UNIVERSITY OF CALIFORNIA Los Angeles New Roles of DNA Methylation in the Epigenome and Developmental Overgrowth Syndrome A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Molecular Biology by Andrew King 2017 © Copyright by Andrew King 2017 ABSTRACT OF THE DISSERTATION New Roles of DNA Methylation in the Epigenome and Developmental Overgrowth Syndrome by Andrew King Doctor of Philosophy in Molecular Biology University of California, Los Angeles, 2017 Professor Michael F. Carey, Chair DNA methylation is an epigenetic mark classically described to repress gene expression in a long and stable manner. DNA methylation plays important roles throughout development as well as throughout life where it maintains many tissue-specific genes in the silent state. DNA methylation is also altered in many diseases. Despite recent interest and innovation in measuring genome-wide DNA methylation, its function in the genome and its role in disease is not well understood. In this body of work, I aim to understand DNA methylation’s role in the cell through perturbation of DNA methylation in two separate contexts: normal mouse embryonic stem cells (mESCs) and a mESC-derived neural stem cell (NSC) disease model. In the first part of my dissertation (Chapter 2), global perturbation of DNA methylation in mESCs are used to address DNA methylation’s relationship with other members of the epigenome, namely histone modifications. Using a series of mESCs with DNA ii methyltransferases knocked-out and subsequently rescued, I dissect the causal effects of DNA methylation. By measuring how histone modifications and gene expression change with respect to global DNA methylation, I address whether DNA methylation is capable of regulating histone modifications and gene expression in mESCs. I find that genome-wide DNA demethylation alters occupancy of histone modifications in multiple genomic contexts. Most interestingly, global remethylation of the genome reverses changes in histone modification occupancy, indicating causal regulation by DNA methylation. In the second part of my dissertation (Chapter 3), highly specific perturbations in DNA methylation are made in mESCs and mESC-derived NSCs to understand what role DNA methylation may play in disease pathogenesis. Mutations recently described in the gene DNMT3A cause an overgrowth syndrome associated with intellectual disability named Tatton- Brown Rahman Syndrome (TBRS). Through genome editing, I establish a cellular model for TBRS where the endogenous Dnmt3a gene contains knock-in of individual missense mutations homologous to those found in TBRS patients. Differentiation of TBRS mESCs into NSCs along with measurements of global DNA methylation, gene expression, and histone modifications reveal downstream consequences of several of these mutations. I find that TBRS-associated DNMT3A mutations largely result in loss of methylation at specific DNMT3A targets. Of particular interest are a group of imprinted genes with known roles in growth and intellectual function. Furthermore, specific DNA methylation changes are also found to associate with changes in histone modification in NSCs. iii The dissertation of Andrew King is approved. Guoping Fan Jason Ernst Harley Kornblum William Lowry Michael F. Carey, Committee Chair University of California, Los Angeles 2017 iv I dedicate this dissertation to my family, without whom I could not have achieved. v Table of Contents List of Figures .............................................................................................................................. viii Acknowledgements ......................................................................................................................... x VITA ............................................................................................................................................. xii Chapter 1 : Introduction to DNA Methylation ............................................................................... 1 Historical Context ............................................................................................................... 3 Epigenetic Landscapes ........................................................................................................ 6 DNA Methylation Mechanisms – Lessons from Biochemistry ........................................ 13 Biological Functions ......................................................................................................... 18 DNA Methylation in Disease ............................................................................................ 21 Relation to this Dissertation .............................................................................................. 26 References ......................................................................................................................... 28 Chapter 2 : Reversible Regulation of Promoter and Enhancer Histone Landscape by DNA Methylation in Mouse Embryonic Stem Cells .............................................................................. 40 Introduction ....................................................................................................................... 41 Dnmt Reconstitution in Demethylated mESCs Restores Global Cytosine Methylation and Causes Various Changes in Histone Modifications .......................................................... 43 DNA Methylation is Required for Maintenance and Reestablishment of Promoter H3K27me3 ........................................................................................................................ 45 Impact of DNA Methylation on Promoter H3K27me3 and Gene Expression Differs Between Bivalent and Silent Promoters ........................................................................... 47 DNA Methylation Maintains and Reestablishes Silent or Primed States at Enhancer Elements ............................................................................................................................ 48 De Novo Enhancers Are Tissue-Specific and Contain Methylation-Sensitive Transcription Factors ........................................................................................................ 51 Regulation of H3K27me3 Depends on 5-Methylcytosine and DNMT Catalytic Activity52 vi DNA Methylation Regulation of H3K27me3 is Mediated by PRC2 Targeting ............... 53 DNMT3 Isoform Differences in Histone Modification Regulation ................................. 54 Discussion ......................................................................................................................... 56 Experimental Procedures .................................................................................................. 60 Figures............................................................................................................................... 68 References ......................................................................................................................... 83 Chapter 3 : Molecular and genomic defects of mutations in DNMT3A-associated Overgrowth Syndrome with Intellectual Disability .......................................................................................... 90 Introduction ....................................................................................................................... 91 Establishing a Cellular Model to Study Overgrowth-Associated DNMT3A Mutations .. 92 DNMT3A Mutants Differ in Functionality in mESC and mNSCs ................................... 93 DNA Methylation Alterations Occur at Sites of de novo Methylation in NSCs .............. 95 Epigenetic Signatures of OG-DMRs and Antagonism of H3K27me3 ............................. 97 Altered Gene Expression in Overgrowth Mutant NSCs ................................................... 99 Structural Interpretation of DNMT3A Mutations ........................................................... 101 Discussion ....................................................................................................................... 103 Experimental Procedures ................................................................................................ 106 Figures............................................................................................................................. 111 References ....................................................................................................................... 123 Chapter 4: Discussion and Concluding Remarks....................................................................... 126 First principles of epigenetics ......................................................................................... 127 Understanding epigenetic alterations in disease context ................................................ 130 References ....................................................................................................................... 133 vii List of Figures Figure 2.1. Alteration of DNA Methylation Causes Selective Genome-wide Changes in Histone Modifications ................................................................................................................................ 68 Figure 2.2. DNA Methylation Causally Regulates the Maintenance and Establishment of Promoter H3K27me3 .................................................................................................................... 69 Figure 2.3. DNA Methylation Regulates Histone Modification Status