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Mécanismes de régulation épigénétique chez l'insecte holocentrique ravageur de culture Spodoptera frugiperd, Lépidoptera, Noctuidae / Epigenetic regulation mecanisms in holocentric pest crop Spdoptera frugiperda, Lepidoptera, NoctuidaeNhim, Sandra 26 November 2018 (has links)
Chez les eucaryotes, l’ADN est empaqueté dans des complexes protéiques d’histones nommés nucléosomes qui assurent sa conformation. Cet arrangement est hétérogène à travers le génome et peut être dynamiquement modifié. La régulation de l’architecture chromatinienne joue un rôle essentiel dans la stabilité des génomes ainsi que la dynamique transcriptionnelle. Certaines régions qualifiées d’ ‘’heterochromatine constitutive’’ sont toutefois connues pour être maintenues à l’état condensé. Régionalisées aux extrémités et centres des chromosomes, l’hétérochromatine constitutive participe des fonctions télomériques et centromériques.Spodoptera frugiperda (S.fru, Lépidoptère, Noctuelle) est un ravageur de culture endémique du continent américain, récemment invasif dans le continent africain. Comme tous les Lépidoptères, S.fru est une espèce holocentrique dont le centromère est réparti le long des chromosomes et non restreint en un point unique. Cette disposition interroge sur l’établissement, la distribution ainsi que la fonction conservée de l’HC puisque cette dernière est principalement décrite pour être majoritairement localisée dans de larges régions péricentriques. Comprendre l’architecture chromatinienne chez S.fru peut avoir un intérêt en lutte biologique mais également permettre d’approfondir les connaissances en épigénétique chez un organisme non-modèle.Dans le cadre de la thèse, nous nous sommes demandés si la diméthylation de la lysine 9 de l’histone 3 (H3K9me2), marqueur de l’hétérochromatine constitutive, possédait un rôle conservé chez S.fru. Pour ce faire, nous avons comparé des données de ChIP-seq d’H3K9me2 sur cellules et larves entières après avoir annoté les gènes et l’ensemble des éléments répétés du génome, susceptibles d’être enrichis par cette marque. Parallèlement, des échantillons d’ARN-seq ont été étudiés afin de questionner le statut répressif de l’hétérochromatine constitutive. Nos résultats suggèrent un invariable maintien d’H3K9me2 dans les régions (sub)télomériques transcriptionnellement inactives ainsi qu’une forte association aux locus répétés d'ADN ribosomal (rDNA). Ces séquences ne constituent toutefois qu’une minorité des régions enrichies, le reste étant retrouvé dans des séquences répétées ainsi que dans le corps des gènes, indifféremment de leur état transcriptionnel. La persistante association d’H3K9me2 aux télomères et rDNA présagerait d’un maintien de la marque à proximité des centromères dont nous proposons un modèle d’établissement.La disposition de l’hétérochromatine constitutive questionne celle des régions euchromatiniennes, pauvres en nucléosomes, transcriptionnellement active et dynamiquement modifiées au cours du développement, du cycle cellulaire et des conditions environnementales. Afin de tester l’antagonisme de ces conformations, nous avons respectivement étudié la répartition des zones ouvertes et fermées du génome de la larve au stade L4 par approches de FAIRE-seq et de MAINE-seq. Ces structures ont été décrites dans la littérature pour être enrichies par de spécifiques modifications d’histones. Ainsi nous avons mis au point le protocole de native ChIP-seq d’H3K4me3 (marque active) et H3K9me2, H3K9me3, H3K27me3 (marques répressives). L’analyse en cours de l’ensemble de ces données de séquençages permettra d’avoir une vue intégrée de l’architecture chromatinienne au stade ravageur. / In eukaryotes, DNA is arranged in histones proteins complexes called nucleosomes that shape its conformation. This arrangement is heterogeneous across genomes and can be dynamically modified. Regulation of chromatin architecture plays an essential role in genome stability and transcription dynamics. Some regions named ‘’constitutive heterochromatin’’ are nonetheless known to remain highly condensed, regardless of conditions. Regionalized at extremities and chromosomes centers, constitutive heterochromatin contributes to telomeric and centromeric functions.Spodoptera frugiperda (S.fru, Lepidoptera, Noctuidae) is major crop pest in the Americas that recently invaded Africa. Like all Lepidopteran, S.fru is holocentric which means that its centromere is spread along chromosome and not restricted to a uniq point. This disposition question about establishment, distribution but also conserved function of constitutive heterochromatin since its usually and mainly localized in large pericentric regions.Deciphering chromatinian architecture in S.fru can be of interest in biological control but also allow to deepen epigenetic knowledge in a non-model organism.During my phD, we questionned the role of histone 3 lysine 9 demethylated (H3K9me2) in S.fru, a histone modification known in other yet described organisms to be a constitutive constitutive heterochromatinian hallmark.We compared H3K9me2 ChIP-seq data on cells and larvae after overall genomic functional annotation, potentially enriched for this mark. In parallel, RNA-seq samples were analyzed to question the putative repressive status of constitutive heterochromatin.Our results suggest an invariant retention of H3K9me2 in (sub)telomeric regions transcriptionally inactive but also a strong association of this mark in repeated ribosomal DNA locus (rDNA).These sequences constitutes nonetheless a minority of enriched regions since most of them regionalize in repeated sequences like transposons and tandem array but also gene bodies, independently of their transcriptional states.Persistent H3K9me2 association to telomeres and rDNA could predict of the conserved expression of this mark near centromeres. Based on literature and bioinformatics analysis, we proposed a model for S.fru holocentromeres.Constitutive heterochromatin questions euchromatin arrangement, described to be nucleosome poor, transcriptionally active and dynamically modified across development, cell cycle and environmental conditions. In order to test these structural antagonisms, we respectively studied open and closed genome conformations by FAIRE-seq and MAINE in larvae. These structures are reported to be associated to specific histones marks. We developed a native ChIP-seq protocol on H3K4me3 (active mark) and H3K9me2, H3K9me3, H3K27me3 (repressives marks). Overall analysis of these NGS data would help to picture an integrative view of chromatin architecture during larval pest stage.
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DISTINCT GENOME WIDE FUNCTIONS OF CHROMATIN REMODELERS IN NUCLEOSOME ORGANIZATION AND TRANSCRIPTION REGULATIONHailu, Solomon Ghebremeskel 01 December 2017 (has links)
AN ABSTRACT OF THE DISSERTATION OF SOLOMON G. HAILU, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry, presented on August 22, 2017, at Southern Illinois University, School of Medicine. TITLE: DISTINCT GENOME WIDE FUNCTIONS OF CHROMATIN REMODELERS IN NUCLEOSOME ORGANIZATION AND TRANSCRIPTION REGULATION MAJOR PROFESSOR: Dr. Blaine Bartholomew Chromatin remodelers are conserved from yeast to humans and are the gatekeepers of chromatin. They regulate transcription by occluding or exposing DNA regulatory elements globally. They are crucial for DNA processes such as DNA replication, repair and recombination. In addition, they are critical in developmental processes and differentiation. Chromatin remodelers are categorized into several families based on their conserved ATPase domain, an essential component required for their DNA translocation ability. In this study, we investigated the role yeast ISWI and SWI/SNF family of chromatin remodelers play on nucleosome rearrangement and transcription regulation by targeted mutagenesis of domains in accessory subunits and at the C-terminus of the catalytic subunit. All members of the ISWI family (ISW1a, ISW1b, ISW2) share a conserved C-terminal HAND, SANT and SLIDE domains, which are important for sensing linker DNA. We find an auto-regulation of ISWI complexes by the SLIDE domain, independent of the histone H4 Nterminal tail. Our protein-protein chemical crosslinking and mass spectrometry (CX-MS) analysis indicate that the SLIDE domain regulates the ATPase core through N terminal domains of the accessory subunit Itc1. Moreover, we show that the accessory subunits of ISWI modulate the ATPase activity and specificity of ISWI complexes. The DNA sensing ability of the SLIDE domain is required for the in vivo nucleosome spacing and transcription regulation by ISWI. We find that while ISW2 primarily regulates transcription at the 5’ end of genes, ISW1a is important in transcription elongation by rearranging nucleosomes starting at the +2 nucleosome and through the rest of the body of genes towards the 3’ end. ISW1b on the other hand rearrange nucleosomes in the gene body to facilitate suppression of cryptic transcription. For the first time, we show the potential division of labor between ISW1a and ISW1b during transcription elongation. On the other hand, SWI/SNF chromatin remodelers are essential epigenetic factors that are frequently mutated in cancer and neurological disorders. They harbor a C-terminal SnAC and AT hook domains that positively regulate their DNA dependent ATPase activity and nucleosome mobilizing capabilities. By deleting the AT hook motifs, we have identified the role of SWI/SNF in organizing the -1 and +1 nucleosomes at transcription start sites flanking the nucleosome free region (NFR). Our RNA-seq analysis shows SWI/SNF positively regulates the bi-directional transcription of non-coding RNA (ncRNA) which are activated when the AT hook motifs are deleted. Moreover, AT hooks regulate such activities of SWI/SNF through direct protein-protein interactions with the ATPase core as evidenced by our chemical crosslinking and mass spectrometry (CX-MS) analysis.
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Genome-wide approaches to explore transcriptional regulation in eukaryotesPark, Daechan 21 August 2015 (has links)
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.
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