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Rna Pol Ii Cleavage Activity Is a Major Effector of Transcription

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Rna Pol Ii Cleavage Activity Is a Major Effector of Transcription RNA POL II CLEAVAGE ACTIVITY IS A MAJOR EFFECTOR OF TRANSCRIPTION DYNAMICS by RYAN MICHAEL SHERIDAN B.S., University of Missouri-St. Louis, 2012 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology Program 2019 This thesis for the Doctor of Philosophy degree by Ryan Michael Sheridan has been approved for the Molecular Biology Program by Sandy Martin, Chair Richard Davis Jay Hesselberth Thomas Blumenthal David Barton David Bentley, Advisor Date: May 17, 2019 ii Sheridan, Ryan Michael (Ph.D., Molecular Biology) RNA Pol II Cleavage Activity is a Major Effector of Transcription Dynamics Thesis directed by Professor David L. Bentley ABSTRACT After transcription initiation, RNA polymerase II (pol II) undergoes several steps that can influence gene expression, including promoter proximal pausing, transcription elongation, and termination. Promoter proximal pausing occurs after pol II has moved 50- 100 bp into the gene body and is induced by negative elongation factors that interact with the polymerase. After release from the 5’ pause, the rate of pol II elongation increases as the polymerase moves further from the TSS. Upon reaching the poly(A) site, transcription termination occurs at a position influenced by a kinetic competition between pol II and termination factor Xrn2. The mechanisms that influence the release of 5’ paused pol II and subsequent elongation rates within gene bodies and downstream of poly(A) sites are poorly understood, but one potential rate-limiting event is pol II backtracking. Pol II backtracking can occur when the polymerase mis-incorporates a base or encounters a roadblock that causes the polymerase to move backwards, dislodging the nascent RNA 3’ end from the active site. To rescue backtracked pol II, the transcription factor TFIIS stimulates the RNA endonuclease activity of the polymerase, which cleaves the nascent RNA to generate a new correctly positioned 3’ end. To investigate the importance of pol II cleavage activity in the transcription cycle, I used a dominant negative TFIIS mutant (TFIISDN) that inhibits this cleavage activity. Using this mutant, I mapped individual pol II pause and backtrack sites and find that these events occur throughout iii transcription but are especially enriched at the 5’ and 3’ ends of genes. I find that inhibition of pol II cleavage activity impairs the release of 5’ paused polymerases and causes transcription to terminate at a more proximal position downstream of the poly(A) site. In addition, expression of TFIISDN results in a ~50% reduction in the rate of elongation and delays the upregulation of hypoxia and heat shock responsive transcripts. These results demonstrate that pol II backtracking is widespread, and the intrinsic RNA cleavage activity of the polymerase influences each post-initiation step of the transcription cycle. The ability to deplete endogenous human proteins is valuable for studying cellular processes. Traditional protein depletion methods such as shRNA-mediated knockdown suffer from off-target effects, incomplete knockdown, and slow kinetics. In addition, if the protein of interest is required for cell viability, a conditional knockdown method must be applied. Protein destabilization domains known as degrons present an appealing strategy for achieving conditional depletion of endogenous factors in a rapid and complete manner. I developed a PCR-based method for tagging endogenous human proteins with an eDHFR degron tag. The stability of eDHFR-tagged proteins can be controlled by modulating the concentration of trimethoprim in the culture media. This approach allows for rapid depletion of eDHFR-tagged proteins that is both reversible and complete. The form and content of this abstract are approved. I recommend its publication. Approved: David L. Bentley iv DEDICATION This thesis is dedicated to my family and friends. To my parents who have always supported my endeavors and pushed me to live a happy, healthy, and balanced life. To my sister who is always willing to help me escape the city and journey into the mountains. To my fiancée, Alexis, who has been with me through this entire strange trip and is never afraid to tell a bad joke to lighten my day. To my loving grandparents who are so good at spoiling me with delicious cooking and good conversation. To my aunts, uncles, and cousins who are always willing to share their homes for our summer retreats. To my friends who have provided me with so much laughter and absurdity, and a countless number of misadventures. These people have supported me so much through this journey and I am forever grateful that they have been a part of my life. v ACKNOWLEDGEMENTS I would like to thank my thesis advisor, David Bentley, who has provided me with so much advice and guidance. Your leadership has fostered such a positive and supportive work environment. I am so proud to have been a part of your lab and I can honestly say that these have been some of the most exciting and rewarding years of my life. I also want to thank members of the Bentley lab including Nova, Michael, Tassa, Ben, and Hyunmin. Nova, not only did you generate the TFIIS cell lines used for my thesis, but you were also responsible for training me in so many techniques. I want to thank Michael and Tassa for their helpful suggestions about the project, and Ben and Hyunmin for their bioinformatics help when I first joined David’s lab. I would also like to thank my thesis committee for their insightful suggestions and for taking the time to keep me on track. During my time as a graduate student I took several courses and workshops that were especially helpful to my project. This included Jay Hesselberth’s Genome Informatics Workshop and a GRO-seq workshop run by Mary Allen and Robin Dowell. I would also like to thank my undergraduate advisor, Teresa Thiel and her lab manager Brenda Pratte. It’s hard for me to imagine that I would be where I am today if not for my experience in the Thiel lab. I would like to thank the Biochemistry and Molecular Genetics Department for bringing together such a great group of scientists and providing an incredibly collaborative work environment. I would like to thank the Molecular Biology Program for taking me in and funding my first three years of graduate school. I would also like to thank the Bolie and Hirs families for providing funding for me to attend so many great conferences. I would like to thank the RNA Bioscience Initiative for all the work they vi have done to advance RNA research and for funding my project for the last two years. Lastly, I would like to thank Jingshi Shen for providing me with the HAP1 cells used for chapter V of my thesis. vii TABLE OF CONTENTS CHAPTER I. INTRODUCTION .................................................................................................. 1 1. Transcription by RNA polymerase II ...................................................................... 1 2. Mechanism of RNA polymerase backtracking ...................................................... 2 3. Regulation of promoter proximal pol II pausing .................................................. 10 4. Pol II pausing and backtracking during transcription elongation ......................... 15 5. Control of transcription termination ..................................................................... 19 6. The transcriptional response to hypoxia ............................................................. 22 7. The transcriptional heat shock response ............................................................ 24 8. Methods for depleting human proteins ............................................................... 25 II. MATERIALS AND METHODS ............................................................................ 30 1. Human cell lines ................................................................................................. 30 2. Antibodies ........................................................................................................... 30 3. Immunoblotting ................................................................................................... 31 4. Flow cytometry ................................................................................................... 31 5. ChIP-seq ............................................................................................................ 31 5A. Immunoprecipitation and library preparation ........................................................ 31 5B. Quantification of ChIP-seq signals ....................................................................... 32 6. Bru-seq ............................................................................................................... 33 viii 7. Calculation of elongation rates ........................................................................... 35 8. mNET-seq .......................................................................................................... 36 8A. Immunoprecipitation and library preparation ........................................................ 36 8B. Identification of pol II pause sites ......................................................................... 37 8C. Cross-correlation analysis .................................................................................... 38 9. GRO-seq ............................................................................................................ 38 10. Calculation of
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