MECHANISMS OF TRANSCRIPTIONAL REGULATION: GENE REPRESSION BY KRAB ZINC FINGER PROTEINS AND GENE INDUCTION BY ESTROGEN RECEPTOR beta by SMITHA P. SRIPATHY Submitted in partial fulfillment of the requirements for a degree in Doctor of Philosophy. Dissertation advisors: David C. Schultz and Monica M. Montano Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY January 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Smitha P. Sripathy for the Pharmacology degree *. (signed) Ruth A. Keri (chair of the committee) John J. Mieyal David Samols Monica M. Montano (date) 07/15/08 *We also certify that written approval has been obtained for any proprietary material contained therein. i Table of Contents List of figures and tables iii Acknowledgement viii List of abbreviations 1 Abstract 3 Chapter I Transcriptional regulation: An overview 6 Gene repression by KRAB zinc finger proteins Chapter II Introduction, review of literature and 17 statement of purpose Chapter III Mechanism of transcriptional repression of 35 KRAB zfps Chapter IV Summary and future directions 117 Gene induction by estrogen receptor beta Chapter V Introduction, review of literature and 134 statement of purpose Chapter VI Mechanism of transcriptional induction of 159 EpRE-genes by ERβ Chapter VII Summary and future directions 201 Bibliography 211 ii List of Tables and Figures Figure I-1 Hox gene clusters regulate head-tail morphology 9 in humans Table I-1 A minimal classification of transcriptional 11 coregulators Figure II-1 Representation of a complex between DNA and 19 the ZIF268 protein, containing 3 zinc finger motifs. Figure II-2 Sequence alignment of the KRAB domain from 22 different KRAB–ZFPs Figure II-3 Schematic representation of the TIF1 protein 26 family Figure III-1 KAP1 is required for KRAB-mediated repression 75 Figure III-2 Stable depletion of KAP1 in HEK293 cells. 77 Figure III-3 Direct tethering of KAP1 to DNA is sufficient to 79 represses transcription Figure III-4 The corepressor activity of KAP1 depends on its 81 interaction with HP1 and a functional PHD finger and bromodomain Figure III-5 Establishment of cell lines with a hormone- 83 iii regulatable reporter transgene Figure III-6 Characterization of cell lines with a chromatinized 85 5XGAL4-TK-luciferase transgene. Figure III-7 Hormone dependent repression of chromatinized 87 luciferase transgenes by ERHBDTM-GAL4 fused repressor proteins Figure III-8 Hormone-dependent repression of the chromatin 89 template by ERHBD-GAL4-KRAB requires KAP1 Figure III-9 Hormone-dependent repression Kinetics of 91 ERHBD-GLA4-KRAB wildtype and mutant cells Figure III-10 Hormone-dependent KRAB repression is 93 dependent on KAP1. Figure III-11 KRAB mediated repression of a chromatin 95 template is independent of TIF1α and TIF1γ. Figure III-12 Hormone-dependent repression of chromatin 97 templates by ERHBD-GAL4-KAP1 Figure III-13 Kinetics of hormone-dependent repression of 99 chromatin templates by ERHBD-GAL4-KAP1 wildtype and mutants Figure III-14 Expression of the KAP1 repression machinery 101 Figure III-15 Chromatin immunoprecipitation analysis of a 103 iv luciferase transgene repressed by ERHBD-GAL4- KAP1. Figure III-16 Hormone dependent repression mediated by 105 KAP1 involves hypoacetylation of histone H3 K9 of the transgenic reporter Figure III-17 Disruption of the KAP1-HP1 interaction fails to 107 induce hormone-dependent changes in RNA polymerase II recruitment, histone occupancy, and histone modifications Figure III-18 Chromatin immnoprecipitation analysis of 109 hormone-dependent changes in mediated by ERHBDTM-GAL4-KAP1, mutated in either the PHD finger or bromodomain, respectively Figure III-19 Hormone-dependent KRAB repression of a 111 chromatin template requires KAP1, HP1, and SETDB1. Figure III-20 Transient knockdown of HP1β expression and its 113 effect on KRAB mediated repression. Figure IV-1 Model of KRAB/KAP1 mediated repression 119 v mechanism Figure V-1 Metabolic activation and deactivation of estradiol 140 Figure V-2 Representation of cDNA variants that encode ERβ 151 isoforms Figure VI-1 hPMC2 interacts directly with ERβ and is involved 182 in mediating Tamoxifen-dependent decrease in ODD levels Figure VI-2 Tamoxifen-dependent recruitment of coactivators 184 to the EpRE sequence of NQO1 Figure VI-3 Both ERβ and hPMC2 are required for tamoxifen- 186 mediated increase in antioxidative enzyme expression and protection against ODD Figure VI-4 Tamoxifen-dependent recruitment of coactivators 188 to the EpRE sequence of NQO1 requires both ERβ and hPMC2. Figure VI-5 Tamoxifen treatment induces increased 190 transcription of antioxidative enzyme levels. Figure VI-6 Knockdown of PARP-1 attenuates tamoxifen- 192 dependent increase in the expression of vi antioxidative enzymes Figure VI-7 PARP-1 down regulation does not significantly 194 affect basal expression levels of antioxidative enzymes. Figure VI-8 Ligand dependent recruitment of ERβ and hPMC2 196 to the ERE region Table VI-1 Sequence of DNA oligos cloned into pSuper- 198 QRshRNA, pSuper-hPMC2shRNA and PCDNA- hPMC2 569miR plasmids respectively. Table VI-2 Primer sequences used in the various PCR 199 reactions described in Chapter VI Table VI-3 Antibodies used in Chapter VI 200 Figure VII-1 Induction of antioxidative genes by tamoxifen 202 liganded ERβ vii Acknowledgements First and foremost I would like to thank my parents who instilled in me a sense of curiosity and wonder, encouraged me to explore the world around and shared in my enthusiasm. These qualities led me to pursue science as a career and were a major factor in my decision to do a Ph.D. They have been a source of constant support and encouragement all through the making of this dissertation. I extend my sincere thanks to my first thesis advisor Dr. David Schultz for starting me out on the path of scientific thinking. I learnt to critically analyze experiments, both others and mine, and think about more than just the most obvious interpretation of a result. He had an invaluable teaching tool that was very effective in getting the message across to me, he simply taught by example. His dedication to order, method, and attention to detail while planning and conducting experiments has made me realize that the “devil is indeed in the details”. I thank him for providing me with the foundations necessary for any student of science. It is a pleasure to thank my current thesis advisor, Dr. Monica Montano whose support and encouragement was key to the completion of this dissertation. I thank her generosity in letting me join her lab and the independence she gave me in conducting my project. She has always been ready viii to discuss any concerns regarding not just my dissertation work, but just about anything. Her open and hands off approach has helped me gain confidence in my ability to successfully generate and test hypotheses, a critical quality in any scientist. It has been a lot of fun being in Monica’s lab, her humor and ready sense of wit has made for many a lively conversation in the lab. In more than 5 years that it took to complete this dissertation, I have been fortunate to meet some wonderful people who have become great friends. I would especially like to acknowledge Bonnie Gorzelle, Ndiya Ogba and Laura O’ Donnell. Bonnie has been a very patient listener, her calmness has helped me many a times in not getting hyper over my negative results (though she is completely unaware of it). I am lucky to have a person like Ndiya for a friend. She has been a great support for me in the lab, a very good source to discuss not only trouble shooting strategies or just about anything. I thank her for being who she is and also for the umpteen number of times she has driven me all over Cleveland at very short notices. Her timely help has enabled me to actually focus on my experiments. Last but not the least, it is with joy that I thank Laura. She is just a great person to be around with and a good friend. I thank her for helping me seek reagents around the lab for the nth time without the slightest trace of irritation. I thank her for her constant bright smile and helping nature that endears her to everyone. Any acknowledgement of Laura is not complete ix without the mention of her amazing cakes and cookies which were frequently to be found outside the lab. x List of Abbreviations 4-OHT 4-Hydroxytamoxifen 8-OHdG 8-Hydroxydeoxyguanine APL Acute Promyelocytic Leukemia ATRA All trans retinoic acid CE Catechol estrogen CE-SQ Catechol estrogen-semiquinone CE-Q Catechol estrogen quinone ChIP chromatin immunoprecipitation DBD DNA binding domain DBS DNA binding site E2 17β-estradiol EpRE electrophile response element ERα Estrogen receptor alpha ERβ Estrogen receptor beta ERE Estrogen response element ERHBD Estrogen receptor hormone binding domain GCSh Gamma-glutamylcysteine Synthase, heavy subunit GSTpi Glutathione-S-transferase H3K9 Histone H3 Lysine 9 HDAC Histone deacetylase 1 HP1 Heterochromatin protein 1 hPMC2 Human homolog of Xenopus laevis prevention of mitotic catastrophe 2 IP Immunoprecipitation KAP1 KRAB associated protein 1 KRAB Kruppel associated box ODD Oxidative DNA damage PHD plant homeodomain QR Quinone reductase RBCC RING B-box Coiled-Coil RING Really Interesting New Gene SETDB1 SET domain bifurcated 1 TOT trans-Hydroxytamoxifen TIF1 Transcription intermediary factor 1 Zfp zinc finger protein 2 Mechanisms Of Transcriptional Regulation: Gene Repression By KRAB Zinc Finger Proteins And Gene Induction By Estrogen Receptor beta Abstract by SMITHA P. SRIPATHY DNA binding transcription factors are key players in the dynamic regulation of distinct gene expression programs, essential for maintenance of cell physiology and differentiation. Transcription factors either activate or repress transcription by sequence-selective binding to cis DNA elements and subsequent recruitment of distinct coregulator complexes.