Mammalian Sexual Determination: Activation of Müllerian
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i MAMMALIAN TESTIS-DETERMINING FACTOR SRY HAS EVOLVED TO THE EDGE OF AMBIGUITY by YEN-SHAN CHEN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Michael A. Weiss Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2013 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of YEN-SHAN CHEN candidate for the Doctor of Philosophy degree. (signer) Hung-Ying Kao, Ph.D. (chair of the committee) Michael A. Weiss, M.D. Ph.D. David Samols, Ph.D. Pieter deHaseth, Ph.D. Shigemi Matsuyama, Ph.D. (date) Jun 4th , 2013 i Table of Contents List of Tables iv List of Figures v Acknowledgements xi List of Abbreviations xiii Abstract xv Chapter I 1 “Introduction” Chapter II 39 “Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold” Chapter III 112 “A binary fate decision in human development is rendered ambiguous by accelerated proteosomal degradation of a transcription factor” Chapter IV 155 “A microsatellite-encoded domain in rodent Sry functions as a genetic capacitor to enable the rapid evolution of biological novelty SRY” Chapter V 220 “Summary and discussion” ii Appendix I 235 “Summary of inherited mutations in human SRY and state of characterizations” Appendix II 237 “Model: CH34 cell” Appendix III 239 “Floppy Sox” Appendix IV 243 “Summary of human XY intersex syndromes” Appendix V 247 “Methods in detail” Appendix VI 259 “Screening an appropriate cell models” Appendix VII 264 “Improving the transient transfection protocol” Bibliography 271 iii List of Tables Table 2.1 Properties of SRY variants 89 Table S2.1 Nucleocytoplasmic trafficking of SOX factors 108 Table S4.1 Construction of chimeric mSry/hSRY proteins 215 Table S4.2 Pairwise differences between HMG boxes 216 Table S4.3 Sox3 conservation 217 Table S4.4 The alignment of HMG boxes of the grass mice with selected Muroidea rodents and non-rodent mammals 218 Table S4.5 ChIP primer sets for qPCR and ChIP (testis-specific enhancer TESCO element) 219 iv List of Figures Figure 1.1 Sex determination mechanisms among selected bony vertebrates 19 Figure 1.2 The ZW sex determination 20 Figure 1.3 The XY sex determination 21 Figure 1.4 Schematic of Y chromosome 22 Figure 1.5 Sox9 testis-specific enhancer 24 Figure 1.6 SRY-Sox9 regulatory axis and related chronological flow 25 Figure 1.7 Human SRY and mouse Sry 27 Figure 1.8 Structure of SRY, SRY-DNA complex, and role of minor wing 29 Figure 1.9 Alignment of selected rodents in Murinae subfamily 31 Figure 1.10 Phylogenetic tree of selected rodents from sister families (Muroidea and Cricetidae) 32 Figure 1.11 Proposed human SRY (typical SRY) functional cascades 33 Figure 1.12 Cantilever substitution in hSRY and the related transcriptional activity 34 Figure 1.13 Box-only transcriptional activity assays by Yeast-One-Hybrid (Y1H) 36 Figure 1.14 A ‘medusa’ network architecture for the gene regulatory v network 37 Figure 1.15 Lorenz attractor model describes sex determination at the edge of ambiguity. 38 Figure 2.1 Domain organization of hSRY and summary of human genetics 76 Figure 2.2 Structure of SRY and outline of SRY-Sox9 regulatory axis 77 Figure 2.3 Transcriptional activity and nuclear localization 79 Figure 2.4 Chromatin immunoprecipitation and interactions with nuclear import-export machinery 81 Figure 2.5 Calmodulin binding and Wnt signaling 83 Figure 2.6 Coupling between nucleocytoplasmic trafficking of SRY and its phosphorylation 85 Figure 2.7 Potential N-terminal phosphorylation sites and NES of primate SRY alleles 87 Figure S2.1 I90M (residues 35 in HMG box) does not perturb folding or stability 91 Figure S2.2 FRET-based equilibrium binding and kinetics 92 Figure S2.3 Emission spectra of free and SRY-bound DNA after excitation at 490 nm 94 vi Figure S2.4 SRY-regulated testicular gene-regulatory network (GRN) and transcriptional activation of Sox9 95 Figure S2.5 MG132 rescues expression of V60L and V60A to achieve levels similar to wild-type SRY 96 Figure S2.6 Subcellular localization of wild-type SRY as analyzed by immunostaining 97 Figure S2.7 SRY-Exportin-4 co-IP assays. Histogram provides a quantitative summary of Western blots repeated in triplicate 98 Figure S2.8 Subcellular localization and CRM1 binding of mammalian SRYs 99 Figure S2.9 Subcellular localization and CRM1 binding of hSRY variants with NES modifications 101 Figure S2.10 V60L and V60A do not perturb binding of calmodulin (CaM) 103 Figure S2.11 Evidence that nucleocytoplasmic trafficking of hSRY does not depend on phosphorylation 105 Figure S2.12 Transient transfection of hSRY in PC-3 cells activates endogenous SOX9 106 Figure S2.13 Luciferase-based co-transfection assay of hSRY and variants 107 Figure 3.1 Domain organization of SRY and summary of clinical vii mutations 141 Figure 3.2 Design and SRY-dependent -galactosidase activity of Y1H-screening system 143 Figure 3.3 Biophysical studies of the free and DNA bound F54S clinical mutant 145 Figure 3.4 SRY-dependent transcriptional activation of Sox9 in per-Sertoli cell model 147 Figure 3.5 Cellular turnover of SRY affects the regulation of SRY-Sox9 central axis 149 Figure 3.6 Truncated SRY mutations accelerated proteosomal degradation, resulting in reduced Sox9 activation 151 Figure 3.7 SRY-regulated testicular gene-regulatory network (GRN) and transcriptional activation of Sox9 153 Figure 4.1 Structures of hSRY and mSry 183 Figure 4.2 Gln-rich domain of mSry contributes to transcriptional activation and TES occupancy 185 Figure 4.3 ChIP analysis of Sox9 testis-specific core enhancer occupancy 187 Figure 4.4 Biochemical differences between HMG boxes of mSry and viii hSRY 188 Figure 4.5 Subcellular localization of mSry, hSRY, and chimeric proteins 190 Figure 4.6 Function of mSry Gln-rich domain in chimeric constructs 192 Figure 4.7 Correlation of CH34 model with sex reversal in transgenic mice 194 Figure 4.8 Rodent Srys with CAG-encoded GRD contain attenuated 196 NES motif Figure S4.1 The phylogenetic tree of selected rodents 198 Figure S4.2 Selected gene expression patterns in CH34 cell line 200 Figure S4.3 Schematic illustration of FRET-based DNA bending probe and stopped-flow experimental design 202 Figure S4.4 A consensus NES motif restores CRM1-mediated nuclear export of mSry 204 Figure S4.5 Studies of SRY phosphorylation and its effects on gene-regulatory activity 206 Figure S4.6 Studies of SRY/Sry-directed sex reversal in XX transgenic mice 208 Figure S4.7 Transfected hSRY/mSry up-regulates endogenous SOX9 in human PC-3 cell line 210 ix Figure S4.8 Transcriptional activity of hSRY/mSry variants in NT2-D1 cells 212 Figure S4.9 Predicted phosphorylation sites of SRY/Sry from selected species 214 x Acknowledgements I would first like to thank my advisor, Michael Weiss, who is my mentor in personal and professional growth. He shapes the wonderful and key concepts presented in my work. He opened a new window for my spirit of independence and motivated my continual learning enthusiasm, which will be the most valuable treasure for my career. I am grateful to thank my thesis committee for their support: Dr. Hung-Ying Kao as my chair of my committee, Dr. David Samols, Dr. Pieter deHaseth, and Dr. Shigemi Matsuyama. This work would not have been completed without your encouragement and assistance. Within in the member of Dr. Weiss Lab, Dr. Nelson Phillips gave me invaluable hands-on support and education, including experience and writing. He is always willing to improve my drafts and provide great personal and professional support, make me confident to reach my next stage of career. Dr. Wan and Dr. Yang have been very kind in helping me operating computer programs. I also want to thank Dr. Hua and Ms. Jia for their warm encouragement for my spirit and career. Last but not the least; I want to present my gratitude to my “team SRY” partner, Joe, thank you for being a wonderful friend. Finally, I want to thank my family for their support. My parents always try their best to understand my difficulties and show me the positive side. Especially, I xi want to present my special gratitude to my fiancée, I-Ju Yeh, my lovely and smart partner for my life. She brought me these wonderful days and her constant love will keep me forward forever. Thank you! xii List of Abbreviations CaM calmodulin CD circular dichroism ChIP chromatin immunoprecipitation CTD C-terminal domain DMEM Dulbecco's Modified Eagle's Medium Exp4 exportin-4 FGF9 fibroblast growth factor 9 FRET fluorescence resonance energy transfer GATA4 GATA binding protein 4 GRD glutamine rich domain GRN gene regulatory network HMG high mobility group HPLC high-performance/pressure liquid chromatography IgG immunoglobulin G LHX9 LIM homeobox 9 LIM1 homeobox 1 LHX1 MIS/AMH Müllerian-inhibiting hormone/anti-Müllerian hormone MYA million years ago xiii NES nuclear export signal NLS nuclear localization signal NRY non-recombining region NTD N-terminal domain PAR pseudoautosomal region PTGDS prostaglandin-H2 D-isomerase SF1 splicing factor 1 SOX Sry-related HMG box SRY sex-determining region Y TBP TATA-box binding protein TDF testis determining factor TES testis-specific enhancer of Sox9 TESCO core of testis-specific enhancer of Sox9 TSPY testis-specific protein on Y chromosome WNT wingless-type WT1 Wilms tumor protein 1 Y1H yeast-one-hybrid ZFY Zinc finger Y-chromosomal protein xiv Mammalian Testis-Determining Factor SRY Has Evolved to the Edge of Ambiguity Abstract by YEN-SHAN CHEN The male sex determination program in eutherian mammals is regulated by Sry, a transcription factor encoded by the Y chromosome.