SETD2/HYPB (SET domain-containing protein 2 / Huntingtin yeast partner B) is the predominant lysine methyltransferase in mammals that mediates histone H3 lysine-36 (H3K36) trimethylation, which is associated with transcription elongation and RNA splicing. SETD2 is further implicated in p53 function, vascular development, cancer progression and, through Huntingtin-interaction, Huntington's disease. Although different transcripts and putative protein isoforms have been detected previously, their identity, function and significance have not been rigorously investigated. This thesis aims to identify and characterise endogenous transcripts and protein isoforms of SETD2 in mouse fibroblasts. Affnity-purified N- and C-terminal antibodies specifically detected the ≈ 290 kDa methyltransferase (p290^SETD2), verified by RNAi, in addition to N terminal-specific ≈ 120 kDa protein, and C terminal-specific forms at ≈ 140 and ≈ 66 kDa (p66), which all appeared too stable to deplete by transient siRNA transfection. Conserved in human and mouse cells, immunodetection of p66 exhibited unusual requirement for denaturation with urea at 95°C. Subcellular fractionation revealed distinct extraction properties of putative isoforms and facilitated partial purification of p66 for proteomic analysis. Co-fractionation and co migration by two-dimensional gel electrophoresis of p66 detected by two independent C terminal antibodies suggested it represents a novel C terminal-specific isoform. Reverse transcription−PCR and DNA-sequencing demonstrated the existence of multiple, alternatively-spliced Setd2 transcripts that plausibly generate truncated proteins. A transcript variant containing a novel complete open-reading-frame, consistent for p66 generation, was identified. Its ectopic expression in mouse fibroblasts produced a distinct SETD2 isoform, whose physical and extraction characteristics were studied in comparison with endogenous immunoforms. In summary, this thesis demonstrates that multiple alternatively-spliced transcripts arise from the Setd2 gene, consistent with immunodetection of several C- and N-terminal-specific putative SETD2 isoforms, additional to the H3K36 methyltransferase. Verification of these isoforms by independent methods would have implications for proposed interactions and function of SETD2 in transcription, epigenetics, cancer development and Huntington’s disease.

Le remodelage de la chromatine et la modification des histones jouent des rôles très importants dans l’établissement et la reprogrammation de l’état de l’expression génique. Il reste largement inconnu concernant les mécanismes de la régulation de ces processus chromatiniens dans le contrôle de l’expression génique impliquée dans le développement de la plante et son adaptation à l’environnement. Mon sujet de thèse se focalise sur l’analyse fonctionnelle d’un facteur de remodelage de la chromatine de type Chromodomain/Hélicase/DNA-binding 1 (CHD1) d’Arabidopsis, appelé CHR5 et une histone démethylase qui est spécifiquement impliquée dans la démethylation de l’histone H3 lysine 4 (H3K4), appelée JMJ15. Dans la première partie de cette étude, nous avons montré que le gène CHR5 est activé au cours de l’embryogénèse et que son expression se maintient élevé dans les tissues/organes en développement. L’analyse de mutants révèle que la perte de fonction de ce gène fait réprimer l’expression de gènes régulateurs de la maturation de l’embryon tels que LEC1, ABI3 et FUS3 pendant le développement des graines, et fait baisser l’accumulation des protéines de réserve. L’analyse de double mutants a permis de démontrer une fonction antagoniste entre CHR5 et PKL, une protéine du groupe « CHD3 », dans l’activité du promoteur de gènes régulateurs du développement de l’embryon et l’accumulation de réserve de graine. Nous avons montré que la protéine CHR5 s’associe directement avec les promoteurs d’ABI3 et FUS3 et que la mutation du gène CHR5 conduit à l’augmentation de présence de nucléosome dans la région du départ de transcription. Ces résultats suggèrent que CHR5 est impliquée dans le positionnement de nucléosome pour stimuler l’expression de gènes de la maturation de l’embryon, ce qui est contrebalancé par l’action de PKL au cours du développement de l’embryon. La deuxième partie de cette étude a permis de montrer que l’expression du gène de l’histone démethylase JMJ15 manifeste une forte spécificité tissulaire. L’analyse de mutants du gène a permis de l’identification de 2 allèles de gain de fonction (avec surexpression du gène), et un allèle de perte de fonction. La surexpression du gène réduit la croissance d’hypocotyle et de tige de la plante avec accumulation de lignine dans la tige, mais le perte de fonction du gène ne produise pas de phénotype apparent. Par ailleurs, la surexpression du gène renforce la tolérance de la plante au stress salin, alors la perte de fonction du gène rend la plante plus sensible. L’analyse du transcriptome a révélé beaucoup plus de gènes réprimés qu’activés par la surexpression du gène JMJ15. Ces gènes réprimés sont préférentiellement marqué la H3K4me2 ou H3K4me3, parmi lesquels beaucoup codent de facteurs de transcription. Ces données suggèrent que l’induction de JMJ15 pourrait réguler le programme de l’expression génique qui coordonne la restriction de la croissance de la plante et la tolérance au stress. Ces travaux de thèse a permis ‘identifier quelques nouveaux éléments dans la compréhension de la fonction de régulateurs chromatiniens dans l’expression génique de la plante. / Chromatin remodeling and histone modification play important roles in the establishment and dynamic regulation of gene expression states. However, little is known regarding to the regulatory mechanism of chromatin modification and remodeling that control gene expression involved in plant development and responses to environmental cues. My thesis work concerns functional analysis of an Arabidopsis Chromodomain/Helicase/DNA-binding 1 (CHD1) type chromatin remodeling gene known as CHR5 and a histone demethylase gene that specifically removes methyl groups from methylated histone H3 lysine 4 (H3K4me), called JMJ15 in regulating chromatin structure or in resetting chromatin modifications that control the expression of plant developmental and stress responsive genes. In the first part of the study we found that CHR5 expression is activated during embryogenesis and remained to be expressed in developing organs/tissues. Analysis of mutants revealed that loss-of-function of the genes led to decreased expression of key embryo maturation genes LEC1, ABI3 and FUS3 in developing seeds and reduced seed storage protein accumulation. Analysis of double mutants revealed an antagonistic function between CHR5 and PKL, a CHD3 gene, in embryo gene promoter activity and seed storage protein accumulation. CHR5 was directly associated with the promoters of ABI3 and FUS3 and chr5 mutations led to increased nucleosome occupancy near the transcriptional start site. The results suggest that CHR5 is involved in nucleosome occupancy to regulate embryo identity genes expression, which is counterbalanced by PKL during embryo development. The second part of this study showed that expression of JMJ15 was restricted to a few tissues during vegetative growth. The jmj15 gain-of-function mutations reduced the length of seedling hypocotyls and inflorescence stems with higher accumulation of lignin in the stem, while the loss-of-function mutants did not show any visible phenotype. The gain-of-function mutants enhanced salt tolerance, whereas the loss-of-function mutants were more sensitive to salt. Transcriptomic analysis revealed a much higher number of genes down-regulated in JMJ15 over-expression plants, which are highly enriched for H3K4me3 and H3K4me2. Among the down-regulated genes, many encode transcription regulators of stress responsive genes. The data suggest that increased JMJ15 levels may regulate the gene expression program that may coordinate plant growth restrains and enhances stress tolerance. Taken together, my thesis work brought a few new elements to the current understanding of chromatin regulators function in plant gene expression.

L'intégrase (IN) du VIH-1 est une enzyme qui catalyse l'intégration du génome du virus dans celui de la cellule infectée. Cette étape d'intégration est cruciale pour le virus pour qu'il puisse se répliquer de manière efficace, l'intégration est donc une cible de choix dans les thérapies antivirales. Comprendre les mécanismes qui participent à l'intégration est donc nécessaire afin de développer des solutions efficaces pour contrecarrer le virus.L’intégration rétrovirale est catalysée par une structure oligomérique d’IN et d’ADN viral bien particulière appelée intasome. Les intasomes rétroviraux catalysent l’intégration préférentiellement sur des nucléosomes, composés d’ADN enroulé de protéines histones, plutôt que sur de l’ADN nu. Ceci est en parti du aux contraintes physiques imposés par la structure de l’intasome, mais également grâce à des facteurs de ciblage cellulaires qui vont interagir avec à la fois l’intasome et des composants du nucléosome.Dans ce projet de thèse, nous avons pu mettre en évidence une nouvelle interaction hôte-pathogène entre l’IN du VIH-1 et la queue d’histone H4 (une des protéines constituant le nucléosome). Ce projet s’est ainsi focalisé autour de cette interaction et a permis de :• Démontrer l’importance de l’interaction entre l’IN du VIH-1 et la queue d’histone H4 lors du cycle viral et plus précisément pour l’étape d’intégration, validant ainsi cette interaction comme une nouvelle interaction hôte-pathogène.• D’identifier que la queue d’histone H4 est un partenaire essentiel de l’intasome du VIH-1 pour qu’il puisse s’ancrer sur le nucléosome.• Développer une nouvelle stratégie antivirale visant à bloquer cette interaction dans les cellules infectées grâce à des composés chimiques. / HIV-1 integrase (IN) catalyzes the insertion of the viral genome into the host cell chromatin. This step is crucial for the virus for its efficient replication, integration is thus of interest to target for antiviral strategies. Understanding the mechanisms involved in integration is important in order to develop efficient tools to fight the virus.Retroviral integration is catalyzed by the intasome, an oligomer of IN and viral DNA. Intasomes integrate onto nucleosomes, composed of DNA wrapped around histone proteins, over naked DNA.In this thesis project, we have identified a new host-pathogen interaction between HIV-1 IN and the H4 histone tail. The topic of the project was then focus on this interaction and has highlighted:• The importance of the HIV-1 IN – H4 histone tail interaction for the viral cycle, especially onto the integration step, validating a new host-pathogen interaction.• The identification of the H4 histone tail as an essential partner for HIV-1 intasome for its anchoring onto nucleosomes.• The development of a novel antiviral strategy aiming to block this interaction in infected cells using chemical compounds

<p>Genomic imprinting is a phenomenon through which a subset of genes are epigenetically marked during gemtogenisis. This mark is maintained in the soma to often manifest parent of origin-specific monoalleleic expresson patterns. Genetics evidence suggests that gene expression patterns in mprinted genes, which are frequently organised in clusters, are regulated by the imprinting control regions (ICR). This thesis is mainly focused on the mechanisms through which the ICRs control the imprinting in the cluster, containing the <i>Kcnq1, Igf2</i> and <i>H19</i> genes, located at the distal end of mouse chromosome 7.</p><p>The <i>H19</i> ICR, located in the 5' flank of the <i>H19</i> gene represses paternal <i>H19</i> and maternal <i>Igf2</i> expression, respectively, but has no effect on <i>Kcnq1</i> expression, which is controlled by another ICR located at the intron 10 of the <i>Kcnq1</i> gene. This thesis demonstrates that the maternal <i>H19</i> ICR allele contains several DNase I hypersensitive sites, which map to target sites for the chromatin insulator protein CTCF at the linker regions between the positioned nucleosomes. The thesis demonstrates that the <i>H19</i> ICR acts as a unidirectional insulator and that this property invovles three nucleosome positioning sites facilitating interaction between the <i>H19</i> ICR and CTCF. The <i>Kcnq1</i> ICR function is much more complex, since it horbours both lineage-specific silencing functions and a methylation sensitive unidirectional chromatin insulator function. Importantly, the thesis demonstrates that the <i>Kcnq1</i> ICR spreads DNA methylation into flanking region only when it is itself unmethylated. Both the methylation spreading and silencing functions map to the same regions.</p><p>In conclusion, the thesis has unraveled and unrivalled complexity of the epigenetic control and function of short strtches of sequences. The epigenetic status of these cis elements conspires to control long-range silencing and insulation. The manner these imprinting control regions can cause havoc in expresson domains in human diseases is hence emerging.</p>

<p>Chromatin structure plays an important role in gene regulation, especially in differentiating the diverse cell types in humans. In this dissertation, we analyze the nucleosome positioning and open chromatin profiles genome-wide and investigate the relationship with transcription initiation, the activity of regulatory elements, and expression levels. We mainly focus on the results of DNase-seq experiments, but also employ annotations from MNase-seq, FAIRE-seq, ChIP-seq, CAGE, and RNA microarrays. Our methods are based on computational approaches including managing large data sets, statistical analysis, and machine learning. We find that different transcription initiation patterns lead to distinct chromatin structures, suggesting diverse regulatory strategies. Moreover, we present a tool for comparing genome-wide annotation tracks and evaluate DNase-seq against a unique assay for detecting open chromatin. We also demonstrate how DNase-seq can be used to successfully predict rotationally stable nucleosomes that are conserved across cell types. We conclude that DNase-seq can be used to study genome-wide chromatin structure in an effort to better understand how it regulates gene expression.</p> / Dissertation

Nearly one half of the human genome is composed of transposable elements (TEs). Once dismissed as 'selfish' or 'junk' DNA, TEs have also been implicated in a numerous functions that serve the needs of their host genome. I have evaluated the role of TEs in mediating the epigenetic mechanisms that serve to regulate human gene expression. These findings can be broadly divided into two major mechanisms by which TEs affect human gene expression; by modulating nucleosome binding in the promoter regions and by recruiting epigenetic histone modifications that enable them to serve as promoters and enhancers. Thus. the studies encompassed in this thesis elucidate the contributions of TEs in epigenetically regulating human gene expression on a global as well as local scale.

Transcriptional regulation is a complicated process controlled by numerous factors such as transcription factors (TFs), chromatin remodeling enzymes, nucleosomes, post-transcriptional machineries, and cis-acting DNA sequence. I explored the complex transcriptional regulation in eukaryotes through three distinct studies to comprehensively understand the functional genomics at various steps. Although a variety of high throughput approaches have been developed to understand this complex system on a genome wide scale with high resolution, a lack of accurate and comprehensive annotation transcription start sites (TSS) and polyadenylation sites (PAS) has hindered precise analyses even in Saccharomyces cerevisiae, one of the simplest eukaryotes. We developed Simultaneous Mapping Of RNA Ends by sequencing (SMORE-seq) and identified the strongest TSS and PAS of over 90% of yeast genes with single nucleotide resolution. Owing to the high accuracy of TSS identified by SMORE-seq, we detected possibly mis-annotated 150 genes that have a TSS downstream of the annotated start codon. Furthermore, SMORE-seq showed that 5’-capped non-coding RNAs were highly transcribed divergently from TATA-less promoters in wild-type cells under normal conditions. Mapping of DNA-protein interactions is essential to understanding the role of TFs in transcriptional regulation. ChIP-seq is the most widely used method for this purpose. However, careful attention has not been given to technical bias reflected in final target calling due to many experimental steps of ChIP-seq including fixation and shearing of chromatin, immunoprecipitation, sequencing library construction, and computational analysis. While analyzing large-scale ChIP-seq data, we observed that unrelated proteins appeared to bind to the gene bodies of highly transcribed genes across datasets. Control experiments including input, IgG ChIP in untagged cells, and the Golgi factor Mnn10 ChIP also showed the strong binding at the same loci, indicating that the signals were obviously derived from bias that is devoid of biological meaning. In addition, the appearance of nucleosomal periodicity in ChIP-seq data for proteins localizing to gene bodies is another bias that can be mistaken for false interactions with nucleosomes. We alleviated these biases by correcting data with proper negative controls, but the biases could not be completely removed. Therefore, caution is warranted in interpreting the results from ChIP-seq. Nucleosome positioning is another critical mechanism of transcriptional regulation. Global mapping of nucleosome occupancy in S. cerevisiae strains deleted for chromatin remodeling complexes has elucidated the role of these complexes on a genome wide scale. In this study, loss of chromodomain helicase DNA binding protein 1 (Chd1) resulted in severe disorganization of nucleosome positioning. Despite the difficulties of performing ChIP-seq for chromatin remodeling complexes due to their transient and dynamic localization on chromatin, we successfully mapped the genome-wide occupancy of Chd1 and quantitatively showed that Chd1 co-localizes with early transcription elongation factors, but not late transcription elongation factors. Interestingly, Chd1 occupancy was independent of the methylation levels at H3K36, indicating the necessity of a new working model describing Chd1 localization.

Eukaryotic genomes are densely compacted into chromatin, so that they can be contained in the nucleus. Despite the tight packaging genes need to be accessible for normal metabolic activities to occur, such as transcription, repair and replication. These processes are regulated by a vast number of proteins but also by the level of compaction of chromatin. The translocation of motor proteins along DNA produces torsional stress which in turn alters chromatin compaction both upstream and downstream. Few single-molecule studies have investigated the behaviour of nucleosomes when subjected to torsion. The inability to measure the applied torque though represented a major limitation to those reports. The implementation of the rotor bead assay, which allows to directly measure the torque applied in magnetic tweezers experiments, has been hindered by a difficult sample preparation procedure. In order to overcome this limitation an efficient protocol for the insertion of chemical or structural modifications in long DNA substrates was developed. This was then further expanded to allow the introduction of labels in multiple loci and/or both strands and has been used successfully in a number of studies. Furthermore this is the first report of tensile experiments performed on nucleosomes with a histone variant. H2AvD nucleosomes were studied due to the interest in the biological role of H2A.Z-family proteins. Interestingly, the variant nucleosomes appear to bind less DNA and to be evicted from the DNA at lower forces than those observed for canonical nucleosomes. These findings show an important role for the H2A-H2B dimers in the mechanical stability of nucleosomes. Furthermore these results are in agreement with recently proposed models of a dynamic nucleosome, in contrast to the long-standing view of nucleosomes as static structures.

The ATP-dependent chromatin remodeling complex SWR1 exchanges a variant histone H2A.Z-H2B dimer for a canonical H2A-H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 Mega-Dalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single hetero-hexameric Rvb1/2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multi-subunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvb1/2 ring and a distinct substrate-handling mode by SWR1, thereby providing the first structural framework for understanding the complex dimer-exchange reaction.

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