Targeting the X for Chromosome-Wide Gene Regulation by Emily L. Petty a Dissertation Submitted in Partial Fulfillment of The
Total Page：16
File Type：pdf, Size：1020Kb
Targeting the X for chromosome-wide gene regulation by Emily L. Petty A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Molecular, Cellular, and Developmental Biology) University of Michigan 2011 Doctoral Committee: Assistant Professor Györgyi Csankovszki Professor Kenneth M. Cadigan Professor Steven E. Clark Assistant Professor Yali Dou © Emily L. Petty 2011 This work is dedicated to my family- Mom, Dad, Eric, Kenny, Gabe, and Celia, to my best friends- Aimee and Evita to the memory of Genevieve Orstadius and Cindy Kessel and to my future husband, Joe. ii Acknowledgments I would first like to thank my mentor Gyorgyi for all of her training and support. I have had many experiments fail during my time in this lab and Gyorgyi has always been encouraging and patient when I struggled with new techniques. I have been lucky to be in a lab where I have had the opportunity to not only attend but also present at scientific meetings. The regular journal clubs and lab meetings have also been extremely helpful in developing my presentation skills. I also really appreciated being able to pop into Gyoryi's office every so often with questions and data (usually both at the same time). I hope that hasn't been too disruptive! Most importantly, the scientific training I have received by being in the Csankovszki lab has been tremendous. I will leave the lab carrying with me a great scientific tool set. Next, I would like to thank all of the members of their lab for their help over the years. I would like to specifically thank Dr. Tim Blauwkamp, for teaching me the ins and outs of qPCR. To the undergraduates I have had the privilege to work with, Alysse Cohen, Emily Laughlin, and Stephanie Campbell, thank you for all of your help. Some of my favorite experiences overall from graduate school have come out of working with Alysse, Emily, and Stephanie. Thank you to all of the friends I have made over the course of my time here, especially Karishma and Yana. Yana and I actually met as roommates during our PIBS interview and it was such a relief to see a friendly face in that iii first week here. Karishma and I started out as bench-mates but quickly became very good friends. I am so thankful that I've been able to work with such a wonderful person and consider myself blessed to have you as one of my friends. Finally, thank you to my friends and family for all of their love and support. To my parents, thank you for always supporting me in my education and thank you for letting me crash back at home these last several months as I've been working toward this goal of graduating. To my friends, thank you for all of the fun and laughter and thank you for being so understanding every time I've told you, "No, I can't go, I have this experiment…,". And Joe, you have been the best surprise. Your love, support, and encouragement have helped me to get back on track when I have been exasperated with my experiments or life in general. I always know that if I call you crying I will hang up laughing. Thank you. iv Table of Contents Dedication……………………………………………………………………………….ii Acknowledgements……………………………………………………………………..iii List of Figures…………………………………………………………………………..ix List of Tables……………………………………………………………………………xv Chapter 1: Introduction…………………………………………………………………1 How do gene regulatory machineries find their targets?……………………1 X-chromosome-wide gene regulation by dosage compensation…………..4 Mammalian X inactivation………………………………………………6 Hyperactivation of the male X in Drosophila………………………....8 Two-fold repression of both X chromosomes in C. elegans……………………………………………………13 The histone variant H2A.Z has diverse functions in eukaryotic genomes………………………………………….....……21 The physical properties of H2A.Z………………………………..….21 Biological function of H2A.Z………………………………………....29 H2A.Z boundary function……………………………………………..34 Function of C. elegans H2A.Z/HTZ-1…………………………….....39 H2A.Z compartmentalizes the genome……………………………..40 The curious case of DPY-30…………………………………………………41 v How is the C. elegans DCC specifically targeted to the X chromosomes?…………………………………………….…48 References………………………………………………………………...…...49 Chapter 2: Three distinct condensin complexes control C. elegans chromosome dynamics………………………………………………………..73 Abstract…………………………………………………………………………73 Introduction……………………………………………………………………..74 Results………………………………………………………………………….78 Discussion………………………………………………………………………90 Experimental Procedures……………………………………………………..94 Acknowledgments……………………………………………………………..96 References……………………………………………………………………..97 Chapter 3: Different roles for Aurora B in condensin targeting during mitosis and meiosis………………………………………119 Abstract……………………………………………………………………….119 Introduction………………………………………………………..................120 Results………………………………………………………………………...125 Discussion…………………………………………………………………….137 Materials and Methods..……………………………………………………..141 Acknowledgments……………………………………………………………144 References……………………………………………………………………145 vi Chapter 4: Restricting Dosage Compensation Complex binding to the X chromosomes by H2A.Z/HTZ-1……………………...164 Abstract…………………………………………………………………….164 Author Summary…………………………………………………………..165 Introduction………………………………………………………………...166 Results……………………………………………………………………...172 Discussion………………………………………………………………....182 Materials and Methods..………………………………………………….190 Acknowledgments…………………………………………………………195 References…………………………………………………………………196 Chapter 5: DPY-30 function in C. elegans dosage compensation is independent of Set1/MLL ………………………………………….…..219 Abstract……………………………………………………………………..219 Introduction………………………………………………………………....220 Results……………………………………………………………..............227 Discussion……………………………………………………………….….236 Materials and Methods..…………………………………………………..244 Acknowledgments…………………………………………………………247 References…………………………………………………………………247 Chapter 6: Conclusions and Future directions………………………..……..…267 Proposed Future Directions………………………………………………274 Aim 1: Utilize ectopic DCC binding in htz-1 to understand DCC binding preferences and link changes in DCC vii binding to changes in gene expression……………………..…274 Aim 2: Does loss of htz-1 lead to expanded heterochromatin in C. elegans as it does in yeast, Arabidopsis, and mammals?.........................................................................276 Aim 3: Determine the effect of DPY-30 on SDC-2 binding and SDC-3 expression………………………………….277 References…………………………………………………………………278 viii List of Figures Figure 1.1 The highly studied dosage compensation strategies of mammals, flies, and worms…………………………………………62 Figure 1.2 Step-wise model of XCI in mammals……………………………...63 Figure 1.3 MSL complex makeup and function in D. melanogaster dosage compensation……………………………………………….64 Figure 1.4 The dosage compensation complex (DCC) localizes to both X chromosomes in the hermaphrodite……………………65 Figure 1.5 Structural differences between H2A and H2A.Z………………….66 Figure 1.6 H2A.Z acetylation is conserved in C. elegans…………………….67 Figure 1.7 Model of euchromatin organization by acetylated H2A.Z………..68 Figure 1.8 Model of heterochromatin formation by hypoacetylated H2A.Z……………………………………………….69 Figure 1.9 Schematic outline of Set1 and MLL domain architecture in budding yeast, fruit fly, and human………………………………70 Figure 1.10 Expansion of H3K4 methyltransferase complexes………………..71 Figure 1.11 Proposed model of a Set1C-dependent role for DPY-30 in C. elegans dosage compensation………………………..72 Figure 2.1 The three condensin model…………………………………………101 Figure 2.2 Condensin subunit interactions identified by immunoprecipitation………………………………………………….102 ix Figure 2.3 Condensin I localizes to mitotic chromosomes in a pattern distinct from condensin IDC or condensin II………………103 Figure 2.4 Condensin I localizes to meiotic chromosomes in a pattern distinct from condensin IDC or condensin II………………104 Figure 2.5 CAPG-1 functions in dosage compensation………………………105 Figure 2.6 Depleting each condensin II subunit results in common chromosomal and developmental defects……………….106 Figure 2.7 Condensin I is required for mitotic and meiotic chromosome segregation………………………………………….108 Figure 2.8 C. elegans CAPG-1 is more closely related to CAP-G proteins, while CAPG-2 is more closely related to CAP-G2 proteins……109 Figure 2.9 IP-western analysis confirms condensin interactions detected by mass spectrometry…………………………….……..110 Figure 2.10 All three CAP subunits of each class show the same localization patterns………………………………………….……..111 Figure 2.11 CAPG-1 shows X chromosome binding relationships with SDC proteins…………………………………………………………112 Figure 2.12 Common chromosomal and developmental defects result from depletion of each condensin II subunit………………….….114 Figure 2.13 Mutational or RNAi-mediated depletion of class I CAP subunits, but not of dpy-27, shows chromosome separation defects…….116 Figure 2.14 Condensin I depletion does not disrupt condensin II localization during mitosis, and RNAi feeding x produces substantial but not complete depletion……………….117 Figure 2.15 Double depletion of a class I and a class II CAP is more severe than either single depletion…………………………….. 118 Figure 3.1 Condensin I and II during mitosis………………………………. .149 Figure 3.2 Condensin I, but not II, depends on AIR-2 for mitotic recruitment…………………………………………………..150 Figure 3.3 A diagram of the adult hermaphrodite gonad…………………….152 Figure 3.4 Condensin I and II in oocyte meiosis……………………….……..154 Figure 3.5 Condensin I and condensin II in sperm meiosis………………….155 Figure 3.6 AIR-2 activity is needed for correct targeting of condensin I, but not condensin II,