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  • 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.
21

The Shape of Silence: The Solution-State Conformation of Sir Heterochromatin: A Dissertation

Swygert, Sarah G. 20 August 2015 (has links)
Heterochromatin is a silenced chromatin region essential for maintaining genomic stability in eukaryotes and for driving developmental processes in higher organisms. A hallmark of heterochromatin is the presence of specialized architectural proteins that alter chromatin structure to inhibit transcription and recombination. Although it is generally assumed that heterochromatin is highly condensed, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, heterochromatin assembly at telomeres and the HM silent mating type loci requires the Sir proteins: Sir3, believed to be the major structural component of SIR heterochromatin, and the Sir2/4 complex, responsible for SIR recruitment to silencing regions and deacetylation of lysine 16 of the histone H4 tail, a mark associated with active chromatin. A combination of sedimentation velocity, atomic force microscopy, and nucleosomal array capture was used to characterize the stoichiometry and conformation of SIR nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome, and that Sir2/4 may additionally bind at a ratio of one per nucleosome. Despite Sir3’s ability to repress transcription in vivo and homologous recombination in vitro in the absence of Sir2/4, Sir3 fibers were found to be significantly less compact than canonical magnesium-induced 30 nanometer fibers. However, heterochromatin fibers composed of all three Sir proteins did adopt a more condensed, globular structure. These results suggest that heterochromatic silencing is mediated both by the creation of more stable nucleosomes and by the steric exclusion of external factors.
22

Recombinational Repair of a Chromosomal DNA Double Strand Break: A Dissertation

Sinha, Manisha 16 March 2009 (has links)
Repairing a chromosomal DNA double strand break is essential for survival and maintenance of genomic integrity of a eukaryotic organism. The eukaryotic cell has therefore evolved intricate mechanisms to counteract all sorts of genomic insults in the context of chromatin structure. Modulating chromatin structure has been crucial and integral in regulating a number of conserved repair processes along with other fundamental genomic processes like replication and transcription. The work in this dissertation has focused on understanding the role of chromatin remodeling enzymes in the repair of a chromosomal DNA double strand break by homologous recombination. This has been approached by recapitulating the biochemical formation of recombination intermediates on chromatin in vitro. In this study, we have demonstrated that the mere packaging of DNA into nucleosomal structure does not present a barrier for successful capture of homologous DNA sequences, a central step of the biochemical pathway of recombinational repair. It is only the assembly of heterochromatin-like more complex nucleo-protein structure that presents additional constraints to this key step. And, this additional constraint can be overcome by the activities of ATP-dependent chromatin remodeling enzymes. These findings have great implications for our perception of the mechanism of the recombinational repair process of a chromosomal DNA double strand break within the eukaryotic genome.
23

ATP-Dependent Heterochromatin Remodeling: A Dissertation

Manning, Benjamin J. 11 September 2015 (has links)
Eukaryotic DNA is incorporated into the nucleoprotein structure of chromatin. This structure is essential for the proper storage, maintenance, regulation, and function of the genomes’ constituent genes and genomic sequences. Importantly, cells generate discrete types of chromatin that impart distinct properties on genomic loci; euchromatin is an open and active compartment of the genome, and heterochromatin is a restricted and inactive compartment. Heterochromatin serves many purposes in vivo, from heritably silencing key gene loci during embryonic development, to preventing aberrant DNA repeat recombination. Despite this generally repressive role, the DNA contained within heterochromatin must still be repaired and replicated, creating a need for regulated dynamic access into silent heterochromatin. In this work, we discover and characterize activities that the ATP-dependent chromatin remodeling enzyme SWI/SNF uses to disrupt repressive heterochromatin structure. First, we find two specific physical interactions between the SWI/SNF core subunit Swi2p and the heterochromatin structural protein Sir3p. We find that disrupting these physical interactions results in a SWI/SNF complex that can hydrolyze ATP and slide nucleosomes like normal, but is defective in its ability to evict Sir3p off of heterochromatin. In vivo, we find that this Sir3p eviction activity is required for proper DNA replication, and for establishment of silent chromatin, but not for SWI/SNF’s traditional roles in transcription. These data establish new roles for ATP-dependent chromatin remodeling in regulating heterochromatin. Second, we discover that SWI/SNF can disrupt heterochromatin structures that contain all three Sir proteins: Sir2p, Sir3p and Sir4p. This new disruption activity requires nucleosomal contacts that are essential for silent chromatin formation in vivo. We find that SWI/SNF evicts all three heterochromatin proteins off of chromatin. Surprisingly, we also find that the presence of Sir2p and Sir4p on chromatin stimulates SWI/SNF to evict histone proteins H2A and H2B from nucleosomes. Apart from discovering a new potential mechanism of heterochromatin dynamics, these data also establish a new paradigm of chromatin remodeling enzyme regulation by nonhistone proteins present on the substrate.
24

Dissecting cis and trans Determinants of Nucleosome Positioning: A Dissertation

Hughes, Amanda L. 14 November 2014 (has links)
Eukaryotic DNA is packaged in chromatin, whose repeating subunit, the nucleosome, consists of an octamer of histone proteins wrapped by about 147bp of DNA. This packaging affects the accessibility of DNA and hence any process that occurs on DNA, such as replication, repair, and transcription. An early observation from genome-wide nucleosome mapping in yeast was that genes had a surprisingly characteristic structure, which has motivated studies to understand what determines this architecture. Both sequence and trans acting factors are known to influence chromatin packaging, but the relative contributions of cis and trans determinants of nucleosome positioning is debated. Here we present data using genetic approaches to examine the contributions of cis and trans acting factors on nucleosome positioning in budding yeast. We developed the use of yeast artificial chromosomes to exploit quantitative differences in the chromatin structures of different yeast species. This allows us to place approximately 150kb of sequence from any species into the S.cerevisiae cellular environment and compare the nucleosome positions on this same sequence in different environments to discover what features are variant and hence regulated by trans acting factors. This method allowed us to conclusively show that the great preponderance of nucleosomes are positioned by trans acting factors. We observe the maintenance of nucleosome depletion over some promoter sequences, but partial fill-in of NDRs in some of the YAC v promoters indicates that even this feature is regulated to varying extents by trans acting factors. We are able to extend our use of evolutionary divergence in order to search for specific trans regulators whose effects vary between the species. We find that a subset of transcription factors can compete with histones to help generate some NDRs, with clear effects documented in a cbf1 deletion mutant. In addition, we find that Chd1p acts as a potential “molecular ruler” involved in defining the nucleosome repeat length differences between S.cerevisiae and K.lactis. The mechanism of this measurement is unclear as the alteration in activity is partially attributable to the N-terminal portion of the protein, for which there is no structural data. Our observations of a specialized chromatin structure at de novo transcriptional units along with results from nucleosome mapping in the absence of active transcription indicate that transcription plays a role in engineering genic nucleosome architecture. This work strongly supports the role of trans acting factors in setting up a dynamic, regulated chromatin structure that allows for robustness and fine-tuning of gene expression.
25

Regulation of replication dependent nucleosome assembly

Gopinathan Nair, Amogh 04 1900 (has links)
Chez les cellules humaines, environ 2 mètres d'ADN est compacté dans le noyau cellulaire par la formation d'une structure nucléoprotéique appelée chromatine. La chromatine est composée d'ADN enroulé à la surface d'un octamère de core histones pour former une structure appelée nucléosome. La structure de la chromatine doit être altérée afin d'accéder à l'information génétique pour sa réplication, sa réparation et sa transcription. La duplication de la chromatine lors de la phase S est cruciale pour la prolifération et la survie des cellules. Cette duplication de la chromatine requière une ségrégation des histones parentales, mais aussi une déposition d'histones néo-synthétisées sur l'ADN. Ces deux réactions résultent en formation de chromatine dès qu'une quantité suffisante d'ADNest générée par la machinerie de réplication. De plus, en raison de conditions intrinsèques et extrinsèques, la machinerie de réplication est souvent confrontée à de nombreux obstacles, sous la forme de lésions à l'ADN qui interfèrent avec la réplication de l'ADN. Sous ces conditions, l'assemblage de nucléosomes et la synthèse d'histones sont étroitement régulées afin d'éviter la production d'un excès d'histones et leurs nombreuses conséquences nuisibles à la cellule. "Chromatin Assembly Factor 1" (CAF-1) est responsable de la déposition initiale des molécules d'H3 et H4 derrière les fourches de réplication. Pour permettre sa fonction d'assemblage de chromatine, CAF-1 est localisée aux fourches de réplication en vertue de sa liaison à une protéine appelée Proliferating Cell Nuclear Antigen (PCNA). Cependant, le mécanisme moléculaire par lequel CAF-1 exerce sa function demeure mal compris. Dans le deuxième chapitre de ma thèse, j'ai exploré comment CAF-1 se lie à PCNA d'une manière distincte des nombreux autres partenaires de PCNA. Grâce à nos collaborateurs, des études de crystallographie ont démontré que CAF-1 se lie à PCNA grâce à une interaction non-canonique entre le "PCNA Interaction Peptide" (PIP) de CAF-1 et une interaction de type cation-pi (π). Nous avons aussi montré qu'une substitution d'un seul acide aminé, unique au PIP de CAF-1, abolit son interaction avec PCNA et sa capacité d'assemblage de nuclésomes. Nous avons aussi montré que le PIP de CAF-1 est situé à l'extrémité C-terminale d'une très longue hélice alpha qui est conservée à travers l'évolution parmi de nombreux homologues de CAF-1. Nos études biophysiques ontmontré que cette longue hélice alpha forme des structures oligomériques de type "coiled-coil", ce qui suggère certains mécanismes pour dédier un anneau de PCNA à l'assemblage de chromatine et ce, en dépit des nombreux intéracteurs de PCNA présents aux fourches de réplication. Dans le troisième chapitre de ma thèse, nos collaborateurs et moi-même avons étudié les mécanismes moléculaires par lesquels les cellules parviennent à maintenir un équilibre délicat entre la synthèse d'ADN et la synthèse d'histones et ce, même en présence de lésions à l'ADN qui interfèrent avec la réplication. Chez Saccharomyces cerevisiae, nous avons montré que les kinases de réponse au dommage à l'ADN, Mec1/Tel1 et Rad53, inhibent la transcription des gènes d'histones en réponse aux liaisons à l'ADN qui interfèrent avec la réplication. Nous avons montré que la répression des gènes d'histones induite par le dommage à l'ADN est médiée par une phosphorylation extensive de Hpc2, l'une des sous-unités du complexe "Histone Gene Repressor" (HIR). Hpc2 contient un domaine qui se lie à l'histone H3. À partir de la structure d'Hpc2, nous avons généré des mutants qui, d'après la structure, sont incapables de se lier à l'histone H3. Nos résultats montrent que l'accumulation d'histones en excès provoquée par le dommage à l'ADN entraîne la phosphorylation d'Hpc2 and la liaison de l'excès d'histone H3 à Hpc2. Ces résultats suggèrent que la répression transcriptionnelle des gènes d'histones induite par le dommage à l'ADN est médiée, du moins en partie, par une simple rétroaction négative impliquant la liaison des histones en excès à la sous-unité Hpc2 du complexe HIR. / In human cells, roughly 2 meters of DNA is compacted into the cell nucleus by the formation of a nucleoprotein complex called chromatin. Chromatin is composed of DNA wrapped around an octamer of core histones to form so-called nucleosomes. Chromatin structure needs to be altered to access genetic information for processes like replication, repair and transcription. Duplication of chromatin during S phase is vital for cell proliferation and viability. Chromatin duplication requires segregation of parental histones, but also deposition of newly synthesized histones onto DNA. This process results in packaging all of the synthesized DNA with histones to form nucleosomes as soon as enough nascent DNA has emerged from the replication machinery. Moreover, as a result of intrinsic and extrinsic conditions, the replication machinery often encounters DNA lesions that impede the continuous synthesis of DNA. Under these conditions, nucleosome assembly and histone synthesis are tightly regulated to prevent the production of an excess of histone proteins and their deleterious consequences. Chromatin Assembly Factor-1 (CAF-1) performs the initial step in chromatin assembly by depositing newly synthesized histone H3-H4 molecules behind replication forks. In order to perform its chromatin assembly function, CAF-1 localizes to DNA replication forks by binding directly to a protein known as the Proliferating Cell Nuclear Antigen (PCNA). However, the exact molecular mechanism by which this is achieved remains poorly understood. Through the second chapter of my thesis, I have explored how CAF-1 binds PCNA in a manner that is distinct from the numerous other binding partners of PCNA. With the help of our collaborators, crystallographic studies demonstrated that CAF-1 binds to PCNA by virtue of a non-canonical PCNA interaction peptide (PIP) and a cation-pi (π) interaction. We have also shown that a single amino acid substitution, unique to the PIP of CAF-1, disrupts its binding to PCNA and chromatin assembly activity. We found that the CAF-1 p150 PIP resides at the extreme C-terminus of a long alpha helix that is evolutionarily conserved among numerous homologues of CAF-1. Our biophysical studies showed that this long alpha-helix is capable of forming higher-order coiled coils, which suggests mechanisms to dedicate one PCNA ring for chromatin assembly despite the presence of multiple PCNA interactors at replication forks. In the third chapter of this thesis, our collaborators and I have addressed the crucial molecular mechanisms by which cells maintain a delicate balance between DNA and histone synthesis despite the presence of DNA lesions that interfere with replication. In Saccharomyces cerevisiae, we showed that the DNA damage response kinases Mec1/Tel1 and Rad53 inhibit histone gene transcription when DNA lesions block DNA replication. We also showed that this repression is mediated by phosphorylation of the Hpc2 subunit of the Histone Gene Repressor complex (HIR). Hpc2 contains a domain that directly binds to histone H3. Interestingly, structure-based mutants of Hpc2 predicted to be incapable of binding H3 are defective in DNA damage-induced transcriptional repression of histone genes in response to DNA damage during replication. Our results indicate that the accumulation of excess histones caused by DNA damage during S phase triggers extensive phosphorylation of Hpc2 and binding of excess H3 to Hpc2. This suggests that DNA damage-induced repression of histone genes is mediated, at least in part, by a simple negative feedback triggered by binding of excess histones to the Hpc2 subunit of the HIR complex.
26

Le rôle de la structure de la chromatine naissante dans la réponse au stress réplicatif

Simoneau, Antoine 12 1900 (has links)
No description available.
27

Biochemical and functional differences of chromatin assembled replication-coupled or independent in Xenopus laevis egg extracts / Biochemische und funktionelle Unterschiede von Chromatin assembliert replikationsabhängig oder -unabhängig in Xenopus laevis Eiextrakten

Stützer, Alexandra 07 June 2011 (has links)
No description available.
28

A Role for Histone Modification in the Mechanism of Action of Antidepressant and Stimulant Drugs: a Dissertation

Schroeder, Frederick Albert 28 December 2007 (has links)
Depression and stimulant drug addiction each result in massive losses of health, productivity and human lives every year. Despite decades of research, current treatment regimes for depression are ineffective in approximately half of all patients. Therapy available to stimulant drug addicts is largely ineffective and moreover, dedicated treatments for drug dependence (including abuse of cocaine) are non-existent. Thus, there is a pressing need to further understanding of the molecular mechanisms underlying these disorders in order to develop novel, targeted therapeutic strategies. Chromatin remodeling, including changes in histone acetylation, has been proposed to play a role in both the etiology and treatment of depression and stimulant abuse. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate numerous cellular processes, including transcription, cell cycle progression and differentiation. Moreover, histone acetylation has been shown to regulate hippocampal neurogenesis, a cellular response associated with the pathogenesis and treatment of depression and stimulant abuse (Hsieh et al., 2004, Yamaguchi et al., 2004, Fischer et al., 2007). Ultimately, such basic cellular processes impact higher order function, namely cognition and emotion. Indeed, recent studies suggest that HDAC activity in selected forebrain regions, including ventral striatum and hippocampus, modulate stimulant- and antidepressantinduced behavior (Kumar et al., 2005, Tsankova et al., 2006a, Fischer et al., 2007). These reports highlight an association between chromatin remodeling and diverse behavioral changes, including changes induced by the pleiotropic HDAC inhibitor, sodium butyrate (SB), (Kumar et al., 2005, Tsankova et al., 2006a, Fischer et al., 2007). However, behavioral, brain-metabolic and molecular effects of SB treatment in the context of rodent models of depression, dopaminergic sensitization and repeated cocaine administration remained unclear. The work described in this thesis illustrates the potential for chromatin modifying drugs in mechanisms underlying the experimental pharmacology of depression and stimulant addiction. Specifically, the data presented here support the view that treatment with the short chain fatty acid, sodium butyrate enhances: (1) antidepressant-like behavioral effects of the selective serotonin reuptake inhibitor (SSRI), fluoxetine (2) locomotor sensitization induced by repeated administration of the dopamine D1/D5 receptor agonist SKF82958; and(3) brain metabolic activation upon repeated cocaine administration as evidenced by fMRI in awake rats. Furthermore, this report provides evidence that these treatment paradigms will result in chromatin modification changes associated with active transcription, in addition to increased mRNA levels of plasticity-associated genes, including brain-derived neurotrophic factor (BDNF) at key brain regions implicated in the pathogenesis of depression and stimulant addiction. To date, little is known regarding the underlying mechanisms of action mediating the enhancing effects of sodium butyrate on the various antidepressant- and stimulantrelated paradigms. Our findings underscore the potential of chromatin-modifying drugs to profoundly affect the behavioral response of an animal to antidepressant and stimulant drugs and warrants consideration in the context of developing novel therapeutic strategies.
29

The Role of Janus-Kinase-3 in CD4<sup>+</sup> T Cell Proliferation and Differentiation: A Dissertation

Shi, Min 27 October 2008 (has links)
Jak3, a member of the Janus family of tyrosine kinases, is essential for signaling via the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These Jak3-dependent cytokines primarily activate STAT5 and are critical for lymphoid generation and differentiation. Using naïve CD4+ T cells from Jak3-deficient mice and wild type CD4+ T cells treated with a pharmacological inhibitor of Jak3, we report that Jak3-dependent cytokine signals are not required for the proliferation of naïve CD4+ T cells. This is illustrated by the similar percentage of divided cells, comparable cell divisions, intact cell cycle progression and unaffected regulation of cell cycle proteins in the absence of Jak3. In contrast to proliferation, differentiation of naïve CD4+ T cells into Th1 effector cells requires Jak3-dependent cytokine signals. In the absence of Jak3, naïve CD4+ T cells proliferate robustly, but produce little IFN-γ after Th1 polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4, nor to impaired up-regulation of the transcription factor T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that while Jak3-dependent signals are dispensable for naïve CD4+ T cell proliferation, Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 differentiation.
30

Chromatin Regulators and DNA Repair: A Dissertation

Bennett, Gwendolyn M. 19 December 2014 (has links)
DNA double-strand break (DSB) repair is essential for maintenance of genome stability. However, the compaction of the eukaryotic genome into chromatin creates an inherent barrier to any DNA-mediated event, such as during DNA repair. This demands that there be mechanisms to modify the chromatin structure and thus access DNA. Recent work has implicated a host of chromatin regulators in the DNA damage response and several functional roles have been defined. Yet the mechanisms that control their recruitment to DNA lesions, and their relationship with concurrent histone modifications, remain unclear. We find that efficient DSB recruitment of many yeast chromatin regulators is cell-cycle dependent. Furthering this, we find recruitment of the INO80, SWR-C, NuA4, SWI/SNF, and RSC enzymes is inhibited by the non-homologous end joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, we find no significant role for H2A.X phosphorylation (γH2AX) in the recruitment of chromatin regulators, but rather that their recruitment coincides with reduced levels of γH2AX. We go on to determine the chromatin remodeling enzyme Fun30 functions in histone dynamics surround a DSB, but does not significantly affect γH2AX dynamics. Additionally, we describe a conserved functional interaction among the chromatin remodeling enzyme, SWI/SNF, the NuA4 and Gcn5 histone acetyltransferases, and phosphorylation of histone H2A.X. Specifically, we find that the NuA4 and Gcn5 enzymes are both required for the robust recruitment of SWI/SNF to a DSB, which in turn promotes the phosphorylation of H2A.X.

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