Local Regulation of DNA Methylation by the Transcription Factor REST Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Juliane Schmidt aus Halberstadt, Deutschland Basel, 2018 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung - Nicht kommerziell - Keine Bearbeitungen 4.0 International Lizenz. Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Dirk Schübeler und Dr. Duncan Odom. Basel, den 21.06.2016 Prof. Dr. Dirk Schübeler Prof. Dr. Jörg Schibler II To my parents and my grandfather Benno. III IV Acknowledgements First and foremost, I want to thank my doctoral thesis supervisor Prof. Dirk Schübeler for his continuous support over the last years. I am deeply thankful for the opportunity to have worked on this intriguing project. I acknowledge his patience and trust, which has allowed me to become a more independent and critical researcher. Moreover, I am thankful to Dr. Arnaud Krebs for his guidance and feedback on my project. His support was instrumental to the success of the project. I acknowledge essential bioinformatics support by Dr. Michael Stadler, Dr. Altuna Akalin, Dr. Arnaud Krebs, Dr. Anais Bardet and Dr. Lukas Burger. In detail, Dr. Altuna Akalin and Dr. Arnaud Krebs created a computational pipeline for analyzing amplicon Bisulfite sequencing data. Dr. Michael Stadler analyzed RNA-seq data, REST interactor ChIP-seqs and was critically involved in many other data sets. Dr. Anais Bardet analyzed the DNase-I seq experiments. Dr. Lukas Burger contributed to data analysis of ChIP-seq experiments. I am thankful to the FMI Genomics Facility, particularly to Dr. Sophie Dessus-Babus for her support with high-throughput sequencing. I thank Christiane Wirbelauer and Leslie Hoerner for technical support within the Schübeler lab. Thanks to Dr. Nicolas Thomä and Dr. Duncan Odom for offering scientific feedback as my doctoral thesis advisors. I am very grateful to Boehringer Ingelheim Fonds for generously funding my PhD project as well as attendance to conferences and courses. I have made invaluable professional and personal connections during that time. Several people have supported me along different steps of my academic career; I want to thank all of them. Special thanks to Prof. Markus Schwaninger, Dr. Gary Parkinson, Dr. Janna Seifried, Dr. Susann Stuttfeld (née Kumpf), Prof. Romeo Ricci, Dr. Stefan Metzler, Prof. Wilhelm Krek and Prof. Dirk Schübeler. Most recently, I thank Dr. Carien Dekker for being an extraordinary mentor as part of the University of Basel Antelope program. More female researchers need to be encouraged by a supportive network. V Personally, I thank Dr. Darko Barisic for being his entertaining and loyal self throughout our time in Basel. And most importantly, thanks to my parents and my brother for everything. VI Summary Transcriptional regulation in eukaryotes is realized through intricate interactions between transcription factors and chromatin. DNA methylation constitutes a chromatin modification that is associated with transcriptional silencing (Deaton and Bird, 2011). Whole-genome methylation profiling in mammals has revealed widespread cytosine methylation with characteristic hypomethylation at cis-regulatory elements. Hypomethylation is typically present within CpG islands and distal CpG-poor regions (Stadler et al., 2011). Previous investigations have shown, that some DNA-binding factors like the RE1-silencing transcription factor (REST) directly reduce methylation at these sites. However, how DNA-binding factors mediate such local methylation changes remains largely unknown. Hence, I studied the regulation of DNA methylation by the transcription factor REST in mouse embryonic stem cells (mESCs). I ectopically expressed different REST mutants and profiled DNA methylation at distal REST binding sites. While the full-length protein is necessary and sufficient to reduce methylation at its binding sites, REST’s DNA-binding domain lacks this ability. Instead, hypomethylation at binding sites required DNA-binding factors with interaction domains. The N-terminal REST mutant for example recruits SIN3A to binding sites and shows strong DNA demethylation ability. These experiments suggest that hypomethylation is not an obligatory consequence of protein binding, but rather requires interaction domains, reflecting the potential involvement of cofactors. I inquired whether TET enzymes contribute to reduced methylation within REST binding sites. Complete Tet1/2/3 deficiency in mouse stem cells caused a strong localized hypermethylation in the immediate vicinity of the REST motif. Whether TET proteins are recruited to REST binding sites through common cofactors or indirect mechanisms remains to be determined. I also characterized chromatin accessibility and nucleosome positioning in the different REST mutant re-expression cells. Interestingly, REST mutants that were competent to decrease DNA methylation also increased chromatin accessibility and nucleosome VII positioning. This could potentially link the chromatin remodeling ability of transcription factors to hypomethylation around binding sites. In summary, the presented study dissected REST induced methylation patterns around binding sites and described several of its required molecular components. This presents an example for a dynamic interplay between genetic and epigenetic information. VIII Table of Contents Acknowledgements, V Summary, VII 1 Chapter: Introduction, 1 1.1 Thoughts on Epigenetics, 1 1.2 Transcription and its Regulation in Eukaryotes, 3 1.3 Functional Organization of the Genome into Chromatin, 4 1.3.1 From Nucleosomes to Higher Order Chromatin Structures, 4 1.3.2 Histone Modifications, 7 1.4 Transcription Factors Act on Chromatin, 9 1.4.1 Transcription Factor Classes, 9 1.4.2 The RE1-silencing Transcription Factor, 11 1.5 DNA Methylation in Mammals, 17 1.5.1 Chemical Modifications of DNA, 17 1.5.2 DNA Methylation Machinery, 19 1.5.3 DNA Demethylation Pathways, 21 1.5.4 DNA Methylation Patterns, 24 1.5.5 DNA Methylation and its Effect on Transcription, 26 1.5.6 DNA Methylation as a Cause or Consequence of Transcription Factor Binding, 28 2 Chapter: Scope of the Thesis, 33 3 Chapter: Results, 35 3.1 Targeted Methylation Profiling by Amplicon Bisulfite Sequencing, 35 3.1.1 Experimental Implementation of Amplicon Bisulfite Sequencing, 36 3.1.2 An R package for Design and Analysis of Amplicon Bisulfite Sequencing Data, 37 3.1.3 Amplicon Bisulfite Sequencing for High-coverage Methylation Profiling, 39 IX 3.2 Regulation of DNA Methylation by the RE1-silencing Transcription Factor, 42 3.2.1 REST is not Required for Pluripotency in Mouse Embryonic Stem Cells, 42 3.2.2 REST is Necessary and Sufficient for Low Methylation of its Binding Sites, 45 3.2.3 REST Binding Overlaps with Several of its Chromatin Modifying Cofactors, 49 3.2.4 REST Does not Show Signs of Methylation Sensitivity, 53 3.2.5 REST’s DNA-binding Domain is not Sufficient to Reduce Methylation at Binding Sites, 55 3.2.6 N-terminal REST Protein Reduces DNA Methylation of REST Binding Sites and Recruits SIN3A, 66 3.2.7 Hypomethylation in the Vicinity of the REST Motif is TET dependen, 70 3.2.8 Chromatin Accessibility and Nucleosome Positioning are Altered in DNA Hypomethylated Cells, 72 3.2.9 The Kinetics of REST Induced Hypomethylation are Slow at a Majority of Cytosines, 76 3.2.10 Methylation Levels of REST Binding Sites Can be Predicted from Binding of REST Interactors, 80 4 Chapter: Discussion and Outlook, 83 4.1 REST Binding Sites are Hypomethylated and Show a Distinct Chromatin Landscape, 83 4.2 Molecular Features of REST That Are Associated with its Demethylating Activity, 87 4.3 Molecular Dissection of Methylation within REST Binding Sites, 92 4.4 Functional Implications of REST Associated Hypomethylation, 95 4.5 Transcriptional Effects of REST in Mouse Embryonic Stem Cells, 96 5 Chapter: Material and Methods, 99 5.1 Published Data Sets Used in Analyses, 99 5.2 Cell Culture, 100 X 5.3 Generation of REST Re-expression Cell Lines, 101 5.4 Amplicon Bisulfite Sequencing, 101 5.5 Amplicon Bisulfite Sequencing Analysis, 105 5.6 RNA-seq Analysis, 106 5.7 REST Motif Insertions, 106 5.8 ChIP-seq of REST Re-expression Cells, 107 5.9 ChIP-seq Analysis of REST Re-expression Cells, 108 5.10 SIN3A ChIP-qPCR, 109 5.11 Test of NTE-DBD Interaction Mutant after Transient Transfection, 109 5.12 NOMe-seq, 111 5.13 Kinetics of REST Binding Site Methylation after Transient REST Transfection, 113 5.14 Random Forest Model to Predict Methylation Levels of Distal REST Binding Sites, 113 List of Abbreviations,115 List of Figures, 117 References, 119 XI XII Chapter 1 Introduction Chapter 1 Introduction 1.1 Thoughts on Epigenetics Only few questions have continuously puzzled humans similarly as the essence of their own making. For scientists this means addressing the question of how a fertilized egg can develop into a complex multicellular organism. The path from a single cell to a unique human being is long; and a tail of decision-making and information integration. One question particularly occupies modern biologists: how can the same genome give rise to hundreds of different human cell types and tissues? How is the unambiguous path of cell type determination possible without writing or deleting information? Why does a muscle cell in our body always stay a muscle cell and does not become a hepatocyte? Answers to these questions might be found in the realms of epigenetics. While linguists would abstractly translate “epi-genetics” into the studies of the mechanisms above genetics, modern biologists seem to struggle with a precise terminology. One of the more frequently agreed upon definitions, states that epigenetics is the “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence” (Russo V.E.A. et al., 1996). In recent years, biologists have fought over the absolute requirement of heritability and what to call “epigenetic”.