters (Figure 1-3). TFIIIA is a Zinc finger containing DNA binding protein that binds to the ICR (Internal Control Region) of type I promoters (139). TFIIIB used in both type 1 and type 2 promoters consists of TBP, del and Brfl whereas the type 3 promoter uses TFIIIB that comprises TBP, del and Brf2 (127, 140, 186, 187, 203, 208, 209, 220,228,233) Figure 1-2: Types of RNA polymerase III promoters. Type 1 promoter in SS rRNA gene has an internal control region (ICR) consisting an A box, intermediate element and C box. Type 2 promoter found in tRNA genes consists of an A box and a B box. Type 3 promoter in the U6 snRNA gene consists of a DSE (Distal Sequence Element), Proximal Sequence Element (PSE) and TATA Box. Tn represents the termination signal. Diagram adapted from (186). 10 Type 1 (5S rRNA) Type 2 (tRNA) Type 3 (U6 snRNA) +1 A C DSE —’ Box IE Box m— L____J Internal Control Region (ICR) +1 A Box BBox T“ +1 -> /—-fl—D—' ,___ PSE TATA Tn Figure 1-3: General transcription machinery for RNA polymerase III transcription. The type I promoter requires TFIIIA, TFIIIC and Brfl -TFIIIB. TFIIIC and Brfl-TFIIIB are required for transcription from type 2 promoters. Type III promoter requires Brfl-TFIIIB, and SNAPc. Octl enhances type 3 transcription but is not essential (186). 12 Type 1 (ss rRNA) Type 2 (tRNA, AdVAI) Type 3 (U6 snRNA) Based on studies carried out on Alu and AdVAI transcription (that use the Brfl -TFIIIB), Chu et al., (32) proposed that RB can inhibit transcription of Pol III-transcribed genes that use Brfl -TFIIIB which are the type 1 and type 2 Pol III promoters, by binding to the Brfl component of TFIIIB via the RB A domain, and to TFIIIC via the RB B domain. The authors proposed a model wherein the binding of either TFIIIB or TFIIIC to RB displaces the other, possibly leading to a failure to recruit TFIIIB to the promoter resulting in transcriptional repression. This study suggests that at the type 1 and 2 Pol III promoters, RB exerts its repressive effect by sequestering transcription factors TFIIIB and/or TFIIIC, but not by directly getting recruited at the target promoter. The observations that RB does not occupy the endogenous SSrRNA (type I) or tRNA genes (type 2) genes are consistent with the above mentioned idea. However, this model does not explain the mechanism for RB repression of U6 transcription, which is independent of TFIIIC and the Brfl component of TFIIIB. In trying to understand the mechanism for RB repression of U6 transcription, Sutcliffe et al., (20]) proposed a model wherein RB interferes with interactions between TFIIIB and Pol III resulting in repression of transcription. Using co-immunoprecipitation results, Sutcliffe et al., (201) demonstrated that RB disrupts interactions between TFIIIB and Pol III in the absence of DNA. Based on evidence that once TFIIIB gets to the promoter, it recruits Pol III via interactions between TFIIIB and Pol III (seen in the tRNA and SSrRNA gene systems) (101) it was reasoned that disruption of interaction between TFIIIB and Pol III by RB can 14 lead to repression of transcription on account of failure to recruit Pol III to the U6 promoter. In contrast, studies done by Hirsch et al., (80) have shown that RB and Pol III co-occupy the U6 promoter, suggesting that RB does not interfere with recruitment of Pol III to the promoter. Furthermore, in contrast to the type 1 and 2 Pol III promoters, RB association with the promoter DNA seems to be important for repression from the U6 promoter, as seen from the observation that truncations in the RB protein that debilitated RB association with the U6 promoter also inactivated RB repression (80). It is possible that RB association with the U6 promoter is important for targeting recruitment of co-repressor proteins which can cause transcriptional repression. Thus, RB may not cause repression by interfering with Pol III recruitment, instead, by recruiting co-repressor proteins that cause chromatin modifications resulting in a chromatin configuration that is non- conducive for polymerase escape into the transcribed region, leading to repression. 7. RB represses U6 snRNA gene transcription Earlier studies have demonstrated RB repression of the human U6 snRN A transcription (79, 80, 114). Factors involved in transcription of the U6 snRNA gene include the SNAP complex (SNAPc) bound to the PSE; a variant form of TFIIIB consisting of TBP, Brf2 and de1 proteins bound to the TATA box; and the Oct-1 protein bound to the DSE (Figure 1-4) (186). SNAPc contains five subunits; designated SNAP190, SNAPSO, SNAP45, SNAP43 and SNAP19 (3, 71-73, 181, 235, 245). SNAP190 binds DNA via its Myb domain. Crosslinking experiments have shown that SNAP190 and SNAP50 are in close contact with 15 Figure 1-4: The U6 snRNA transcription machinery. The U6 snRNA promoter is a type 3 RNA Polymerase III promoter. The key promoter elements comprise the TATA Box, PSE (Proximal Sequence Element) and DSE (Distal Sequence Element). The Bri2 containing TFIIIB complex containing also TBP and del binds the TATA box. The multi-protein snRNA activator protein complex (SNAPc) consisting of SNAP190, SNAPSO, SNAP45, SNAP43 and SNAP19 subunits binds the PSE. The Octl activator protein binds the DSE (186). 16 RNAPIII U6 snRNA Brfl-TFIIIB l7 DNA (71, 141, 244) suggesting that SNAP190 and SNAPSO are the DNA binding components of the SNAP complex. The DSE is bound by the POU domain containing Octl activator protein. The endogenous human U6 promoter contains a positioned nucleosome between the DSE and the PSE, thereby promoting interactions between the Octl POU domain and SNAPc leading to transcriptional activation of U6 (200, 251). RB co-occupies the U6 promoter with SNAP43, TBP and Pol III (80). RB also interacts with U6 transcription machinery components SNAP43, SNAPSO, TBP and del (79, 80). These results suggest that RB interactions with U6 transcription factors at the U6 promoter might interfere with the transactivation functions of these factors, contributing to repression of transcription. In addition, as discussed in section 6, RB repression can involve recruitment of co-repressor proteins to the U6 promoter to cause transcriptional repression 8. RB co-repressor proteins Additional mechanisms proposed for RB-mediated repression of target gene transcription involve the role of co-repressor proteins in causing chromatin modifications that impede transcription (50). Studies have revealed functional interactions between RB and other co-repressor proteins during repression of target genes (50). DNA methyltransferases, methyl CpG binding proteins, histone deacetylases, components of the SWI/SNF chromatin remodeling complex, histone methyltransferases, Polycomb group proteins (40, 90, 91) and DNA topoisomerases (14) are among those factors known to regulate RB function. 18 Sequence analysis of the nine human U6 copies that have identical coding region sequence revealed an enrichment of CpG dinucleotides in the functional U6 copies compared to the non-functional copies. CpG dinucleotides are frequents targets of methylation in mammalian cells. This suggested a role for DNA methylation and DNA methyltransferases in U6 regulation. As will be discussed in Chapter Two, I have investigated the role of DNA methylation in U6 transcriptional regulation and also analysed the correlation between RB function and DNA methylation in regulating U6 transcription. Evidence for RB directed recruitment of DNA methyltransferases and DNA methylation to the U6 promoter is presented, suggesting involvement of DNMT activity as an important aspect of the mechanism for RB repression of U6 transcription. In mammalian cells, there are three functional DNA methyltransferase enzymes that have been identified; DNMT], DNMT3A and DNMT3B. The following subsection 8.1 focuses on DNA methyltransferases and DNA methylation. 8.1. DNA methyltransferases DNA methylation in mammalian cells is regulated by three DNA methyltransferases (DNMTs); designated DNMTI, DNMT3A and DNMT3B (174) (Figure 1-5). Another methyltransferase, DNMT2, has been cloned and characterized but lacks catalytic activity (154, 242). DNMTs target cytosines in CpG dinucleotide sequences and methylate the cytosine at the 5 position. This reaction is catalysed by an active site cysteine (I l). DNMT] was the first 19 Figure 1-5: Schematic representation of the DNMT family proteins. DNMTs 1, 3A and 3B contain a regulatory domain and catalytic domain. Conserved motifs among the DNMT proteins are indicated as I, IV, VI, IX, X. DNMT] contains domains for nuclear localization (NLS), replication foci targeting and a Zinc- binding cysteine rich region. DNMTs 3A and B contain a cysteine- rich PHD domain in their regulatory domain. Diagram adapted from (174). 20 x x. _> >_ . .233 «.520 :=I=.l.n x x. _> >_ _ 1. l .2 as $529 ‘ w . r 1.-._ -._ _|1l_ 59:8 are 8:85 x x. _> >_ _ fill. .83. 435.520 5:1- -_EE _H_ 1 - _ .....-.._ Ml“ 59:8 aczoemfioon 5.8.30”. x x. _> >_ _ _ _ €38: FFEZD DE .1- l. _Efl.1-:.I: - n r. HIIHu \ / .L \ x 52.8 .23 £38 353:8 8.23-5 53.8.. .UQ. 0% 023 §g1w>0 uflO-ODZ EmEov ozzmfio EmEon bofizmom 21 methyltransferase to be discovered (10) followed by the discovery of the DNMT3 family of methyltransferases (153). DNMT] is considered to be the maintenance methyltransferase that recognizes and binds hemi-methylated CpG to methylate the unmethylated cytosine in the complementary DNA strand (162, 163). DNMT] is the most abundant methyltransferase in somatic cells (176). DNMT] is thought to copy methylation patterns after replication as seen from its ability to localize to replication foci and to interact with PCNA (33, 119, 125). DNMTl is required for proper embryonic development, imprinting and X-inactivation as observed from effects in DNMT] knockout mice. DNMT] depletion arrests embryonic development and causes a 70% reduction in genomic 5meCpG content (8, 123, 124). However, DNMTl knockdown in adenocarcinoma cells showed about 80% normal methylation and did not suffer remarkable growth defects. This suggested that DNMT3 family methyltransferases may also be involved in maintenance methylation in certain situations (172). Alternatively, other DNMTs that are yet to be discovered might perform redundant methyltransferase function. Studies done by Robertson et al., (175) have found that DNMT] can interact with RB both in vivo and in vitro and that the C706F mutation in RB abolished interaction with DNMTl and also RB repression activity. DNMT] co- purifies with RB, E2F] and HDACl and cooperates with RB to repress E2F- dependent transcription. Expression of DNMT] enhanced repression of transcription by RB when RB was tethered to an E2F responsive promoter. Similarly, expression of RB enhanced the repressive effect of DNMTl when 22 DNMT] was tethered to the same promoter, suggesting a cooperative functional interaction between RB and DNMT] in repressing transcription from an E2F target gene. Repression by either tethered RB or tethered DNMTl was relieved by the addition of the HDAC inhibitor TSA. Expression of RB, DNMT] and E2Fl in combination led to the most efficient transcriptional repression from a natural E2F responsive promoter, in comparison to transcriptional repression seen with expression of E2F] and RB or with E2F1, RB and DNMT] expressed individually. Consistently, transcription from an E2F unresponsive promoter remained unaffected even upon combined expression of RB, DNMT] and E2F] (175). Genes encoding the DNMT3 family of methyltransferases were first cloned and characterized by Okano et al., in 1998 (153). Mice knockouts for the DNMT3 family methyltransferases showed loss of de novo methylation following embryo implantation (152). In vitro studies as well as studies done in DNMT3 knockout mice revealed that DNMT3 enzymes have an equal preference for hemi- methylated and unmethylated DNA substrates, leading to their classification as de novo methyltransferases (153). DNMT3A knockout mice die at 4 weeks after birth and DNMT3B knockout mice are not viable (152). In vitro assays performed with recombinant proteins expressed in baculovirus showed that DNMT3A was more active than DNMT3B. DNMT3B was found to be involved in maintenance of DNA methylation of minor satellite repeats adjacent to centromeres. Mutations in the catalytic domain of the human DNMT3B are associated with the ICF syndrome (Immunodeficiency, Centromere instability and Facial anomalies 23 syndrome), a rare autosomal recessive disorder. Patients with ICF syndrome suffer from immunodeficiency, chromosomal abnormalities and facial abnormalities (62, 152, 239). DNMTl methyltransferase is considered to be the maintenance methyltransferase while the DNMT3 family of methyltransferases are thought to perform the de novo methylation. Studies done by Lei et al., (118) showed that DNMTl knockout embryonic cells retained de novo methylation. Expression of DNMT] and DNMT3A in Drosophila melanogaster showed that DNMT] had no denovo methylation property whereas expression of DNMT3A led to low levels of methylation (131). Also, in ES cells, homozygous deletions of Dmnt3a and Dnmt3b led to no alterations in pre-existing methylation patterns but homozygous deletion of Dnmtl led to about 70% reduction in cytosine methylation (124, 152). However, recent evidence support a reinterpretation of these observations. Earlier studies by Vertino et al., (214) showed that overexpression of DNMTl in cancer cell lines resulted in de novo methylation. Also, Rhee eta1., (172) reported that 80% normal methylation patterns are retained in somatic cells that lack DNMT] but contain normal expression levels of DNMT3A and DNMT3B. It is possible that other methyltransferases can compensate for the loss of DNMTl function. It is also possible that all three DNMTs have both de novo and methyltransferase activities, but the different methlytransferases are involved in methylating DNA in certain genomic regions via their interactions with other DNA binding proteins (174). 24 8.1.1. DNA methylation mechanism The following reaction mechanism for cytosine methylation by DNA methyltransferases has been proposed (Figure 1-6) (11, 26, 183). A cysteine thiolate group in the enzyme active site adds covalently to the C6 position of the target cytosine residue, pushing electrons to the C5 position to make the carbanion, which attacks the methyl group of S-adenosyl methionine (AdoMet, SAM). After methyl transfer, abstraction of a proton from the C5 position allows reformation of the C5-C6 double bond and release of the enzyme. DNMT3 have a conserved prolycysteinyl active site dipeptide that provides the cysteine thiolate. Motifs I and X form the SAM binding site. Motif IV contains the active site prolyl-cysteinyl active site. Motif VI contains an important glutamyl residue for proton abstraction process in the enzyme catalysis mechanism. There is a motif VIII downstream of motif VI (not indicated in Figure l-5).The target recognition domain is usually located between motifs VIII and IX and is involved in making base-specific contacts in the major groove of DNA. Motif IX is involved in maintaining the structure of the target recognition domain (1 1). 8.1.2. DNA methylation and cancer Aberrations in methylation patterns are frequently observed in cancers (48, 58, 75, 196). Global hypomethylation along with region-specific hyperrnethylation is often seen in tumor cells (7, 93). Considering that maintenance of normal DNA methylation patterns is important for tumor suppression (detailed in the following paragraphs in this section), and that RB can 25 Figure 1-6: Proposed mechanism for DNA methylation. (A). Cysteine thiolate of DNMT attacks carbon 6 of the cytosine residue, pushing electrons to the C5 position. (B). Enamine attack on the methyl group of Adomet (SAM) occurs. (C). Methyl group transfer to carbon 5 and abstraction of a proton from carbon 5 follows (26, 183). (D). Reformation of the C5-C6 double bond followed by release of the methyltransferase enzyme occurs. Diagram adapted from Ref (1 1). 26 NH2 2 1 CAN H /4 N 3 5 Cytosine 6 /k ' S-DNMT + S AdoMet NH2 / (I;H3 H 27 DNA direct DNA methylation to target gene during repression (discussed in Chapter Two), RB functional association with DNMT activity could be an important aspect of the tumor suppression mechanism of RB. A majority of the hypomethylation events associated with cancer are seen in repetitive and parasitic elements which are normally heavily methylated, possibly leading to an increase in transcription from these elements causing genomic instability and malignant growth. One idea is that the ancestral function of DNA methylation is to prevent the spread of these parasitic elements to protect the genome against unrestricted transpositions (13, 243). This genome defense system could later have evolved as a gene regulatory system (174). CpG methylation plays a role in X-chromosome inactivation in females and in genomic imprinting. Silencing associated with promoter hypermethylation is seen in the case of several genes such as those involved in tumor suppression for e.g., RB, DNA mismatch repair, cell adhesion and DNA damage protection mechanisms (174). Promoter hypermethylation at the RB gene (196) is observed in familial cases of retinoblastoma and similarly, the VHL (von Hippel Lindau) gene promoter is hyperrnethylated in renal cancer (74). In sporadic cases of colorectal carcinomas exhibiting microsatellite instability, elevated levels of promoter hypermethylation of the mismatch repair gene hMLHl was observed (75). Aberrant hypermethylation can occur early in tumorigenesis, leading to misregulated gene regulation predisposing cells to malignant transformation. Aberrant methylation patterns were observed in pre-neoplastic lesions and the 28 frequency of aberrant DNA methylation events increased with disease progression. (9, 150, 234). This supports the idea that DNA hypermethylation can directly contribute to tumorigenesis. Tumors exhibit misregulated DNA methylation with both hypomethylation as well as region-specific hypermethylation occurring in different genomic regions in the same cell (174). The global loss of DNA methylation also plays an important role in the cellular transformation process. Primary tumor samples from humans and rodents show demethylation and re- expression of transposable elements (52, 60). DNA methylation can contribute to genome stability by inhibiting homologous recombination between repeats (37). Deleterious consequences of recombination between repeats in humans have been reported (165, 180, 195). In the fungal species Ascobolus immerses, induced methylation of a recombination hotspot reduced the frequency of crossing-over in this region by several hundred fold (134). In mammalian cells, V(D)J recombination is reduced more than 100-fold when the recombining genomic region is methylated (84). Repetitive elements are found to be demethylated in tumors and the degree of hypomethylation correlated with disease progression (168). Some potential mechanisms by which DNA methylation might downregulate homologous recombination include masking the recombination initiation site, maintenance of a highly condensed chromatin structure, destabilization of the recombination intermediate or interfering with the assembly of the recombination machinery (l 74). 29 8.1.3. DNA methylation and transcriptional repression DNA methylation has been associated with transcriptional silencing (12, 35, 93, 100, 164, 249). Promoters enriched for cytosine methylation are usually transcriptionally silent and condensed into nuclease-resistant chromatin structures that contain hypoacetylated histones (45, 93). In some promoters, CpG methylation can interfere with transcription factor binding, resulting in transcriptional repression (207). An alternative model for transcriptional repression can involve methyl CpG binding proteins (MeCPs) (detailed in section 8.1.6). MeCP2 contains a methyl-CpG binding domain (MBD) that recognizes and binds to symmetrically methylated CpG dinucleotides, and a transcriptional repression domain (TRD) (122, 142, 218). Other methyl CpG binding proteins, MBDs 1-4, have also been identified (174). Evidence for MeCP2-mediated recruitment of HDAC machinery to repress transcription has been reported (94, 144). Methyl CpG binding proteins recognize and bind methylated DNA, directing recruitment of HDAC activity which can result in tighter chromatin packaging and possible prevention of access to transcription factors and/or polymerase escape (94, 144, 177). In Xenopus, the Mi2 chromatin remodeling complex contains MBD3 and de3 (HDAC) (216). Evidence suggesting that MBD2, HDACl and HDAC2 are components of the MeCPl repressor complex in HeLa cells has been reported (148). The combined effect of DNA methylation and HDAC function has been demonstrated by robust activation of target gene expression in the presence of inhibitors of both DNA methylation and HDAC activity, whereas using either one of these inhibitors alone had little to no effect 30 on gene activation (22). Also MeCP2 directed recruitment of histone methyltransferase activity catalyzing H3K9 methylation during transcriptional repression by MeCP2 has been demonstrated (55). Another mechanism proposed for the mechanism of transcriptional repression by DNA methylation is that the steric obstruction caused by binding of methyl CpG binding proteins prevents transcription factor recruitment (174). 8.1.4. DNA demethylation DNA methylation is involved in transcriptional silencing by establishing a chromatin state that does not support transcription. DNA methylation is thought to play an important role in X chromosome inactivation, genomic imprinting and tissue-specific gene-silencing, where the methyl marks are made permanently (l 6, 136). DNA methylation is also involved in transcriptional repression of genomic regions that need to be permanently silenced, such as the parasitic elements and transposons, to prevent their amplification in the genome (13, 174, 243). However, DNA methylation is also involved in silencing genes that need to be switched between ON and OFF states. This necessitates a reversal mechanism for DNA methylation. DNA methylation is subject to reprogramming especially during development (16, 171, 193). Cyclical DNA methylation and demethylation events have been observed in a transcriptionally active promoter (138). Genome-wide demethylation and remethylation are seen during gametogenesis and post- fertilization. Also, localized DNA demethylation occurs at specific genes during differentiation (l6, 17, 29, 110). DNA demethylation is important for epigenetic 31 reprogramming in Xenopus oocytes (193). Although there is abundant information about DNA methylation, there is much less known about DNA demethylation. DNA demethylation can be either a passive or an active process, or a combination of both (99). Passive demethylation involves a lack of DNMT activity through cycles of replication, whereas active demethylation involves enzymatic functions that demethylate DNA (16, 110). There are at least three proposed mechanisms for active DNA demethylation (99). The first mechanism involves the direct replacement of the methyl group with a hydrogen atom. The human MBD2 protein was thought to demethylate DNA by this mechanism (15, 169). However, this reaction is thermodynamically unfavorable as it involves the breakage of a carbon-carbon covalent bond. This result has not been reproduced by others (16, 110, 148, 205). Moreover studies done by Kress et al., (109) have demonstrated that active cytosine demethylation involved DNA strand breaks. This observation does not agree with the proposed mechanism that involves the direct replacement of the methyl group with a hydrogen atom in the C5 position of the methylated cytosine, because this mechanism does not involve the formation of DNA strand breakage. The remaining two proposed mechanisms involve DNA strand breaks and repair processes as part of the demethylation process (95, 104). The second mechanism involves DNA glycosylases, which cleave the methyl cytosine base from the deoxyribose moiety (base-excision model) (95, 96). For example, a G/T mismatch repair DNA glycosylase initiates DNA demethylation by breaking the 32 glycosidic bond of 5-methyl cytosine, leaving an abasic site that is processed by an AP endonuclease and other DNA repair enzymes (254). Human MBD4 has 5- methylcytosine DNA-glycosylase activity (5-MCDG) that leads to DNA demethylation in vitro. Overexpression of a human 5-MCDG in human embryonic kidney cells led to promoter demethylation of a hormone-regulated reporter gene (252-254). The third mechanism proposed for DNA demethylation involves the removal of the methylated nucleotide by nucleotide excision and then replacement with unmethylated cytosine by DNA repair mechanisms (223, 224). Recent studies by Barreto et al., (5) demonstrated that Gadd 45 alpha (grth arrest and DNA-damage inducible protein alpha), which is involved in nucleotide excision repair, leads to gene activation by active demethylation of target promoter. Evidence for functional co-operation between Gadd 45 alpha and XPG, which is a DNA repair endonuclease, in DNA demethylation was also presented in this study (5). 8.1.5. Non-CpG methylation in mammalian DNA As will be discussed in Chapter 2, evidence for RB-directed methylation of cytosine residues in a non CpG but in a CNG sequence (at the U6 start site) (i.e., the outside cytosine in the CCGG sequence) was observed. Also, data suggesting that cytosine methylation in a non CpG sequence context can also contribute to transcriptional repression is presented in Chapter 2. This observation revealed a novel link for RB-directed methylation of cytosines in non CpG 33 sequences to transcriptional repression. This section will focus on elaborating non CpG methylation in mammalian cells. DNA methylation of cytosines at non CpG sequences were frrst identified by Salomon and Kaye in 1970 (182). Analysis of DNA isolated from newborn mice and mouse embryo cultures showed that 5meCpG dinucleotides were not the only methylated species, because 5meCpT, 5meCpC and 5meCp5meC species were also found (182). Studies by Jones and colleagues (151) have shown that upon induction of hypermethylation by treatment with DNA synthesis inhibitors, apart from CpG methylation, methylation of cytosines in CpA, CpT and CpC sequences was also observed in hamster fibrosarcoma cells. Woodcock et al., (236) found that human spleen DNA contains 5meCpA, 5meCpT and 5meCpC dinucleotides. - Methylation of cytosines in CprG sequences have been reported by Clark et al., (34). In plant genomes, the existence of methylated cytosines in CpG dinucleotides and CprG sequences is well known (61). But in mammalian DNA, much focus was given to cytosine methylation in CpG dinucleotides only. Only few studies have emphasized the presence of cytosine methylation in CprG sequences and also in other asymmetric sequences (156). Evidence for ‘ methylation of cytosines in CpApG, CpCpG, CprG and CpGpG sequences is available (34). Furthermore, bisulfite sequencing analysis of stably transfected pre-methylated DNA revealed efficient maintenance of CprG methylation, and also de-novo methylation of previously unmethylated CprG sites have been found (34). 34 Studies by Pradhan et al., (162) found that the human DNMT] enzyme can methylate CpCpG, CprG and CpApG sequences in vitro, in addition to CpG, as seen from DNMT] crosslinking to these duplex oligonucleotides in the presence of SAM. The efficiency of complex formation was in the order CpG > CpCpG > CpApG = CprG. Ramsahoye et al., (170) reported that DNMT3A enzyme can methylate cytosines in CpA and CpT dinucleotides. These studies indicate that it is possible that cytosine methylation can occur outside the context of the cognate CpG sequence. My results indicate a novel link to methylation of cytosines in a CNG sequence context to transcriptional repression (in Chapter 2). 8.1.6. Methyl CpG binding proteins Methyl CpG binding proteins are involved in transcriptional repression, by binding to methylated CpG possibly by targeting the recruitment of co- repressor proteins such as sin 3 and HDACs (94, 144) and components of the SWI/SNF chromatin remodeling machinery (67). The first Methyl CpG binding proteins to be identified were MeCPl and MeCP2. MeCP] was identified as a nuclear factor that can discriminate between methylated and unmethylated DNA (137). MeCP2 was another factor that bound specifically to methylated CpG (143). MeCP2 is a chromatin-associated nuclear protein that binds to chromosomes that contain methylated DNA (142). A short region in the N-terrninus of MeCP2 (of about 70 amino acid residues) contains the methyl CpG binding domain (MBD) (143). 35 A sequence homology search using the MBD of MeCP2 led to the identification of MBD] (38). MBDl binds methylated DNA and can repress transcription in vitro. Later database searches led to the discovery of MBD2, MBD3 and MBD4 (69). The sequence similarity between MeCP2 and MBDs 1-4 is largely limited to their MBD region (4). MBD] is a 70 kDa protein that has cysteine-rich regions (CXXC motifs) related to those in DNMT] (38). MBD2 is thought to have DNA demethylase function, but the findings remain controversial (15, 148, 216). MBD3 shares high homology (80% similar, 72% identical) to MBD2b which is the shorter splice variant form of MBD2 (216, 250). MBD4 has sequence homology to bacterial DNA repair enzymes at the C-terrninus including E. coli Mut Y (an 8-oxoGA mispair-specific adenine-glycosylase), Mig from Methanobacterium thermoautotrophicum (a GT-mismatch specific glycosylase), Endo III from E. coli (a thymine glycol glycosylase) and UV-endonuclease of Micrococcus luteus (4). Splice variant forms of MBDs 1-4 have also been reported (70). Studies done by Hendrich and Bird (69) have found that MBDl, MBD2 and MBD4 can bind to methylated DNA in vitro. MBD3 did not bind methylated DNA. GFP-tagging and cellular localization studies have identified that MBDs l, 2 and 4 preferentially localized to genomic regions that were rich in methylated DNA such as major satellite DNA. On the other hand, MBD3 did not localize to methylated regions despite its high homology to MBD2b (69). Studies to understand the sequence specificities of these methyl-CpG binding proteins support the proposal that differences in CpG density of various genomic regions 36 and differences in dissociation constants of these methyl CpG binding proteins with methylated DNA serve to distinguish binding of these various methyl binding proteins to specific regions of the genome (4). In mouse, MeCP2 localizes to pericentromeric chromatin whereas MBDI is localized to the hyperrnethylated region of chromosome lq12 (54). MeCP2 does not require prior disruption of nucleosomal chromatin to bind to the genome. MeCP2 can bind nucleosomal DNA (24, 145). These findings lead to a model wherein MeCP2 and possibly the other methyl CpG binding MBDs can access chromatin first to target co-repressor complexes to cause chromatin modifications, leading to repression (4). Methyl CpG binding proteins have been associated with transcriptional repression in several studies (94, 142, 147, 148, 216, 250). MeCP2 represses transcription in vitro and in vivo from methylated promoters but not from non- methylated promoters (98, 142). The transcription repression domain (TRD) of MeCP2 was found to associate with co-repressor complexes containing Sin3 and HDAC activity (94, 144). MeCP2-mediated silencing was relieved by TSA, suggesting a relationship between DNA methylation and HDAC function in transcriptional repression (94, 144). Similar studies have found that MBDl, MBD2 and MBD3 also associate with histone deacetylases (147, 148, 216, 250). MBDl can affect transcription from both methylated and unmethylated promoters. One of the three CXXC motifs can bind DNA regardless of the methylation status. MBDl represses transcription though the co-operation between its MBD, CXXC motifs and TRD (54). MBD2 is part of an MeCP2 repressor complex that contains HDAC] and 2 and RbAp48/46 the latter of which are histone binding proteins (4, 37 38, 142, 148). MBD2b was also shown to repress transcription in a deacetylase- dependent manner (142). MBD3 has been identified as part of the Mi2/NuRD complex which has both histone deacetylase fimction and nucleosome remodeling activity indicating a potential link between DNA methylation, histone deacetylation and nucleosome remodeling activities (216, 217, 250). The presence of different cellular MBDs in the cell suggests the binding of different MBDs to different genomic regions governed by CpG density and the binding affinities for these Methyl CpG containing proteins to methylated DNA. Despite a conserved MBD, significant differences in the functions, cellular localization and binding specificities to cofactors, and to DNA have been observed. It is possible that the regions outside the MBD also regulate important functional aspects of these proteins. MeCP2 is found to associate with DNA more stably than the MBD2/MeCPl complex. MeCP2 binds single methylated CpG but MeCPl can bind only to heavily methylated DNA. MBD] can affect transcription form both methylated and unmethylated promoters. The molecular mechanism underlying these unique aspects of the seemingly similar Methyl-CpG binding proteins remains to be understood (4). 8.2. Histone deacetylases and RB-mediated transcriptional repression Considering the presence of a positioned nucleosome which contributes to U6 transcriptional activation (200, 251), the activity of chromatin modifiers such as HDACs and the SWI/SNF chromatin remodeling machinery which can modulate the location of this nucleosome can play a role in U6 transcriptional 38 regulation. Also the presence of HDACs l and 2 and the BRGl component of the SWI/SNF complex on the endogenous U6 promoter in RB positive cells suggests a role for these factors in U6 transcriptional regulation (discussed in the Appendix). Additional links between RB function and the activity of HDACs (discussed in this section) and the SWI/SNF complex (discussed in section 8.3) reported in studies by other groups suggests an important role for HDACs and the SWI/SNF complex in RB repression of U6 transcription. Also the presence of HDACs l and 2 and the BRGl component of the SWI/SNF complex on the endogenous U6 promoter in RB positive cells suggests a role for these factors in U6 transcriptional regulation. Histone deacetylases comprise seven families of which HDACs 1-3 are known to interact with RB (20, 28, 65, 130, 132). In a recent study to analyze the acetylation status of histones H3 and H4 at several cell-cycle regulated genes containing E2F sites, hypoacetylation of H3 and H4 was observed in quiescent cells but hyperacetylation of H3 and H4 was observed in late GI and into S phase, indicating the importance of histone acetylation status of E2F target genes in cell cycle regulation (204). RB-directed recruitment of HDACs to target genes led to histone deacetylation and correlated with transcriptional repression (130). Inhibition of HDAC activity with Trichostatin A (TSA) relieved RB repression of certain genes. HDAC activity was found be important for RB repression of cyclin B and DHFR genes (required for G1 to S phase progression) during early to mid Gl phase, suggesting that HDAC activity is crucial for RB to induce G1 arrest (247). HDACs l and 2 contain the LXCXE motif that is found in many RB- 39 binding proteins including viral oncoproteins such as EIA and E7 (115). However, HDAC 3, which also binds to RB, does not contain this motif (64). E2Fs do not contain an LXCXE motif, and interact with RB via a distinct site than that which interacts with HDACs. This can allow the formation of an RB-E2F-HDAC complex that gets recruited to E2F site containing promoters (20, 28, 39, 130, 132). In some studies, LXCXE binding mutants of RB (defective for EIA and E7 binding) were unable to bind HDACl and HDAC2, and were defective in active transcriptional repression and in induction of growth arrest of long term assays (39). However, upon transient overexpression of the RB mutants, there was an observed increase in percentage of cells that were in G1 (39). There is also evidence suggesting that similar RB mutants that were defective for E7 binding could still bind HDACl to mediate transcriptional silencing and induce G1 arrest (42). In another study, an RB mutant for HDAC] binding was as effective as wild type RB in arresting growth of RB negative cells, however the mutant RB was defective in the ability to cause irreversible growth arrest in differentiated myocytes (28). Nevertheless, in each of these studies, the ability of RB to interact with HDACs correlated with its ability to induce growth arrest or maintain differentiation, highlighting the importance of RB-HDAC interaction in RB function (246). 40 8.3. SWI/SNF and RB-mediated repression ATP-dependent chromatin remodeling complexes constitute an important component of the chromatin modulatory machinery in the cells (106). The yeast SWI/SNF chromatin remodeling complex was discovered first among a group of many chromatin remodeling complexes which contain an ATPase catalytic subunit. The human homologs of SWI2/SNF2 are the BRGI and hBRM proteins which along with BAFs (BRGl associating factors) constitute the human SWI/SNF (hSWI/SNF) (157). In vitro, SWI/SNF and other chromatin remodeling complexes are able to catalyze the interconversion between ‘closed’ and ‘remodeled’ chromatin states in an ATP-dependent manner (128, 185). SWI/SNF- like complexes are known to be involved in both transcriptional activation and repression, as observed in a genetic study where disruption of the SWI/SNF complex led to transcriptional activation of one set of genes and to repression of another set of genes (82). RB can interact with both BRGl and hBRM (43, 199). There is evidence linking HDAC function and BRGl-containing nucleosome remodeling complexes in co-operative involvement with RB-mediated transcriptional repression (43). Overexpression of BRGl in cells deficient in BRGl and hBRM led to growth arrest, and this was dependent on the ability to interact with RB (43). Overexpression of a dominant negative form of BRGl or hBRM that is mutant for the ATPase catalytic domain inhibited RB-mediated growth arrest (43, 198) suggesting that the chromatin remodeling function of SWI/SNF (which is ATPase dependent) can play an important role in RB-mediated repression. In C33A cells 41 that are deficient for BRGI, hBRM and RB, expression of BRGl was essential for ectopically expressed RB to arrest cell growth (197, 247). While RB was sufficient for repression of transcription from a transiently transfected reporter, repression of a stably integrated identical reporter required both RB and BRGl activities, suggesting a requirement for chromatin remodeling by BRGI for RB repression (247). Although BRGl has an LXCXE motif, it does not seem to require it for binding to RB, thereby possibly allowing the association of RB, HDAC and SWI/SNF in a single complex which can lead to transcriptional repression (247). 8.4. Other co-repressor proteins 8.4.1. Histone Methyltransferases RB transcriptional repression also involves the histone methyltransferase SUV39H1, which is responsible for histone H3K9 methylation. Studies done by Nielsen et al., (149) have found that RB targets H3 methylation and HP] (heterochromatic protein that binds methyl lysine) to the cyclin B gene during repression. Interesting links between histone methylation and DNA methylation have also been reported (108, 205). Studies done in Neurospora have indicated that chromatin modifications facilitated by histone methylation were necessary for DNA methylation to occur, although the mechanism by which this occurs is unclear. Also, the observed co-localization between DNMT3 and HPl suggests a functional link between histone methylation and DNA methylation (2, 108, 205). 42 8.4.2. Topoisomerases Topoisomerases (Topo) are ATP-dependent enzymes that remove positive supercoils in DNA by causing DNA strand breakage and re-ligation (l4). Topoisomerases are important for several cellular functions such as transcription, transcription, chromatin segregation, cell cycle progression and DNA repair (21). Topo 1 makes a single-stranded break whereas Topo 11 causes double-stranded breaks (21). In mammalian cells, Topo II is present as two isoforms Topo Ila and Topo IIB encoded by different genes (206). Studies by Bhat et al., (14) have found a functional interaction between RB and Topo Ila, including physical association between RB pocket domain and Topo Ho and inhibition of TopoIIa by RB, both in vitro and in vivo (14) although the mechanism for inhibition of Topo Ila activity by RB remains uncharacterized. My studies have shown the presence of Topo Ila and Topo 1113 on the endogenous U6 promoter in RB positive cells (discussed in Appendix). These findings suggest a possible RB repression mechanism involving also the inhibition of topoisomerase activity. 8.4.3. Polycomb (PcG) group proteins PcG proteins are classically known for their repressive effect on Hox genes. This repression coupled with transcriptional activation by trithorax proteins is important for establishing the pattern of Hox expression required for proper embryonic development (40, 59, 104, 121, 161, 188). Apart from Hox gene expression, PcG proteins are also involved in cell cycle regulation (90, 120, 215). 43 Mice that lack Bmi-l show severe defects in lymphoid proliferation (213). Bmi-l represses expression of p16 which is a cdk4/6 inhibitor. The absence of Bmi-l leads to accumulation of pl 6 which blocks Cyclin D/cdk4 activity and consequent accumulation of hypophosphorylated RB and growth arrest (90, 91). Another member of the PcG proteins M33/CBX2/HPC1 is required for normal cellular proliferation. In contrast to Bmi-l and M33, other PcG proteins such as BED and HPC2 act as negative regulators of the cell cycle (184). Studies done by Dahiya et al., (40) have found that the polycomb group protein HPCl in association with Ringl protein acts as an HDAC-independent co- repressor for RB, causing repression of cdc2 and cyclin A genes leading to G2 arrest. This suggests co-operativity between PcG proteins and RB in transcriptional repression (40). 8.5. Summary Deducing from studies discussed in this chapter, a model for RB repression of Pol III transcription can be proposed. Considering the observation that the presence of RB on the U6 promoter is important for transcriptional repression, it is possible that RB recruitment to the U6 promoter directs recruitment of other co-repressor proteins that can lead to inhibition of transcription. The observed enrichment of CpG dinucleotides in the functional U6 copies compared to the non-functional U6 copies, indicates possible involvement of DNMTs in RB repression of Pol III transcription. 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Genes Dev 7:1111-25. 75 CHAPTER TWO THE RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN REPRESSES U6 snRNA TRANSCRIPTION BY A MECHANISM INVOLVING PROMOTER DNA METHYLATION ABSTRACT The Retinoblastoma tumor suppressor (RB) protein functions as a cellular checkpoint at the G1 to S phase transition, thereby guarding against unregulated cell growth and division characteristic of tumors. RB represses transcription of gene products that facilitate the biosynthesis of cellular products whose levels determine the rate of cellular growth and concomitant cell division. RB function as a transcriptional repressor can be an important aspect of its tumor suppressor function. RB represses global Po] 1 and Pol III transcription as well as specific Pol II transcribed genes that are E2F regulated. The study presented herein analyzes the mechanism for RB repression of transcription from a type 3 RNA Polymerase III promoter, using the human U6 snRNA gene as a model system. My results demonstrate a role for DNA methyltransferase function in repression of U6 transcription by RB. I present in vitro and in vivo evidence that RB directs increased methylation of a conserved start site CpG at the U6 promoter. Moreover, RB directed recruitment of the DNA methyltransferases DNMTl and DNMT3A to the U6 promoter. Depletion of DNMTI, 3A and 3B by si-RN A led to enhanced U6 transcription, demonstrating a repressive role for DNA methyltransferases in 76 U6 transcription. Consistently, transcription from a methylated U6 reporter was diminished compared to unmethylated template. This study provides previously unreported evidence for RB directed DNA methylation at a target gene during transcriptional repression. Considering that the role of RB as a transcriptional repressor is linked to its role as a tumor suppressor, the results presented in this study can be useful towards our understanding about RB function in tumor suppression. Introduction RB functions as a cellular safeguard against unregulated cellular growth by regulating the G1 to S phase transition phase of the cell cycle. RB is a transcriptional repressor of genes whose products are important for progression into S phase (14, 40). Some of the P01 II-transcribed genes that are targets of E2F- mediated transactivation, such as c-myc, B-myb, cdc2, dihydrofolate reductase and thymidine kinase, are repressed by RB (5, 26). RB also acts as a general repressor of Pol III and P01 1 (4, 44). Products of Pol III and P01 I transcription include ribosomal RNAs and tRNAs, the structural components of the protein synthesis machinery, and uridine rich small nuclear RNAs that are structural components of the splicing machinery. Unregulated cellular growth observed in tumors requires elevated levels of these Pol III and P01 I products, necessitating the cell to have a mechanism to repress Pol III and P01 I transcription to suppress tumor development. This raises the possibility that RB might exert its tumor suppressive effects by acting as a repressor of Pol III and P01 I transcription (43). 77 Several lines of evidence suggest a potential link between the tumor suppressive capacity and the Pol III and P01 I repressor functions of RB. Naturally occurring RB mutations in tumors that rendered RB inactive as a tumor suppressor also debilitate its ability to repress Pol III and P01 I transcription (19, 40, 45), whereas the adenoviral EIA oncoprotein that binds and inactivates RB also interferes with its ability to repress Pol III transcription (44). Moreover, the functional domains within RB required for transcriptional repression and for tumor suppression largely coincide (4, 44). The minimal region of the RB protein required for tumor suppression comprises amino acids 378-928, which includes the A and B pocket domains and the C-tenninal end of the protein (46). Studies done by Hirsch eta1., have revealed that this region was also the minimal region for efficient repression of Pol III transcription (17). RB is known to be a master regulator of the cell cycle and a key tumor suppressor that represses diverse types of genes that require distinct transcription machinery. Consequently, RB uses distinct mechanisms at different types of target genes in order to cause gene-silencing. RB repression of Pol II transcribed E2F target genes, by binding to and inactivating E2F proteins thereby inhibiting the E2F transactivation function, is a mechanism for regulating G1 to S phase transition and cell cycle progression by RB (1, 6, 9, 19, 41, 42). Studies done by Cavanaugh et al., (4) have found that RB represses Pol I transcription by binding to and inactivating the UBF transcription factor that is involved in rDNA transcriptional activation. Studies directed towards understanding RB repression 78 of Pol III transcription have indicated that RB interferes with critical transcription factors. Studies carried out on Alu and AdVAI transcription repression mechanisms have pr0posed that RB represses Alu and AdVAI genes which are type 2 Pol III promoters via interactions with TFIIIB and TFIIIC (7). It is possible that at type I and type 11 Pol III promoters, RB exerts its repressive effect by binding to and sequestering the transcription factors TFIIIB and TFIIIC but not by getting directly recruited to the promoter. In support, RB does not occupy the endogenous SSrRNA (type I) or tRNA genes (type 2) genes although it represses these genes (17). In contrast, RB occupied the U6 promoter (17). Truncations in the RB protein that debilitated RB association with the U6 promoter also inactivated RB repression (17) indicating that RB association with the promoter DNA is important for transcriptional repression from the U6 promoter. These results suggest that RB might use distinct mechanisms at different types of Pol III promoters to repress transcription. With respect to type 3 Pol III promoter system exemplified by the U6 snRNA gene, White et al., (38) proposed that RB might cause repression of transcription by interfering with interaction between TFIIIB and Pol III, resulting in an inability to recruit Pol III to the promoter. On the contrary, studies done by Hirsch et al., (17) have found that RB and Pol III co-occupy the U6 promoter, suggesting that RB does not exclude the polymerase from recruitrnent to the promoter. This raises the possibility that RB might be using a different 79 mechanism to repress U6 transcription that does not exclude Pol 111 from getting recruited to the promoter. Alternatively, RB could direct recruitment of co- repressor protein(s) to the U6 promoter to lead to transcriptional repression. In this study, I present evidence that DNA methylation contributes to RB repression of the U6 snRNA gene. Promoter CpG methylation and the function of DNA methylation have previously been associated with gene silencing (18, 31). RB interacts with the DNA methyltransferase DNMT] , and the co-operative role of DNMTI is thought to be important for RB repression of specific E2F-regulated RB target genes that are transcribed by Pol II (30). Methylation of rDNA repeat sequences leading to inhibition of UBF transcription factor binding to the rRNA core promoter DNA has been proposed as a mechanism for repression of rDNA transcription by P01 1 (34). The study presented here shows that DNA methylation plays a role in silencing a Pol III promoter. Specifically, my results indicate that RB directly employs the cellular DNA methylation machinery to cause promoter- proxirnal methylation at the U6 gene to cause gene-silencing. This study illuminates a previously undescribed role for DNA methylation as a repressive mechanism targeting Pol III-dependent transcription and lends fiirther support to previous findings that have hinted at a close cooperative fimction for DNMT3 with RB function in Pol II and P01 I transcriptional repression (30, 34). 8O Materials and Methods Images in this thesis are presented in color. Chromatin Immunoprecipitation (ChIP): Chromatin immunoprecipitation reactions were done as described previously (17). Chromatin was collected from HeLa, MCF 7 and 184B5 cells that were grown to approximately 75% confluency. After harvesting by trypsinization, cells were fixed with 1% formaldehyde for 30 mins followed by washing with PBS, buffer I (10 mM HEPES pH 6.5, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and buffer II (10 mM HEPES pH 6.5, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Cells were then suspended in 1 ml lysis buffer per 108 cells (Lysis Buffer comprises 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 0.5 uM phenylmethylsulfonyl fluoride (PMSF), 1 uM pepstatin A, 1 mM sodium bisulfite, 1 mM benzarnidine, 1 mM DTT) followed by sonication to obtain soluble chromatin. Immunoprecipitation reactions were set up in dilution buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1% Triton X-100, 0.5 uM PMSF, 1 mM DTT) with chromatin equivalent of 107 cells using 1 pg antibody in a total volume of 1 ml. Immunocomplexes were recovered using Protein-G agarose beads. The beads were washed once with TSE (20 mM Tris pH 8.0, 0.1% SDS, 2 mM EDTA, 1% Triton X-100), TSE with 250 mM NaCl, TSE with 500 mM NaCl, Buffer III (10 mM Tris pH 8.0, 1 mM EDTA, 0.25 M LiCl, 1% NP-40, 1% deoxycholate) and TE (20 mM Tris pH 8.0, 2 mM EDTA). Complexes were eluted from beads with elution buffer (0.1 M NaHCO3 + 1% SDS) and then reverse crosslinked by incubating at 65° C overnight. DNA was then recovered 81 after phenol-chloroforrn extraction followed by ethanol precipitation. Primers for PCR amplification of the U6, U1, U2 and GAPDH loci were described previously (17). For amplification of SS rRNA loci the primers used were 5’ GGCCATACCACCCTGAACGC 3’ and 5’ CAGCACCCGGTATTCCCAGG 3’. The 7SK promoter region was amplified using the following primers. 5’ TT'ITGGGAATAAATGATATITG 3’ and S’ GAGGTACCCAGGCGGCGCACAAG 3’ PCR products were then separated on a 2% 0.5X TBE agarose gel and images recorded with Kodak imaging software. In vitro transcription: In vitro transcription reactions were performed as described previously (17). Transcript levels were analyzed using the body- labelled riboprobe protection method (17). 250 ng of pU6/Hae/RA.2 plasmid DNA was incubated with HeLa nuclear extract with appropriate recombinant proteins. Transcription was done at 30 °C for 30 minutes and was stopped by adding stop mix (0.3 M sodium acetate pH 7.0, 0.5% SDS, 2.5 mM EDTA). Proteinase K digestion (20 ug/ml) was then done at 37° C for 1 hr. Nucleic acids were recovered by phenol extraction and ethanol precipitation. The nucleic acid pellet was then resuspended in 30:11 FAHB (80% forrnarnide, 400 mM NaCl, 40 mM PIPES pH 6.4 and 1 mM EDTA pH 8.0) containing 300,000 cpm riboprobe (body-labelled with or-P32 CTP) complementary to the transcripts whose synthesis was driven from the U6 promoter. Hybridization was done overnight at 61° C, followed by digestion with T1 RNAase (at IOU/ml) for 30 mins at 30° C to select 82 for U6 promoter-driven transcripts protected from hybridization to the riboprobe. The T1 RNAse was then inactivated by treatment with SDS (0.5% final concentration) and Proteinase K (20 ug/ml). Protected RNA transcripts were recovered by phenol extraction and ethanol precipitation and the RNA pellet was dissolved in FALB (90% forrnarnide, 0.1 % Bromophenol blue and 0.1 % xylene cyalanol) and were analysed by fractionation on a 6% urea — polyacrylamide denaturing gel followed by exposure to film. Transient transfections: HeLa cells were plated at 3x106 cells per plate onto 15 cm diameter tissue culture plates in DMEM containing 5% F BS and antibiotics (penicillin and streptomycin). Transfection was done 24 hrs after plating. 10 ul Lipofectamine 2000 (Invitrogen) was mixed with 250 pl DMEM lacking serum and antibiotics and incubated at room temperature for 15 rrrinutes. This solution was then mixed with 250 pl DMEM lacking serum and antibiotics, containing appropriate amount of plasmid DNA (pCMV-RB or pCMV-empty vector) and incubated for 15 minutes at room temperature. This DNA mix (total volume 500 pl) containing lipofectamine and plasmid DNA in DMEM was then added to cells in fresh 10 m1 DMEM free of serum and antibiotics. Cells were incubated at 37° C, 5% C02 for 5 to 7 hours and then 10 ml media containing serum and antibiotics was added. After 24 hours, cells were harvested by scrapping and analyzed by Westen blotting for RB expression. 83 Methylation analysis (in vivo): Genomic DNA was harvested from HeLa or MCF7 cells that were resuspended in 10 mM Tris—HCl (pH 8.0), 10 mM EDTA at 107 cells/ml. Cells were treated with SDS (final concentration 0.5%) and Proteinase K (200 ug/ml) followed by incubation at 55° C for 2 hrs. Sodium chloride was added to a final concentration of 0.2 M and the resultant mixture extracted with phenol twice, followed by one extraction with chloroform. RNAase A (final concentration 25 ug/ml) was added to a final concentration of 25 ug/ml for 1 hr at 37° C. The DNA samples were extracted with phenol : chloroform (1:1) once followed by extraction with chloroform only. DNA was precipitated with 1.5 volumes of ethanol and resuspended in TE (10 mM Tris-HCI (pH 8.0), 1 mM EDTA) 100 ng of genomic DNA was incubated with either Taa I (F errnentas), prCH4 III, Ava II (New England Biolabs (NEB)) or no restriction enzyme overnight at either 37° C (prCH4 III and Ava II) or 65° C (Ta 1). Digested DNA was recovered by phenol extraction and ethanol precipitation, followed by PCR analysis. Primer sequences used for amplification of the region spanning the U6 start site that contained 1 Ta I/prCH4 111 site were 5’ AAGTATTTCGATTTCTTGGC 3’ and 5’ AATATGGAACGCTTCACG 3’. Primers for amplification of the GAPDH region exon 2 that had no Taa I/prCH4 III site are 5’ AGGTCATCCCTGAGCTGAAC 3’ and 5’ GCAATGCCAGCCCCAGCGTC 3 ’. 84 For methylation analysis after transient RB transfections, genomic DNA was harvested from HeLa cells transiently transfected with RB expression plasmid (as described earlier in this section) and restriction digestion was done as explained above. PCR analysis was carried out with the following primers for the U6, GAPDH region 1 and GAPDH region 2. U6: S’AAG TAT TTC GAT TTC ITG GC 3’ and 5’ AAT ATG GAA CGC TTC ACG 3’ GAPDH region 1: 5’ CAT CAA GAA GGT GGT GAA GCA GGC 3’ and 5’ GCA ATG CCA GCC CCA GCG TC 3’ GAPDH region 2: 5’ CAT TGA CCT CAA CTA CAT GO 3’ and 5’ CCT GGA AGA TGG TGA TGG G 3’ Methylation analysis (in vitro): In vitro transcription was performed as explained previously in this section. DNA was recovered and resuspended in 51 pl water and 6 p1 restriction digestion buffer (NEB buffer 1). Each sample was split into three aliquots of 19 pl each and 1 pl of restriction enzyme (Hpa II/ Msp I, purchased from NEB) or water (for no restriction enzyme control) was added and incubated overnight at 37° C. DNA was recovered by phenol extraction and ethanol precipitation and resuspended in 20 pl water. Each sample was diluted 1:250 in water and 3 pl from this was used for PCR analysis. Primer sequences used for PCR analysis of regions R1, R2 and R3 in Figure 2-4D are as follows. 85 R1: 5’ TTTCTT GGG TAG TTT GCAG3’ and 5’ GTC CTC TGC TGC CTT CAG TG 3’ R2: 5’ AAG CAA CCA TAG TAC GCG CCC 3’ and 5’ GGT CGA GGT GCC GTA AAG CAC 3’ R3: 5’ CCC ATG ATI‘ CCT TCA TAT TTG C 3’ and 5’ CAA GTT ACG GTA AGC ATA TG 3’ RNAi: MCF 7 cells were plated at a density of 2x105 cells per well in 6 well plates. Transient transfection was carried out with serum and antibiotic-free DMEM. siRNA targeting DNMTl, DNMT3A and DNMT3B (at a final concentration of 200 nM) or equivalent amount of control siRNA and reporter plasmid DNA was mixed with 250 pl DMEM. 2.5 p1 Lipofectamine 2000 (Invitrogen) was mixed with 250 pl DMEM and incubated at room temperature for 15 rrrins. Solutions containing lipofectamine and the siRNA and plasmid reporter were then mixed and incubated at room temperature for 15 mins. This mix was then added to cells containing 500 p1 DMEM. Cells were incubated at 37 °C, 5% CO2 for 5 to 7 hours and then 1.5 ml serum and antibiotic containing media was added. After 24 hrs following transfection, RNA was isolated using Trizol reagent. siRNA information: DNMTl-siRNA 1: Oligo ID- HSSlO2859 (Invitrogen) Oligo 1: 5’ AAA GAU GGA CAG CUU CUC AUU UGU C 3’ Oligo 2: 5’ GAC AAA UGA GAA GCU GUC CAU CUU U 3’ 86 DNMTl-siRNA 2: Oligo ID- HSSlO2861 (Invitrogen) Oligo l: 5’ UUC CUU GAU GGA CUC AUC CGA UUU G 3’ Oligo 2: 5’ CAA AUC GGA UGA GUC CAU CAA GGA A 3’ DNMT3A-siRNA 1: Oligo ID- HSSl41868 (Invitrogen) Oligo 1: 5’ UAC ACC AGC CGC UCU CUU GUG CGC U 3’ Oligo 2: 5’ AGC GCA CAA GAG AGC GGC UGG UGU A 3’ DNMT3A-siRNA 2: Oligo ID- HSSl41870 (Invitrogen) Oligo l: 5’ UUC UUU GGC AUC AAU CAU CAC AGG G 3’ Oligo 2: 5’ CCC UGU GAU GAU UGA UGC CAA AGA A 3’ DNMT3B-siRNA 1: Oligo ID- HSSlO2865 (Invitrogen) Oligo 1: 5’ UAG GAG ACG AGC UUA UUG AAG GUG G 3’ Oligo 2: 5’ CCA CCU UCA AUA AGC UCG UCU CCU A 3’ DNMT3B-siRNA 2: Oligo ID- HSSlO2867 (Invitrogen) Oligo l: 5’ UUG AGA UGC CUG GUG UCU CCC UUC A 3’ Oligo 2: 5’ UGA AGG GAG ACA CCA GGC AUC UCA A 3’ Negative Control siRNA: Cat. No. 12935-300 (Invitrogen) RNA isolation: Cells were washed in 2 m1 PBS following removal of medium. Cells were lyzed in 1 m1 Trizol and were resuspended well by repeated pippetting. The contents were transferred to a centrifuge tube followed by addition of 200 pl chloroform. The tubes were vortexed well and spun at 4° C for phase separation. 87 The upper aqueous phase was transferred to a new tube followed by isopropanol precipitation. The pellets were washed with 75% ethanol and then air dried before suspension in 25 p1 TE. RNA quantifications were performed using Nanodrop spectrophotometer (Thermo Scientific) and by agarose gel electrophoresis. 800 ng RNA was hybridized to riboprobe for RNAase protection analysis. Reverse Transcriptase PCR (RT-PCR): 0.2 pg RNA was mixed with 1 pl RNase free DNase (lOU/ pl) and the final volume was adjusted to 10 p1 with water and incubated at 37° C for 10 minutes to digest DNA. Following this, DNase inactivation was done at 75° C for 10 minutes. First strand DNA synthesis was performed by adding 0.5 pg oligo (dT) 12-18 (from Invitrogen) and lpl IOmM dNTP mix. Samples were then incubated at 65° C for 5 minutes and then tubes transferred to ice. 4 pl 5X first strand buffer (Invitrogen) and 2 p1 0.1 M DTI (Invitrogen) were added and incubated at 42° C for 2 minutes. Tubes were placed on ice and 1 pl Superscript II (Reverse Transcriptase, 200 U/pl, Invitrogen) was added, the contents mixed thoroughly (total volume now was 20 pl) and then incubated at 42° C for 1 hour after reverse transcription. The tubes were incubated at 70° C for 15 minutes to inactivate the reverse transcriptase. From this cDNA stock, 1 pl was used as template for PCR amplification with 10 pmoles appropriate primers in a 50 pl PCR reaction. The primers used for amplification of DNMT3A cDNA was 5’ CGT TGG CAT CCA CTG TGA ATG A 3’ and 88 5’ TTA CAC ACA CGC AAA ATA CTC CTT 3’ and those for beta-actin amplification were 5’ CCA TCG AGC ACG GCA TCG TCA CCA 3’ and 5’ CTC GGT GAG GAT CTT CAT GAG GTA GT 3’ (27). Tissue culture: HeLa and MCF 7 cells were grown in DMEM containing 5% fetal bovine serum (Gibco) and penicillin-streptomycin. SAOS2 and UZOS cells were grown in DMEM containing 10% fetal bovine serum (Gibco) and penicillin- streptomycin. The SOASZ derived cell line inducible for RB induction was a gift from Dr. Liang Zhu (Albert Einstein College of Medicine of Yeshiva University). These cells were normally cultured in 15 cm diameter cell culture plates in DMEM containing 10% FBS and penicillin-streptomycin in lpg/ml tetracycline. RB expression was induced by removing media containing tetracycline, washing the plates twice with 10 ml PBS followed by addition of media that is free of tetracycline. In vitro methylation: pU6/Hae/RA.2 was methylated by incubating with 4 units M.Sss I methylase (M0226 NEB) per pg plasmid along with methylase buffer (NEB) and SAM (final concentration 160 pM) in a total volume of 20 p1. Tubes were incubated at 37° C overnight. DNA was then recovered by phenol extraction and ethanol precipitation and the DNA pellet was resuspended in water. The final DNA concentration was quantified by spectrophotometry and also by agarose gel 89 electrophoresis. The indicated amounts of this methylated DNA were used in vitro transcription reactions. Methylation with Msp I methylase was performed similarly. In the case of methylation with both methylases, the methylation reactions were performed sequentially. Plasmid DNA was first methylated with M.SssI, DNA recovered by phenol extraction and ethanol precipitation. DNA was then methylated with Msp I methylase (M0215 NEB) followed by phenol extraction and ethanol precipitation and used in transcription reactions. Protein expression and Purification: GST-RB (379-928) was expressed and purified as described previously (16). Plasmid constructs: pU6/Hae/RA.2 was used as the U6 reporter (23) and pBS-Y1-997 containing the Y1 promoter and the reverse beta-globin reporter were used in this study. Antibodies: anti-SNAP43 (C848), anti-TBP (SL2) are previously described (15, 33). IgG (Gibco), anti- DNTMl (Imgenex IMG-26l), anti- DNMT3A (Imgenex IMG-268A), anti-DNMT3B (Abcarn ab2851) were used in this study. 90 Results In mammalian genomes, DNA methylation at the 5 position of cytosine residues is often used as a mechanism to silence transcription. CpG dinucleotide enrichment seen in many functional promoters in the genome suggests an important role for DNA methylation in gene regulation. In H. sapiens, the U6 snRNA gene is present as nine copies, five of which are functional (U6- 1, 2, 7, 8 and 9) for U6 transcription and four are non-functional (U6- 3, 4, 5, 6). All the nine U6 copies have identical coding regions but vary only at the promoter region (10). The non-functional U6 copies — U6-3, 4, 5 and 6 lack the TATA Box, PSE and DSE (Figure 2-1). Sequence analysis (from -300 to +200) of all the nine copies of the human U6 gene revealed that the promoter proximal regions of all the functional copies were comparatively more enriched in CpG dinucleotides than the non-functional copies (Figure 2-1). The ratio of the number of CpG dinucleotides to the number of GpC dinucleotides was also higher for U6 copies 1, 2, 7, 8 and 9 than U6- 3, 4, 5 and 6 (Figure 2-1). CpG dinucleotides are frequent targets of methylation at the 5 position of cytosine, catalysed by the DNMT family of proteins DNMT], DNMT3A and DNMT3B. Considering the pre- existing body of knowledge that .suggests a link between RB and DNA methyltransferases (however, demonstrated only in Pol II and P01 1 systems), and the role of DNA methylation in gene silencing, I hypothesized that CpG dinucleotides could be playing an important role in regulating U6 transcription. Therefore, I examined the potential role for DNA methylation and DNA methyltransferases in RB repression of U6 transcription. Firstly, I was 91 Figure 2-1: CpG plot of all the nine U6 copies. The positions of CpG dinucleotides (represented by a red dot) were plotted for all the nine U6 genes copies. The general promoter structure of the U6 genes is depicted. The non- functional copies U6-3, 4, 5 and 6 lack the U6 promoter elements DSE (green box), PSE (blue box) and TATA Box (pink box) (10). The U6-9 gene lacks a PSE consensus but recruits SNAPc (10). The transcriptional start site is indicated by the green line. The position of the termination sequence is indicated as ‘t’. The ratio of the number of CpG dinucleotide to GpC dinucleotide contained between - 300 to +1 for each of these copies is indicated. 92 3N... cognac 3... w “-1- -8 ‘7 -§ L§ 6.9 3.: 8.: 3.: to: 8.8 .2 .1 . Y T 8: 8.8 6.: 0868 mg 03 5.0: mgn 15m «.8 s «.3 Te: 93 interested in knowing whether there was any link between the presence of CpG methylation and RB activity status. For this, I analyzed the methylation status of a highly conserved start site CpG dinucleotide in cells that either have active or inactive RB. 1 have analysed the U6-1 gene in all the experiments presented in this study. The conserved U6 start site CpG is methylated in cells containing active RB but not in cells lacking RB activity To examine whether promoter CpG methylation is linked to RB function, a comparative analysis of the methylation status of the start site CpG was done using genomic DNA harvested from cells containing active RB and those that lack functional RB. Methylation status of the start site CpG was analysed in HeLa (lacking functional RB) and MCF 7 cells (that contain functional RB) using a restriction digestion-based approach. The U6-1 start site contains the sequence ACCGT, which is cleaved by isoschizomers Taa I and prCH4 III depending on the methylation status of the second cytosine in the recognition sequence (Figure 2-2A). Methylation at the 5 position of the second cytosine impairs digestion by Ta 1 but not prCH4 III (Figure 2-2B). Restriction digestion by either enzyme was measured by PCR amplification across this restriction site. Effective cutting would lead to the absence of PCR product but protection from cutting by methylation would result in a PCR product. Restriction digestion of the ACCGT sequence at the U6 start site by Taa I was impaired in MCF7 genomic DNA (Figure 2-2C, lane 8) but not in HeLa genomic DNA, whereas digestion by 94 Figure 2-2: The U6 start site CpG is methylated in vivo in MCF7 but not HeLa cells. (A) Nucleotide sequence (from -10 to +10) of the U6 snRNA gene. The U6 start site region contains a recognition sequence for isoschizomers Taa I and prCH4 III. (B) Methylation of the indicated cytosines at the 5 position impairs Taa I digestion but not prCH4 III digestion. (C) Restriction digestion of genomic DNA from MCF7 or HeLa cells using Ta 1 or prCH4 III enzymes followed by PCR amplification of the U6 snRNA gene (lanes 5 to 8) or the GAPDH exon 2 region (negative control)(lanes l to 4). Lanes 1 to 3 and lanes 5 to 7 (Input titrations: 100%, 10%, 1%) are negative control reactions in which the genomic DNA was incubated with no restriction enzyme at either 65 °C (optimum temperature at which Taa I digestion was done) or at 37°C (optimum temperature at which prCH4 III digestion was done), respectively. (D) Ratio of normalized U6 PCR product intensity of Taa I to prCH4 III treated DNA was calculated for HeLa and MCF7 cells. Normalization was done to corresponding 10% input. Quantitation from three experimental measurements was performed and the ‘p value’ for MCF7 in comparison to HeLa is indicated. ‘p value’ calculations were done using two-tailed student T-test. 95 +1 5' . .ACGAAAACCG crcecrr. . 3' U6 template (in viv0) i Taal & prCH4||I recognition sequence . 5m 5' . . . A C O G T . . . . 3' Taal digestion impaired 3', , . , T G G C A. , , , 5' prCH4lll digestion occurs 5m GAPDH exon2 U6 1 8 I F .8 1 Input a Input 3 ‘I 6 — '6 — - n _ m . Taal HeLa "" -- ""'" "- ~- prCH4Ill P w tun-w ~ ~ “II-”'- n Taal MCF7 '- --- _ "" prCH4lll 1 2 3 4 5 6 7 8 96 Figure 2-2 contd. 0.02 p Cell Line HeLa 5. 2 5. 1 a... q 2 1 0 <20 .038: 5 Eu .3: 2 _ E Co E235 63:9: 6.: w: :0 0:3. 97 prCH4 III was not impaired in either of the two cell lines, indicating that the start site CpG is methylated only in cells containing active RB. This suggests a positive correlation between U6 promoter methylation and RB functional status. PCR amplification across GAPDH exon 2 that has no Taa I/prCH4 III restriction site serves as a control for presence of equal amounts of total DNA (Figure 2-2C, lanes 1-4). Quantitation results (Figure 2-2 D) from three experimental measurements showed approximately 2.7 fold enhanced methylation in MCF7 cells when compared to HeLa cells. Transient RB expression in cells lacking RB function results in start site CpG methylation The presence of promoter CpG methylation only in cells containing active RB but not in cells lacking functional RB suggests a potential link between DNA methylation and RB function at the U6 gene. However, it was unclear whether RB is the factor that is responsible for directing CpG methylation at the U6 promoter. Therefore, to address whether RB can direct DNA methylation at the U6 start site, RB was transiently expressed in HeLa cells (which lack functional RB), followed by analysis of the U6 start site methylation status. Panel A of Figure 2-3 is a Western blot showing RB expression in HeLa cells transfected with the RB expression vector pCMV-RB. Genomic DNA from HeLa cells that were either untreated or transfected with pCMV-RB or empty vector (pCMV-EV) was analysed for start site methylation status using restriction digestion with Taa I and prCH4 III enzymes. PCR amplification across the U6 start site or GAPDH 98 Figure 2-3: The U6 start site CpG gets methylated in vivo in HeLa cells in response to transient RB expression. (A) Western Blot showing RB expression in HeLa cells transiently transfected with the RB expression vector (pCMV-RB) (lane 2). (B) Schematic representation of the number of restriction enzyme sites in the genomic regions analyzed by PCR amplification. (C) Restriction digestion of genomic DNA from HeLa cells that were either untreated (lanes 1 to 4), or transfected with pCMV-RB (lanes 5 to 8) or pCMV-EV (lanes 9 to 12) using Taa I or prCH4 III or Ava II enzymes, followed by PCR amplification of the U6 snRNA gene or GAPDH region 1 or region 2. (D) Ratio of normalized PCR product intensity for Taa I to pr CH4 III treated DNA was calculated for the U6 locus, GAPDH region 1 and GAPDH region 2 for untreated HeLa cells and HeLa cells treated with either pCMV-RB or pCMV-EV. Quantitation from two experimental repeats was performed. Normalization was done to the corresponding Ava II digested sample. The respective ‘p values’ for the U6 locus, GAPDH region 1 and GAPDH region 2 for HeLa cells treated with pCMV-RB or pCMV-EV were calculated in comparison to HeLa cells that were untreated. ‘p value’ < 0.05 is indicated with an asterisk C“). ‘p values’ were calculated using two-tailed student T-test. 99 $30.5 echo-Swot E9: :ofioEQEm mom NP 2 9 m m C. o w. v m N F II ll 11 I...- II 1.. I. ll. Ncofimm 'I " I... I'll. '11'Fco_oom l in... I i I ' ' Ilr.‘ ' 1.1 I. V H V l. V H V I. V H H _ H _ _ L _ I _ >m.>s_oa - 32.. 3.22% - 32.. 852.5 - So: :05 ESmmxS m> o o o = < mm _H ma m m , r o A .. em. 3 __=V_._o>ax\_m£ d .a n m m M... « o« A A 9 .~. .~. new c2638”: .02 100 Figure 2-3 cond. N c063". IOQ I . .mIOD l :cwma NEE-$3.. + I I + + ..._. + + + .+ + ...+ + I - — — _ _ _ — \— — — . _ uofiozc: x82 .85. + .35. _ $0.2 8822:: _ mwm._>_ .Enoo 0N onE 116 Figure 2-6 contd. A pus/Hae/RA2 + + + + +g pY1-Bglobin C Untreated H "-‘T’ . U66. ”carp—.- Y1-5' Methylated '* *' "' ““‘U5'5' (M.Sss l + Mspl) P—u—mc—N-W -5' Mock T" fl-“ ‘U6'5' 1" .0 h. :1 'YI _51 b 1 2 f 4... “”5 2.5" I) 2‘ / Untreated U6 transcription 5‘. 1 I 1-1 ——I +, 0.5- \- Methylated 0 r T l l l I so 100 150 200 250 Mass DNA (ng) 117 reporter only (lanes 12 and 13, Figure 2-6B) or Y1 reporter only (lane 14, Figure 2-6B) are also shown. ‘IC’ represents the loading control band. To make sure that we were carrying out these transcription reactions with template amounts that were not limiting for transcription (which can affect the fold differences in transcription seen), I titrated the U6 template (from 0.95ng to 250 ng in two-fold increments). The indicated amounts of U6 template that was either treated with both methylases or mock treated or untreated plasmid were used in transcription reactions (Figure 2-6C, shown only 7.5 ng to 125 ng titration). Quantitation of this data presented in panel D of Figure 2-6 again confirmed the result presented in panel B of Figure 2-6. At template amounts (from 50 ng and higher) that were not limiting for transcription, I saw at least 3- fold reduction in U6 transcription from template that was treated with both methylases in comparison to the mock treated and untreated templates. These results indicate that methylation of the outside cytosine at the start site CCGG sequence along with methylation of all the CpG dinucleotides in the U6 reporter plasmid further enhanced the repressive effect that CpG methylation had on U6 transcription. This suggests that RB could be employing CprG methylation along with CpG methylation at the U6 promoter to cause efficient repression of transcription. The observations that CprG and CpG methylation can repress U6 transcription and that CprG methylation adds to the repressive effect of CpG methylation lead us to propose that RB repression can be mediated by the recruitment of methyltransferase activity that leads to methylation of 118 cytosine residues in the CpG as well as non-CpG sequence contexts. Reports suggesting that cytosine methylation in CpCpG contexts can be catalysed by DNMTl (28) and that DNMT3A can methylate cytosines in CpA and CpT dinucleotides (29) lend support to the idea that RB can direct DNMT activity to catalyze cytosine methylation at CpG and at non CpG sequences to cause transcriptional repression. However, methylation of only start—site cytosines (using Hpa II and Msp I methylases) without methylating the other CpG dinucleotides seemed to have no effect on U6 transcription (data not shown). These results suggest that the combined methylation of the start site cytosines and the other promoter Cst is important for exerting the repressive effect on U6 transcription. DNMT] and DNMT3A occupy the endogenous U6 promoter in RB positive cells but not cells lacking RB activity Many lines of evidence presented here suggest a correlation between RB repression of U6 transcription and DNA methylation at the U6 promoter. In mammalian cells DNA methylation is carried out primarily by the DNMTs 1, 3A and 3B. So I questioned whether these DNA methyltransferases occupied the U6 promoter and analysed for possible correlation between RB functional status and DNMT occupancy at the U6 promoter. Robertson et al., (30, 32) have suggested that DNMT], which is a maintenance methyltransferase and the most abundant DNMT in somatic cells, cooperates with RB to repress target gene transcription by RNA polymerase 11. Therefore, I investigated the presence of DNMT] at 119 endogenous U6 promoters. To examine potential correlation between RB functional status and DNMT occupancy status at the U6 promoter, we carried out comparative ChIP analyses with MCF7 (that has active RB) and HeLa cells (which lack functional RB). Results presented in Figure 2-7B show that DNMT] occupies the U6 promoter only in MCF7 cells but not in HeLa cells. DNMT] did not occupy the U1 promoter (which is not an RB target gene) in either cell line. Interestingly, DNMTl did not occupy the SS rRNA gene although RB represses SS rRNA transcription. Although the SS rRNA gene is subject to RB regulation, RB does not occupy the 5S rRNA promoter. On the other hand, the U6 gene is sensitive to RB regulation and also is targeted for occupancy by RB (Figure 2-7A). Therefore, DNMT] occupancy on the U6 promoter but not 5S rRNA promoter (lane 6, Figure 2-7B) indicated that DNMTl occupancy on target genes correlates with RB sensitivity and RB occupancy on the target gene. GAPDH exon 2 serves as a negative control for PCR amplification. Immunoprecipitations done with antibodies against SNAP 43 (lane 5, Figure 2-7B) and TBP (lane 7, Figure 2-7B) serve as positive controls. Negative control immunoprecipitation was done with IgG. Figure 2-7C shows quantitation of the data shown in Figure 2-7B. In MCF7 cells, approximately S-fold enrichment of the signal corresponding to DNMT] occupancy on U6 was observed compared to the IgG control. The signal corresponding to DNMTI occupancy on the U6 gene was negligible in HeLa cells. No significant signal intensity was observed for DNMT] occupancy on the U1 and 5S rRNA genes in both cell lines. Chromatin immunoprecipitation from 18435 cells (derived from normal mammary tissue) also showed the occupancy 120 Figure 2-7: DNMT] association with the endogenous U6 promoter coincides with RB activity status. (A) Promoter structure of the U6, U1 snRNA and 5S rRNA genes, their RNA Polymerase specificity, sensitivity to RB repression and the occupancy status of RB on these genes. (B) Chromatin from MCF7 or HeLa cells and was used in immunoprecipitation reactions with or-SNAP43 (lane 5) or or- DNMT] (lane 6) or oc-TBP (lane 7) or non-specific IgG (negative control) (lane 4). PCR analysis was done to test the enrichment of DNA fragments containing U6, U1 or SSrRNA promoter regions in the immunoprecipitated DNA. GAPDH exon 2 served as the negative control. Lanes 1-3, input titration (1%, 0.1% and 0.01). (C) Quantitation of the ChIP result in B. PCR product intensity was normalized to 1% Input. Results for the U6, U1 and 5S rRNA loci for the MCF7 and HeLa cells are shown. (D) Chromatin harvested from 184B5 cells was used in immunoprecipitation with antibodies against SNAP43 (lane 6), DNMT] (lane 7), TBP (lane 8) or IgG (lane 5). PCR amplification was done for U6, U1, U2, 7SK, 5S rRNA and GAPDH loci. Input titrations 10%, 1%, 0.1%, 0.01% are in lanes 1, 2, 3 and 4 respectively. (B) Quantitation of ChIP results from two experimental repeats from 184B5 cells. Results for immunoprecipitations of the U6, U1, U2, 5S rRNA and GAPDH loci with IgG, anti-SNAP43 and anti-DNMT] antibody from 184B5 cells are shown. PCR product intensity was normalized to the corresponding 1% input. The ‘p value’ for the U6 immunoprecipitation with anti-DNMTI antibody was calculated in comparison to the ‘IgG control’ using two-tailed student T-test. 121 I + 5 “22¢ I i = $2: + + =_ a(2¢ 6:838 32:28 £6589 a. a: .22: (2% mm (2:5. 5 (2x5. 0: L L mmn. Ella 122 Figure 2-7 contd. B ,3 r: 3 3 a Input g 0. ,2 - aura—- - - ' ~ ‘ “ MCF7 U6 — _ - an. HeLa u - — all-n MCF7 U1 0.— -—- — .— HeLa m .- .. MCF7 SSrRNA [* a m " HeLa ._ ., MCF7 GAPDH u 4‘ HeLa exonz 1 2 3 4 5 6 7 Chromatin Immunoprecipitation 123 Figure 2-7 contd. 19375543240 000000000 C 38:35 _mcm_w mom HeLa MCF7 HeLa MCF7 5225 .25: mo: SS rRNA .5235 .29:- mo: HeLa MCF7 124 Figure 2-7 contd. (g .— g. Q. g E a- Input U.) D. g K9 .5 .5 .5 — 9 g g g *‘ - " “ — ~ g u m a ’* - u q... (II-I C-ID a... an.- ,,,,, A 7 , 1 2 3 4 5 6 7 8 Chromatin Immunoprecipitation 125 U6 U1 U2 75K SSrRNA GAPDH PCR signal intensity (1% Input) Figure 2-7 contd. IgG anti—SNAP43 18485 cell line 126 anti-DNMT1 of DNMTI on the U6 promoter and not U1, U2, 7SK or SS rRNA promoters (Figure 2-7D). Quantitation of two experimental repeats of chromatin immunoprecipitation with IgG, anti-SNAP43 or anti-DNMT] antibodies using 184B5 cells are shown in Figure 2-7E. Approximately 2.7 fold enrichment of the signal corresponding to DNMT] occupancy on the U6 gene was observed when compared to the ‘IgG control’. However, the ‘p .value’ measured was 0.1, indicating a 90% confidence level for this result. No significant signal intensity corresponding to DNMT] occupancy on the U1, U2, SS rRNA and 78K genes was observed. Following this, I extended the analysis to look for the occupancy of the other major methyltransferases DNMT3A and DNMT3B on the endogenous U6 promoter and whether there was any correlation with RB functional status. For this, chromatin immunoprecipitation was done from two osteosarcoma cell lines SAOSZ and U208 cells of which SAOSZ does not contain RB activity whereas U208 is positive for RB activity (Figure 2-8A). I observed that along with RB, DNMTl and DNMT3A occupied the endogenous U6 promoter only in U208 cells but not in SAOSZ cells, indicating a positive correlation between RB functional status and occupancy of DNMT] and DNMT3A at the endogenous U6 promoter. Quantitation of chromatin immunoprecipitation results from two experimental repeats is shown in the lower panel of Figure 2-8A. At least 3-fold enrichment of the DNMT] signal and a 4-fold enrichment of the DNMT3A signal in comparison to IgG was observed. The ‘p values’ corresponding to DNMTI and 127 DNMT3A were 0.1 and 0.06 respectively. At least 90% confidence level can be attributed to this result. Transient expression of RB results in recruitment of DNMT] and 3A on the endogenous U6 promoter The results mentioned above led us to speculate that RB directs DNMT recruitment to the U6 promoter. To examine whether RB directs recruitment of DNMTs to the U6 promoter, RB expression was induced in cells lacking RB and DNMT association with the U6 promoter was analysed by chromatin immunoprecipitation. A SAOSZ derived inducible cell line was induced to express RB upon tetracycline removal. Figure 2-8B is a Western blot showing RB eXpression upon tet removal. Following induction of RB expression, chromatin immunoprecipitation was done to look for RB recruitment to the U6 promoter as a first step. Panel C of Figure 2-8 shows recruitment of RB to the endogenous U6 promoter upon induction of RB expression by Tet removal. GAPDH exon 2 serves as the negative control. Then occupancy status of the DNMTs 1, 3A and 3B were examined after induction of RB expression. Figure 2-8D shows that upon induction of RB expression by tet removal, RB and also DNMT3 1 and 3A get recruited to the U6 promoter, suggesting that RB can direct the recruitment of DNMT] and DNMT3A to the U6 promoter in vivo. The DNMT occupancy profile from ChIP analysis after induction of RB expression is in agreement with comparative analysis among U208 and SAOSZ cells (Figure 2-8A). Quantitation of the result in Figure 2-8D is shown in its lower panel. Approximately 3-fold 128 Figure 2-8: RB recruits DNMT] and DNMT3A to the endogenous U6 promoter: (A) Chromatin immunoprecipitation from two osteosarcoma cell lines SAOSZ (RB negative) and U208 (RB positive) was performed with antibodies raised against RB (lane 5), DNMT] (lane 6), DNMT3A (lane 7) and DNMT3B (lane 8). Immunoprecipitation performed with IgG (lane 3) and anti-TBP (lane 4) antibody serve as the negative and positive controls respectively for immunoprecipitation of crosslinked DNA. Lanes 1 and 2 contain input titration from 0.1% to 0.01% respectively. PCR amplification of the U6 promoter or GAPDH exon 2 (negative control for immunoprecipitation) was done. Lower Panel. Quantitation from two experimental repeats of the representative result shown in the top panel. PCR product intensity was normalized to the corresponding 0.1% input and the respective ‘p values’ were calculated in comparison to ‘IgG’ control and are indicated. (B) The SAOSZ derived inducible cell line was cultured in media containing Tetracycline (1 ug/ml). Western blot analysis shows expression of RB (by tet removal) after 48 hours after induction. (C) 8A082 inducible cells were induced for RB expression, chromatin harvested at 48 hrs after RB induction, and immunoprecipitation was done with antibodies against TBP (lane 4), RB (lane 5) or non-specific IgG (lane 3). PCR amplification of U6 promoter region or GAPDH exon 2 region (negative control for immunoprecipitation) was done. Lanes 1 and 2 contain 0.1% and 0.01% input respectively. Chromatin immunoprecipitation was also done from cells that were not induced for RB expression (panels labelled as +TET). (D) RB expression was induced as explained above and chromatin immunoprecipitation was done with antibodies raised against TBP (lane 4), RB (lane 5), DNMTl (lane 6), DNMT3A (lane 7), DNMT3B (lane 8) or non-specific IgG (lane 3). PCR amplification of U6 promoter DNA was done. Lanes 1 and 2 contain 0.1% and 0.01% input respectively. Lower Panel. Quantitation of results in the top panel. PCR product intensity was normalized to corresponding 0.1% input. 129 mmbzzoé _20 N_20 F_Z0