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Showing 51 to 55 of 55 (0.125 seconds)Spelling suggestions: "subject:"chromatin immunoprecipitation"" "subject:"chromatin mmunoprecipitation""
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The GRAS Protein SCL14 and TGA Transcription Factors Interact to Regulate Stress-Inducible Promoters / Das GRAS-Protein SCL14 und TGA-Transkriptionsfaktoren interagieren bei der Regulation stress-induzierbarer PromotorenFode, Benjamin 08 May 2008 (has links)
No description available.
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Caractérisation de la fonction des complexes histone déacétylases Rpd3S et Set3CDrouin, Simon 05 1900 (has links)
La chromatine est essentielle au maintien de l’intégrité du génome, mais, ironiquement, constitue l’obstacle principal à la transcription des gènes. Plusieurs mécanismes ont été développés par la cellule pour pallier ce problème, dont l’acétylation des histones composant les nucléosomes. Cette acétylation, catalysée par des histones acétyl transférases (HATs), permet de réduire la force de l’interaction entre les nucléosomes et l’ADN, ce qui permet à la machinerie transcriptionnelle de faire son travail. Toutefois, on ne peut laisser la chromatine dans cet état permissif sans conséquence néfaste. Les histone déacétylases (HDACs) catalysent le clivage du groupement acétyle pour permettre à la chromatine de retrouver une conformation compacte.
Cette thèse se penche sur la caractérisation de la fonction et du mécanisme de recrutement des complexes HDACs Rpd3S et Set3C. Le complexe Rpd3S est recruté aux régions transcrites par une interaction avec le domaine C-terminal hyperphosphorylé de Rpb1, une sous-unité de l’ARN polymérase II. Toutefois, le facteur d’élongation DSIF joue un rôle dans la régulation de cette association en limitant le recrutement de Rpd3S aux régions transcrites. L’activité HDAC de Rpd3S, quant à elle, dépend de la méthylation du résidu H3K36 par l’histone méthyltransférase Set2.
La fonction du complexe Set3C n’est pas clairement définie. Ce complexe est recruté à la plupart de ses cibles par l’interaction entre le domaine PHD de Set3 et le résidu H3K4 di- ou triméthylé. Un mécanisme indépendant de cette méthylation, possiblement le même que pour Rpd3S, régit toutefois l’association de Set3C aux régions codantes des gènes les plus transcrits.
La majorité de ces résultats ont été obtenus par la technique d’immunoprécipitation de la chromatine couplée aux biopuces (ChIP-chip). Le protocole technique et le design expérimental de ce type d’expérience fera aussi l’objet d’une discussion approfondie. / Chromatin is essential for the maintenance of genomic integrity but, ironically, is also the main barrier to gene transcription. Many mechanisms, such as histone acetylation, have evolved to overcome this problem. Histone acetylation, catalyzed by histone acetyltransferases (HATs), weakens the internucleosomal and nucleosome-DNA interactions, thus permitting the transcriptional machinery access to its template. However, this permissive chromatin state also allows for opportunistic DNA binding events. Histone deacetylases (HDACs) help restore a compact chromatin structure by catalyzing the removal of acetyl moieties from histones.
This thesis focuses on the characterization of the function and of the recruitment mechanism of HDAC complexes Rpd3S and Set3C. The Rpd3S complex is recruited to actively transcribed coding regions through interactions with the hyperphosphorylated C-terminal domain of Rpb1, a subunit of RNA polymerase II, with the DSIF elongation factor playing a role in limiting this recruitment. However, the HDAC activity of Rpd3S depends on H3K36 methylation, which is catalyzed by the Set2 histone methyltransferase.
The Set3C complex’ function is still not clearly defined. It is recruited to most of its targets through the interaction between the Set3 PHD domain and di- or trimethylated H3K4. However, Set3C recruitment to genes displaying high RNA polymerase II occupancy is independent of H3K4 methylation. The mechanism by which Set3C is recruited to this gene subset is under investigation.
These results have mostly been obtained through chromatin immunoprecipitation coupled to tiling microarrays (ChIP-chip). The protocol and experimental design challenges inherent to this technique will also be discussed in depth.
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Caractérisation de la fonction des complexes histone déacétylases Rpd3S et Set3CDrouin, Simon 05 1900 (has links)
La chromatine est essentielle au maintien de l’intégrité du génome, mais, ironiquement, constitue l’obstacle principal à la transcription des gènes. Plusieurs mécanismes ont été développés par la cellule pour pallier ce problème, dont l’acétylation des histones composant les nucléosomes. Cette acétylation, catalysée par des histones acétyl transférases (HATs), permet de réduire la force de l’interaction entre les nucléosomes et l’ADN, ce qui permet à la machinerie transcriptionnelle de faire son travail. Toutefois, on ne peut laisser la chromatine dans cet état permissif sans conséquence néfaste. Les histone déacétylases (HDACs) catalysent le clivage du groupement acétyle pour permettre à la chromatine de retrouver une conformation compacte.
Cette thèse se penche sur la caractérisation de la fonction et du mécanisme de recrutement des complexes HDACs Rpd3S et Set3C. Le complexe Rpd3S est recruté aux régions transcrites par une interaction avec le domaine C-terminal hyperphosphorylé de Rpb1, une sous-unité de l’ARN polymérase II. Toutefois, le facteur d’élongation DSIF joue un rôle dans la régulation de cette association en limitant le recrutement de Rpd3S aux régions transcrites. L’activité HDAC de Rpd3S, quant à elle, dépend de la méthylation du résidu H3K36 par l’histone méthyltransférase Set2.
La fonction du complexe Set3C n’est pas clairement définie. Ce complexe est recruté à la plupart de ses cibles par l’interaction entre le domaine PHD de Set3 et le résidu H3K4 di- ou triméthylé. Un mécanisme indépendant de cette méthylation, possiblement le même que pour Rpd3S, régit toutefois l’association de Set3C aux régions codantes des gènes les plus transcrits.
La majorité de ces résultats ont été obtenus par la technique d’immunoprécipitation de la chromatine couplée aux biopuces (ChIP-chip). Le protocole technique et le design expérimental de ce type d’expérience fera aussi l’objet d’une discussion approfondie. / Chromatin is essential for the maintenance of genomic integrity but, ironically, is also the main barrier to gene transcription. Many mechanisms, such as histone acetylation, have evolved to overcome this problem. Histone acetylation, catalyzed by histone acetyltransferases (HATs), weakens the internucleosomal and nucleosome-DNA interactions, thus permitting the transcriptional machinery access to its template. However, this permissive chromatin state also allows for opportunistic DNA binding events. Histone deacetylases (HDACs) help restore a compact chromatin structure by catalyzing the removal of acetyl moieties from histones.
This thesis focuses on the characterization of the function and of the recruitment mechanism of HDAC complexes Rpd3S and Set3C. The Rpd3S complex is recruited to actively transcribed coding regions through interactions with the hyperphosphorylated C-terminal domain of Rpb1, a subunit of RNA polymerase II, with the DSIF elongation factor playing a role in limiting this recruitment. However, the HDAC activity of Rpd3S depends on H3K36 methylation, which is catalyzed by the Set2 histone methyltransferase.
The Set3C complex’ function is still not clearly defined. It is recruited to most of its targets through the interaction between the Set3 PHD domain and di- or trimethylated H3K4. However, Set3C recruitment to genes displaying high RNA polymerase II occupancy is independent of H3K4 methylation. The mechanism by which Set3C is recruited to this gene subset is under investigation.
These results have mostly been obtained through chromatin immunoprecipitation coupled to tiling microarrays (ChIP-chip). The protocol and experimental design challenges inherent to this technique will also be discussed in depth.
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Kallikrein Gene Regulation in Hormone-Dependent Cancer Cell LinesMyers, Stephen Anthony January 2003 (has links)
Hormone-dependent cancers (HDCs), such as those of the prostate, ovary, breast and endometrium, share characteristics that indicate similar underlying mechanisms of carcinogenesis. Through steroid hormone signalling on "down-stream" target genes, the growth, development and progression of HDCs are regulated. One such family of target genes, highly expressed in HDCs and regulated by steroid hormones, are the tissue kallikreins (KLKs). The KLKs are a multigene family of serine proteases involved in physiological processes such as blood pressure regulation, inflammation, and tumour development and progression via the hydrolysis of specific substrates. Although the KLK gene family is clearly implicated in tumourigenesis, the precise roles played by these genes are largely unknown. Additionally, except for the androgen-responsive genes, KLK2 and KLK3, the mechanisms underlying their hormonal regulation in HDCs are yet to be identified. The initial focus of this thesis was to examine the regulation of the kallikreins, KLK1 and KLK4, by estradiol and progesterone in endometrial and breast cancer cell lines. From these studies, progesterone clearly regulated KLK4 expression in T47D cells and therefore, the focus of the remaining studies was to further examine this regulation at the transcriptional level. An overview of the results obtained is detailed below. Human K1 and hK4 protein levels were increased by 10 nmol/L estradiol benzoate, progesterone, or a combination of the two, over 48 hours in the endometrial cancer cell line, KLE. However, these same treatments resulted in no change in KLK1 gene or hK1 protein levels in the endometrial cancer cell lines, HEC1A or HEC1B (only hK1 analysed). Progesterone treatment (0-100 nmol/L) over 24 hours resulted in a clear increase in KLK4 mRNA at the 10 nmol/L dose in the breast cancer cell line, T47D. Additionally, treatment of T47D cells with 10 nmol/L progesterone over 0-48 hr, resulted in the rapid expression of the hK4 protein at 2 hr which was sustained for 24 hr. Further analysis of this latter progesterone regulation with the antiprogesterone, RU486, over 24 hours, resulted in an observable decrease in hK4 levels at 1 µmol/L RU486. Although the estrogen and progesterone regulation of the hK1 protein was not further analysed, the data obtained for hK4 regulation in T47D cell lines, supported the premise that this gene was progesterone-responsive. The rapid expression of hK4 protein by progesterone at two hours suggests that KLK4 transcription is directly coupled to progesterone regulation, perhaps through progesterone receptor (PR) binding to progesterone-responsive regions within the KLK4 promoter or far "up-stream" regions. Thus, the following further studies were performed. To test this hypothesis, the transcription initiation site (TIS) and 5' flanking regions of the KLK4 gene in T47D cells were interrogated. Primer extension and 5' RACE identified the TIS 78 bp 5' of the putative ATG site for translation as identified by Korkmaz et al. (2001). This KLK4 gene transcript consists of only four exons, and thus excludes the pre/pro signal peptide. Although a TATA-box is not present within -25 to -30 bp 5' of the identified TIS, a number of consensus binding motifs for Sp1 and estrogen receptor half-sites were identified. It is possible that the Sp1 sites are involved in the basal levels of transcription for this gene. Additionally, a putative progesterone response element (PRE) was identified in the far "up-stream" regions of the KLK4 gene. Basal levels of transcription were observed within the KLK4 proximal promoter region when coupled to a luciferase reporter gene and transfected into T47D cell lines. Additionally, the KLK4 proximal promoter region did not induce the luciferase reporter gene expression when progesterone was added to the system, however, estradiol was inhibitory for luciferase gene expression. This suggests that the proximal promoter region of the KLK4 gene could contain functional EREs but not PREs. In keeping with this hypothesis, some ER half-sites were identified, but PR sites were not obvious within this region. The identified PRE in the far "up-stream" region of the KLK4 gene assembled the progesterone receptor in vitro, and in vivo, as assessed by electromobility shift assays and chromatin immunoprecipitation assays (EMSAs and ChIPs), respectively. The binding of the PR to the KLK4 PRE was successfully competed out by a PR antibody and not by an androgen receptor antibody, and thus confirms the specificity of the KLK4 PRE-PR complex. Additionally, the PR was recruited and assembled onto and off the progesterone-responsive KLK4 region in a cyclic fashion. Thus, these data strongly suggest that the PR represents one of the core components of a transcription complex for the KLK4 gene, and presumably also contributes to the expression of this gene. Moreover, these data suggest a functional coordination between the PR and the KLK4 progesterone-responsive region in T47D cells, and thus, provide a model system to further study these events in vivo.
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Role of Ring1B in ephitelial to mesenchimal transition, invasion and migration of mammary epithelial cellsBosch Gutiérrez, Almudena 21 December 2009 (has links)
The Polycomb group (PcG) family of proteins form chromatin-modifying complexes essential for embryonic development, and stem cell renewal and are commonly deregulated in cancer. There are several reports that address the possible implication of PcG proteins in tumor progression and metastasis, but very little is known about the specific role of these proteins in tumor progression and invasion. On the other hand, the molecular processes of the worst cancer prognosis, metastasis, which leads to an incurable disease, are yet incompletely elucidated. Here we show a role for Ring1B, a PcG protein, in three processes related to metastasis: in the Epithelial-mesenchymal transition (EMT), a critical morphogenic event that occurs during embryonic development and during the progression of various epithelial tumors, an in the migration and the invasion of mammary epithelial cells. / Las proteínas del grupo Polycomb (PcG) forman complejos modificadores de la cromatina esenciales en el desarrollo embrionario y en la renovación de las células madre, y su desregulación ha sido asociada al cáncer. Varios estudios muestran la posible implicación de las proteínas de PcG en la progresión tumoral y en la metástasis, pero a pesar de ello se sabe muy poco de los procesos moleculares en los que estas proteínas están participando. Por otro lado, los procesos moleculares responsables del peor pronóstico en cáncer, la metástasis, que continua siendo una enfermedad incurable, siguen sin estar completamente elucidados. En esta disertación mostramos el papel de Ring1B, una proteína del PcG, en tres procesos implicados en la metástasis: en la transición epitelio-mesénquima (EMT), un proceso morfogénico crítico en el desarrollo embrionario y durante la progresión de varios cánceres epiteliales, y en la migración y la invasión de las células epiteliales mamarias.
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