Northwestern University Dynamic Transcription Factor-Mediated Recruitment of Genes to the Nuclear Pore Complex in Saccharomyces cerevisiae. A dissertation SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS For the degree DOCTOR OF PHILOSOPHY Field of Biological Sciences By Carlo Randise-Hinchliff EVANSTON, ILLINOIS March 2017 2 © Copyright by Carlo Randise-Hinchliff 2016 All Rights Reserved 3 ABSTRACT Dynamic Transcription Factor-Mediated Recruitment of Genes to the Nuclear Pore Complex in Saccharomyces cerevisiae. Carlo Randise-Hinchliff In yeast, inducible genes such as INO1, PRM1 and HIS4 reposition from the nucleoplasm to nuclear periphery upon activation. This leads to a physical interaction with nuclear pore complex (NPC), interchromosomal clustering, and stronger transcription. Repositioning to the nuclear periphery is controlled by cis-acting transcription factor (TF) binding sites located within the promoters of these genes and the TFs that bind to them. Such elements are both necessary and sufficient to control positioning of genes to the nuclear periphery. We have identified four TFs capable of controlling the regulated positioning of genes to the nuclear periphery in budding yeast under different conditions: Put3, Cbf1, Gcn4 and Ste12. Gcn4 and Ste12 are also sufficient when tethered to an ectopic site to recruit chromatin to the nuclear periphery. For each TF, we have defined the molecular basis of regulated relocalization to the nuclear periphery. Put3- and Cbf1-mediated targeting to nuclear periphery is regulated through local recruitment of Rpd3(L) histone deacetylase complex by transcriptional repressors. Rpd3(L), through its histone deacetylase activity, prevents TF-mediated gene positioning by blocking TF binding. Yeast transcriptional repressors were capable of blocking Put3-mediated recruitment; 11 of these required Rpd3. Thus, it is a general function of transcription repressors to regulate TF-mediated recruitment. However, Ste12 and Gcn4-mediated recruitment is regulated independently of 4 Rpd3(L) and transcriptional repressors. Ste12-mediated recruitment is regulated by phosphorylation of an inhibitor called Dig2, and Gcn4-mediated gene targeting is up-regulated by increasing Gcn4 protein levels. Gcn4-mediated gene targeting genetically requires NPC (Nup2), SAGA (Gcn5 & Spt20), Mediator (Med31), Mex67, however of these only the NPC is directly involved in recruitment of HIS4 to the nuclear periphery. Finally, by iterative deletion from amino and carboxyl termini, a 27 aa Positioning Domain (PD) of Gcn4 was identified. The PD of Gcn4 is sufficient to reposition and cluster chromatin at the nuclear periphery. The ability of transcription factors to mediate recruitment to the NPC and interchromosomal clustering of genes represents a novel function. This ability allows cells to alter the organization of the genome in a directed and regulated manner. 5 Acknowledgments I would like to express my deepest gratitude to my advisor and mentor, Dr. Jason Brickner. He has been truly inspirational as both a mentor and a scientist. Like many, I believe graduate school is not only about results. It is about developing and honing skills that will translate into a successful career in science. These skills are complex and diverse, which make them hard to obtain. Jason’s extraordinary mentoring capacity has given students like me the optimal chance to succeed in both graduate school and in our careers moving forward. Reflecting back on the last 6 years, I have seen growth in multiple areas including formatting and presentation techniques as well as my scientific writing. In lab, he has taught me how to develop and test a hypothesis in the strictest but most efficient ways. Also he has also taught me how to resolve technical issues using the correct controls or alternative approaches. Jason has always shown genuine interest in my progress and has offered consistent encouragement. He has made me the scientist I am today. All I can say is thank you. I would like to thank my committee, Dr. Curt Horvath, Dr. Richard Morimoto, and Dr. Sadie Wignall for helpful discussions and feedback during my yearly review meetings. I am very fortunate to have an exemplary group of scientists supporting my progression through graduate school. Curt, you were an excellent committee chair. Our meetings together has provided me with important and encouraging feedback. I will cherish our interactions together as well as my interactions with the rest of my committee members. I would also like to thank Dr. Donna Brickner for being a wonderful lab manager and colleague. Donna has provided support on many of my projects and has provided numerous 6 yeast strains. I also want to thank you for persuading Jason to accept me into the lab. I know that without you I would have not had the opportunity to be part of the Bricknerds. Thank you. Varun, you were an amazing colleague and friend over the years. You are a wonderful person with a brilliant mind. Graduate school would have been a completely different experience without you. You answered so many of my questions, and I have always felt like I could come to you for advice and support not only in science but also in life. You are the hardest worker I know and I see a great future in science for you. I will miss our scientific discussions, your delicious snacks you offered me, and the constant support. Good luck in all your endeavors. Agustina, you are inspirational. Whether it is in science or personal health you devote yourself completely and whole-heartedly, and I admire that. The last six years together has been a great experience for me, and I will miss you a lot. I would say good luck moving forward, but you don’t need it, you have passion, intellect, and skill. Finally, I want to thank all of the current and former Bricknerds, I want to specifically thank Rob Coukos, Michael Sumner, Stefan Zdraljevic, Lauren Meldi, Sara Ahmed, and Heidi Schmit for their countless hours of hard work and scientific discussions. 7 Dedication I would like to dedicate my dissertation to my grandmother, Joanne Davis Hinchliff. From a young age she believed in me, inspired me, and instilled in me a love of knowledge and a desire to explore the world. Every summer we traveled the United States and immersed ourselves in history and geography. We traced along the 3,700 mile Lewis and Clark trail, from the banks of the woods river in Illinois all the way to the coast of Oregon. By helicopter, we flew over the forest of Alaska landing on glaciers and staring down the deep crevasses. We also shared countless memories tent camping in beautiful landscapes of state and national parks. These adventures were memorable, but my grandma meant so much more. She believed in me when others did not and I would not be in graduate school without her. I will always love her and miss her. This is for you, Joanne. 8 Table of Contents Abstract 3 Acknowledgements 5 Dedication 7 Table of Contents 8 List of Figures and Tables 12 Chapter 1. Introduction 16 1.A. Introduction 16 1.B. Spatial organization of the yeast genome 18 1.C. Composition of NPC 19 1.D. Nuclear pore complex interacts with the genome 21 1.E. Nups influence transcription 25 1.F. Interchromosomal clustering at the NPC 28 1.G. Gene recruitment and clustering through the cell cycle 29 1.H. Transcription Memory 30 1.I. Molecular mechanism of INO1 transcriptional memory 34 Chapter 2: Transcription factor-mediated gene recruitment to the NPC 37 2.A. Introduction 37 2.B. Transcription factor-dependent and stimulus-specific recruitment of INO1, 38 PRM1 and HIS4 to the nuclear periphery. 2.C. Transcription factor binding sites function as DNA zip codes 4543 9 2.D. TF-mediated gene recruitment occur under specific stimuli. 4543 2.E. Conclusion 4646 Chapter 3: Rpd3(L) histone deacetylase regulates zip-code dependent 4848 recruitment to the nuclear periphery and interchromosomal clustering 3.A. Introduction 4848 3.B. Upstream Repressing and Upstream activing sequences regulate INO1 4848 gene recruitment to the nuclear periphery 3.C. Trans-acting factors regulate INO1 gene recruitment to the nuclear 5050 periphery 3.D. Rpd3(L) histone deacetylase regulates INO1 gene recruitment to the 5151 nuclear periphery 3.E. Rpd3(L) histone deacetylase regulates Put3 binding through local histone 5353 acetylation 3.F. Rpd3 (L) regulates interchromosomal clustering of INO1 5252 3.G. Conclusion 5555 Chapter 4: A general role of transcriptional repressors in regulating zip-code 5757 function. 4.A. Introduction 5757 4.B. Opi1 and Ume6 are sufficient to block GRS I and GRS II function 5757 4.C. A survey of transcriptional repressors in regulating zip code-mediated 6060 targeting to the nuclear periphery 10 4.D. Artificially tethering Mig1 and Sfl1 to the URA3 locus is sufficient to 6464 cause URA3 to reposition to the nuclear periphery 4.E. Conclusion 6464 Chapter 5: Identifying multiple mechanisms involved in regulating zip-code 6666 activity 5.A Introduction 6666 5.B. Regulation of Ste12-mediated gene positioning by MAP kinase signaling 6868 5.C. Independent of DNA binding, Ste12 is sufficient to induce peripheral 6969 positioning 5.D. Increased peripheral gene positioning through regulated TF synthesis 7070 5.E. Different regulatory strategies provide dynamic control of the yeast 7171 genome through different time scales 5.F. Conclusion 7676 Chapter 6: Identifying factors directly mediating zip-code dependent 7979 requirement 6.A. Introduction 7979 6.B. Mediator and SAGA complexes function in INO1, PRM1 and HIS4 8181 recruitment to the nuclear periphery 6.C. Genetic epistasis analysis of factor’s function downstream of Gcn4 to 8282 mediate targeting to the nuclear periphery 6.D. Conditional inactivation of factors by anchor away 8484 6.E Tethered Gcn4 leads to a physical association of Nup2 and Gcn5 at URA3 8888 11 6.F.