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SUMO-1 mapping in the human genome and its implications for transcription control DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Hui-wen Liu Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2014 Dissertation Committee: Jeffrey D. Parvin, M.D., Ph.D., Advisor Ching-shih Chen, Ph.D., Mark Parthun, Ph.D., Amanda Toland, Ph.D. Copyright by Hui-wen Liu 2014 Abstract SUMOylation, a post-translational modification with SUMO proteins covalently conjugated to a variety of proteins, regulates a range of cellular processes, including cell proliferation and maintenance of genome stability. In this study, we investigated how SUMO-1 functions as a chromatin mark on the human genome during cell cycle progression by ChIP-seq approach. Surprisingly, despite the known repressive role of SUMOylation on histones, we found that SUMO-1 localizes to the promoters of constitutively active genes involved in protein translation and proliferation during interphase. For example, ribosomal protein genes; and SUMO-1 marks on these promoters were absent during mitosis. In addition, SUMO-1 association on the promoters recruits RNAPII, and depletion of SUMO-1 leads to down regulation of those ribosomal protein genes, suggesting a positive role of SUMO-1 in gene activation. To further elucidate how SUMOylation regulates transcription process related to protein synthesis, we identified that SUMO-1 marks the promoters via the Scaffold Associated Factor B (SAFB) protein. The results showed that SAFB is SUMOylated, and depletion of SAFB caused the decrease of SUMO-1 marks on the promoters of those housekeeping genes transcribed by RNAPII. In addition, depletion of SAFB decreased the splicing of the mRNAs and disrupted the organization of Cajal body, which is important for snRNP and snoRNP biogenesis. All these findings suggested that SUMOylation plays an ii important role in the regulatory process for transcription initiation and splicing of mRNA of ribosomal protein genes. iii Dedication This dissertation is dedicated to my family. iv Acknowledgements I would like to thank my advisor, Dr. Jeffrey Parvin for supporting and guiding me throughout the past 5 years. Whenever I feel lost in my research, you are always willing to help. You have set a great example to be an outstanding researcher, mentor, and role model. I am truly grateful to have such an excellent mentor in my life. I also thank my thesis committee members, Dr. Mandy Toland, Dr. Mark Parthun, and Dr. Ching-shih Chen. Not only your time and patience, but also your invaluable feedback and discussion, have guided me on the path as a researcher. I am also thankful to have my lab-mates, Zeina, Mansi, Muhtadi, Grace, Cindy, Shweta, Derek, Alaina, Eliana, and Ian. You have been very helpful and fun to work with. I will easily miss the time we hanging out (and working in the lab, of course!). I thank my friends for all the support and suggestions, and it is always a great pleasure to hanging out with you guys. I am a person who gets homesick a lot, but you guys make me feel like home, and I really appreciate that. Last but not least, I would like to thank my family and my husband Aaron Chen, for unconditional love and support. There are times when I feel frustrated, and you are always there for me. This journey would not have been possible without all the support from everyone, and I am truly thankful for everything I have. v Vita 2008-Present…...…………….PhD Candidate, The Ohio State University, Columbus, OH 2005………... MS, Microbiology and Biochemistry, National Taiwan University, Taiwan 2003…………………...BS, Agricultural Chemistry, National Taiwan University, Taiwan Publications 1. Liu HW, Zhang J, Heine GF, Arora M, Ozer HG, Onti-Srinivasan R, Huang K, Parvin JD, Chromatin modification by SUMO-1 stimulates the promoters of translation machinery genes. Nucleic acid research. 40(20): 10172-86, 2012. 2. Arora M, Zhang J, Heine GF, Liu HW, Ozer G, Huang K, Parvin JD. Chromatin ubiquitination: a bookmark for transcription and DNA replication through mitosis. Nucleic acid research. 40(20): 10187-202, 2012. 3. Zhang J, Lu K, Xiang Y, Islam M, Kotian S, Kais Z, Lee C, Arora M, Liu HW, Parvin JD, Huang K. Weighted Frequent Gene Co-expression Network Mining to Identify Genes Involved in Genome Stability. PLOS Computational biology. 8(8): e1002656, 2012 4. Shen YF, Chen YH, Chu SY, Lin MI, Wu PY, Hsu HT, Wu CJ, Liu HW, Lin FY, Lin G, Hsu PH, Yang AS, Cheng SH, Wu YT, Wong CH, Tsai MD. E339…R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors. Proc Natl Acad Sci USA. 108(40):16515-20, 2011 5. Chen SC, Liu HW, Lee KT, Yamakawa T. High-efficiency Agrobacterium rhizogenes-mediated transformation of sHSP18.2-GUS in Nicotiana tabacum. Plant Cell Reports. 26: 29-37, 2007. Fields of Study Major Field: Molecular, Cellular and Developmental Biology vi Table of Contents Abstract ...…...................................................................................................................... .ii Dedication......... ................................................................................................................. iv Acknowledgements ............................................................................................................. v Vita......................................................................................................................................vi Table of Contents .............................................................................................................. vii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Chapter 1: Introduction ..................................................................................................... 1 1.1 Chromatin functions in eukaryotes ........................................................................ 1 1.1.1 Chromatin structures are well organized in eukaryotes ................................... 1 1.1.2 Epigenetics and histone dynamics ................................................................... 2 1.1.3 Histone modifications ...................................................................................... 4 1.2 SUMO pathway ...................................................................................................... 9 1.2.1 Enzymes involved in SUMOylation ................................................................ 9 1.2.2 SUMO proteases ............................................................................................ 11 1.2.3 SUMO proteins .............................................................................................. 12 vii 1.2.4 SUMO-Interaction Motif ............................................................................... 13 1.3 The role of SUMOylation in chromatin remodeling ............................................ 14 1.3.1 SUMO localization on chromatin .................................................................. 14 1.3.2 SUMO modification of Histones and HDACs .............................................. 15 1.3.3 Crosstalk between histone methylation and SUMOylation ........................... 17 1.4 SUMOylation and transcription regulation .......................................................... 17 1.4.1 Transcription repression ................................................................................ 17 1.4.2 Transcription activation ................................................................................. 18 1.4.3 SUMO, transcription, and chromatin structure ............................................. 19 1.5 SUMO function in subnuclear structure ............................................................... 20 1.5.1 Polycomb bodies ............................................................................................ 20 1.5.2 PML bodies ................................................................................................... 21 1.5.3 Nucleolus and speckles .................................................................................. 22 1.6 Interactions between SUMO and mRNA biogenesis ........................................... 23 1.7 The role of SUMOylation in genome stability and tumorigenesis ....................... 24 1.7.1 SUMOylation regulates cell cycle progression ............................................. 24 1.7.2 SUMOylation regulates DNA damage response ........................................... 25 1.7.3 Deregulation of SUMO system causes tumorigenesis .................................. 25 Chapter 2: Rationale ........................................................................................................ 30 viii Chapter 3: Chromatin modification by SUMO-1 stimulates the promoters of translation machinery genes ............................................................................................ 32 3.1 Abstract ................................................................................................................ 33 3.2 Introduction .......................................................................................................... 34 3.3 Materials and Methods ........................................................................................
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