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X-Chromosome Inactivation

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X-Chromosome Inactivation Autosomal random asynchronous replication is analogous to X-chromosome inactivation By MAS HUSETTS INSTITUTEI Alexander Wilson Ensminger OF TECHNOLOGY B.S. Biology FEB 1 2006 Haverford College Haverford, PA 19041 LIBRARIES Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology at the 4*'CHIVES Massachusetts Institute of Technology February, 2006 © 2005 Alexander W. Ensminger. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created. Signature of A uthor .......... r........................... ..... Department of Biology October 28, 2005 Certified by.. .......................... Andrew Chess Associate Professor of Biology Thesis Supervisor A ccepted by ..............................------ ..................................... Stephen P. Bell Chair, Committee on Graduate Students Department of Biology Autosomal random asynchronous replication is analogous to X-chromosome inactivation by Alexander Wilson Ensminger Submitted to the Department of Biology on October 28, 2005 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Abstract A number of mammalian genes are expressed from only one of two alleles in either an imprinted or random manner. Those belonging to the random class include X-linked genes subject to X inactivation, as well as a number of autosomal genes, including odorant receptors, immunoglobulins, T-cell receptors, interleukins, natural killer-cell receptors, and pheromone receptors. Random asynchronous replication of DNA in S-phase represents an epigenetic mark that often parallels monoallelic expression. All randomly monoallelically expressed genes discovered to date replicate asynchronously in S-phase, though not all of the genes contained within asynchronous domains are monoallelically expressed. The focus of my work has been on understanding this random choice that cells make between two sequence-identical alleles. Using two-color fluorescent in situ hybridization (FISH) analyses, the random asynchronous replication of a large number of human and mouse genes appears to be coordinated at the level of entire chromosomes. This regulatory scheme is reminiscent of random X-chromosome inactivation, the dosage compensation machinery in mammals. We have shown that autosomal coordination responds to trisomy in a fashion similar to X inactivation, with one copy of the trisomic chromosome marked for early replication and the other two rendered late replicating. These observations raise the intriguing possibility that the mechanistic underpinnings of X inactivation and autosomal coordination may also be similar. Furthermore, the existence of chromosome- wide epigenetic differentiation between autosomes has evolutionary implications concerning the establishment of X inactivation as the approach to mammalian dosage compensation. A crucial event in X inactivation is the random monoallelic expression of a noncoding RNA, Xist from one of the two X chromosomes. Noncoding RNA transcripts are enticing candidates for regulating chromatin structure within the mammalian nucleus. We have initiated a screen for novel nuclear, noncoding RNA transcripts. Using expression array profiling, we have identified several broadly expressed nuclear enriched transcripts. In addition to Xist, this approach identified two noncoding transcripts, NEATI and NEAT2 that are located near one another on human chromosome 1I and chromosome 19 of mice. Using a variety of techniques, including RNA FISH and RNA-mediated interference, we have explored the potential regulatory functions of these transcripts. Thesis Supervisor: Andrew Chess Title: Associate Professor of Biology 2 Dedicated to Julie Claycomb and William and Karen Ensminger 3 Acknowledgements There are a number of people who have contributed to my progress as a student over the years and have helped me to find this path. I have always felt that things work out ok if you just wait long enough. Graduate school has certainly had its share of challenges, both personal and professional. The ability to look at the opportunities afforded by temporary defeat and to keep things in perspective has been indispensable. The feeling that everything is going to be okay if you just stick it out is probably part genetic and part environment. Surely, that level of optimism is reinforced if things really do turn out okay in the end. I want to thank my parents for not only instilling that belief, but also for being there to make sure that it comes true. I certainly have had my share of ups and downs over the past several years, but they were there in the peaks and the valleys, helping to keep things in perspective. I feel very lucky to be their son. One of the major peaks for me during graduate school was the development of some great friendships over these years. One of these friendships, with my wife, Julie Claycomb, deserves particular highlighting. We started out as classmates in 7.50. Years passed (as they often do in grad school) and we became good friends. It is a remarkable experience to have such a partner in every facet of life. We have helped each other through the hard times and celebrated together when experiments actually work. She brings balance to my life and so much more. Julie works harder than anyone I know. Sometimes trying to keep up with her wears me out, but I thrive off of her energy. A life with Julie is worth a thousand lives with anyone else. Julie is not the only friend I have made in graduate school. Nelson Lau and I have been great friends since our first year at MIT. He has been a great sounding board for life's trials and tribulations over the last several years. He and his wife, Dianne Schwarz, are among the nicest, most generous people I've met. Another extremely dependable friend over the years has been Tate Kauffman, who I met while working in the lab of Phil Meneely at Haverford College. He and I thought we might be distant cousins once, due to the potential for shared ancestry in central Pennsylvania. Regardless of the biology, Tate, along with his wife Lara, will always be family to me (even if they are both M.D.s). I would like to thank some of the other friends I have had the pleasure to work with the past several years. Alexander Gimelbrant has been a great lab neighbor for the last 5 years. I will miss his humor and his advice. Nandita Singh did a wonderful job of teaching me the FISH technique. I appreciate all of the work that she and Sasha Ebrahimi did with respect to the first characterization of coordination that laid the groundwork for the rest of my thesis. Michael Tackett and I have suffered together at times, but I have been glad to have someone to complain to. John Hutchinson and Guilherme Neves have been wonderful colleagues and friends, brightening everyone's path that crosses theirs. These are not the only friends I have made in lab. I would like to express my deepest thanks to Andy Chess for being not only and advisor but a friend. I expect that both of us will be in this business for quite some time and it's nice to know that I will have a very creative friend and advisor to call upon when times get tough. My thesis committee members, David Page and Paul Garrity have provided me with great support and guidance over the years. I will miss our discussions and the reassurance of having them in my corner through all these years. I also would like to thank the Howard Hughes Medical Institute for supporting me as a predoctoral fellow. It has meant a lot to me to be a part of such a great program. I would also like to thank my college advisor and genetics professor, Philip Meneely. Phil showed me that biology is a dynamic science. Without him, I know that I would have been an economics major and my life would have taken a totally different path. So I guess what I'm trying to say is that this is all Phil's fault. 4 TABLE OF CONTENTS Chapter One: Introduction: Monoallelic Expression and Asynchronous Replication 7 The comparative value of haploidy versus diploidy 8 Dosage compensation mechanisms in worms and flies 10 Dosage compensation in mammals: X-chromosome inactivation 12 A number of autosomal genes are monoallelically expressed in mammals 19 Asynchronous replication coincides with monoallelic expression 28 Prevalence of asynchronous replication and monoallelic expression 35 Coordination of asynchronous replication between disparate loci 36 Summary of Thesis 39 References 43 Chapter Two: Coordinated replication timing of monoallelically expressed genes along human autosomes. 59 Abstract 60 Introduction 61 Results 63 Asynchronous replication in humans 63 Coordination of asynchronous replication in human cells 68 Asynchronous replication in trisomies 75 Discussion 81 Materials and Methods 85 Acknowledgements 87 References 88 Chapter Three: A search for Xist-like nuclear transcripts in the human genome identifies two conserved noncoding RNAs on Chromosome 11 90 Abstract 91 Introduction 92 Results 95 An array-based approach to identify ubiquitously expressed nuclear RNAs 95 Two noncoding RNAs enriched in the nuclei of human cells 97 RNA FISH of Xist, NEAT], and NEAT2 100 Examining transcription around NEATI and NEAT2 104 Functional analysis of NEAT] and NEAT2 using RNA interference 105 Comparative genomic analysis of hNEATI and hNEAT2 106 Discussion 114 Materials and Methods 117 References
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