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E2f6 Is a Common Repressor of Meiosis-Specific Genes

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E2f6 Is a Common Repressor of Meiosis-Specific Genes E2F6 IS A COMMON REPRESSOR OF MEIOSIS-SPECIFIC GENES By SARAH M. KEHOE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1 © 2007 Sarah M. Kehoe 2 To my dad, who instilled in me his lifelong passion for learning 3 ACKNOWLEDGMENTS I would first like to thank my mentor, Dr. Naohiro Terada, for always being optimistic, no matter how confusing science may seem at times. His positive attitude, encouragement, and perseverance will not be forgotten. Dr. Terada’s office door being always open for scientific discussions and his willingness to meet regularly with his graduate students despite his busy schedule, exemplifies his dedication. I would also like to thank all of the members of the Terada lab, both past and present, for their support. In particular, I’d like to thank lab manager, Amy Meacham, for her frequent assistance, her reliable smile, and her friendship. I would especially like to thank former graduate student, Amar Singh, for serving as my mentor at the lab bench; he was a wonderful teacher who answered my many questions and helped me to get my feet off the ground. I am also grateful for my committee members, Dr. Jörg Bungert, Dr. Hideko Kasahara, Dr. Paul Oh, and Dr. Jim Resnick, for their helpful suggestions, feedback, and expertise. I would like to thank our collaborator, Dr. Stefan Gaubatz at the University of Wurzburg for providing invaluable materials and sound advice for my project. Without him, I would not have had the resources necessary to come to the conclusions that I did. Further, I want to thank my parents, Robert and Patricia, and sister, Karen, for their unconditional love and support. Likewise, I’d like to thank my husband, Joe, for always being there for me at the end of every day. His humor and encouragement have been important to surviving graduate school. Lastly, I'd like to thank my horse, Sundance, for keeping my life balanced and my stress levels at bay. Without being able to escape to the barn and ride my horse on days when the complexity of science seemed too great to ever make headway, I would never have made it this far. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT...................................................................................................................................10 CHAPTER 1 INTRODUCTION ..................................................................................................................12 Transcriptional Control of Gene Expression ..........................................................................12 The General Transcription Machinery ............................................................................13 Gene Regulatory Proteins................................................................................................15 The E2F Family of Transcription Factors ..............................................................................16 The Activating E2Fs........................................................................................................18 Pocket Protein-Dependent Repressive E2Fs ...................................................................20 Pocket Protein-Independent Repressive E2Fs.................................................................22 Summation..............................................................................................................................25 2 MATERIALS AND METHODS ...........................................................................................30 Cell Culture and Creation of Stable Cell Lines ......................................................................30 Gel Mobility Shift Assay........................................................................................................30 Chromatin Immunoprecipitation ............................................................................................31 Real-Time Quantitative PCR..................................................................................................32 Plasmid Construction and Site-Directed Mutagenesis ...........................................................33 Transient Transfection and Reporter Assays..........................................................................34 Immunoblotting ......................................................................................................................35 Reverse Transcription-PCR....................................................................................................35 Computational Analysis..........................................................................................................36 Bisulfite Sequencing and Combined Bisulfite Restriction Analysis (COBRA).....................37 Immunostaining ......................................................................................................................38 3 REPRESSION OF ANT4 GENE EXPRESSION BY E2F6..................................................41 Motivation...............................................................................................................................41 Background.............................................................................................................................41 Results.....................................................................................................................................46 Discussion...............................................................................................................................51 5 4 REPRESSION OF ADDITIONAL MEIOSIS-SPECIFIC GENES BY E2F6.......................74 Motivation...............................................................................................................................74 Background.............................................................................................................................74 Results.....................................................................................................................................77 Discussion...............................................................................................................................79 5 THE ROLE OF E2F6 DURING MALE MEIOSIS AND SPERMATOGENESIS...............91 Motivation...............................................................................................................................91 Background.............................................................................................................................91 Results.....................................................................................................................................96 Discussion...............................................................................................................................97 6 CONCLUSIONS AND SIGNIFICANCE............................................................................102 LIST OF REFERENCES.............................................................................................................104 BIOGRAPHICAL SKETCH .......................................................................................................117 6 LIST OF TABLES Table page 2-1 Primers used for analyzing ChIP experiments with semi-quantitative PCR .....................39 2-2 Primers used for semi-quantitative RT-PCR .....................................................................40 4-1 Locations of E2F6 TFBS (TCCCGC) within upstream promoter regions of meiosis- specific genes relative to their transcription initiation sites...............................................86 7 LIST OF FIGURES Figure page 1-1 Five ways in which regulatory proteins can inhibit transcription......................................27 1-2 The E2F family proteins and their core functional domains..............................................28 1-3 Changes in the composition and location of E2F complexes as cells enter the cell cycle. ..................................................................................................................................29 3-1 Ant4 encodes a novel isoform of adenine nucleotide translocase......................................59 3-2 The conservation of Sp1 binding sites in the Ant4 promoter.............................................60 3-3 The proximal promoter region of the Ant4 gene has a conserved E2F6 binding site........61 3-4 Gel mobility shift assay of E2F6 binding to the Ant4 promoter........................................62 3-5 The proximal promoter region of Ant4 is bound by endogenous E2f6 in undifferentiated WT R1 ES cells. ......................................................................................63 3-6 E2F6-mediated repression of Ant4 requires an intact transcription factor binding site. ...64 3-7 E2F6-mediated repression of Ant4 requires an intact C-terminal and DNA binding domain................................................................................................................................65 3-8 Ant4 transcription is repressed by stable E2F6 overexpression
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