Approaches to Determining the Three-Dimensional Structure and Dynamics of Bacterial Chromosomes

Approaches to Determining the Three-Dimensional Structure and Dynamics of Bacterial Chromosomes by Matthew A. Wright B.A. Chemistry, B.M. Music Performance University of Southern Maine, 1999 Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY in Physical Chemistry at the Massachusetts Institute of Technology September 2005 © Massachusetts Institute of Technology, 2005 All rights reserved A/ A I A Signature of Author I _ _ _ _ I Department of Chemistry /J7 September 1,2005 ( Certified by V George M. Church A Professor of Genetics, Harvard Medical School Thesis Supervisor Accepted by. -l N ) Robert W. Field MASSACHUSETTSINSTIT Chairman, Departmental Committee on Graduate Students OF TECHNOLOGY OCT ! 1 2005 OiL, Nt/ke LIBRARIES This doctoral thesis has been examined by a Committee of the Department of Chemistry as follows: --------Professor Keith A. Nelson . - X/ nCommittee Chairman ( Professor George M. Church Prssor of-Genetics, Harvard Medical School I Professor of Genetics, Harvard Medical School Thesis Supervisor A ,, Professor Leonid Mirny 1 _ _ Associate Professor of Physics, Massa tts Institute of Technology Committee Member Approaches to Determining the Three-Dimensional Structure and Dynamics of Bacterial Chromosomes by Matthew A. Wright Submitted to the Department of Chemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Chemistry Abstract The information in genomes is only partially contained in the linear sequence of their nucleotides. Their folding into dynamic three-dimensional structures creates spatial relationships between loci that likely play important functional roles. Yet so far only the broad outlines of this spatial organization have been discerned. In chapter 2 of this thesis I describe a general constraint-based framework for defining the configuration space of chromosomes. Analogous to protein structure determination through NMR, such a framework allows the quantitative reduction of the conformation space down to the level of a single structure or an ensemble of structures. It is compatible with both experimentally determined and theoretical constraints, particularly those motivated by evolutionary optimality. In chapter 3., I describe the first method to search for signals of large-scale three- dimensional structure in genome sequences. The results suggest that there is strong selection for three-dimensional relationships within the chromosome, particularly those related to transcription. The signals generated recapitulate both known structural data from microscopy and functional data on genome-wide transcription levels. Moreover, a detailed analysis of these signals in E. coli suggests previously unknown structural features including chromosome-long periodic looping and an axis of high transcriptional activity. There are immediate applications to other bacteria and potentially to eukaryotes. Thesis Supervisor: George M. Church Title: Professor of Genetics, Harvard Medical School 3 Table of Contents Chapter 1 Introduction 10 Chapter 2 Constraint-based determination of 27 chromosome structure Chapter 3 "Just in place" functional organization 59 of bacterial chromosome structure Chapter 4 Conclusions and future directions 112 Appendix 1 On the complete determination of 122 biological systems Appendix 2 An open-source oligomicroarray 138 standard for human and mouse 4 I dedicate this thesis to my parents, Bonnie and Tom Wright, for their love and encouragement, to my grandfather, Robert E. Arnot, and uncle, Robert B. Arnot, who ignited in me a passion for medical science, and to my undergraduate chemistry advisor, Henry J. Tracy, who first showed me the beauty of basic scientific research. 5 Acknowledgements It was my grandfather, Robert Arnot, who introduced me to science, through my first scientific tool, a calculator, given to me at age 2. Although it was the buttons that fascinated me, I like to think that a bit of arithmetic wore off during the months that followed while the calculator never left my side. My grandfather was an enormous influence on me, through his example as a fine physician and remarkable human being, and through his passion for science as a means for human good. He brought the mindset of a researcher to the care of patients long after he left his own formal work in psychopharmacology. It is my father from whom I inherited a love for mathematics. He is among the few people I know who takes on a glow at the mere mention of the words "linear algebra" and can spend an evening discussing abstract multidimensional spaces. For better or for worse I seem to have inherited these traits. Moreover, he has a seemingly insatiable curiosity, evidenced by a constant and ever-changing stack of scientific texts heaped upon his desk. His curiosity serves as a reminder to always inquire. I thank my mother who has been a solid support for me and for my entire family through many trials; additionally she lovingly guided me from music into science. I thank my piano teacher Laura Kargul who both enriched my life through 7 years of teaching and helped to open me to possibilities outside of music. I thank my family as a whole, my grandmother, Mary Arnot, my siblings,Tom Doug and Emily, and my uncle Robert, an important support. I thank also many friends, in particular Brad Coull, David Goodman, Andrew Butterworth, Tracey Orick, and Janice Sullivan, Cathy O'Neil and Jen Johnson, 6 close friends and inspiring mathematicians, and Serhan Altunata, an excellent physicist. I thank the entire Sievers family, quite a scientific clan, in particular Kathryn for her quick mind, LeRoy, for his wonderful practicality as a physicist/engineer, and Charlie, for frequent conversations on physics and Fourier analysis. Stephen MacDonald first introduced me to the beauty of higher mathematics, in a calculus course that I originally took as a core math requirement in college. His brilliant teaching, somehow allowed his students to always see one step ahead in the web of mathematical logic and convinced me to take one, then two, then three more courses with him while still a piano performance major. Ultimately this led me to embrace science as a career. John Ricci's, patient and clear teaching of general chemistry and then physical chemistry introduced me to the mathematics of chemical structure. And his long talks with me illuminated the possibilities of a life in science. It was Henry Tracy, though, who first introduced me to scientific research. He inspired in me a true passion for science by his enormously enthusiastic classroom teaching and through his guidance in my first independent research project, a year-long organo-metallic synthesis, which, for him, given my lack of synthetic ability was a true labor of love. Outside of chemistry, he exposed me to a broad range of science, from quantum mechanics to astronomy, which he introduced me to through his own telescope from the fields outside his home in Maine. He became a mentor and a good friend. In my brief stay at Caltech, I had the privilege to work with a truly great theorist, Rudolf Marcus, and to listen as he worked through the complex problem of ion-transport across interfaces. During this time, I gained an appreciation for the multiple angles a 7 theorist must approach a problem from and the multiple fields a theorist must therefore draw upon. I was also fortunate to work with Niles Pierce and ErikWinfree, both highly interdisciplinary scientists who encouraged me to be broad in choosing problems. It is George Church and all of the scientists of the Church lab who have been the enablers of my doctoral work and who have been the teachers that have nurtured my growth as a scientist over the past 5 years. I thank everyone I've known in the lab but must mention a few in particular. Tzachi Pilpel, was my first mentor here and offered me important encouragement and guidance. He is a wonderful scientist, with a keen mind, amazing clarity of thought, and an amazing ability to communicate. He continues to serve as a model and inspiration. Kun Zhang, a fabulous experimentalist, has been both a patient teacher and friend. Farren Isaacs, has been an excellent editor and a sharp mind. Xliao Xia Lin has been a great support. Peter Kharchenko, an excellent and critical colleague. Kyriacos Leptos, an avid conversationalist on language and music. Dana Pe'er has been both an inspiring example, and a source of wonderful encouragement. Mark Umbarger, a strong support, and a rigorous critic. Doug Selinger a fellow philosopher and important friend. Yonatan Grad, an inspiration as a scientist, as a humanist, and also as friend. Jay Shendure, has been an important example, a startlingly quick mind which both critically grasps and will not relinquish the important details of a problem but which also views these details within the compass of the whole. His is a mind simultaneously critical and visionary, and practical. Nikos Reppas has managed to somehow be both a roommate and a bay-mate for three years. He has been my educator in the lab, fielding an average of 1 question every 5 minutes while I attempted experimental work, a constant editor and a fabulous example of both an amazing work ethic and extraordinary scientific rigor. 8 Daniel Segr&deserves pages of his own. He has been my closest mentor. Indeed much of the work of this thesis is as much his as it is my own. His mind is a philosopher scientist's and serves as a model of creativity. He fosters a work environment which simultaneously encourages free expression of ideas and hones these ideas critically to maturity. From him I have learned the process of theoretical science and been guided through this process during our chromosome structure work. His like-mindedness has made him both a scientific colleague and an intellectual colleague for talks on differential geometry, language, the vast span of physics, and music.

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