Les cellules souches embryonnaires (cellules ES) constituent un excellent système modèle pour étudier les mécanismes épigénétiques contrôlant la transcription du génome mammifère. Un nombre important de membres de la famille des facteurs de remodelage de chromatine ATP-dépendants ont une fonction essentielle pour l’auto-renouvellement des cellules ES, ou au cours de la différentiation. On pense que ces facteurs exercent ces rôles essentiels en régulant l’accessibilité de la chromatine au niveau des éléments régulateurs de la transcription, en modulant la stabilité et le positionnement des nucléosome.Dans ce projet, nous avons conduit une étude génomique à grande échelle du rôle d’une dizaine des remodeleurs (Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Ep400, Brg1, Smarca3, Smarcad1, Smarca5, ATRX et Chd1l) dans les cellules ES. Une double stratégie expérimentale a été utilisée : Des expériences d’immunoprécipitation de la chromatine suivi par un séquençage à haute-débit (ChIP-seq) sur des cellules ES étiquetées pour les différents remodeleurs, pour étudier leur distribution sur le génome, et un approche transcriptomique sur des cellules déplétées de chaque remodeleur par traitement avec des vecteurs shRNA (knockdown). Nous avons établi les profils de liaison des remodeleurs sur des éléments régulateurs (promoteurs, enhancers et sites CTCF) sur le génome, et montré que ces facteurs occupent toutes les catégories d’éléments régulateurs du génome. La corrélation entre les données ChIP-seq et les données transcriptomiques nous a permis d’analyser le rôle des remodeleurs dans les réseaux de transcription essentiels des cellules ES. Nous avons notamment démontré l’importance particulière de certains remodeleurs comme Brg1, Chd4, Ep400 et Smarcad1 dans la régulation de la transcription chez les cellules ES. / The characteristics of embryonic stem cells (ES cells) make them one of the best models to study the epigenetic regulation exerted by different actors in order to control the transcription of the mammalian genome. Members of the Snf2 family of ATP-dependent chromatin remodeling factors were shown to be of specific importance for ES cell self-renewal and during differentiation. These factors are believed to play essential roles in modifying the chromatin landscape through their capacity to position nucleosomes and determine their occupancy throughout the genome, making the chromatin more or less accessible to DNA binding factors.In this project, a genome-wide analysis of the function of a number of ATP-dependent chromatin remodelers (Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1, Ep400, ATRX, Smarca3, Smarca5, Smarcad1 and Alc1) in mouse embryonic stem (ES) cells was conducted. This was done using a double experimental strategy. First, a ChIP-seq (Chromatin Immunoprecipitation followed by deep sequencing) strategy was done on ES cells tagged for each factor in the goal of revealing the genomic binding profiles of the remodeling factors. Second, loss-of-function studies followed by transcriptome analysis in ES cells were performed in order to understand the functional role of remodelers. Data from both studies were correlated to acquire a better understanding of the role of remodelers in the transcriptional network of ES cells. Specific binding profiles of remodelers on promoters, enhancers and CTCF binding sites were revealed by our study. Transcriptomic data analysis of the deregulated genes upon remodeler factor knockdown, revealed the essential role of Chd4, Ep400, Smarcad1 and Brg1 in the control of transcription of ES cell genes. Altogether, our data highlight how the distinct chromatin remodeling factors cooperate to control the ES cell state.

Cells within multicellular organisms share the same genetic information, yet their shape and function can differ dramatically. This diversity of form and function is established by differential use of the genetic information. Early embryonic development describes the processes that lead to a fully differentiated embryo starting from a single fertilized cell - the zygote. Interestingly, in metazoan species this early development is governed by maternally provided factors (nutrients, RNA, protein), while the zygotic genome is transcriptionally inactive. Only at a specific developmental stage, the zygotic genome becomes transcriptionally active, and zygotic transcripts drive further embryonic development. This major change is called zygotic genome activation (ZGA). While major regulators of activation of early zygotic genes could be identified recently, the molecular mechanisms that contribute to robust global genome activation during embryonic development is not fully understood. In this study, I investigated whether the establishment of histone H3 lysine 4 trimethylation (H3K4me3) is involved in zebrafish zygotic transcription activation and early embryonic development. H3K4me3 is a chromatin modification that is implicated in transcription regulation. H3K4me3 has been shown to be enriched at Transcription Start Sites (TSS) of genes prior to their activation, and is postulated facilitate transcription activation of developmentally important genes. To interfere with H3K4me3 establishment, I generated histone methyltransferase mutants. I further inhibited H3K4me3 establishment by introduction of histones with lysine 4-to-methionine (K4-to-M) substitution, which act as dominant-negative inhibitors of H3K4me3 establishment. Upon H3K4me3 reduction, I studied the resulting effect on early transcription activation. I found that H3K4me3 is not involved in transcription activation during early zebrafish embryogenesis. Finally I analyzed possible cues in DNA sequence and chromatin environment that might favor early H3K4me3 establishment. These studies show that H3K4me3 is established during ZGA, yet it is not involved in transcription activation during early zebrafish development. Establishment of H3K4me3 might be a consequence of histone methyltransferase recruitment during a permissive chromatin state, and might be targeted to CpG-rich promoter elements that are enriched for the histone variant H2A.z.:Frontmatter II Acknowledgements VII Thesis Summary (English) IX Thesis Summary (German) X Table of Contents XIV List of Figures XVI List of Tables XVII List of Abbreviations XXIII 1 Introduction 1 1.1 Transcription regulation 2 1.1.1 Promoter elements - genetic information that guides transcription initiation 2 1.1.2 Enhancers - fine-tuning of transcription by distal DNA elements 3 1.1.3 CpG islands - DNA sequences that allow for epigenetic regulation 4 1.2 Chromatin 4 1.2.1 Histone variants 7 1.2.2 Posttranslational histone modifications 7 1.2.3 Histone Lysine methylation 8 1.2.4 H3K4me3 in embryonic development 10 1.3 Establishment and removal of H3K4me3 10 1.3.1 Set1 homologs - Set1a and Set1b 11 1.3.2 Trithorax homologs - Mll1 and Mll2 11 1.3.3 Homologs of Trithorax-related - Mll3 and Mll4 13 1.3.4 COMPASS complex proteins 13 1.3.5 H3K4me3 removal 14 1.4 Transcription activation in embryos 14 1.4.1 Zebrafish early embryonic development 15 1.4.2 H3K4me3 during early zebrafish development 17 1.5 Thesis aim 17 2 Materials and Methods 19 2.1 Materials 19 2.2 Methods 36 2.2.1 Zebrafish husbandry and care 36 2.2.2 Generation of zebrafish knock-out lines by TALEN mutagenesis 36 2.2.3 Generation of plasmids for mRNA production 38 2.2.4 Microinjection 39 2.2.5 Germline transplantation 39 2.2.6 Western Blot Assays 40 2.2.7 RNA extraction and quantification assays 41 2.2.8 Chromatin immunoprecipitation (ChIP) 43 2.3 Bioinformatics Analyses 46 2.3.1 Quality control, alignment and peak calling 46 2.3.2 Lambda normalization 46 2.3.3 Differential ChIP enrichment analysis 47 2.3.4 Data integration 47 2.3.5 Gene classification 48 3 Results I: H3K4me3 interference by Histone methyltransferase mutation 49 3.1 Generation and phenotypic description of histone methyl-transferase mutants 49 3.1.1 HMT TALEN mutagenesis workflow 49 3.1.2 Ash2l TALEN mutation does not result in a larval or adult phenotype 52 3.1.3 Mll2 mutation results in increased larval mortality, while adult fish are healthy and fertile 54 3.1.4 Mll1 mutation results in increased larval mortality and a severe adult phenotype 56 3.2 HMT mutations do not affect global H3K4me3 levels in early zebrafish embryos 60 3.3 Mll1 mutation results in local H3K4me3 reduction of a small subset of genes 62 3.4 Early embryonic transcription is not altered in mll1 maternal-zygotic mutants 67 3.5 Conclusion 70 4 Results II: H3K4me3 interference by introduction of HMT inhibitors 71 4.1 Establishing a Western Blot assay to monitor H3K4me3 reduction 71 4.2 Overexpression of H3K4-specific histone demethylases does not result in global H3K4me3 reduction 73 4.3 Global reduction of H3K4me3 could not be achieved by small-molecule inhibition of HMT activity 75 4.4 Overexpression of K4-specific methylation-defective H3 results in global H3K4me3 reduction 76 4.4.1 Overexpression of H3K4-to-E constructs does not affect global H3K4me3 establishment 76 4.4.2 H3K4-to-M constructs act as dominant-negative substrate for H3K4me3 establishment 77 4.5 H3K4me3 levels at gene promoters are reduced upon introduction of methylation-defective Histone H3 79 4.6 Early transcription activation is not altered upon K4M overexpression 88 4.7 Conclusion 92 5 Results III: Promoters rich in CpG and H2A.z gain H3K4me3 early 93 5.1 H3K4me3 levels increase over developmental time at all gene classes 93 5.2 H3K4me3 is gained at CpG-rich elements 98 5.3 H2A.z marks overlaps with H3K4me3 at promoters of non-transcribed genes 100 5.4 High CpG density and H2A.z enrichment are predictive for H3K4me3 establishment 101 5.5 Maternally provided genes are enriched for H2A.z and CpG content 103 5.6 Conclusion 104 6 Discussion 105 6.1 Neither Mll1 nor Mll2 are the main histone methyltransferase for H3K4me3 establishment in early zebrafish development 106 6.2 H3K4me3 reduction does not affect transcription initiation during genome activation 107 6.3 The timing of H3K4me3 establishment might be determined by a permissive chromatin state 109 6.4 H3K4me3 potentially gains importance during later developmental stages 111 6.5 CpG-content and H2A.z enrichment might be predictive for H3K4me3 establishment during genome activation 112 6.6 Conclusion 115 Appendix 117 Bibliography 139 Authorship Declaration 159 / Jede Zelle eines multizellulären Organismus enthält dieselbe Erbinformation, und doch können Form und Funktion von Zellen untereinander sehr unterschiedlich sein. Diese Diversität wird durch unterschiedliches Auslesen - Transkribieren - der Erbinformation erreicht. Embryogenese beschreibt den Prozess, der aus einer einzelnen Zelle - der Zygote - einen multizellulären Embryo entstehen lässt. Interessanterweise laufen frühe Stadien der Embryogenese ohne Transkription der embryonalen Erbinformation ab, sondern werden durch maternal bereitgestellte Faktoren ermöglicht. Erst nach einer spezies-spezifischen Entwicklungsphase wird das Erbgut der Zygote aktiv transkribiert und ermöglicht die weitere Embryonalentwicklung. Obwohl bereits wichtige Regulatoren dieser globalen Genomaktivierung identifiziert werden konnten, sind viele molekulare Mechanismen, die zur Aktivierung des zygotischen Genoms beitragen, bisher unbekannt. In der hier vorliegenden Doktorarbeit habe ich die Rolle von Histon H3 Lysin 4 Trimethylierung (H3K4me3) während der frühen Embryogenese des Zebrafischs untersucht. H3K4me3 ist eine Chromatinmodifikation, die mit aktiver Transkription in Verbindung gebracht wird. H3K4me3 ist an Transkriptions-Start-Stellen von aktiv ausgelesenen Genen angereichert und es wird vermutet, dass diese Modifikation das Binden von Transkriptionsfaktoren und der Transkriptionsmaschinerie erleichtert. Während meiner Arbeit habe ich durch Mutation verschiedener Histon-Methyltransferasen beziehungsweise die Überexpression eines dominant-negativen Histonsubstrats versucht, die Etablierung von H3K4me3 in frühen Entwicklungsstadien des Zebrafischs zu verhindern. Anschliessend habe untersucht, welchen Effekt H3K4me3-Reduktion auf Tranksriptionsaktivität entsprechender Gene hat. Allerdings konnte ich keinen Zusammenhang zwischen H3K4me3-Reduktion und Transkriptionsaktivität beobachten. Um herauszufinden, weshalb H3K4me3 dennoch während früher Embryonalstadien etabliert wird, habe ich nachfolgend untersucht, ob möglicherweise bestimmte DNASequenzen oder Chromatin-Modifikationen zur Etablierung von H3K4me3 wahrend der Embryogenese des Zebrafischs beitragen. Aus der hier vorliegenden Arbeit lässt sich schlussfolgern, dass H3K4me3 in Tranksriptionsaktivierung während früher Embryonalstadien des Zebrafischs nicht involviert ist. Möglicherweise wird H3K4me3 in diesen Stadien in einer permissiven Chromatinumgebung etabliert, bevorzugt an Promotoren mit starker H2A.z-Anreicherung und CpG-reichen DNA-Elementen.:Frontmatter II Acknowledgements VII Thesis Summary (English) IX Thesis Summary (German) X Table of Contents XIV List of Figures XVI List of Tables XVII List of Abbreviations XXIII 1 Introduction 1 1.1 Transcription regulation 2 1.1.1 Promoter elements - genetic information that guides transcription initiation 2 1.1.2 Enhancers - fine-tuning of transcription by distal DNA elements 3 1.1.3 CpG islands - DNA sequences that allow for epigenetic regulation 4 1.2 Chromatin 4 1.2.1 Histone variants 7 1.2.2 Posttranslational histone modifications 7 1.2.3 Histone Lysine methylation 8 1.2.4 H3K4me3 in embryonic development 10 1.3 Establishment and removal of H3K4me3 10 1.3.1 Set1 homologs - Set1a and Set1b 11 1.3.2 Trithorax homologs - Mll1 and Mll2 11 1.3.3 Homologs of Trithorax-related - Mll3 and Mll4 13 1.3.4 COMPASS complex proteins 13 1.3.5 H3K4me3 removal 14 1.4 Transcription activation in embryos 14 1.4.1 Zebrafish early embryonic development 15 1.4.2 H3K4me3 during early zebrafish development 17 1.5 Thesis aim 17 2 Materials and Methods 19 2.1 Materials 19 2.2 Methods 36 2.2.1 Zebrafish husbandry and care 36 2.2.2 Generation of zebrafish knock-out lines by TALEN mutagenesis 36 2.2.3 Generation of plasmids for mRNA production 38 2.2.4 Microinjection 39 2.2.5 Germline transplantation 39 2.2.6 Western Blot Assays 40 2.2.7 RNA extraction and quantification assays 41 2.2.8 Chromatin immunoprecipitation (ChIP) 43 2.3 Bioinformatics Analyses 46 2.3.1 Quality control, alignment and peak calling 46 2.3.2 Lambda normalization 46 2.3.3 Differential ChIP enrichment analysis 47 2.3.4 Data integration 47 2.3.5 Gene classification 48 3 Results I: H3K4me3 interference by Histone methyltransferase mutation 49 3.1 Generation and phenotypic description of histone methyl-transferase mutants 49 3.1.1 HMT TALEN mutagenesis workflow 49 3.1.2 Ash2l TALEN mutation does not result in a larval or adult phenotype 52 3.1.3 Mll2 mutation results in increased larval mortality, while adult fish are healthy and fertile 54 3.1.4 Mll1 mutation results in increased larval mortality and a severe adult phenotype 56 3.2 HMT mutations do not affect global H3K4me3 levels in early zebrafish embryos 60 3.3 Mll1 mutation results in local H3K4me3 reduction of a small subset of genes 62 3.4 Early embryonic transcription is not altered in mll1 maternal-zygotic mutants 67 3.5 Conclusion 70 4 Results II: H3K4me3 interference by introduction of HMT inhibitors 71 4.1 Establishing a Western Blot assay to monitor H3K4me3 reduction 71 4.2 Overexpression of H3K4-specific histone demethylases does not result in global H3K4me3 reduction 73 4.3 Global reduction of H3K4me3 could not be achieved by small-molecule inhibition of HMT activity 75 4.4 Overexpression of K4-specific methylation-defective H3 results in global H3K4me3 reduction 76 4.4.1 Overexpression of H3K4-to-E constructs does not affect global H3K4me3 establishment 76 4.4.2 H3K4-to-M constructs act as dominant-negative substrate for H3K4me3 establishment 77 4.5 H3K4me3 levels at gene promoters are reduced upon introduction of methylation-defective Histone H3 79 4.6 Early transcription activation is not altered upon K4M overexpression 88 4.7 Conclusion 92 5 Results III: Promoters rich in CpG and H2A.z gain H3K4me3 early 93 5.1 H3K4me3 levels increase over developmental time at all gene classes 93 5.2 H3K4me3 is gained at CpG-rich elements 98 5.3 H2A.z marks overlaps with H3K4me3 at promoters of non-transcribed genes 100 5.4 High CpG density and H2A.z enrichment are predictive for H3K4me3 establishment 101 5.5 Maternally provided genes are enriched for H2A.z and CpG content 103 5.6 Conclusion 104 6 Discussion 105 6.1 Neither Mll1 nor Mll2 are the main histone methyltransferase for H3K4me3 establishment in early zebrafish development 106 6.2 H3K4me3 reduction does not affect transcription initiation during genome activation 107 6.3 The timing of H3K4me3 establishment might be determined by a permissive chromatin state 109 6.4 H3K4me3 potentially gains importance during later developmental stages 111 6.5 CpG-content and H2A.z enrichment might be predictive for H3K4me3 establishment during genome activation 112 6.6 Conclusion 115 Appendix 117 Bibliography 139 Authorship Declaration 159

Chromatin structure is intricately involved in the mechanisms of eukaryotic gene regulation. In general, the compact nature of chromatin blocks DNA accessibility such that components of the transcriptional machinery are unable to access regulatory sequences and gene activation is repressed. These repressive effects can be overcome or augmented by the actions of chromatin remodeling enzymes. Numerous studies highlight two classes of these enzymes: those that covalently modify nucleosomal histones and those that utilize energy derived from ATP hydrolysis to destabilize the histone-DNA contacts within the nucleosome (13, 14, 92). Members of each of these groups of chromatin remodeling enzymes play pivotal roles in modulating chromatin structure and in facilitating or blocking the binding of transcription factors. Mutations in genes encoding these enzymes can result in transcriptional deregulation and improper protein expression. Therefore, the regulation of chromatin structure is critical for precise regulation of almost all aspects of gene expression. Consequently, enzymes regulating chromatin structure are important modulators of cellular processes such as cell viability, growth, and differentiation. There remain many uncharacterized members of the ATP-dependent class of remodeling enzymes; characterization of these proteins will further elucidate the cellular functions these enzymes control. Here, we focus primarily on the ATP-dependent remodeling complexes, specifically the chromodomain helicase DNA-binding (CHD) family. The CHD proteins are distinguished from other ATP-dependent complexes by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. These proteins also contain a SNF2-like ATPase motif and are further classified based on the presence or absence of additional domains. Genetic, biochemical, and structural studies demonstrate that CHD proteins are important regulators of transcription and play critical roles during developmental processes. Numerous CHD proteins have also been implicated in human disease. The first CHD family member, mChd1, was identified in 1993 in a search for DNA-binding proteins with an affinity for immunoglobin promoters. Since then, additional CHD genes have been identified based on sequence and structural homology to mChd1. Despite an increase in the number of studies relating to CHD proteins, the function of most remains unknown or poorly characterized. Using embryonic stem (ES) cells containing an insertional mutation in the murine Chd2 locus, we generated a Chd2-mutant mouse model to address the biological effects of Chd2 in development and disease. The targeted Chd2 allele resulted in a stable Chd2-βgeo fusion protein that contained the tandem chromodomains, the SNF2-like ATPase motif, but lacked the C-terminal portion of the DNA-binding domain. We demonstrated that the mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis as well as perinatal lethality. Similarly, heterozygous mice showed a decreased neonatal viability. Moreover, the surviving heterozygous mice showed a general growth delay during the neonatal period and increased susceptibility to non-neoplastic lesions affecting multiple organs, most notably the kidneys. We further examined the connection between Chd2 and kidney disease in this murine model. Our findings revealed that the kidney phenotype observed in Chd2 mutant mice led to the development of membranous glomerulopathy, proteinuria, and ultimately to impaired kidney function. Additionally, serum analysis revealed decreased hematocrit levels in the Chd2-mutant mice, suggesting that the membranous glomerulopathy observed in these mice is associated with anemia. Lastly, we investigated whether the type of anemia observed in the Chd2-mutant mice. Red blood cell (RBC) indices and morphological examination of the RBCs indicated that the anemia seen in the Chd2-mutant mice can be classified as normocytic and normochromic. Further analyses have been initiated to determine if the anemia is due to an intrinsic effect in erythropoiesis or a secondary consequence of the glomerular disease. In summary, our findings have contributed to our understanding of the putative chromatin remodeling enzyme Chd2. Although much remains to be studied, these findings demonstrate a role for Chd2 in mammalian development and have revealed a link between Chd2 and disease.

No description available.

Covalent histone modifications such as methylation, acetylation as well as differential incorporation of histone variants are shown to coincide with different chromatin compartments and mark active or repressed genes. Msc1 is one of the seven JmjC Domain Proteins (JDPs) in Fission Yeast. JDPs are known to function in chromatin and some act as histone demethylases. We found that Msc1 is a member of Swr1 Complex which is known to exchange histone H2A variant H2A.z in nucleosomes. We purified H2A.z as a member of Swr1 Complex and its interaction with Swr1 Complex depends on Swr1. We’ve shown that histone H4 Lysine 20 trimethylation (H4 K20 Me3) is lost in h2A.z and msc1 deletion strains and these strains are sensitive to UV. Deletion strain of h2A.z is sensitive to Camptothecin. Histones H3 and H4 are obtained in Msc1 and H2A.z purifications and we’ve shown that histone H4 from these purifications has low level of Lysine 16 acetylation (H4 K16 Ac). Deletion strains of h2A.z, swr1 and msc1 are shown to be sensitive to TSA, a histone deacetylase (HDAC) inhibitor suggesting that H2A.z cooperates with HDACs. TSA treatment of wild type cells cause an increase in H4 K16 Ac and a decrease in H4 K20 Me3. Gene expression profiles of h2A.z, swr1 and msc1 are significantly similar and upregulated genes in deletion strains localize at chromosome ends (a region of 160 kb for each end). The number of stress or meiotic inducible genes is increased in deletion strains suggesting that H2A.z has a role in regulation of inducible genes. We suggest that H2A.z, in cooperation with HDACs, functions in regulation of chromatin accessibility of inducible promoters.

Die Geschlechtsbestimmung bei Säugetieren erfolgt über gegensätzliche Netzwerke von ovariellen und testikulären Genen, die recht gut charakterisiert sind. Die epigenetischen Mechanismen, insbesondere diejenigen, die die 3D-Chromatinorganisation beeinflussen, sind jedoch größtenteils unbekannt. Ich habe die 3D-Chromatinlandschaft der Geschlechtsbestimmung in vivo untersucht, indem ich FACS-sortierte embryonale Mausgonadenpopulationen vor und nach der Geschlechtsbestimmung in beiden Geschlechtern analysierte. Dabei wurde eine begrenzte Variation in der dreidimensionalen Chromatindynamik beobachtet, insbesondere bei den Topologically Associating Domains (TADs). Konventionelle Hi-C-Analysemethoden sind hauptsächlich auf vordefinierte 3D-Strukturen ausgerichtet und könnten potenziell andere Veränderungen in der Chromatinorganisation übersehen, die für die Genregulation relevant sein könnten. Um diese Einschränkungen zu überwinden, wurde METALoci eingesetzt - ein innovatives Werkzeug, das Hi-C- und ChIP-seq-Daten integriert und räumliche Autokorrelationsanalyse nutzt, um dreidimensionale Enhancer-Hubs im gesamten Genom zu identifizieren. METALoci zeigte eine deutliche Umverdrahtung von Chromatininteraktionen während der Geschlechtsbestimmung, die die regulatorischen Landschaften von Hunderten von Genen beeinflusste. Darüber hinaus führte die Vorhersagekraft von METALoci in Kombination mit funktionalen Validierungen an transgenen Mäusen zur Identifizierung eines neuen Fgf9-regulatorischen Hubs. Die Deletion dieses Hubs führte zu teilweisem Geschlechtsumkehr von männlich zu weiblich, mit einer Hochregulierung ovarieller spezifischer Marker und der Einleitung der Meiose. So erweist sich die räumliche Autokorrelationsanalyse als eine effektive Strategie zur Identifizierung von regulatorischen Netzwerken, die mit biologischen Prozessen verbunden sind, und zur anschließenden Charakterisierung der funktionalen Rolle des dreidimensionalen Genoms. / Mammalian sex is determined by opposing networks of ovarian and testicular genes that are relatively well characterized. Yet, the epigenetic mechanisms governing sex determi- nation, in particular those involving 3D chromatin organization, remain largely unknown. This gap of knowledge constrains our understanding of a fundamental process for species reproduction and perpetuation. Here, I explored the 3D chromatin landscape of sex deter- mination in vivo, by profiling FACS-sorted embryonic mouse gonadal populations, prior and after sex determination, in both sexes. Using conventional Hi-C analysis tools, limited variation in the 3D chromatin dynamics was observed, especially at the level of Topolog- ically Associating Domains (TADs). This contrasts with the broad transcriptional differ- ences occurring during sex determination. Yet, conventional Hi-C analysis methodologies are largely focused on predefined 3D structures, potentially overlooking other types of changes in chromatin organization that might be relevant for gene regulation. To ad- dress these limitations, METALoci was applied- an innovative tool that integrates Hi-C and ChIP-seq data and relies on spatial auto-correlation analysis to identify 3D enhancer hubs distributed throughout the genome. METALoci uncovered a prominent rewiring of chromatin interactions during sex determination, affecting the regulatory landscapes of hundreds of genes. Furthermore, METALoci ’s predictive capacity, in combination with functional validations in transgenic mice led to the identification of a novel Fgf9 regulatory hub, which deletion resulted in partial male-to-female sex reversal with the upregulation of ovarian-specific markers and the initiation of meiosis. Thus, spatial auto-correlation anal- ysis proves to be an effective strategy to identify regulatory networks linked to biological processes and to subsequently characterize the functional role of the 3D genome.

Die Chromatinstruktur spielt eine bedeutende Rolle bei der Stadien- und Gewebs-spezifischen Genexpression. Der epigenetische Status von Chromatindomänen wird mit Hilfe einer Anzahl von Proteinen und Histonmodifikationen etabliert und aufrechterhalten. Das Ziel dieser Arbeit ist die Untersuchung der Bedeutung und Funktion des Chriz Protein Komplexes bei Drosophila, der spezifisch in dekondensierten Regionen von Interphase Chromosomen gebunden ist. Meine RNAi Experimente an embryonalen S2 Zellen zeigten, dass die Rekrutierung von Z4 und der Kinase Jil-1 an das Chromatin sowie dessen H3S10 Phosphorylierung während der Interphase von Chriz abhängt. Diese Ergebnisse lieferten einen starken Hinweis auf eine Ähnlichkeit bei der Bildung des Komplexes in polytänen Zellen und diploiden S2 Zellen. Ich führte eine vergleichende Analyse der Bindungsprofile von Proteinen des Chriz Komplex zwischen Speicheldrüsen im 3.Larvenstadium und S2 Zellen im chromosomalen Intervall 61C7-8 durch. Ich fand heraus, dass die Chriz Bindungsprofile im proximalen Teil der Domäne konserviert waren. Dagegen beobachtete ich eine veränderte Chriz Bindung im distalen Teil, die mit einem veränderten Transkriptionsprofil in dieser Region übereinstimmte. Chriz und Z4 RNAi Experimente in S2 Zellen führten zu einer Veränderung der Expression vieler Chriz- und Z4- bindender Gene. Mittels Co-Immunpräzipitation und Protein „Pull-down“ konnte ich die Bindung der Insulator Proteine BEAF-32 und CP190 im Chriz-Komplex nachweisen und die für die Protein-Protein Interaktion notwendigen Proteinabschnitte grob kartieren. Schließlich überprüfte ich die Möglichkeit einer Rekrutierung des Chriz Komplexes durch BEAF-32 durch Induktion von Punktmutationen in bekannten BEAF-32 Bindemotiven. Meine Ergebnisse wiesen eine Wechselwirkung zwischen Chriz und Insulator Proteinen nach und zeigten, dass der Chriz-Komplex eine wichtige Bedeutung bei der strukturellen Organisation von Chromatin Domänen besitzt. / Chromatin structure is important for the correct stage and tissue-specific expression of the genetic material. The epigenetic state of chromatin domains is established and maintained by a number of proteins and histone modifications. The aim of current thesis is to investigate the role and function of Chriz protein complex, specifically bound to decondensed regions of interphase chromosomes in Drosophila. Several proteins were known to compose Chriz complex - chromodomain protein Chriz, zinc finger protein Z4 and H3S10 kinase Jil-1. I performed RNAi experiments on S2 cells which demonstrated that recruitment of Z4 and Jil-1 kinase to chromatin and interphase H3S10 phosphorylation are dependent on the presence of Chriz. I accomplished the comparative analysis of binding profiles of Chriz complex components between 3rd instar larvae salivary glands and S2 cell culture within 61C7-8 chromosomal interval. I found that Chriz binding profiles between two tissues are conserved at proximal part, however variant Chriz binding in distal part of 61C7-8 domain coincides with differences in transcription profile in the same region. Publicly available genome-wide data shows a tendency of Chriz to bind near Transcription Start Sites (TSS) and promoter regions of active genes. Chriz and Z4 RNAi experiments, performed in S2 cells resulted in expression changes of many Chriz- and Z4-binding genes. Using co-immunoprecipitation and pull-down assays, I identified insulator proteins BEAF-32 and CP190 to be present in the Chriz complex and performed rough mapping of interacting domains. Using BEAF-32 RNAi and introduced point mutations to BEAF-32 binding motifs I examined the possibility for recruitment of Chriz complex by BEAF-32. The obtained results revealed the interplay between Chriz and insulator proteins and point to important architectural role of Chriz complex in organizing chromatin domains.

Der pRB/E2F-Signalweg ist ein zentraler Regulator der Proliferationskontrolle in Säugerzellen, der in fast allen auftretenden Tumoren dereguliert ist. Durch unterschiedliche Mutationen in Komponenten dieses Signalwegs kommt es letzten Endes zu einer erhöhten Aktivität der E2F-Transkriptionsfaktoren und somit zu einer verstärkten Transkription von E2F-Zielgenen in diesen Tumoren. Um die molekularen Mechanismen der Rolle von E2F3 in der Zellzykluskontrolle und der Tumorigenese besser zu verstehen, wurden in dieser Arbeit per GST-Pulldown mit anschließender Massenspektrometrie neue potenzielle Interaktions-partner von E2F3 identifiziert. Ein identifizierter Interaktionspartner war die SNF2-ähnliche Helikase HELLS. HELLS interagiert in vitro und in vivo spezifisch mit der Marked Box-Domäne von E2F3, aber nicht mit anderen untersuchten E2F-Transkriptionsfaktoren, wie durch GST-Interaktionsstudien und Ko-Immunpräzipi-tationsexperimente demonstriert werden konnte. Durch Chromatin-Immunpräzipitation konnte zusätzlich gezeigt werden, dass E2F3 für die Rekrutierung von HELLS an E2F-regulierte Promotoren wie z. B. CDC6 oder p107 verantwortlich ist. Die shRNA-vermittelte Depletion von HELLS führte zu einer stark verminderten Induktion von allen untersuchten E2F-Zielgenen nach Serumstimulation und einem verspäteten Eintritt in die S-Phase der HELLS-depletierten Zellen, was zeigt, dass HELLS essenziell für die Induktion von E2F-Zielgenen ist. Bei der immunhistochemischen Untersuchung der E2F3- und HELLS-Expression in humanen Prostatakarzinomen zeigte sich, dass sowohl E2F3 als auch HELLS in späten aggressiven Stadien dieser Tumore sehr stark exprimiert sind, jedoch nur sehr schwach in den weniger aggressiven Tumoren. Diese Versuche zeigen, dass es sich bei HELLS um einen neuen Bestandteil des pRB/E2F-Signalwegs handelt, der eventuell in der Entstehung gewisser Tumorarten eine Rolle spielt und somit ein neues potenzielles Ziel für neuartige Krebstherapien darstellt. / The pRB/E2F pathway is a key regulator of proliferation in mammalian cells and is commonly mutated in human tumors. These mutations in the components of the pRB/E2F pathway lead to deregulated activity of the E2F transcription factors resulting in increased expression of E2F target genes. To further understand the molecular mechanisms of E2F3 in cell cycle control and its role in tumorigenesis new interaction partners for E2F3 were identified in the course of this thesis with the help of a GST-Pulldown approach coupled to mass spectrometric analysis. One of the identified interaction partners was the SNF2-like helicase HELLS. With the help of GST-interaction studies and Co-Immunoprecipitation assays it could be demonstrated that HELLS interacts specifically with E2F3 via its Marked Box domain but does not bind to the other investigated E2F transcription factors. HELLS could be detected at E2F target genes like p107 and CDC6 in vivo with the help of Chromatin-Immunoprecipitation assays. Furthermore, the forced recruitment of E2F3 to E2F target genes led to an enhanced binding of HELLS to these promotors suggesting that HELLS is recruited to E2F target genes via protein-protein interaction with E2F3. The shRNA-mediated depletion of HELLS led to a strongly reduced induction of E2F target genes and a delay in S-phase entry, showing that HELLS is essential for the induction of E2F target genes. During the immunohistochemical analysis of human prostate cancer specimens it became evident that both E2F3 and HELLS are strongly expressed in the more aggressive late stages but only weakly expressed in the early stages of this tumor type. These findings demonstrate that HELLS is a new component of the E2F/pRB pathway which might play a role in the development of certain tumors and might represent a new target for novel cancer therapies.

Die Beschreibung genregulatorischer Ereignisse ist entscheidend um Zelldifferenzierung und -entwicklung zu verstehen. Dynamische Vernderungen der Chromatinstruktur, Histonmodifikationen und das Binden von Transkriptionsfaktoren an Enhancer und Promotoren, koennen mit Hilfe von genomweiten Hochdurchsatz-Sequenziertechniken wie ChIP-Seq, DNase-Seq, ATACSeqund RNA-Seq untersucht werden. In dieser Arbeit entwickele ich mehrere probabilistische Modelle fuer die Analyse von genomweiten Sequenzierungsdaten. Diese umfassen 1. einen Peak-Finder fuer ChIP-/DNase-/ATAC-Seq-Daten, der sich Replikate zunutze macht und praezise Peak-Weiten berechnet, 2. eine Pipeline um das Genom in hoher Aufloesung in eindeutige Klassen von Kombinationen von Histonmodifikationen zu segmentieren, 3. ein Bayes-Netzwerk-Modell welches multiple zeitlich aufgelste Histonmodifikations-ChIP-seq-Daten kombinatorisch clustert Klassen von regulatorischen Elementen zu identifizieren. Mit Hilfe dieser Modelle untersuchen wir die Promotorumgeben und zeigen einen Zusammenhang zwischen Chromatinstruktur und Promotordirektionalitaet. Darueber hinaus verwenden wir ein Modell zur direkten Reprogrammierung von Stammzellen in Motorneuronen durch die gezielte Expression von Transkriptionsfaktoren und analysieren die dadurch induzierten zeitlichen Vernderungen der Chromatinstruktur und Transkriptionsfaktorbindedynamik. Wir beobachten, dass Promotoren verschiedenen Chromatin-Dynamiken zur Aktivierung und Repression folgen, die mit den Chromatin-Dynamiken von Enhancer-Elementen korrelieren. Enhancer hingegen werden durch kooperatives Verhalten direkt induzierter Transkriptionsfaktoren und anderen Faktoren, die in den Stammzellen zu Beginn vorhanden waren oder im Verlaufe der Differenzierung aktiviert wurden, kontrolliert. Diese Arbeit zeigt wie wichtig Chromatin-Dynamik und ihre Beziehung zur Logik von Transkriptionsfaktoren ist, um die Veraenderungen der Genexpression zu verstehen. / Delineating transcription regulatory events is crucial to understand cell differentiation and development. Dynamic changes of chromatin structure, histone modifications and transcription factor binding to enhancers and promotors can be investigated with the aid of genome-wide high-throughput sequencing technologies such as ChIP-Seq, DNase-Seq, ATAC Seq and RNA Seq. In this thesis, I develop several probabilistic models for the analysis of genome-wide sequencing data. These include: 1. a peak finder for ChIP-Seq, DNase-Seq and ATAC Seq data, which exploits biological replicates and accurately demarcates peak widths, 2. a pipeline for high-resolution genome segmentation into unique classes of combinations of histone modifications and 3. a Bayesian network model that can co-cluster multiple time-course histone modification ChIP-Seq data sets into distinct classes of regulatory elements. With the aid of these models we investigate the promoter chromatin environment and show a link between chromatin state and transcription initiation directionality. In addition, we use a system for direct reprogramming of stem cells in motor neurons by the targeted expression of transcription factors to analyse changes in chromatin state and transcription factor dynamics during differentiation. We observe that promoters follow different chromatin dynamics for activation and repression that correlate with the chromatin dynamics of enhancer elements. Enhancers are controlled by cooperative behavior of directly induced transcription factors and other factors present in the stem cells initially, or activated in the course of differentiation. Overall, this work demonstrates the importance of understanding chromatin dynamics and their relationship to transcription factors logic in order to better explain changes in gene expression.

The chromatin structure, consisting of DNA and histones, changes dynamically during the cell cycle and cell differentiation. DNA can only be transcribed and replicated when it is packaged loosely, whereas tight packaging allows for more efficient storage. Chromatin remodelling is therefore one of the tools of gene expression control. The chromatin remodelling factors recognise chromatin with varying specificity and have an effect on the interaction between DNA and the histones. One of these factors is the Smarca5 protein. This study investigates the role of Smarca5; its goal is to create a mouse model with the ability to trigger Smarca5 overproduction in specific tissues. This model will be used to study the effect of a high, unregulated dose of Smarca5 on the physiological function of the protein. Previous studies have shown that non-physiological expression of a chromatin-remodelling factor can lead to malignant transformation. Our model can help to understand this process. Another goal of this study is to investigate some phenotype aspects of the mouse model with conditional deletion of Smarca5 in T and B cells, in particular the effects of this deletion on progenitor cell differentiation. Our results show that Smarca5 has an important role in lymphocyte development, and we have observed that...