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Investigating the role of human HAT (histone acetyltransferase) containing complexes, ATAC and SAGA, in living cells / Etude du rôle des complexes HAT (histone acetyltransferase) humains, ATAC et SAGA, dans les cellules vivantesVosnakis, Nikolaos 16 December 2014 (has links)
Les complexes acétyltransférases (HAT), SAGA et ATAC, sont des régulateurs de la transcription des gènes. Cependant, peu d’études ont été menées sur la dynamique de ces complexes au niveau cellulaire et sur les mécanismes régulant leur assemblage. Au cours de mes travaux de thèse, j’ai utilisé des approches d’imagerie sur cellules vivantes, afin de déterminer la mobilité de ces complexes en comparaison avec celle d’autres régulateurs transcriptionnels. Les résultats ont montré que les sous-unités de SAGA et ATAC interagissent de manière transiente avec la chromatine. En complément, nous avons montré que les sous-unités spécifiques de SAGA et ATAC (ADA2b et ADA2a) ont des propriétés dynamique intracellulaire différentes et que GCN5, affecte la distribution d’ADA2a. Des analyses protéomique menées sur le comportement de ces protéines au niveau endogène, ont permis de montrer que les voies d’assemblage de ces deux complexes étaient différentes au niveau cytoplasmique et nucléaire. / Human SAGA and ATAC, are histone acetyltransferase (HAT) containing complexes that share a set of subunits and facilitate RNA polymerase II (Pol II) transcription. Little is known for the dynamics of the complexes in living cells and the regulation of their assembly. In this work, we used live-cell imaging to characterise the mobility of the two complexes and compare it with other actors of Pol II transcription. All tested ATAC and SAGA subunits exhibit very transient interactions with chromatin, a property that explains certain aspects of the function of the complexes. Moreover, we showed that overexpressed ATAC- and SAGA-specific HAT-module subunits (ADA2a and ADA2b respectively) have different intracellular dynamics and that the abundance of the shared subunit GCN5, affects the distribution of ADA2a. Quantitative proteomic analysis expanded our findings on endogenous proteins and provided evidence that the cytoplasmic and nuclear assembly pathways of SAGA and ATAC are different.
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Analyse bioinformatique des modifications post-traductionnelles du domaine carboxyl-terminal de l'Arn polymérase II / Bioinformatic analysis of post-translational modifications of the carboxy-terminal domain of RNA polymerase IIDescostes, Nicolas 12 December 2014 (has links)
Le processus transcriptionnel par l'ARN polymérase II (Pol II) chez les eucaryotes se déroule en trois étapes : L'initiation, l'élongation et la terminaison. De nombreux facteurs de transcription, des modifications de la chromatine (épigénétique) et des éléments régulateurs distants interviennent dans ce processus. La sous-unité RPB1 de l'ARN Pol II contient un domaine carboxyle terminale (CTD) composée d'une répétition de sept acides-aminés. Au travers de différentes modifications biochimiques, ce domaine coordonne le processus transcriptionnel par le recrutement de différents facteurs. Le CTD est également impliqué dans la coordination de la transcription au niveau de l'initiation, de l'élongation et de la terminaison par le biais de modifications épigénétiques et nucléosomales, mais aussi par l'action de régulateurs distants (enhancers) et probablement de changements de conformation tridimensionnelle du génome. Mon travail de thèse a consisté en l'étude de deux modifications biochimiques du CTD de l'ARN Pol II par traitement bioinformatique de données issues du séquençage haut-débit. J'ai pu montrer que la phosphorylation de la thréonine 4 influence l'élongation de la transcription chez l'humain. J'ai également montré que la phosphorylation de la tyrosine 1 est présente durant l'initiation, est préférentiellement localisée dans la direction anti-sens, est hyper-phosphorylée aux enhancers transcrits et tissus spécifiques et est une marque caractéristique de ces modules génomiques. Ce travail de doctorat a constitué une contribution à la compréhension du processus transcriptionnel chez l'humain par l'utilisation de méthodes bioinformatiques innovantes. / The biggest subunit of eukaryotic RNA polymerase II contains a carboxy-terminal domain (CTD) that consists in a repetition of seven amino-acids ranging from 26 in yeast to 52 in mammals. Specific biochemical modifications of CTD residues have been linked to specific stages of the transcriptional process. The CTD acts as a recruitment platform for processing factors that are involved in initiation, promoter proximal pausing, early and productive elongation (alternative splicing), 3' processing, termination and epigenetics.During my PhD, I used bioinformatics and high-throughput sequencing data to study two novel biochemical modifications of the CTD in human. I showed, in collaboration with biologists and bioinformaticians, that threonine 4 phosphorylation is important for proper elongation and probably termination of transcription. I showed also that tyrosine 1 phosphorylation is present during early transcription, antisense transcription (at divergent promoters) and is hyperphosphorylated at transcribed and tissue specific enhancers.Overall my doctorate has contributed to the understanding of the transcriptional process in human through the use of innovative bioinformatic methods.
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RNA Polymerase II identifies enhancers in different states of activationCaglio, Giulia 15 May 2019 (has links)
Enhancer regulieren die Transkription ihrer Zielgene und deren Expression. Sie bieten eine Bindestelle für verschiedenste Transkriptionsfaktoren (TF) und RNA Polymerase II (RNAPII) und unterstützen die Gentranskription durch das Zustandekommen von Chromatinkontakten. Zusätzlich transkribiert RNAPII in Enhancer-Regionen kurze, non-polyadenylierte Transkripte, die man Enhancer-RNA (eRNA) nennt. Der Mechanismus der RNAPII-Rekrutierung und –Regulation an Enhancern ist bisher wenig verstanden, insbesondere wie das Vorhandensein von RNAPII-Modifikationen den Chromatinstatus, -faltung sowie die Genaktivierung beeinflusst.
In dieser Arbeit wurden verschiedene Ansätze der Enhancer-Bestimmung miteinander verglichen. Während eine klare Bestimmung des besten Ansatzes sich als komplex erwies, konnte gezeigt werden, dass die Bindung von RNAPII an regulatorische Regionen in Zusammenhang mit TF eine universelle Konstante darstellte. Weiterhin wurden der Status der Enhancer-gekoppelten RNAPII-Aktivierung und deren Transkriptionsaktivität untersucht. Als Hauptergebnis ergab sich, dass der RNAPII-Status mit der Enhancer-Aktivität und daraus folgend mit veränderter Transkriptionsaktivität korreliert ist. Weiterhin konnte gezeigt werden, dass das Vorhandensein extragenischer RNAPII ein neues Werkzeug zur Identifikation von regulatorischen Regionen ist. Erfolgreich konnten regulatorische Regionen in embryonalen Stammzellen der Maus sowie während der neuronalen Differenzierung vorhergesagt und mittels Enhancer-Aktivität in-vivo bestätigt werden. Dabei zeigte sich, dass im Laufe der der neuronalen Differenzierung extragenische RNAPII-Bindung spezifische Aktivierungsmuster aufweist: ihr Transkriptionslevel wird durch Kinasen feinmaschig reguliert und es werden verschiedene Formen maturierter RNA erzeugt.
Zusammenfassend konnte RNAPII als Werkzeug zur Identifikation und Charakterisierung regulatorischer Regionen in verschiedenen Zelltypen ausgemacht werden. Selbst mit minimalen RNAPII-Datensätzen ist es möglich, gleichzeitig regulatorische Regionen zu identifizieren als auch ihren eigenen Aktivierungsstatus sowie den ihrer kodierender Genpromotoren zu bestimmen. / Enhancers regulate transcription of target genes and gene expression. They act as recruitment sites for multiple transcription factors (TFs) and RNA polymerase II (RNAPII) and favour transcription of target genes through chromatin contacts. RNAPII at enhancer regions transcribes short and mostly non-polyadenylated transcripts, called enhancer RNAs (eRNAs).
The mechanisms of RNAPII recruitment and regulation at enhancers remain ill understood, in particular how signalling through RNAPII modifications may influence chromatin states, looping and gene activation. In this study, I compare enhancer lists defined with different approaches and find that their relation is very complex. However, I find that RNAPII binding co-occurs with TF binding at regulatory regions, independently of the identification approach used. I characterize the state of RNAPII activation at enhancers and its transcriptional activity. I find that RNAPII state reflects enhancer activation state and correlates with different transcriptional outputs. In addition, I demonstrate that extragenic RNAPII is a novel tool to identify regulatory regions. I successfully identified putative regulatory regions in mESC and during neuronal differentiation, with enhancer activity in vivo. Extragenic RNAPII regions have specific activation patterns during neuronal differentiation, are finely regulated at the transcriptional level by kinases and transcribe differently mature RNAs.
In conclusion, I establish RNAPII as a tool to identify and characterise regulatory regions in a cell type of interest. With minimal RNAPII datasets it is possible to simultaneously identify regulatory regions, infer their state of activation, and the state of activation of coding gene promoters.
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Role of R-loops in pause-dependent transcriptional termination of RNA polymerase IISkourti-Stathaki, Konstantina January 2012 (has links)
Transcription termination of RNA polymerase II (Pol II) in mammals requires a functional poly(A) signal and either downstream pause sites or co-transcriptional cleavage (CoTC) sequences together with 3’transcript degradation by the nuclear 5’-3’ exonuclease Xrn2. However the molecular mechanism of pause-dependent transcriptional termination is not yet fully understood. This thesis investigates the molecular role of R-loop structures in pause-dependent transcriptional termination of mammalian genes. The results described in Chapters 3 and 4 indicate that nascent transcripts form RNA/DNA hybrid structures (R-loops) behind elongating Pol II and are especially prevalent over G-rich pause sites positioned downstream of gene poly(A) signals. Senataxin, a helicase protein and the human homologue of the yeast Sen1, acts to resolve these R-loop structures and by so doing allows access of Xrn2 at 3’ cleavage poly(A) sites. This ultimately leads to efficient Pol II termination. In effect R-loops formed over G-rich pause sites, followed by their resolution by senataxin, are required for efficient pause-dependent transcriptional termination. In addition to this, the 3’ end processing factor, Pcf11 is also involved in this process. Experiments presented in the final part of this study reveal a link between R-loops and RNAi-dependent H3K9me2 formation over G-rich termination regions. Overall my results suggest that R-loop structures and the H3K9me2 repressive mark over pause regions are important features of Pol II pause-dependent transcriptional termination of mammalian genes.
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Role promotoru při regulaci RNA sestřihu / Role of promoter in the regulation of alternative splicingKozáková, Eva January 2014 (has links)
It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...
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Investigation of the role of essential proteins in gene silencing at the centromere of Schizosaccharomyces pombeDobbs, Edward January 2012 (has links)
The centromeres of eukaryotes have a region on which the kinetochore is assembled, flanked by heterochromatin which provides cohesion between the sister chromatids during cell division. When centromeric heterochromatin is lost chromosomes no longer segregate evenly into the daughter cells during cell division. In the fission yeast Schizosaccharomyces pombe (S. pombe) RNA interference (RNAi) is responsible for maintaining this heterochromatin. The pathway is part of a feedback loop whereby siRNAs generated from non-coding centromere transcripts are loaded into an Argonaute complex. The siRNAs guide the complex to the homologous centromere repeats in order to recruit Clr4 which modifies histone H3 with the heterochromatin mark H3K9me. A previous screen to find factors affecting centromere silencing isolated 13 loci termed centromere: suppressor of position-effect (csp) 1-13. Several csp mutants have been identified to be RNAi components. In this investigation the csp6 locus has been identified to be the Hsp70 gene ssa2+. It has been demonstrated that Argonaute proteins from plants and flies require Hsp70/90 chaperone activity for loading of siRNA. It therefore seems likely that Hsp70 may play a similar role in fission yeast. Genetic and biochemical techniques have been used in this study to investigate if the csp6 alleles are affecting siRNA loading in S. pombe. RNA Polymerase II (RNAPII) transcribes the pre-siRNA transcripts from the centromere repeats. csp3 was identified to be an allele of the RNAPII subunit rpb7+. rpb7-G150D was found to cause a silencing defect in the centromeric heterochromatin through a defect in transcription. Another RNAPII mutation, rpb2-m203, was found to have strong silencing defects caused by an unidentified non-transcriptional role in RNAi-mediated heterochromatin formation at the centromere. In order to gain more insight into the role of RNAPII in heterochromatin assembly I performed a screen in which the subunits rpb3 and rpb11 were subjected to random mutagenesis. Several mutants were isolated and characterisation of phenotypes regarding heterochromatin at the centromere has been carried out for nine of the mutants. As a result a novel phenomenon of RNAi-independent silencing at the centromere has been discovered.
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Structural and Functional Investigation of Promoter Distortion and Opening in the RNA Polymerase II CleftDienemann, Christian 09 April 2018 (has links)
No description available.
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Investigating the role of mRNA capping enzyme in C-MYC functionLombardi, Olivia January 2017 (has links)
C-MYC is a transcription factor and a potent driver of many human cancers. In addition to regulating transcription, C-MYC promotes formation of the mRNA cap which is important for transcript maturation and translation. However, the mechanistic details of C-MYC-dependent mRNA capping are not fully understood. Since anti-cancer strategies to directly target the C-MYC protein have had limited success, enzymatic co-factors or effectors of C-MYC present attractive alternatives for therapeutic intervention of C-MYC-driven cancers. mRNA capping enzyme (CE) initiates mRNA cap formation by catalysing the linkage of inverted guanosine via a triphosphate bridge to the first transcribed nucleotide. The involvement of CE in C-MYC-dependent mRNA capping and C-MYC function has not yet been explored. Therefore, I sought to determine whether C-MYC regulates CE, and whether CE is required for C-MYC function. I found that C-MYC promotes CE recruitment to RNA polymerase II (RNA pol II) transcription complexes and to regions proximal to transcription start sites on chromatin. Consistently, C-MYC increases RNA pol II-associated CE activity. Interestingly, cells driven by C-MYC are highly dependent on CE for C-MYC-induced target gene expression and cell transformation, but only when C-MYC is overexpressed; C-MYC-independent cells or cells retaining normal control of C-MYC expression are insensitive to CE inhibition. C-MYC expression is also dependent on CE. Taken together, I present a bidirectional regulatory relationship between C-MYC and CE which is potentially therapeutically relevant. Studies here strongly suggest that inhibiting CE is an attractive strategy to selectively target cancer cells which have acquired deregulated C-MYC.
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Regulation of MYC Activity by the Ubiquitin-Proteasome System / Regulation der MYC Aktivität durch das Ubiquitin-Proteasom-SystemJänicke, Laura Annika January 2015 (has links) (PDF)
The oncogenic MYC protein is a transcriptional regulator of multiple cellular processes and is aberrantly activated in a wide range of human cancers. MYC is an unstable protein rapidly degraded by the ubiquitin-proteasome system. Ubiquitination can both positively and negatively affect MYC function, but its direct contribution to MYC-mediated transactivation remained unresolved.
To investigate how ubiquitination regulates MYC activity, a non-ubiquitinatable MYC mutant was characterized, in which all lysines are replaced by arginines (K-less MYC). The absence of ubiquitin-acceptor sites in K-less MYC resulted in a more stable protein, but did not affect cellular localization, chromatin-association or the ability to interact with known MYC interaction partners.
Unlike the wild type protein, K-less MYC was unable to promote proliferation in immortalized mammary epithelial cells. RNA- and ChIP-Sequencing analyses revealed that, although K-less MYC was present at MYC-regulated promoters, it was a weaker transcriptional regulator. The use of K-less MYC, a proteasomal inhibitor and reconstitution of individual lysine residues showed that proteasomal turnover of MYC is required for MYC target gene induction. ChIP-Sequencing of RNA polymerase II (RNAPII) revealed that MYC ubiquitination is dispensable for RNAPII recruitment and transcriptional initiation but is specifically required to promote transcriptional elongation. Turnover of MYC is required to stimulate histone acetylation at MYC-regulated promoters, which depends on a highly conserved region in MYC (MYC box II), thereby enabling the recruitment of BRD4 and P-TEFb and the release of elongating RNAPII from target promoters. Inhibition of MYC turnover enabled the identification of an intermediate in MYC-mediated transactivation, the association of MYC with the PAF complex, a positive elongation factor, suggesting that MYC acts as an assembly factor transferring elongation factors onto RNAPII. The interaction between MYC and the PAF complex occurs via a second highly conserved region in MYC’s amino terminus, MYC box I.
Collectively, the data of this work show that turnover of MYC coordinates histone acetylation with recruitment and transfer of elongation factors on RNAPII involving the cooperation of MYC box I and MYC box II. / Der Transkriptionsfaktor MYC ist an der Regulation einer Vielzahl biologischer Prozesse beteiligt ist und spielt bei der Tumorentstehung und des Tumorwachstum eine entscheidende Rolle. MYC ist ein kurzlebiges Protein, das durch das Ubiquitin-Proteasom-System abgebaut wird. Die Ubiquitinierung von MYC hat auch einen stimulierenden Einfluss auf dessen transkriptionelle Aktivität. Dabei blieb jedoch der Mechanismus, der dieser Beobachtung zugrunde liegt, bislang ungeklärt.
Um den direkten Einfluss von Ubiquitinierung auf die Aktivität von MYC zu untersuchen, wurde in der vorliegenden Arbeit eine MYC Mutante analysiert, in der alle Lysine zu Argininen mutiert wurden (K-less MYC). Die Mutation der Ubiquitin-Verknüpfungsstellen resultierte in einem stabileren Protein, hatte jedoch keinen Einfluss auf die zelluläre Lokalisation oder Assoziation mit bekannten Interaktionspartnern. Im Vergleich zu Wildtyp (WT) MYC war K-less MYC jedoch in der Vermittlung MYC-induzierter biologischer Phänotypen stark beeinträchtigt. Mittels RNA- und ChIP-Sequenzierungen konnte gezeigt werden, dass K-less MYC zwar an MYC-regulierte Promotoren bindet, in der transkriptionellen Aktivität aber stark beeinträchtigt ist und diese Zielgene nicht aktivieren kann. Dabei war K-less MYC noch in der Lage, RNA Polymerase II (RNAPII) zu den Zielpromotoren zu rekrutieren und die Transkription dort zu initiieren, jedoch war der Übergang zur Elongation blockiert. Die Verwendung eines Proteasom-Inhibitors sowie die Rekonstitution einzelner Lysine in K-less MYC zeigten, dass der proteasomale Abbau von MYC für die Aktivierung von Zielgenen benötigt wird. Der proteasomale Abbau ist für die Histon-Acetylierung von Bedeutung, die von einer hoch konservieren Region in MYC, der MYC Box II, abhängt. Durch die WT MYC-vermittelte Induktion der Histon-Acetylierung können folglich die Proteine BRD4 und P-TEFb an die Promotoren rekrutiert werden. Diese Proteine spielen bei dem Übergang der initiierenden RNAPII zur elongierenden RNAPII eine essentielle Rolle.
Darüber hinaus ermöglichte die Inhibition des MYC Abbaus die Identifizierung eines Zwischenschritts der MYC-abhängigen Transaktivierung: die Assoziation von MYC mit dem positiven Elongationskomplex, dem PAF-Komplex. Dieser wird über eine zweite hochkonservierte Region in MYC, der MYC Box I, rekrutiert. Somit kann angenommen werden, dass MYC als eine Verbindungsstelle fungiert, die positive Elongationsfaktoren auf die RNAPII transferiert.
Zusammenfassend resultieren die Daten dieser Arbeit in einem Model, nach dem der proteasomale Abbau von MYC die Histon-Acetylierung mit der Rekrutierung und dem Transfer von Elongationsfaktoren auf die RNAPII koordiniert, was der Kooperation von MYC Box I und MYC Box II bedarf.
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Endogenous gypsy insulators mediate higher order chromatin organization and repress gene expression in DrosophilaZhang, Shaofei 01 August 2011 (has links)
Chromatin insulators play a role in gene transcription regulation by defining chromatinboundaries. Genome-wide studies in Drosophila have shown that a large proportion of insulator sites are found in intergenic DNA sequences, supporting a role for these elements as boundaries. However, approximately 40% of insulator sites are also found in intragenic sequences, where they can potentially perform as yet unidentified functions. Here we show that multiple Su(Hw) insulator sites map within the 110 kb sequence of the muscleblind gene (mbl), which also forms a highly condensed chromatin structure in polytene chromosomes. Chromosome Conformation Capture assays indicate that Su(Hw) insulators mediate the organization of higher-order chromatin structures at the mbl locus, resulting in a barrier for the progression of RNA polymeraseII (PolII ), and producing a repressive effect on basal and active transcription. The interference of intragenic insulators in PolII progression suggests a role for insulators in the elongation process. Supporting this interpretation, we found that mutations in su(Hw) and mod(mdg4) also result in changes in the relative abundance of the mblD isoform, by promoting early transcription termination. These results provide experimental evidence for a new role ofintragenic Su(Hw) insulators in higher-order chromatin organization, repression of transcription, and RNA processing.
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