• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 500
  • 122
  • 71
  • 35
  • 30
  • 18
  • 10
  • 9
  • 6
  • 3
  • 3
  • 3
  • 3
  • 3
  • 3
  • Tagged with
  • 1027
  • 189
  • 188
  • 179
  • 175
  • 174
  • 141
  • 130
  • 128
  • 117
  • 108
  • 88
  • 86
  • 86
  • 79
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
361

Functional long non-coding RNA transcription in Schizosaccharomyces pombe

Ard, Ryan Anthony January 2016 (has links)
Eukaryotic genomes are pervasively transcribed and frequently generate long noncoding RNAs (lncRNAs). However, most lncRNAs remain uncharacterized. In this work, a set of positionally conserved intergenic lncRNAs in the fission yeast Schizosaccharomyces pombe genome are selected for further analysis. Deleting one of these lncRNA genes (ncRNA.1343) exhibited a clear phenotype: increased drug sensitivity. Further analyses revealed that deleting ncRNA.1343 also disrupted a previously unannotated lncRNA, termed nc-tgp1, transcribed in the opposite orientation of the predicted ncRNA.1343 gene and into the promoter of the phosphate-responsive permease gene tgp1+. Detailed analyses revealed that the act of transcribing nc-tgp1 into the tgp1+ promoter increases nucleosome density and prevents transcription factor access. Decreased nc-tgp1 transcription permits tgp1+ expression upon phosphate starvation, while nc-tgp1 loss induces tgp1+ in repressive phosphate-rich conditions. Notably, drug sensitivity results directly from tgp1+ expression in the absence of nc-tgp1 transcription. Similarly, lncRNA transcription upstream of pho1+, another phosphate-regulated gene, increases nucleosome density and prevents transcription factor binding to repress pho1+ in phosphate-replete cells. Importantly, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription occurs in the absence of RNAi and heterochromatin components. Instead, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription resembles examples of transcriptional interference reported in other organisms. Thus, tgp1+ and pho1+ are the first documented examples of genes regulated by transcriptional interference in S. pombe.
362

DNAase I hypersensitive site and their correlation to the differential expression of exogenous thymidine kinase gene

Unknown Date (has links)
by Jose Victor Lopez. / Typescript. / Thesis (M.S.)--Florida State University, 1988. / Bibliography: leaves 98-109.
363

Role transkripčních faktorů PU.1 a GATA-1 v leukemické diferenciaci / The role of transcription factors PU.1 a GATA-1 during leukemia differentiation.

Burda, Pavel January 2011 (has links)
Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...
364

ProteÃnas espermÃticas e dinÃmica da cromatina em ruminantes: relaÃÃo com a fertilidade em touros e com o uso de castanha de caju na dieta de ovinos / Sperm proteins and chromatin dynamics in ruminants : relationship with fertility in bulls and with the use of cashew nuts in the diet of sheep

Rodrigo Vasconcelos de Oliveira 05 July 2013 (has links)
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The ruminant fertility is influenced by intrinsic sperm factors, like chromatin or proteins. Considering that the reproductive efficiency is dependent on a balanced and feasible nutrition, the cashew nut meal (CNM) is a low cost byproduct that must be analyzed for possible effects on sperm chromatin and proteins.Study 1:. The objectives of study 1 were to determine failures of chromatin condensation, expression levels and cellular localizations of histones; H3.3, H2B and H4, respectively in spermatozoa from low (LF) vs. high fertility (HF) bulls. The data were analyzed by t test and Pearson correlation (P < 0.05). We demonstrated that aniline blue staining was different within LF (1.73 (0.55, 0.19)) and HF Groups (0.67 (0.17, 0.06) (P < 0.0001), which was also negatively correlated with in vivo bull fertility (r = -0.90; P < 0.0001). Although those histones were consistently immune-detectable and specifically localized in bull sperm, this was not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and conserved among bovine, mouse and human. The H2B variants were more conserved between bovine and human than those of mouse. In conclusion, we showed that H2B, H3.3 and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related with bull fertility. Study 2: The objectives of study 2 were evaluate the effects of 13% of CNM inclusion in the diet of Morada Nova rams on the semen parameters, chromatin integrity and sperm proteins. Twenty rams were distributed in two equal groups: cashew nut group (CNG) and control group (COG) that received 13% and 0% of CNM in the diet for 90 days, respectively. The groups were compared for live weight, scrotal circumference, seminal parameters, chromatin integrity and sperm protein profile at 0, 45 and 90 days of the experiment. The data were evaluated by GLM for repeated measures (P < 0.05). At 90 days, CNG (69.00% (7.38; 2.33)) presented percentage of motile sperm superior than control group (60,00% (9,43; 2,98)) (P<0.05). There was not effect from the diet with CNM on chromatin integrity. But, the percentages of protein expression from ODF1 and H2B were larger in the CNG (P<0.05). The proteins: ODF1, GPX4, FTL and H2B were negatively correlated with sperm chromatin quality. In conclusion, the cashew nut meal did not affect negatively the semen quality. / A fertilidade em ruminantes à influenciada por fatores intrÃnsecos espermÃticos como a cromatina e as proteÃnas. Considerando que a eficiÃncia reprodutiva do macho depende de uma nutriÃÃo balanceada e viÃvel, o farelo de castanha de caju (FCC) à um subproduto de baixo custo que deve ser analisado quanto a possÃveis efeitos na integridade cromatÃnica e proteica dos espermatozoides. Estudo 1: O estudo 1 teve como objetivos determinar: falhas na condensaÃÃo da cromatina, nÃveis de expressÃo e localizaÃÃo celular das histonas: H3.3, H2b e H4B, respectivamente, em espermatozoides de touros de baixa (BF) e alta fertilidade (AF). Os dados foram avaliados pelo teste t e correlaÃÃo de Pearson (P < 0,05). Os resultados do teste do azul de anilina foram diferentes entre os grupos BF (1.73 (0.55, 0.19)) e AF (0,67 (0,17, 0,06) (P < 0,0001), os quais tambÃm foram negativamente correlacionados com a fertilidade in vivo de touros (r = -0,90; P < 0,0001). Apesar das histonas terem sido consistentemente imunodetectadas e localizadas nos espermatozoides, estas nÃo apresentaram diferenÃas entre os grupos. As proteÃnas H3.3 e H4 apresentaram 100% de identidade e foram conservadas entre bovinos, murinos e seres humanos. Entretanto, as variantes H2B foram mais conservadas entre touros e humanos do que entre humanos e camundongos. Em conclusÃo, as proteÃnas H2B, H3.3 e H4 foram detectÃveis em espermatozoides de touros e a condensaÃÃo da cromatina espermÃtica, alterada pela retenÃÃo de histonas, à relacionada com a fertilidade de touros. Estudo 2: O estudo 2 objetivou avaliar os efeitos da inclusÃo de 13% de FCC na dieta de carneiros Morada Nova sobre as caracterÃsticas seminais, integridade de cromatina e perfil das proteÃnas espermÃticas. Vinte carneiros foram divididos em dois grupos: castanha (GCA) e controle (GCO).que receberam na dieta 13% e 0% de FCC durante 90 dias, respectivamente. Os grupos foram comparados quanto ao peso vivo, circunferÃncia escrotal, parÃmetros seminais, integridade de cromatina e perfil das proteÃnas espermÃticas aos 0, 45 e 90 dias de experimento. Os dados foram analisados pelo mÃtodo GLM para medidas repetidas (P < 0,05). Aos 90 dias o GCA (69,00% (7,38; 2,33) apresentou porcentagem de espermatozoides mÃveis superior ao GCO (60,00% (9,43; 2,98)) (P<0.05). NÃo houve efeito da dieta contendo FCC sobre a integridade da cromatina. PorÃm, os percentuais das proteÃnas ODF1 e H2B foram mais elevados nos carneiros do GCA (P < 0,05). As proteÃnas: ODF1, GPX4, FTL e H2B foram negativamente correlacionadas com a qualidade da cromatina espermÃtica. Em conclusÃo, a inclusÃo de FCC na dieta de carneiros nÃo afetou negativamente a qualidade seminal.
365

Aspectos da estrutura da cromatina e da atividade de transcrição induzida na glândula salivar de Rhynchosciara americana (Diptera: Sciaridae) / Aspects of chromatin structure and induced transcription activity on salivary glands of Rhynchosciara americana (Diptera: Sciaridae)

Alejandra Badaracco 10 December 2014 (has links)
Os cromossomos politênicos da glândula salivar dos dípteros proporcionam condições excepcionalmente favoráveis à observação, em microscopia óptica, da estrutura da cromatina bem como da atividade gênica. Neste trabalho, a distribuição de modificações epigenéticas e de marcadores associados à atividade de transcrição foi estudada em cromossomos politênicos de larvas Rhynchosciara americana em condições normais de crescimento e também submetidas a tratamentos que levam ao estresse, um dos quais (anestesia por éter dietílico) realizado pela primeira vez nesta espécie. Na recuperação do estresse por anestesia, o locus do gene hsp70 apareceu transcricionalmente ativo embora, frequentemente, a marcação de RNA polimerase II não ocupava todo o volume do pufe como ocorre usualmente na resposta ao estresse por choque térmico, sugerindo descondensação parcial do locus. A localização cromossômica da proteína Sex-lethal, postulada como um fator de splicing genérico em espécies da família Sciaridae, foi estudada pela primeira vez em larvas submetidas ao estresse. A ausência da mesma em loci cromossômicos que estão ativos em transcrição em condições estressantes sugere que Sex-lethal não seja um fator de splicing genérico. Além disto, a localização desta proteína em regiões transcricionalmente inativas durante o estresse sugere que Sex-lethal cumpra funções ainda não descritas. Entre os dados de localização de histona H3 metilada em lisina 4, marcador epigenético usualmente associado à transcrição, destacou-se a forte marcação de uma região particular dentro da secção cromossômica A-9. Esta região, entretanto, apresenta sinais muito fracos de anti-híbrido DNA/RNA e de RNA polimerase II, sugerindo atividade transcricional muito baixa ou ausente. Observou-se também que o marcador epigenético conservado de heterocromatina, H3me3K9 (histona H3 trimetilada em lisina 9), não está presente na mesma; mas, por outro lado, detectou-se na região sinal significativo da proteína da heterocromatina de Sciara coprophila. Com o objetivo de conhecer sequências de DNA presentes na região, foi produzida uma biblioteca plasmidial produzida a partir de microdissecção cromossômica do sub-setor de A-9 seguida de DOP-PCR. O produto de amplificação foi usado como sonda em hibridação in situ e marcou, além da região microdissecada, extremidades cromossômicas não centroméricas, indicando que estas regiões compartilham sequências repetitivas. Algumas puderam ser identificadas após o seqüenciamento, tais como repetições em tandem e elementos genéticos móveis enquanto que outras não apresentaram similaridade significativa com sequências disponíveis em bancos de dados. Os dados obtidos sugerem que o sub-setor de A-9 analisado assemelha-se à heterocromatina, porém composta de uma combinação aparentemente não usual de marcadores epigenéticos / Polytene chromosomes of dipteran salivary glands provide optimal visualization of chromatin structure as well as gene activity at the cytological level. In this work, the distribution of epigenetic and transcription-associated markers was studied in polytene chromosomes of Rhynchosciara Americana larvae, either under during normal growth or stressful treatments, one of which (anaesthesia by ether) has been observed for the first time in this species. During the recovery from the anesthesia, the hsp70 gene locus was transcriptionally active although RNA polymerase II detection did not occupied the entire volume of the puff -as usually happens during the heat shock response-, suggesting partial puff decondensation. The chromosomal localization of the sciarid Sex-lethal protein, which was supposed to be a general splicing factor, was studied for the first time in stressed larvae and was not detected in transcriptionally active loci. Hence, the Sex-lethal protein may not be a general splicing factor. Moreover, Sex-lethal localization in transcriptionally inactive regions during stress situations suggests it plays still unknown roles. Antibodies to histone H3 methylated at lysine 4 -an epigenetic marker of transcription- labeled strongly at a particular region within chromosome section A-9. This region, in contrast, displays very low levels of both DNA/RNA hybrid and RNA polymerase II, suggesting, if any, poor transcriptional activity. Interestingly, the conserved epigenetic heterochromatin marker H3me3K9 (histone H3 trimethylated at lysine 9) is not significantly present in this region, even though the Sciara coprophila heterochromatin protein was clearly detected. In order to sample DNA sequences contained in this region, a plasmid library was produced by performing a microdissection of A-9 sub-section followed by DOP-PCR. The total microdissection product was used as a probe, and hybridized, in addition to the microdissected region, to non-pericentric chromosome ends, indicating that repetitive sequences are shared by these regions. Some sequences could be identify, after sequencing, such as tandem repeats and mobile elements, but others failed to display significant similarity to sequences deposited in databases. The results suggest that the sub-section analyzed resembles heterochromatin but being composed of an apparently unusual combination of epigenetic markers
366

The role of Chd7 & Chd8 chromatin remodelers in oligodendrogenesis and (re)myelination / Le rôle de Chd7 & Chd8 facteurs du remodelage de la chromatine dans l'oligodendrogenese et la (re)myélinisation

Marie, Corentine 29 September 2017 (has links)
Les oligodendrocytes (OLs) sont les cellules myélinisantes du système nerveux central, s’enroulant autour des axones et permettant la conduction saltatoire du potentiel d’action. Dans la Sclérose en Plaques, des gaines de myélines sont détruites et l’efficacité de la remyélinisation par les précurseurs d’oligodendrocytes (OPCs) diminue avec la progression de la maladie. Une meilleure compréhension du mécanisme qui contrôle la génération des OPCs et leur différentiation est donc essentielle pour développer des thérapies efficaces de remyélinisation. L’oligodendrogenèse, qui comprend les étapes de génération des OPCs, de différenciation et de maturation des OLs, est un processus contrôlé par des facteurs de transcription spécifiques incluant Ascl1, Olig2 and Sox10 mais le mécanisme impliqué est encore peu connu. Sachant que les facteurs du remodelage de la chromatine sont des régulateurs nécessaires à la formation de la boucle promoter-enhancer permettant l’initiation de la transcription, nous nous sommes focalisé sur Chd7 (Chromodomain-Helicase-DNA-Binding 7), un membre de la famille de protéine CHD. Dans une première étude, nous avons montré que Chd7 est hautement enrichi dans le lignage oligodendroglial avec un pic d’expression pendant la différenciation des OLs. Nous avons également montré que la délétion conditionnelle de Chd7 diminuait la différentiation des OLs pendant la (re)myélinisation. Dans un seconde étude, nous avons utilisé des techniques de génomique sur les OPCs purifiés pour étudier la régulation par Chd7 de gènes impliqués dans la différenciation, la survie et la prolifération des OPCs. Dans ce but, nous avons générer des délétions inductible de Chd7 spécifiquement dans les OPCs (Chd7iKO) et nous avons analysé le transcriptome (RNA-seq) d’OPCs purifiés à partir de cerveaux de souris P7 comparé à des contrôles. Nous avons trouvé que Chd7 activait l’expression des gènes impliqué dans la différenciation des OPCs et la myélinisation et inhibait l’apoptose, sans montré de défaut de prolifération. Pour aller plus loin, nous avons étudié Chd8, un paralogue de Chd7, et nous avons montré qu’il est exprimé dans le lignage oligodendrocytaire avec un pic d’expression dans les OL en différenciation, similairement à Chd7. Les données de fixation (ChIP-seq) de Chd7 et Chd8 indiquent que ces deux facteurs du remodelage de la chromatine se fixent sur des gènes communs reliés au processus de différenciation, de survie et de prolifération des OPCs. Intégrant ces données avec celles de facteurs transcriptionnels clés dans l’oligodendrogenèse (Olig2, Ascl1 et Sox10), nous avons construit un modèle de la régulation de l’expression de gènes contrôlés dans le temps et impliqué dans chacune des étapes de la différenciation des oligodendrocytes. / Oligodendrocytes (OLs) are myelin-forming cells of the central nervous system wrapping axons and allowing the saltatory conduction of action potentials. In Multiple sclerosis (MS), myelin sheath is destroyed and effective remyelination by oligodendrocyte precursor cells (OPCs) diminishes with disease progression. Therefore, a better understanding of the mechanisms controlling OPC generation and differentiation is essential to develop efficient remyelinating therapies. Oligodendrogenesis, involving the steps of OPC generation, OPC differentiation and maturation of OLs, is a process controlled by specific transcription factors including Ascl1, Olig2 and Sox10 but the mechanisms involved are poorly understood. As it is known that chromatin remodelers are regulatory factors necessary in the formation of the promoter-enhancer loop prior to transcription, we focused our study on Chd7 (Chromodomain-Helicase-DNA-Binding 7), a member of the CHD protein family. In a first study, we showed that Chd7 is highly enriched in the oligodendroglial lineage cells with a peak of expression during OL differentiation and that Chd7 OPC-conditional deletion impairs OL differentiation during (re)myelination. In a second study, we used unbiased genome wide technics in purified OPCs to study Chd7 regulation of genes involved in OPC differentiation, proliferation and survival. To this aim, we have generated OPC-specific inducible Chd7 knock-out (Chd7-iKO) and analyse the transcriptome (RNA-seq) of purified OPCs from P7 mouse cortices compared to control littermates. We found that Chd7 promote the expression genes involved in OPC differentiation and myelination and inhibits apoptosis, without affecting OPC proliferation. Furthermore, we investigated Chd8, a paralog of Chd7, showing that it is expressed in the oligodendroglial lineage with a peak of expression in differentiating oligodendrocytes, similar to Chd7. Genome wide binding (ChIP-seq) profiling for Chd7 and Chd8 indicate that these two chromatin remodelers bind to common genes related to OPC differentiation, survival and proliferation. Integrating these datasets with other key transcriptional regulators of oligodendrogenesis (Olig2, Ascl1 & Sox10), we have built a model accounting for the time-controlled regulate expression of genes involved in each step of OL differentiation.
367

Expanding the network of enzymes affecting methylation at H3K4 (histone 3 lysine 4) during Caenorhabditis elegans embryogenesis

Wilkins, Elizabeth January 2016 (has links)
Post translational modifications (PTMs) of histone tails are an important determinant of chromatin structure, and can act as key regulators of DNA-dependent processes. Methylation of histone 3 at lysine 4 (H3K4) is one of the most widely studied PTMs because of its correlation with transcription. Three methylation states exist at H3K4: mono-, di, and tri-methylation (H3K4me1, -me2, and -me3, respectively). Each methylation state occupies a distinct genomic position, supporting the view that the extent of methylation at H3K4 has a functional significance. However, the exact biological function of these three marks are not well understood. H3K4 methylation is written by SET domain-containing enzymes that function within SET/COMPASS/MLL complexes. Our lab has previously identified the SET-16 enzyme as writing H3K4me3 in C. elegans. The other well-characterised H3K4-specific methyl transferases in the worm is SET-2, an enzymes responsible for bulk H3K4me2/me3 levels. Using targeted RNAi screens, we have characterised the full landscape of SET domain enzymes affecting all three methylation states at H3K4 during embryogenesis in C. elegans (Chapter 3). Unexpectedly, many previously uncharacterised enzymes were identified as preferentially affecting each of the methylation states, including SET-19 that can deposit all three marks, and several candidates that preferentially affect H3K4me1: SET-30, SET-27, MES-2, and MES-4. During the project, Greer et al. 2014 independently identified two enzymes with activities targeting H3K4, SET-17 and SET-30, which were also candidates from our RNAi screens. With a focus on enzymes acting on H3K4me1, we demonstrate that H3K4me1 candidates can show different patterns of temporal regulation and also have roles in regulating soma versus germline cell-fate decisions (Chapter 4). Finally, we demonstrate a novel role for MES-2 (a methyltransferase enzyme with a highly conserved role in depositing repressive H3K27 methylation) in acting alongside the SPR-5 H3K4me2 demethylase to regulate levels of H3K4me1 during embryogenesis (Chapter 5).
368

Study of histone variants and chromatin dynamics in the preimplantation mouse embryo / Etude des variantes d'histones et dynamique de la chromatine dans l'embryon préimplantatoire de souris

Boskovic, Ana 28 July 2014 (has links)
Comment le zygote acquiert la totipotence à partir de deux cellules complètement différenciées, et comment les décisions du destin cellulaire sont faites plus tard dans le développement sont des questions biologiques essentielles. Les études menées au cours de la première partie de mon doctorat ont contribué à l'annotation de la composition de la chromatine embryonnaire en ce qui concerne les variantes des histones et des modifications post-traductionnelles. L'expression ectopique de H2A.Z après la fécondation réduit la progression du développement, ce qui suggère que l'absence de H2A.Z au début du développement pourrait être importante pour l'organisation de la chromatine embryonnaire nouvellement formée. Deuxièmement, j'ai étudié la dynamique des histones dans l'embryon de souris en développement. La reprogrammation épigénétique après la fécondation est accompagnée par une étonnante forte mobilité des histones dans le noyau. Ma thèse a contribué à la compréhension des événements dynamiques affectant la chromatine embryonnaire pendant le remodelage épigénétique après la fécondation. / How the zygote acquires totipotency from two differentiated cells, and how cell fate decisions are made later in development is a pivotal biological question. The studies conducted during the first part of my doctorate contributed to the annotation of embryonic chromatin composition with regards to histone variants and PTMs, and more specifically those correlated with active chromatin regions. The histone variant H2A.Z was shown to be present on embryonic chromatin in a stage-specific manner. Ectopic expression of H2A.Z after fertilization reduced developmental progression, suggesting that absence of H2A.Z at the onset of development might be important for the organization of the newly formed embryonic chromatin. Secondly, I investigated histone dynamics in the developing mouse embryo. Our work represents the first report on histone mobility during early mouse embryogenesis. My thesis contributed to the understanding of the dynamic events affecting embryonic chromatin during epigenetic remodeling after fertilization.
369

Identification et caractérisation de HIRIP3 comme nouveau chaperon d'histone H2A / Identification and characterization of HIRIP3 as a novel histone H2A chaperone

Ignatyeva, Maria 31 May 2017 (has links)
Le génome des cellules eucaryotes est empaqueté dans la chromatine, dont l’établissement et la maintenance nécessitent des processus d’assemblage et de remodelage. Ce travail de thèse a été consacré à la caractérisation de deux facteurs de la machinerie d’assemblage de la chromatine. Le premier facteur étudié dans ce travail était HIRIP3, un homologue mammifère de la levure H2A.Z chaperon Chz1. Nous voulions vérifier si HIRIP3 est une chaperon d'histone par elle-même. Pour commencer, nous avons décrit l'interaction de HIRIP3 avec les histones in vivo. Ensuite, nous avons étudié la spécificité structurale de cette interaction in vitro. Nous avons caractérisé HIRIP3 comme une nouvelle chaperon d'histone H2A qui utilise le motif CHZ pour sa fonction. La deuxième partie de ce travail a été axée sur le complexe de remodelage de la chromatine SRCAP. Nous avons cherché à décoder son réseau d'interaction et à décrire ses sous-complexes. Nous avons reconstitué le complexe de base YL1, SRCAP, TIP49A, TIP49B et H2A.Z / H2B en utilisant le système d'expression chez baculovirus. Notre protocole nous a permis de purifier un complexe de base adapté aux futures études structurelles par microscopie cryo-électronique. / The genome of eukaryotic cells is packaged into chromatin, which establishment and maintenance require mechanisms of assembly and remodelling. This thesis work was dedicated to the characterization of two factors of chromatin assembly machinery. The first factor studied in this work was HIRIP3, a mammalian homologue of yeast H2A.Z chaperone Chz1. We aimed to test whether HIRIP3 is a histone chaperone by itself. At first, we established HIRIP3 interaction with histones in vivo. After then, we studied the structural specificity of this interaction in vitro. We have characterized HIRIP3 as a novel H2A histone chaperone that utilizes the CHZ motif for its function. The second part of this work was focused on SRCAP chromatin remodelling complex. We aimed to decipher its interaction network and to describe its sub-complexes. We have reconstituted YL1, SRCAP, TIP49A, TIP49B and H2A.Z/H2B core complex using baculovirus expression system. Our protocol allowed us to purify core complex suitable for future structural studies by cryo-electron microscopy.
370

Dynamiques chromatiniennes au cours de la photomorphogenèse chez Arabidopsis thaliana / Chromatin dynamics during photomorphogenesis in Arabidopsis thaliana

Bourbousse, Clara 25 June 2012 (has links)
Les états chromatiniens peuvent être étudiés à l’échelle des unités transcriptionnelles par des approches moléculaires ou à l'échelle plus globale de l'hétérochromatine structurée au sein de chromocentres par des approches cytogénétiques. Ces deux niveaux d’organisation de la chromatine sont dynamiques et influencent l'ensemble des processus nucléaires. L’objectif de cette thèse était d'avancer la compréhension des dynamiques chromatiniennes à ces deux échelles chez la plante modèle Arabidopsis thaliana, en se focalisant sur une transition développementale majeure, la photomorphogenèse. Le processus de dé-étiolement implique la reprogrammation de l’expression de centaines de gènes en réponse à la lumière, constituant ainsi un excellent modèle d'étude. La première partie des travaux montre que la reprogrammation de l’expression du génome au cours de la photomorphogenèse est associée à des dynamiques de l’hétérochromatine qui sont régulés de façon différentielles dans les hypocotyles et les cotylédons. Ces dynamiques à grande échelle ont des conséquences localement, car les états décompactés sont associés à la réactivation d'éléments hétérochromatiniens répétés. Dans une deuxième partie, le répresseur transcriptionnel DE-ETIOLATED-1 (DET1) a été utilisé afin de rechercher l'implication de régulateurs de la photomorphogenèse dans les mécanismes chromatiniens. Ce répresseur majeur de la photomorphogenèse peut lier l'histone H2B et influence le niveau global de sa modification par mono-ubiquitination (H2Bub). Dans le cadre de ma thèse, j'ai révélé d'une part l’existence d’interactions génétiques entre DET1 et les gènes contrôlant l’homéostasie de H2Bub et d'autre part un défaut de la régulation chromatinienne des variants des gènes ribosomiques 5S et 45S dans le mutant det1-1. L’ensemble de ces données permet de proposer un modèle impliquant DET1 dans la régulation de H2Bub de façon différentielle dans l’euchromatine et l’hétérochromatine, constituant ainsi le premier lien entre régulateurs de la photomorphogenèse et modifications des histones. La marque H2Bub étant directement liée à l'activité transcriptionnelle chez divers eucaryotes, l'impact de H2Bub sur l'expression des gènes durant la photomorphogenèse a été analysé. La combinaison d’approches épigénomiques et transcriptomiques a permis de montrer que le gain de H2Bub est associé à l’induction des gènes. L’utilisation du mutant hub1 dans lequel le dépôt de H2Bub est aboli a également permis de révéler le rôle de cette marque pour une régulation rapide de l’induction et de la répression de nombreux gènes. De façon générale, ce travail a révélé des dynamiques chromatiniennes impliquant des réorganisations massives au niveau cytologique ainsi que des variations fines des modifications d'histones au niveau des gènes de l'euchromatine, ainsi que le rôle de DET1 dans la régulation de ces processus. Il ouvre donc la voie à l'étude des connections entre ces deux échelles de dynamiques pour la régulation de l'activité transcriptionnelle, liant compartimentation nucléaire et activité des gènes dans le contexte global de la réponse aux signaux lumineux. / Chromatin states can be studied both at the level of individual transcriptional units by molecular approaches or at the larger scale of heterochromatin by cytogenetic approaches. These two levels of chromatin organization are dynamic and influence all nuclear processes. The objective was to enhance the understanding of chromatin dynamics at these two scales in the model plant Arabidopsis thaliana, focusing on a major developmental transition, photomorphogenesis. The process of de-etiolation involves the reprogramming of the expression of hundreds of genes in response to the perception of light therefore constituting an excellent experimental system. The first part of the work shows that reprogramming of genome expression during photomorphogenesis is associated with heterochromatin dynamics that is differentially regulated in the hypocotyls and the cotyledons. These widespread dynamics have local consequences, as the decompacted states are associated with reactivation of heterochromatic repeat elements. In the second part, the transcriptional repressor DE-ETIOLATED-1 (DET1) was used to investigate the involvement of photomorphogenesis regulators in chromatin mechanisms. This major repressor of photomorphogenesis can bind histone H2B and influences the overall level of mono-ubiquitinated H2B (H2Bub). As part of my thesis, I uncovered the existence of genetic interactions between DET1 and the genes controlling H2Bub homeostasis and also a defect in the regulation of the chromatin around the 45S and 5S ribosomal genes in the mutant det1-1. These data have led me to propose a model involving DET1 in the differential regulation of H2Bub in heterochromatin and euchromatin, thus constituting for the first time a link between photomorphogenesis regulators and histone modifications. Because the H2Bub mark has been directly linked to transcriptional activity in a diverse range of eukaryotes, I analysed the impact of H2Bub on gene expression during photomorphogenesis in the third part of my thesis. The combination of transcriptomic and epigenomic approaches showed that the gain of H2Bub is associated with gene induction. The use of a hub1 mutant in which H2Bub deposition is abolished also revealed the role of this mark for the rapid control of many genes. In general terms, this work has revealed both dynamic chromatin changes that result in major genome reorganizations at the cytological scale and fine variations of histone modifications on euchromatic genes, as well as the role of DET1 in regulating these changes. My study paves the way for further studies on the connections between these two scales of dynamics and their function in the nuclear localization and changes in expression of genes in the overall context of light signaling.

Page generated in 0.1907 seconds