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Biology of Maintenance and De Novo Methylation Mediated by DNA Methyltransferase-1

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Biology of Maintenance and De Novo Methylation Mediated by DNA Methyltransferase-1 Biology of maintenance and de novo methylation mediated by DNA methyltransferase-1 Olga Yarychkivska Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2017 © 2017 Olga Yarychkivska All rights reserved ABSTRACT Biology of maintenance and de novo methylation mediated by DNA methyltransferase-1 Olga Yarychkivska Within the past 70 years since the discovery of 5-methylcytosine, we have acquired considerable knowledge about genomic DNA methylation patterns, the dynamics of DNA methylation throughout development, and the enzymatic machinery that establishes and perpetuates genomic methylation patterns. Nonetheless, in the field of epigenetics major questions remain open about the mechanisms of spatiotemporal control that exist to ensure the fidelity of methylation patterns. This thesis aims to decipher the regulatory logic and upstream pathways influencing one of the DNA methyltransferases by leveraging the diverse resources of molecular genetics, biochemistry, and structural biology. The primary subject of my research, DNA methyltransferase 1 (DNMT1), is crucial for maintaining genomic methylation patterns upon DNA replication and cell division. In addition to its C-terminal catalytic domain, mammalian DNMT1 harbors several N-terminal domains of unknown function: a succession of seven glycine-lysine (GK) repeats, resembling histone tails, and two Bromo-Adjacent Homology (BAH) domains that are absent from bacterial DNA methyltransferases. The work I present in this thesis characterizes the role of these hitherto enigmatic domains in regulating DNMT1 activity. In my studies, I found that mutation of the (GK) repeats motif leads to de novo methylation by DNMT1 specifically at paternally imprinted genes. Conventionally, de novo methylation is thought to be undertaken by complete different enzymes, DNMT3A and DNMT3B, whereas DNMT1 is limited to perpetuating the patterns these other methyltransferases had set down. Recombinant DNMT1 had been previously shown to efficiently methylate unmethylated DNA substrate in vitro, but this is the first time its de novo methyltransferase capability has been observed in vivo. Based on these data, I propose a new model in which DNMT1 is the enzyme responsible for laying down de novo methylation patterns at paternally imprinted genes in the male germline, explaining the previously observed non-essential role of other DNA methyltransferases in the establishment of paternal imprints. Furthermore, I demonstrated that acetylation of the (GK) repeats motif inhibits this de novo methyltransferase activity of DNMT1, making this particular motif an essential regulatory platform for controlling the diverse in vivo functions of the enzyme. Though the (GK) repeats motif had previously been proposed to regulate the stability of DNMT1 protein through its interaction with the deubiquitinase USP7, I tested the biological relevance of this interaction and found that USP7 deletion does not alter DNMT1 protein levels. In fact, USP7 appears to play no part in regulating maintenance DNA methylation, as I present evidence that USP7 localization to replication foci is entirely independent of DNMT1. Finally, I demonstrated that the tandem BAH domains of DNMT1 are required for its maintenance methyltransferase activity as they are involved in targeting the enzyme to replication foci during S phase. Based on biochemical data supporting an interaction between DNMT1's BAH1 domain and histones, I propose that this targeting could occur through BAH1's recognition of specific histone modifications, thus providing a potential mechanistic link between maintenance DNA methylation and chromatin markings. This thesis identifies DNMT1 as a novel de novo methyltransferase in vivo and also characterizes the regulatory functions of the enzyme's BAH domains and the (GK) repeats. These results elucidate the multiple regulatory mechanisms within the DNMT1 molecule itself that control its functions in mammalian cells, thereby providing critical insights as to how the DNA methylation landscape takes shape and yielding surprising revelations about the parts that well- studied proteins have to play in this process. TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................................... iv LIST OF ABBREVIATIONS .................................................................................................................... v CHAPTER I. GENERAL INTRODUCTION ............................................................................................. 1 I.1. Patterns and Machinery of DNA Methylation .................................................................................... 3 a. Overall composition of the mammalian genome .............................................................................. 3 b. Methylation patterns and different genomic compartments .............................................................. 6 c. DNA methyltransferases ................................................................................................................... 7 I.2. Functions of DNA Methylation ........................................................................................................ 11 a. Defense against transposable elements ........................................................................................... 11 b. Genomic stability and apoptosis ..................................................................................................... 11 c. Imprinting ........................................................................................................................................ 13 d. X chromosome inactivation ............................................................................................................ 15 I.3. Methylation Dynamics ..................................................................................................................... 17 a. Developmental overview................................................................................................................. 17 b. Establishment of sex-specific genomic methylation in the germline .............................................. 18 c. Correlation with chromatin landscape ............................................................................................. 20 I.4. Role and Regulation of DNMT1 ...................................................................................................... 26 a. DNMT1’s discovery and genetic studies ........................................................................................ 26 b. Structural insights into DNMT1 regulation .................................................................................... 29 i. RFTS and targeting to sites of DNA replication ......................................................................... 29 ii. Autoinhibitory role of CXXC domain ....................................................................................... 32 iii. BAH domains ........................................................................................................................... 32 iv. (GK) repeats .............................................................................................................................. 34 v. Methyltransferase domain and mechanism of methylation ....................................................... 34 c. Regulation of DNMT1 by other proteins ........................................................................................ 36 i. UHRF1 is essential for maintenance DNA methylation ............................................................. 36 ii. Acetylation-ubiquitination regulation by USP7 and TIP60 ....................................................... 37 I.5. Outline of Thesis .............................................................................................................................. 39 CHAPTER II. ACETYLATED (GK) REPEATS PREVENT DNMT1 FROM CARRYING OUT DE NOVO METHYLATION AT PATERNALLY-IMPRINTED GENES IN EMBRYONIC STEM CELLS .................................................................................................................................................................... 40 II.1. Rationale and Summary .................................................................................................................. 40 II.2. Materials and Methods .................................................................................................................... 41 a. Cell lines and cell culture ................................................................................................................ 41 i b. Immunoblotting ............................................................................................................................... 42 c. Methylation analysis ....................................................................................................................... 42 d. Southern blots ................................................................................................................................. 42 e. Bisulfite sequencing ........................................................................................................................ 43 f. RT-qPCR ........................................................................................................................................
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