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Dynamic chromatin associated ubiquitination with cell cycle progression in human cancer cells DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Mansi Shyam Arora Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2014 Dissertation Committee: Dr. Jeffrey Parvin, Advisor Dr. Anita Hopper Dr. Mark Parthun Dr. Robin Wharton Copyright by Mansi Shyam Arora 2014 Abstract In this dissertation work, we have analyzed the pattern of ubiquitin conjugates on human chromatin and its changes with the progression of cell cycle. Our work shows that during interphase, ubiquitination marks the transcribed regions of the genome. This ubiquitination correlates with the ubiquitination of H2B, is dependent on active transcription and is removed during mitosis. We had anticipated that all the ubiquitin associated with the transcribed regions would be removed from chromatin during mitosis, but contrary to our expectation, we found that at the promoters of active genes chromatin ubiquitination levels actually increase thus implying this modification as a possible mitotic bookmark. In the second half of this project, we set out to identify the substrate modified by this post translational modification at these promoters and the enzymes involved in its deposition and removal before and after mitosis respectively. Our results show the surprising involvement of the SAGA associated deubiquitinase USP22 and the polycomb complex proteins BMI1 and RING1A in the regulation of this bookmark during mitosis. The polycomb complex proteins are thought to primarily regulate gene expression by transcriptional repression. Although some previous studies have implied the involvement ii of the polycomb proteins in the regulation of active genes, their association with the transcriptional regulation of active genes during the mitosis to G1 transition has not been described before this work. Our data reveal that BMI1 and RING1A regulate the mitotic bookmarking by ubiquitination and their expression once the cells exit mitosis and enter G1. They are also required for the progression of the cells through mitosis and entry into G1 as well as progression through G1. We also show that lack of RING1A (and thus mitotic bookmarking by ubiquitination) is deleterious to the survival and proliferation of tissue culture cell lines thus presenting as a potential for novel targets for cancer therapy. iii Dedication Dedicated to my husband and my family iv Acknowledgments First and foremost, I would like to express my deep gratitude towards my advisor Dr. Jeffrey Parvin. This thesis would have been inconceivable without his invaluable guidance throughout the course of my time in his lab. Jeff has a great mentor, a good scientist and great person and I find myself extremely fortunate to be provided an opportunity to know him and work under his guidance. He not only helped me develop my research skills and values, but his faith and confidence in me has also helped me become an independent scientist. I would also like to thank my committee members - Dr. Robin Wharton, Dr. Mark Parthun and Dr. Anita Hopper for their helpful discussions during committee meetings and for always encouraging me to think critically of my work and to strive for higher academic and research standards. I will be forever grateful to Dr. Anita Hopper for her excellent career guidance, her eternally optimistic attitude and her kind words of encouragement and support whenever I needed them. I would like to thank my lab mates and good friends, Zeina Kais and Huiwen Liu for their insightful suggestions and comments, and other members of the lab (past and present) who made the times in the Parvin Lab always fun and exciting. v The journey through grad school and life in Columbus would not have been as much fun had it not been for the awesome company of friends I was lucky enough to meet in this city. I thank you all for being there for me, always. I dedicate this thesis to my parents who, despite coming from a traditional background, have gone against the tide and always encouraged me to achieve academic excellence and personal independence. Last but not the least; I would like to express my deepest love and gratitude to my dear husband who from the time we met almost ten years ago and until now remains my best friend, biggest emotional support, academic critic, and inspiration to be my best. It would have been impossible to be where I am without him. vi Vita 2007-Present……………….PhD Candidate, The Ohio State University, Columbus, OH 2006………………………...M.Sc., Biochemistry, M.S. University, Vadodara, India 2004………………………...B.Sc., Biochemistry, Gujarat University, Ahmedabad, India Publications 1. Arora M, et al. (2012) Nucleic Acids Res. 40(20):10187-202. (Featured Article) 2. Liu HW, Zhang J, Heine GF, Arora M, et al. (2012) Nucleic Acids Res. 40(20):10172-86. (Featured Article) 3. Zhang J, Lu K, Xiang Y, Islam M, Kotian S, Kais Z, Lee C, Arora M, et al. (2012) PLoS Comp Biol. 8(8):e1002656. 4. Johnson N, Cai D, Kennedy RD, Pathania S, Arora M, e al. (2009) Mol Cell. 14; 35(3):327-39. 5. Parvin JD, Kais Z, Arora M, et al. (2009) Proceedings of the Ohio Collaborative Conference on Bioinformatics (OCCBIO): 71-75. vii Fields of Study Major Field: Molecular, Cellular and Developmental Biology viii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments ............................................................................................................... v Vita .................................................................................................................................... vii Publications ....................................................................................................................... vii Fields of Study ................................................................................................................. viii Table of Contents ............................................................................................................... ix List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv Chapter 1: Dynamic ubiquitination of chromatin associated proteins through the cell cycle .................................................................................................................................... 1 1.1 Chromatin: What is it? ........................................................................................ 1 1.2 Epigenetic Vs. Dynamic: .................................................................................... 1 1.3 Post translational modifications: Writers, readers and erasers ........................... 3 1.4 Polycomb repressive complex ............................................................................ 18 ix 1.4 Epigenetic inheritance through cell cycle ......................................................... 23 1.5 Cell cycle associated changes in ubiquitination ............................................... 29 Chapter 2: Rationale ......................................................................................................... 31 Chapter 3: Ubiquitination of chromatin associated proteins changes through the cell cycle ........................................................................................................................................... 33 3.1 ABSTRACT ................................................................................................................ 34 3.2 BACKGROUND ........................................................................................................ 35 3.3 MATERIALS AND METHODS ................................................................................ 37 Cell culture, cell cycle synchronization, transfection and reagents .............................. 37 Chromatin Fractionation ............................................................................................... 38 FACS analysis ............................................................................................................... 38 Chromatin Affinity Purification and Immuno Precipitation ......................................... 38 ChAP DNA preparation for Illumina GAII Sequencing ............................................... 40 Data analysis ................................................................................................................. 41 3.4 RESULTS ................................................................................................................... 44 3.4.1 Conjugation of ubiquitin to chromatin changes during the cell cycle: ................ 44 3.4.2 Ubiquitination mark redistributes in the genome during mitosis: ........................ 54 3.4.3 Ubiquitination is enriched at active genes throughout the cell cycle: .................. 56 3.4.4 Ubiquitination at transcribed regions is sensitive to transcriptional inhibition: ... 60 x 3.4.5 RNF20 is required for ubiquitination of H2B associated with active transcription: ......................................................................................................................................
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