Crosstalk between DNA Methylation and Histone Modifications GARWIN PICHLER Dissertation an der Fakultät fur Biologie der Ludwig-Maximilians-Universität München vorgelegt von Garwin Pichler aus Oberhausen München, den 5. März 2012 Erstgutachter: Prof. Dr. Heinrich Leonhardt Zweitgutachter: Prof. Dr. Peter Becker Tag der mündlichen Prüfung: 22.05.2012 CONTENTS Contents Summary 1 1 Introduction 2 1.1 Epigenetic Gene Regulation . 2 1.1.1 DNA Methylation . 3 1.1.2 Histone Modifications . 6 1.1.3 Nucleosome Positioning . 9 1.1.4 Crosstalk between Epigenetic Modifications . 10 1.2 DNA Methyltransferases . 12 1.2.1 Structure and Function Dnmt1 . 14 1.2.2 Regulation of Dnmt1 by Interactions . 16 1.2.3 Regulation of Dnmt1 by Post-translational Modifications . 19 1.3 The Uhrf Family of Proteins . 21 1.3.1 Phylogenetic Tree of Evolution . 21 1.3.2 Structure and Function of Uhrf Proteins . 23 1.3.3 Regulation of Uhrf Proteins . 25 2 Aims of this work 26 3 Results 27 3.1 Fluorescent protein specific Nanotraps to study protein-protein interac- tions and histone tail peptide binding . 27 3.2 Versatile Toolbox for High-Throughput Biochemical and Functional Studies with Fluorescent Fusion Proteins . 43 3.3 The multi-domain protein Np95 connects DNA methylation and histone modification . 56 3.4 Cooperative DNA and histone binding by Uhrf2 links the two major repres- sive epigenetic pathways . 75 3.5 DNMT1 regulation by interactions and modifications . 93 3.6 Cell Cycle-dependent Interactions of Dnmt1 . 105 4 Discussion 117 4.1 Versatile Toolbox for High-Throughput Biochemical and Functional Studies with Fluorescent Proteins . 117 4.2 Uhrf proteins connect different pathways . 120 4.2.1 Uhrf1 coordinates repressive epigenetic pathways . 120 4.2.2 Uhrf2 coordinates repressive epigenetic pathways . 121 4.2.3 Diverse function of Uhrf proteins . 123 4.3 Dynamic interactions of Dnmt1 . 129 ii CONTENTS 5 Annex 130 5.1 References . 130 5.2 Abbreviations . 157 5.3 Contributions . 160 5.4 Declaration . 162 5.5 Acknowledgement . 163 5.6 Publications . 165 iii LIST OF FIGURES AND TABLES List of Figures 1 Three levels of epigenetic regulation . 3 2 Overview of DNA modifications . 5 3 Histone post-translational modifications and their binding partners . 8 4 Schematic outline of DNA Methyltransferases . 12 5 Structural insights into the DNA methyltransferases 1 . 15 6 Interactom of Dnmt1. 16 7 Regulation of Dnmt1 by post-translational modifications . 20 8 Phylogenetic Tree of Life for Dnmts and Uhrf proteins . 22 9 Multi-domain proteins Uhrf1 and Uhrf2 . 23 10 Workflow of SILAC-based quantification of cell cycle-dependent interac- tions of Dnmt1 . 108 11 Initial testing of different Dnmt1 binder . 110 12 Immunoprecipitation of Dnmt1 with the Dnmt1 binder . 111 13 Cell cycle synchronization of HeLa cells using mimosine . 112 14 Dynamic Dnmt1 interactome visualized by a heat map and one-way hi- erarchical clustering . 115 15 SILAC ratios for Dnmt1, PCNA and H2A . 116 16 Schematic drawing of the GFP-multiTrap . 118 17 Radioactive in vitro DNA methyltransferase assay . 124 18 Diverse functions of Uhrf proteins . 128 List of Tables 1 Interaction partners of Dnmt1 . 113 2 Interaction partners of Uhrf1-GFP and Uhrf2-GFP . 125 3 Uhrf proteins associate with a broad range of proteins . 126 iv SUMMARY Summary DNA methylation and histone modifications play a central role in the epigenetic regu- lation of gene expression and cell differentiation. DNA methyltransferase 1 (Dnmt1) is the most ubiquitously expressed DNA methyltransferase and is essential for maintaining DNA methylation patterns during semi-conservative DNA replication and DNA repair. The fidelity and processivity of this process is crucial for genome stability and is based on the specific recognition of hemimethylated CpG sites emerging at the replication forks. Remarkably, a variety of proteins have been reported to interact with Dnmt1, regulating the activation, stabilization and recruitment of Dnmt1 to specific sites and regions. Recently, Uhrf1 has been found to interact with Dnmt1, indicating together with genetic studies a central role in the maintenance of DNA methylation. However, the exact mechanism how Uhrf1 plays a role in DNA methylation remains elusive. Uhrf1 is a multi-domain protein harbouring distinct functional domains, which specifi- cally recognize epigenetic marks such as DNA methylation and histone modifications. To investigate the histone-tail binding specificities of Uhrf1, we developed an in vitro histone-tail peptide binding assay using GFP-fusion proteins. Moreover, we expanded this assay to 96-well micro plates, allowing high-throughput screening for histone-tail peptide binding, DNA binding, protein-protein interactions, detection of endogenous binding partners and quantification of post-translational modifications. Using this as- say, we demonstrated that Uhrf1 preferentially binds to H3K9me3 via a tandem Tudor domain. The binding is mediated by three highly conserved aromatic amino acids that form an aromatic cage similar to the chromodomain of HP1b. Furthermore, we characterized the second member of the Uhrf family, Uhrf2. Binding assays demonstrate a cooperative interplay of Uhrf2 domains that induces preference for hemimethylated DNA and enhances binding to H3K9me3 heterochromatin marks. Localization and binding dynamics of Uhrf2 in vivo require an intact tandem Tudor do- main and depend on H3K9 trimethylation but not on DNA methylation. Remarkably, both genes showed an opposite expression pattern and ectopic expression of Uhrf2 in uhrf1-/- ESCs did not restore DNA methylation at major satellites indicating functional differences. Finally, we used a combination of SILAC and mass spectrometry to quantify protein interactions of Dnmt1 over the cell cycle. Therefore, we immunoprecipitated endoge- nous Dnmt1 from HeLa cells cultured in "light" or "heavy" media synchronized in either G1, early S or late S phase of the cell cycle. With this approach, we could accurately identify interactions of endogenous Dnmt1 and characterize the cell cycle-dependent dynamics. In conclusion, this work contributes to the further elucidation of the complex and dy- namic mechanisms of Dnmt1 regulation by interacting factors and demonstrates the role of Uhrf proteins in mediating epigenetic crosstalk. 1 INTRODUCTION 1 Introduction 1.1 Epigenetic Gene Regulation Multicellular organisms comprise a variety of highly specialized cells that contain the same genetic information, the DNA sequence, but differ dramatically in morphology and function. In simple terms, large ranges of different phenotypes arise from the same genotype. The formation of organs and tissues of morphological and functional distinct cell types throughout development are controlled by spatial and temporal changes of gene activation and repression. One of the most important control mech- anisms to ensure proper development is transcriptional regulation by a complex pro- tein network that results in gene transcription by the RNA polymerase II. Basically, the regulation of transcription initiation is accomplished by three different mechanisms: (i) Architecture of the core promoter including regulatory elements, multiple start sites and alternative promoter usage (ii) control of DNA accessibility by altering the chro- matin structure (Berger, 2007) and (iii) general and sequence-specific transcription fac- tors (Malik and Roeder, 2005). Transcription factors are crucial determinants for the regulation of gene expression that activate or repress specific genes through sequence-specific interactions with their promoters. Interaction of transcription factors with their cognate DNA sequence trig- gers a chain of events, often involving changes in the structure of chromatin, leading to the assembly of an active transcription complex. However, due to the complexity of genomic functions in eukaryotes, transcription factors are not sufficient for full es- tablishment and long-term stability of transcriptional states. This probably accounts for the very low efficiency of cloning animals from the nuclei of differentiated cells and that cloned animals generated from the same donor DNA are not identical to their donor. Even more drastically, cloned animals by nuclear transfer often develop diseases with various defects (Rideout et al., 2001). It became clear that stable cell- type specific gene expression pattern are established and maintained by a complex interplay between transcription factors and epigenetic regulators. In particular, epi- genetic processes like DNA methylation, post-translational histone modifications and nucleosome positioning play an essential role in the regulation of gene expression (Fig- ure 1). These modifications are by definition epigenetic, since epigenetics in its classic definitions describes heritable modifications of DNA or chromatin that do not alter the primary nucleotide sequence (Bird, 2002; Jaenisch and Bird, 2003). These epigenetic processes control the composition, structure and dynamics of chromatin and thereby regulate gene expression by regulating the condensation and accessibility of genomic DNA (Bird, 2002; Kouzarides, 2007; Reik, 2007). 2 INTRODUCTION DNA Methylation Histone Modifications Nucleosome Positioning Ac Me Me Chromatin Remodelling Me Me Histone Variant P Figure 1: Three levels of epigenetic regulation Epigenetic regulation depends on the interplay of DNA methylation (PDB ID code: 3BSE), histone modi- fications (PDB ID code: 1AOI) and nucleosome positioning. Figure is adapted from (Portela and Esteller, 2010) 1.1.1 DNA Methylation