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NOVEL GENE REGULATORY MECHANISMS WITH IMPLICATIONS IN CANCERKaja, Amala 01 May 2022 (has links)
Eukaryotic gene expression to proteins is a complex process that begins with transcription which is regulated by numerous regulatory factors and signals. Alterations in these regulatory factors that modulate gene expression are linked with a multitude of cellular pathologies including cancers. Thus, it is important to delineate these transcriptional regulatory mechanisms of gene expression. Therefore, a large number of studies have been aimed at understanding the mechanism of transcription at the level of initiation, elongation, and termination. In line with this, my dissertation work is focused towards elucidating novel regulatory mechanisms of transcription initiation and elongation. Our results illuminate genetically how TOR (target of rapamycin) signaling pathway regulates transcription initiation and hence, transcription, in response to nutrients. The process of transcription initiation at the promoter is followed by RNA polymerase II (Pol II) pausing at the promoter-proximal site for mRNA capping/quality control. Such promoter-proximal pausing of Pol II (paused Pol II) plays an important role in regulating transcription elongation. Our results unveil how paused Pol II is released to engage into productive elongation for mRNA synthesis. We show that the capping enzyme, Cet1, targets a transcription factor known as FACT (facilitates chromatin transcription) which subsequently recruits a transcription elongation factor, Paf1C (RNA polymerase II- associated factor 1 [Paf1] complex), to release the paused Pol II for productive transcription elongation for mRNA synthesis. During such transcription elongation, histones need to be evicted in front of Pol II and reassembled in the wake of Pol II, and this dynamic histone disassembly and reassembly are coordinated by a number of histone chaperones. The aforementioned transcription factor, FACT, is one such histone chaperone that plays a key role in histone reassembly during transcription elongation. Importantly, we find a new regulation of FACT, by the ubiquitin-proteasome system (UPS), and hence, histone dynamics at the coding sequence and transcription. Specifically, the Spt16 component of FACT is ubiquitinated by the E3 ubiquitin ligase San1, and subsequently degraded by the 26S proteasome in yeast. Such proteasomal regulation of Spt16 subunit of FACT regulates transcription, and impairment of this UPS regulation alters transcription, leading to cellular pathologies. Indeed, SPT16 has been found to be associated with a lot of cancers, and our results show that this proteasomal degradation of SPT16 is impaired in cancer cells. Further, upregulation of SPT16 is associated with alterations in transcription of genes linked to cancer. Subsequent to its synthesis, mRNA needs to be exported to cytoplasm for translation to proteins. Importantly, transcription elongation has been found to be coupled to mRNA export, and like elongation, mRNA export is also controlled by UPS. Our findings demonstrate the role of active transcription in the proteasomal degradation of a key mRNA export factor, Sub2, mediated via Mdm30 (an F-box protein), thus, enhancing our understanding of the UPS regulation of mRNA export. Taken together, my dissertation work elucidates novel regulatory mechanisms of gene expression in response to nutrients and UPS, with implications in cellular pathologies including cancers.
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Structural investigation of the histone chaperone complex FACT using genetically encoded crosslinkers in Saccharomyces cerevisiaeHoffmann, Christian 01 December 2014 (has links)
No description available.
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Histone H2B-R95A mutant identifies the pheromone pathway that signals cell cycle arrest during rapamycin responseAyachi, Sami 12 1900 (has links)
La rapamycine est un immunosuppresseur utilisé pour traiter plusieurs types de maladies dont le cancer du rein. Son fonctionnement par l’inhibition de la voie de Tor mène à des changements dans des processus physiologiques, incluant le cycle cellulaire. Chez Saccharomyces cerevisiae, la rapamycine conduit à une altération rapide et globale de l’expression génique, déclenchant un remodelage de la chromatine. Nous proposons que les modifications des histones peuvent jouer un rôle crucial dans le remodelage de la chromatine en réponse à la rapamycine. Notre objectif principal est d’identifier d’une banque de mutants d’histone les variantes qui vont échouer à répondre à la rapamycine dans une tentative de réaliser une caractérisation des modifications d’histone critiques pour la réponse à cette drogue. Ainsi, nous avons réalisé un criblage d’une banque de mutants d’histone et identifié plusieurs mutants d‘histone dont la résistance à la rapamycine a été altérée. Nous avons caractérisé une de ces variantes d’histone, à savoir H2B, qui porte une substitution de l’alanine en arginine en position 95 (H2B-R95A) et démontré que ce mutant est extrêmement résistant à la rapamycine, et non à d’autres drogues. Des immunoprécipitations ont démontré que H2B-R95A est défectueux pour former un complexe avec Spt16, un facteur essentiel pour la dissociation de H2A et H2B de la chromatine, permetant la réplication et la transcription par les ADN et ARN polymérases, respectivement. Des expériences de ChIP-Chip et de micropuce ont démontré que l’arginine 95 de H2B est requise pour recruter Spt16 afin de permettre l’expression d’une multitude de gènes, dont certains font partie de la voie des phéromones. Des évidences seront présentées pour la première fois démontrant que la rapamycine peut activer la voie des phéromones et qu’une défectuosité dans cette voie cause la résistante à cette drogue. / Rapamycin is an immunosuppressant used for treating many types of diseases such as kidney carcinomas. It works by inhibiting the Tor signaling pathway leading to changes in physiological processes, including cell cycle arrest. In Saccharomyces cerevisiae, rapamycin leads to a rapid and global alteration in gene expression, prompting chromatin remodeling. We propose that histone modification(s) might play a crucial role in remodeling of the chromatin in response to rapamycin. Our main objective is to identify from a histone mutant collection variants that fail to respond to rapamycin in an attempt to characterize histone modifications critical for this drug response. As such, we conducted a screen of the histone mutant collection and identified several hits that showed resistance to rapamycin. We characterized one of the histone variants, namely H2B, carrying alanine substitution at arginine 95 (H2B-R95A) and show that it is extremely resistant to rapamycin, but not to other drugs. Pull downs demonstrated that H2B-R95A was defective in forming a complex with Spt16, an essential factor that is required to disassociate H2A and H2B from the chromatin in order to allow replication and transcription by DNA and RNA polymerases, respectively. ChIP-Chip and microarray experiments showed that arginine 95 of H2B is required to recruit Spt16 to allow expression of several genes, a subset of which are involved in the pheromone signaling pathway. Evidence will be presented to show for the first time that rapamycin can activate the pheromone pathway and that defects in this pathway cause resistance to the drug.
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