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Structural and functional characterization of histone acetyltransferase-1Mersfelder, Erica Lee Paul, January 2008 (has links)
Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 104-115).
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Expression, purification and characterisation of recombinant chromatin assembly factor 1Royle, Nikki January 2013 (has links)
Chromatin Assembly Factor 1 (CAF-1) is the only known replication dependant histone chaperone, responsible for the deposition of the histone H3/H4 tetramer onto DNA. Found in all eukaryotes, CAF-1 consists of three subunits, p150, p60 and p48. Since its identification work on CAF-1 has mainly focused on in vivo studies due to the lack of a reliable method to produce large quantities of recombinant protein for biochemical studies. Herein the cloning, production and purification of the three subunits of recombinant CAF-1 is described. The proteins were expressed as complexes and individually in insect cells and Escherichia coli, optimised protocols are described for maximum protein recovery and purity. Constructs of p150 and p60 were also produced and used to analyse the binding regions and modes of both the p48 and p60 proteins to p150. It is shown that the two smaller subunits of CAF-1 do not interact in the absence of p150 and that the p150 subunit of CAF-1 acts as a scaffold for assembly of the complex, binding directly to both p48 and p60. The stoichiometry of the CAF-1 complex was also investigated and a basis for further work, including structural studies, discussed.
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Organisation nucléaire et régulation transcriptionnelle dans les lymphomes / Nuclear Organisation and Transcription Regulation in LymphomasMarkozashvili, Diana 09 December 2015 (has links)
Le lymphome des cellules du manteau (LCM) est un lymphome d’une rare agressivité causée par la translocation chromosomique t(11; 14)(q13; q32) juxtaposant le locus de la cycline D1 (CCND1) sur le chr 11 avec le locus de la chaîne lourde de l'immunoglobuline (IgH) sur le chr 14. En conséquence, une cycline D1 proto-oncogène devient active alors qu’elle n’est pas exprimée dans les cellules-B normales. L’hypothèse initiale semble indiquer une influence directe du fort enhancer IgH sur le promoteur du gène CCND1 afin de surexprimer sa transcription. Quoi qu’il en soit, le locus CCND1 peut être éloigné jusqu'à 200kb du point de cassure du chromosome. Nous avons montré que le locus 11q13 relocalise depuis la périphérie du noyau jusque au centre actif de transcription et au nucléole (Allinne et al., 2014). Ce phénomène qui mène à l’activation du locus entier, suggère un mécanisme epigénétique de régulation des gènes dans les LCM plutôt que simplement un simple effet enhancer-promoteur. Plusieurs nouveaux traitements contre le MCL ont été proposés, y compris les inhibiteurs d’histone deacetylase (HDACis) qui impliquent des mécanismes épigénétiques. Dans les lignées cellulaires LMC, les HDACis sont décrites comme aillant des effets antiprolifératifs, et paradoxalement, le niveau d’expression protéique de la cycline D1 dans la cellule diminue. Jusqu'à présent, il n'y a pas une compréhension claire de ce phénomène, de même que les mécanismes d’action des HDACis reste inconnus. Pour ces raisons, une étude du « paysage épigénétique » sur les loci 11q13 et 14q32 devrait fortement améliorer notre connaissance sur les mécanismes de surexpression de la cycline D1 dans les LMC. L’objectif de ce travail est d'étudier la structure de la chromatine dans le locus réarrangé (11; 14)(q13; q32) dans des cellules LMC par rapport au locus 11q13 et 14q32 dans les lymphocytes humains normaux. Nous avons ensuite étudié l'effet de différentes HDACis sur le locus réarrangé (11; 14)(q13; q32) à plusieurs niveaux: l'acétylation des histones / la méthylation de la chromatine ainsi que sa conformation et l'expression des gènes. Nous avons montré que t(11:14)(q13; q32) conduit à la surexpression de CCND1 avec un groupe de gènes couvrant plus de 15 Mb autour du point de translocation. Les mêmes gènes, sensibles à la dérégulation par la translocation t(11; 14, réagissent au traitement HDACi en augmentant leur expression. Nos résultats indiquent que bien que HDACi stimule la désagrégation de l'hétérochromatine sur l'ensemble du génome, les promoteurs de gènes restent à l'abri de ces effets. / Mantle cell lymphoma (MCL) is a rare aggressive lymphoma caused by the chromosome translocation t(11;14)(q13;q32) juxtaposing the cyclin D1 (CCND1) locus on chr 11 with the immunoglobulin heavy chain (IgH) locus on chr 14. As a result, a proto-oncogene cyclin D1 which is not expressed in normal B-cells, becomes active. The initial hypothesis favored direct influence of the strong IgH enhancer on CCND1 gene promoter to upregulate its transcription. However, the CCND1 locus may be as far as 200 kb from the chromosome breakpoint. We have shown that 11q13 locus relocalizes from the nucleus periphery towards the transcriptionally active center and nucleolus (Allinne et al., 2014). This may lead to activation of the entire locus, suggesting an epigenetic mechanism of gene regulation in MCL, rather than just simple enhancer-promoter effect.Several new treatments are proposed for MCL, including histone deacetylase inhibitors (HDACis) with epigenetic mechanism of action. In MCL cell lines, HDACis were shown to have antiproliferative effects, and paradoxically, they decreased the cyclin D1 protein level in the cells. Until now, there is no clear understanding of this phenomenon, nor of HDACis mechanism of action. Therefore, a study of «epigenetic landscape» in 11q13 and 14q32 loci should significantly advance our knowledge about the mechanisms of cyclin D1 upregulation in MCL.The purpose of the present work was to study chromatin structure in the rearranged (11;14)(q13;q32) locus in MCL cells as compared to the 11q13 and 14q32 loci in normal human lymphocytes. We then studied the effect of different HDACis on the rearranged (11;14)(q13;q32) locus at several levels: histone acetylation / methylation, chromatin conformation and gene expression.We have shown that t(11:14)(q13;q32) translocation leads to overexpression of CCND1 along with a group of genes spanning over 15 Mb around the translocation point. The same genes, sensitive to deregulation by t(11;14) translocation, react to the HDACi treatment by increasing their expression. Importantly, while HDACi stimulates genome-wide disaggregation of heterochromatin, genes’ promoters stay shielded from its effect.
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Développement d'une approche méthodologique basée sur la biotinylation in vivo de protéines de la chromatine - Application à l’étude des interactions entre des domaines chromosomiques et une protéine de l'enveloppe nucléaire dans des cellules individuelles / Development of a methodological approach based on in vivo biotinylation of chromatin proteins - Application to the study of interactions between chromosomal domains and a nuclear envelope protein in individual cellsJurisic, Anamarija 18 October 2016 (has links)
Les arguments en faveur d’un rôle important de l'architecture des chromosomes en interphase pour la régulation des gènes et la maintenance du génome s’accumulent rapidement. Au cours de l'interphase, les chromosomes sont positionnés de façon non aléatoire l’un par rapport à l'autre et fournissent ainsi des points de repère nucléaires. Deux types d'interactions contribuent probablement à ce positionnement non aléatoire: (i) des domaines subchromosomiques interagissent avec des structures nucléaires telles l'enveloppe nucléaire (EN) et (ii) des interactions intrachromosomiques s’établissent entre des loci situés de façon linéairement distante en cis sur un même chromosome. Contribuant à l’expansion de ce domaine de recherche, nous avons poursuivi le développement d’une technique préalablement établie au laboratoire pour détecter des interactions protéine-protéine. Le développement de cette technique nouvelle a constitué une part de ce travail de thèse accompli sur des cellules humaines. Elle se base sur le marquage par la biotine de composants de la chromatine qui en interphase se trouvent à proximité immédiate de l’EN. Les cellules ont été traitées pour exprimer (i) la biotine ligase BirA fusionnée à l’émerine, une protéine de l’EN, conjointement avec (ii) une variante d’histone, l’histone macroH2A, en fusion avec un peptide accepteur de biotine. L'étiquette biotine déposée sur l’histone macroH2A pendant l'interphase est ensuite détectée par microscopie à fluorescence sur des cellules en mitose étalées sur lames. Les chromosomes mitotiques marqués peuvent en outre être caractérisés par des techniques plus classiques de caryotypage. Nous avons nommé cette technique «topokaryotypage» car elle peut fournir des informations d’ordre à la fois topologique et caryotypique. Son développement pas à pas a nécessité la production d'une lignée cellulaire ad hoc et une optimisation fine du protocole. Ce travail de thèse peut déboucher sur des questions biologiques explorées sur cellules uniques. A titre d’application, une analyse comparative a été réalisée par topokaryotypage sur des cellules cultivées in vitro dans diverses conditions de stress expérimentales. L’utilisation du topocaryotypage pourrait fournir des informations précieuses sur les mécanismes à la base de l’organisation et de la dynamtique des noyaux cellulaires. / Evidence is rapidly accumulating that the architecture of interphase chromosomes is important for both gene regulation and genome maintenance. During interphase, chromosomes are nonrandomly positioned with respect to each other and thus they provide nuclear landmarks. Two kinds of interactions are likely to contribute to this nonrandom positioning: (i) subchromosomal domains interact with nuclear structures such as the nuclear envelope (NE) and ii) intrachromosomal interactions take place between linearly distant loci positioned in cis on the same chromosome. As a contribution to this expanding research domain, we have built upon an existing approach previously established in the laboratory to detect protein-protein interactions. The new technique was developed in human cells as part of the present PhD research. It is based on biotin labelling of chromatin components which are in close proximity with the nuclear envelope (NE) in interphase cells. Cells were made to express (i) the biotin ligase BirA fused to the NE protein emerin together with (ii) a fusion between a biotin acceptor peptide and macroH2A, a variant core histone. The biotin label deposited on the macroH2A histone during interphase is then detected by fluorescence microscopy on mitotic cells spread on slides. The biotin-labelled mitotic chromosomes can be further characterized using more classical karyotyping techniques. We refer to this new technique as “Topokaryotyping” since it can provide both topological and karyotypic information. Its step-by-step development has required the establishment of an ad hoc cell line and a fine protocol optimization. This PhD work could pave the way for biological questions explored at a single cell level. As an illustration, a comparative topokaryotyping analysis was performed on cells cultivated in vitro in various experimental stress conditions. It is envisioned that using this technique can provide valuable mechanistic insights relevant to the organization and dynamics of cell nuclei.
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Studies on Poly(ADP-ribose) Metabolism and Chromatin StructureCárdenas-Corona, María E. (María Elena) 08 1900 (has links)
In these studies, a procedure which allowed the in vivo labeling and detection of poly(ADP-ribose) was combined with nuclear fractionation techniques to analyze the nuclear distribution of ADP-ribose polymers. The results from these studies suggest the occurrence of poly(ADP-ribose) metabolism in two compartments of chromatin; one that is nuclear matrix-associated and one that is not. The biological significance of this compartmentalization is conceptualization in a model. This model postulates that, under some physiological conditions, poly(ADP-ribose) metabolism accomplishes the reversible targeting of specific regions of chromatin to the nuclear matrix domain by modulating DNA-protein and or protein-protein interactions.
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Differential chromatin topology and transcription factor enhancer binding regulate spatiotemporal gene expression in limb developmentWilliamson, William Iain January 2013 (has links)
Many developmental genes are located in gene-poor genomic regions and are activated by long-range enhancers located up to 1Mb away. Modification and reorganisation of chromatin structure is pivotal to such long-range gene regulation. A prerequisite for enhancer activity is the binding of transcription factors and co-factors with the interplay between activating and repressive factors determining tissue, spatial and temporal specificity. Spatiotemporal control of sonic hedgehog (Shh) and the 5′ Hoxd genes (especially Hoxd13) is crucial for vertebrate limb anterior-posterior (A-P) axis and autopod patterning. Shh tissue specificity is controlled by multiple enhancers throughout an adjacent gene desert. The ~0.8Mb-distant limb enhancer (ZRS) bypasses nearby genes to activate only Shh. In contrast, limb-specific HoxD expression is regulated by multiple enhancers, with the ~200kb-distant global control region (GCR) regulatory element the most characterised. In this thesis I investigated the mechanisms of ZRS and GCR regulation of Shh and Hoxd13 respectively. The model system used was immortalised cell lines derived from the anterior and posterior distal forelimb buds of E10.5 and E11.5 mouse embryos. Cell line data were confirmed in dissected limb tissue. Increased expression of the 5′ Hoxd genes, particularly Hoxd13, correlated with the loss of the repressive, polycomb catalysed, histone modification H3K27me3 and decompaction of chromatin structure over the HoxD locus at the distal posterior forelimb bud at stage E10.5. Moreover, I show that the GCR spatially co-localises with the 5′ HoxD locus at the distal posterior region of E10.5-11 embryos. These data are consistent with the formation of a chromatin loop between Hoxd13 and the GCR at the time and place of distal limb bud development when the GCR is required to initiate 5′ Hoxd gene expression. This is the first example of A-P differences in chromatin compaction and local folding in the limb. Point mutations within the ZRS cause ectopic (anterior) Shh expression, which results in preaxial polydactyly (PPD). The ZRS contains multiple canonical ETS transcription factor binding motifs, and point mutations in two families with PPD results in the formation of additional ETS binding sites. The point mutations cause the loss or reduction of ETV4/5 transcription factor binding at a non-canonical ETS binding site and enable additional binding instead of ETS1. I show that ETV4/5, ETS1 and another ETS protein GABPα all bind to the ZRS. This work has revealed the differential effect on Shh expression of two groups of ETS factors mediated through the ZRS. The binding of ETS1/ GABPα determines the posterior Shh expression domain while ETV4/5 restricts anterior Shh expression. Two point mutations alter the ETS-binding profile, creating an additional ETS1/ GABPα site that is sufficient to drive ectopic Shh expression. DNA FISH on E11.5 forelimb and floorplate tissue sections revealed that the Shh-ZRS genomic locus is in a compact chromatin conformation in both Shhexpressing and non-expressing cells. However, I show that the ZRS co-localises with Shh to a significantly greater extent in the distal posterior limb bud and the floorplate compared with cells where Shh is not expressed. This thesis presents novel research into long-range gene regulation during limb development, elucidating the role of chromatin re-organisation and how spatial-specific enhancer activity is determined by opposing sets of binding factors.
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On key modulators of higher-order chromatin structureFaure, André Jean January 2014 (has links)
No description available.
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Phosphorylation of linker histones by cdc2 kinaseHarris, Ruth V. January 1994 (has links)
No description available.
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The use of the yeast two-hybrid system as a means of identifying protein interactors of the human protein, BMI-1Holgate, Robert George Edward January 1999 (has links)
No description available.
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Surface mapping of the higher order structure of chromatinBunn, Nicholas Reynolds January 1990 (has links)
No description available.
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