REGULATION OF YEAST TRANSCRIPTION FACTORS SKO1 AND CST6 BY SUMOYLATION VERONI SARATHA SRI THEIVAKADADCHAM A DISSERTATION SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOLOGY YORK UNIVERSITY TORONTO, ONTARIO February 2020 © Veroni Saratha Sri Theivakadadcham, 2020 ABSTRACT Sumoylation is a post-translational modification that plays an essential role in cellular processes, including transcriptional regulation. Transcription factors represent one of the largest groups of proteins that are modified by the SUMO (Small Ubiquitin-like Modifier) protein. In this study, we focused on finding roles for sumoylation in regulating two gene-specific bZIP transcription factors, Sko1 and Cst6, in Saccharomyces cerevisiae. Sko1 plays a unique bifunctional role in regulating transcription: it is a repressor during normal growth, by interacting with co-repressor complexes, and an activator during osmotic stress, via interaction with Hog1 Kinase. We show that Sko1 is poly-sumoylated at Lys 567 but its sumoylation is not regulated by stress. Along with sumoylation, Sko1 also undergoes phosphorylation, by PKA and Hog1, and our experiments show that these two modifications are not interdependent. We find that DNA binding is a requirement for Sko1 sumoylation and genome-wide chromatin immunoprecipitation (ChIP-seq) analysis shows that Sko1 sumoylation controls the occupancy level of Sko1 on target promoters and is involved in preventing Sko1 from binding to non-target promoters. Moreover, blocking sumoylation attenuated the interaction between Sko1 and Hog1 on target promoters. Cst6, on the other hand, is required for survival during ethanol stress and has roles in the utilization of carbon sources other than glucose. In this study, we show that Cst6 is multi-sumoylated at Lys residues 139, 461 and 547. The level of Cst6 sumoylation increases in ethanol and oxidative stress conditions, but decreases if ethanol is used as the sole carbon source. Unlike Sko1, protein levels of SUMO-deficient Cst6 were moderately reduced compared to the wild-type form, implying that sumoylation promotes Cst6 stability. ChIP experiments suggests that sumoylation is important for the timely recruitment of Cst6 to its target promoters. In addition, we provide evidence that Cst6 sumoylation reduces the expression of some target genes, during non-stress and ethanol stress ii conditions. Taken together, our studies suggest that the specific effects of sumoylation in regulating transcription factors are target specific. Nevertheless, SUMO plays a general role by controlling the transcription factor-DNA interaction to maintain proper gene expression. iii DEDICATION I dedicate this dissertation to my mother, Annette Sri Theivakadadcham. I pursued with PhD mainly to fulfill my mother’s dream. Her constant support and motivation has given me the strength to travel through my PhD journey. Thank you mum, without your love, advice and support, this would not have been possible. iv ACKNOWLEDGMENTS First and foremost, I would like to thank my family members for all their love and support. Along with my mother, my Godfather, Dunstan Egbert, has shown his love, care, and support from day one. I would not have been able to complete this dissertation without your constant advice and support. I would like to thank my better half, my husband, Vinoth Matthews. Although you entered midway into my journey, you are one of the main reasons for the successful completion of my PhD. You are my pillar of strength. I thank my siblings for their love and support and in particular, to my sister, Antonette Nicholas, for being another mother to me. Your support in my early education has set the right foundation for my present success. I would like to thank my supervisor Dr. Emanuel Rosonina for giving me the opportunity to work in the Rosonina Lab. You have been a great mentor during my work. Thank you for your support, guidance and encouragement. I am thankful for the opportunities you have given me over the years to present my research at various conferences. I am also grateful to my supervisory committee members, Dr. John McDermott and Dr. Peter Cheung. I was able to improve my projects with your excellent advice, suggestions and support. Additionally, I would like to express my sincere gratitude to Dr. Bridget Stutchbury for her support, understanding, and advice. Your assistance has helped me a lot. I would also like to thank Dr. Yi Sheng, Dr. Derek Wilson and Dr. Brian Raught for agreeing to be on my examining committee, for their time and effort. I would also like to thank all the members of the Rosonina lab, past and present: John, Yimo, Marjan, Akhi, Justin, Farhaan, Russell, Jessica Petricca, Giovanni, Areeb, Ben, Bryan, Mostafa, Mohammad, and Sukyong. You have made my time in the Rosonina lab so enjoyable and memorable. Special thanks to John for being my best friend. You were the first person I have always approached whenever I needed someone to talk to. Thank you for all the help with proof reading my work. Although they belonged to different labs, special thanks to Jessica D’Angelo, and Shailee Jani for being two great friends. You were there supporting me through all the ups and downs I have faced. Together, we had many good times and those memories will never be forgotten. A special thanks to all my other friends from the 3rd Floor of LSB, Dr. Rahima Khatun, Dr. Mohamed Salem, Dr. Dayana D’amoura, Dr. Queenie Hu, Dr. Jyotsna Vinayak, Dr. Stefanie Bernaudo and Dr. Yara Zayed. Thank you all for being a good friend and a mentor. Your advice and guidance have helped me to finish my PhD on time. v TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii DEDICATION.............................................................................................................................. iv ACKNOWLEDGMENTS ............................................................................................................ v TABLE OF CONTENTS ............................................................................................................ vi LIST OF TABLES ..................................................................................................................... viii LIST OF FIGURES ..................................................................................................................... ix LIST OF ABBREVIAITONS ..................................................................................................... xi CHAPTER 1: LITERATURE REVIEW .................................................................................. 1 1.1 Post-translational modification ............................................................................................. 1 1.2 Sumoylation .......................................................................................................................... 3 1.2.1 Sumoylation Pathway .................................................................................................... 7 1.2.2 De-sumoylation .............................................................................................................. 9 1.2.3 SUMO acceptor sites ................................................................................................... 11 1.2.4 SUMO chain formation ............................................................................................... 12 1.2.5 Molecular consequences of sumoylation ..................................................................... 14 1.2.6 The SUMO Enigma ..................................................................................................... 17 1.2.7 SUMO and stress response .......................................................................................... 19 1.3 Biological functions of SUMO ........................................................................................... 21 1.3.1 Non-nuclear sumoylation ............................................................................................. 21 1.3.2 Nuclear sumoylation .................................................................................................... 22 1.4 Sumoylation and transcription ............................................................................................ 23 1.4.1 Transcriptional repression by SUMO .......................................................................... 24 1.4.2 Transcriptional activation by SUMO ........................................................................... 25 1.5 Transcription ....................................................................................................................... 26 1.5.1 DNA cis-regulatory elements ...................................................................................... 26 1.5.2 Transcription overview ................................................................................................ 29 1.6 Transcription factors ........................................................................................................... 31 1.6.1 Transcription Factor binding motifs and DNA binding specificity ............................. 31 1.6.2 Characterization of transcription factors based on DBD ............................................. 32 1.7 bZIP transcription factors ..................................................................................................