Dissection of Xist Functional Elements Involved in X-Chromosome Inactivation
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Dissection of Xist Functional Elements Involved in X-Chromosome Inactivation The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Colognori, David A. 2019. Dissection of Xist Functional Elements Involved in X-Chromosome Inactivation. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029476 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Dissection of Xist functional elements involved in X-chromosome inactivation A dissertation presented by David A. Colognori to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachusetts May 2019 © 2019 by David A. Colognori All rights reserved. Dissertation Advisor: Jeannie T. Lee David A. Colognori Dissection of Xist functional elements involved in X-chromosome inactivation Abstract X-chromosome inactivation (XCI) is the epigenetic process of silencing one of the two X chromosomes in female mammals to balance X-linked gene dosage with that of males. This process is governed by the long noncoding RNA Xist. Xist is expressed from and coats one X chromosome in cis, leading to recruitment of various protein factors to silence gene expression. However, the functional RNA elements and molecular mechanisms involved in Xist coating and silencing remain ambiguous. The rationale for this work was thus to perform a systematic deletional analysis to identify functional motifs within the endogenous Xist locus in female cells. The screen identified regions important for correct splicing of the Xist transcript, such as the Repeat A region. Others, including Repeat F, were required for proper expression and/or RNA stability. Repeat E was crucial for restricting Xist localization to the inactive X (Xi) territory. Finally, Repeat B was necessary for recruitment of Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2) across the Xi, as well as for proper Xist coating. To gain mechanistic insight into Polycomb targeting and Xist localization, I identified protein trans factors associated with the Repeat B and E motifs. Repeat B function in Polycomb recruitment and Xist coating was shown to be mediated through direct interaction with the RNA- binding protein HNRNPK. Meanwhile, Repeat E function in Xist localization was found to be mediated through interaction with the nuclear matrix protein CIZ1. iii Just as Xist RNA is necessary to spread Polycomb complexes across the Xi, I found that Polycomb complexes are in turn necessary to properly spread Xist RNA. In addition, PRC1 and PRC2 occupancy on Xi was discovered to be mutually interdependent. Hence, Xist, PRC1, and PRC2 require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading has significant consequences, as deleting Repeat B during de novo XCI establishment causes failure of X- linked gene silencing and disrupts architectural reconfiguration of the X from and active to inactive chromosomal structure. iv Table of Contents Abstract iii Table of Contents v List of Figures and Tables vii Acknowledgements viii Dedication x Chapter 1 Introduction 1 Epigenetics and chromatin 2 Dosage compensation and X-chromosome inactivation 3 The X-inactivation center and X-inactive specific transcript 6 XCI as a paradigm of epigenetic regulation by RNA 7 Features and mechanisms of Xist RNA 8 Xist spreading 9 Xist-mediated gene silencing 11 Polycomb complexes and their function 12 Polycomb recruitment, spreading, and maintenance 16 Xist-mediated Polycomb recruitment 18 Unique chromosomal architecture of the Xi 21 Xist-mediated changes to chromosomal architecture 23 Preface 24 Chapter 2 Xist deletional analysis reveals an interdependency between Xist 26 RNA and Polycomb complexes for spreading along the inactive X Summary 28 Introduction 28 Results 30 Discussion 66 Materials and Methods 68 Chapter 3 Repeat E anchors Xist RNA to the inactive X chromosomal 89 compartment through CDKN1A-interacting protein (CIZ1) Summary 91 Introduction 91 Results 92 Discussion 110 Materials and Methods 112 Chapter 4 Conclusion 120 Controversy over Xist functional domains 121 Advantages of CRISPR/Cas9 technology and cell lines used in this work 122 Xist regions affecting RNA abundance and splicing: 123 Repeats A, F, exons 2-6, 7a/d Xist regions affecting RNA localization: Repeat E 125 v CIZ1 interaction with Repeat E 125 Xist regions affecting RNA coating and Polycomb recruitment: Repeat B 126 HNRNPK interaction with Repeat B 126 Repeat B-HNRNPK interaction is required for Polycomb recruitment 127 Role of Repeat A in Polycomb recruitment 128 Role of Polycomb in Xist-mediated gene silencing 130 Mechanism of Repeat B/HNRNPK-mediated Polycomb recruitment 130 Xist co-opts general factors for XCI 132 Independent versus interdependent recruitment of Polycomb complexes 134 Role of Polycomb in Xist RNA coating 135 Role of Repeat B in architectural reconfiguration of the Xi 137 References 140 Appendix 162 vi List of Figures and Tables Chapter 1 Figure 1.1 – Features of Xist 7 Figure 1.2 – Composition and interplay between PRC1 and PRC2 15 Chapter 2 Figure 2.1 – CRISPR/Cas9 deletion screen identifies Xist functional domains 31 Figure 2.2 – Xist deletions affecting splicing 33 Figure 2.3 – The Repeat B motif affects Xist localization in a CIZ1-independent manner 36 Figure 2.4 – H3K27me3/H2AK119ub IF and Xist RNA FISH for deletions without 37 phenotype Figure 2.5 – Repeat B is required for Xist RNA spreading and Polycomb maintenance 39 Figure 2.6 – Identification of HNRNPK as a Repeat B-interacting protein 42 Figure 2.7 – Direct Repeat B-HNRNPK interaction is required for Xist spreading and 45 Polycomb maintenance Figure 2.8 – Xist and Polycomb complexes depend on each other to spread across the Xi 48 Figure 2.9 – Diffuse Xist cloud morphology is not due to large-scale decompaction of Xi 51 Figure 2.10 – Independent and interdependent recruitment of PRC1 and PRC2 to the Xi 54 Figure 2.11 – Repeat B is required for complete Xist-mediated gene silencing 57 Figure 2.12 – Repeat B is required for proper Xa to Xi architectural reconfiguration 62 Chapter 3 Figure 3.1 – ASH2L antibody exhibits cross-reactivity to an unknown Xi-localizing protein 93 Figure 3.2 – CIZ1 is a novel Xi-localizing protein 96 Figure 3.3 – Xist RNA is required for CIZ1 recruitment to Xi 99 Figure 3.4 – CIZ1 is critical for maintenance of Xist cloud and Xi heterochromatin marks 102 Figure 3.5 – CIZ1 interacts with Xist RNA through the Repeat E region 105 Figure 3.6 – CIZ1 interacts with Xist RNA independently of HNRNPU 108 Chapter 4 Figure 4.1 – Repeat B secondary structure 127 Appendix Table S1 – Sanger sequencing information 163 Table S2 – Mass spectrometry data 198 Table S3 – Guide RNA sequences 215 Table S4 – FISH probe sequences 216 Table S5 – Primer sequences 224 vii Acknowledgements This dissertation would not have been possible without the love and support of countless individuals in my life. Science is intended to be a collaborative effort, and the long journey toward a Ph.D.—both at and away from the bench—accurately reflects that. First and foremost, I would like to thank my family. I am grateful to my mother for recognizing and encouraging my scientific curiosity and creativity from a young age, as well as her patience with my many idiosyncrasies. I thank my sister, the other Ph.D. in our family, for her guidance throughout this difficult journey. I also thank my brother-in-law, Kevin, and my recent nephews, John and Christopher, for making my visits home so enjoyable. I was also fortunate to have several relatives near Boston that went out of their way to make me feel at home and provide a respite from work. Finally, I am grateful to my father for always believing in me and helping me get back up again when I fall. Although he is no longer with us, he is the reason I chose to pursue biological research in the first place. I miss you dad. I was lucky to have amazing friends and housemates in Boston who made these years fly by: Diana Cai, Luvena Ong, Terence Wong, Tim Whipple, Jarom Chung, Shela Durresi, Bing Han, and Bernie Kuan. Thanks for all the game nights, potlucks, and sharing your lives with me. I would also like to thank my high school and college friends for their support from afar, as well as my online gaming friends for all the laughs we shared over the internet. I am indebted to my undergraduate research advisor, Joan Steitz, for starting me down the path of RNA biology. She accepted me into her lab with no prior research experience and taught me the fundamentals of doing good science. I owe my mentors in the Steitz lab, Andrei Alexandrov and Kasandra Riley, for teaching me everything I knew upon entering graduate school. I am grateful to all past and present members of the Lee lab for their advice and support over the years, both scientific and personal. I am especially grateful to Hongjae Sunwoo, Stefan viii Pinter, Cathy Cifuentes-Rojas, Yesu Jeon, and Brian del Rosario for their close mentorship on various projects. Thank you for putting up with all my whining and shenanigans. To my fellow BBS graduate students in the Lee lab (Chen-Yu Wang, John Froberg, Andrea Kriz, Lin Wang, and Johnny Kung), thanks for making my time here so enjoyable.