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Venkataraman, Shanmugasundaram. "Histone acetylation and nucleosome dynamics." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/23234.
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In this report, I will describe purification of core histone octamers from chicken blood, HeLa nuclei and yeast cells, along with preparation of DNA fragments containing the 208 bp 5S rDNA gene and the adult beta (bA)-globin gene promoter. In vitro experiments studying the effect of histone acetylation on the positioning and mobility of nucleosomes on the sea urchin 5S rDNA gene and the chicken bA-globin gene promoter will be described. The former provides a well studied nucleosome positioning and mobility model system, while the latter is a developmentally regulated gene, with globin gene switching through the early stages of the lifetime of the chicken, and a proposed involvement of positioned nucleosomes in its regulation. The aim was to determine the difference between hypoacetylated and hyperacetylated core histones in terms of their influence upon nucleosome positioning and mobility. In earlier studies, it was noted that there was a difference in relative positioning intensities between the two forms (ie. hypoacetylated core histones preferentially positioned at certain sites, while hyperacetylated core histones positioned at the same sites but with different relative affinities). Therefore, acetylation affects where a nucleosomes is able to position. I have carried on this work to further characterize nucleosome positioning and to study the implications of histone acetylation on nucleosome mobility. I have found subtle differences in the thermodynamics and kinetics of hyperacetylated nucleosomes compared to hypeoacetylated nucleosomes: hyperacetylated nucleosomes appear to have a lower threshold in both these parameters when studied using the 208 pb rDNA fragment. Experiments involving two other types of core histones, trypsinized chicken core histone octamers and chicken core histone tetramers will also be described, which will be placed into the context of the results found with the other types of core histones. Finally, I will describe the effect of reconstituting hyperacetylated core histones with methylated DNA, long known to be a mediator of transcriptional repression, in the form of the chicken bA-globin gene promoter.
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Dorigo, Benedetta. "Studies of nucleosome array structure and dynamics /." Zürich, 2004. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=15710.
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Hu, Zhenhua. "Nucleosome positioning dynamics in evolution and disease." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25399.
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Nucleosome positioning is involved in a variety of cellular processes, and it provides a likely substrate for species evolution and may play roles in human disease. However, many fundamental aspects of nucleosome positioning remain controversial, such as the relative importance of underlying sequence features, genomic neighbourhood and trans-acting factors. In this thesis, I have focused on analyses of the divergence and conservation of nucleosome positioning, associated substitution spectra, and the interplay between them. I have investigated the extent to which nucleosome positioning patterns change following the duplication of a DNA sequence and its insertion into a new genomic region within the same species, by assessing the relative nucleosome positioning between paralogous regions in both the human (using in vitro and in vivo datasets) and yeast (in vivo) genomes. I observed that the positioning of paralogous nucleosomes is generally well conserved and detected a strong rotational preference where nucleosome positioning has diverged. I have also found, in all datasets, that DNA sequence features appear to be more important than local chromosomal environments in nucleosome positioning evolution, while controlling for trans-acting factors that can potentially confound inter-species comparisons. I have also examined the relationships between chromatin structure and DNA sequence variation, with a particular focus on the spectra of (germline and somatic) substitutions seen in human diseases. Both somatic and germline substitutions are found to be enriched at sequences coinciding with nucleosome cores. In addition, transitions appear to be enriched in germline relative to somatic substitutions at nucleosome core regions. This difference in transition to transversion ratio is also seen at transcription start sites (TSSs) genome wide. However, the contrasts seen between somatic and germline mutational spectra do not appear to be attributable to alterations in nucleosome positioning between cell types. Examination of multiple human nucleosome positioning datasets shows conserved positioning across TSSs and strongly conserved global phasing between 4 cancer cell lines and 7 non-cancer cell lines. This suggests that the particular mutational profiles seen for somatic and germline cells occur upon a common landscape of conserved chromatin structure. I extended my studies of mutational spectra by analysing genome sequencing data from various tissues in a cohort of individuals to identify human somatic mutations. This allowed an assessment of the relationship between age and mutation accumulation and a search for inherited genetic variants linked to high somatic mutation rates. A list of candidate germline variants that potentially predispose to increased somatic mutation rates was the outcome. Together these analyses contribute to an integrated view of genome evolution, encompassing the divergence of DNA sequence and chromatin structure, and explorations of how they may interact in human disease.
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Pohl, Andy 1979. "Nucleosome dynamics and analysis in breast cancer cells." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/328416.
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Genome-wide analysis of the nucleosome positioning and histone H1 isoform content of the T47D breast cancer cell line has found a number of observations, namely that with a gentle digestion of microccocal nuclease (MNase), a nucleosome is visible just upstream of the transcription start site, in the region known as the “nucleosome-free region” (NFR). H1 isoforms bind to chromatin mainly in a redundant manner, but H1.2 and H1.3 show some specificity while H1.5 increases its binding dramatically after a progesterone stimulus. In the course of these studies, a general-purpose software package was developed for the manipulation and analysis of bigWig files, a data format for storing continuous signal data assigned to genome coordinatesEn el meu estudi genòmic sobre el posicionament de nucleosomes i sobre elcontingut de les isoformes de la histona H1 en cèl•lules de càncer de mama T47D he dut a terme una sèrie d'observacions. Específicament he trobat que amb una digestió suau amb nucleasa micrococcal, es pot identificar un nucleosoma just abans del lloc d'inici de transcripció, en la regió coneguda com a "regió lliure de nucleosomes". També he vist que les diferents isoformes somàtiques de la histona H1 (H1.0-H1.5, H1x) s'uneixen a la cromatina de manera redundant, però que la H1.2 i la H1.3 presenten certa especificitat, mentre que la H1.5 mostra un augment de la unió generalitzat després d'estimular les cèl•lules amb progesterona. En el decurs de la meva recerca, he desenvolupat un programari general per la manipulació i l'anàlisi d'arxius amb format bigWig, un format per a l'emmagetzematge de dades de senyals continus al llarg de les coordenades del genoma.
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Hada, Arjan. "DYNAMICS OF NUCLEOSOME REMODELING BY ATP-DEPENDENT CHROMATIN REMODELERS." OpenSIUC, 2017. https://opensiuc.lib.siu.edu/dissertations/1431.
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Chromatin is highly regulated nucleoprotein complex facilitating the dynamic balance between genome packaging and accessibility. The central workhorses regulating the dynamic nature of chromatin are ATP-dependent chromatin remodelers- ISWI, SWI/SNF, INO80, and CHD/Mi2. All chromatin remodelers transduce the energy from ATP hydrolysis to translocate on DNA, break histone-DNA contacts, and mobilize nucleosomes. However, the final outcomes of nucleosome remodeling are diverse - nucleosome sliding, dimer exchange, nucleosome disassembly, and nucleosome conformation alteration. This study sheds light on how different chromatin remodelers catalyze various structural transformations. We provide novel insights into the nucleosome dynamics, the role of histone octamer dynamics on nucleosome remodeling by ISW2, mechanism of dimer exchange by INO80 and mechanism of nucleosome disassembly by the coordinated action of RSC and histone chaperone Nap1. We also provide insights on how aberrant SWI/SNF complexes affect fundamental enzymatic properties such as ATPase and processive nucleosome remodeling. ISW2 remodelers sense and respond to the length of linker DNA separating the nucleosome and centers nucleosome. Histone octamers are perceived as a mostly static structure whereas DNA deforms itself to fit nucleosome. We have found change in histone octamer conformation as a novel step in ISW2 mobilizing DNA through the nucleosome. We provide evidence for an induced fit mechanism where histone-histone and histone-DNA interactions change in respond to remodeler, and these changes promote DNA entry into the nucleosome. Our data supports a model in which DNA translocation causes the change in histone octamer conformation, followed by DNA entry into nucleosome and resetting of the histone octamer core. We also move a step ahead and show that SANT domain promotes the entry of DNA into nucleosome and resets the histone octamer core allowing processive nucleosome mobilization. INO80 nucleosome remodeling provides two outcomes- nucleosome centering and dimer exchange. INO80 exchanges H2A.Z-H2B dimer for H2A-H2B. We show that INO80 is incredibly slow at centering nucleosome compared to ISW2. We also provide evidence for a mechanism where INO80 persistently displaces DNA from the dimer interface, unlike ISW2, facilitating dimer exchange. In another instance, we show that kinetic step sizes are modulated by a combination of enzyme and DNA sequence properties, and are not hardwired into the enzyme. ISW2 has been previously shown to translocate DNA with a kinetic step sizes of ~7bp and ~3bp. We show that kinetic step sizes may vary depending on nucleosomal location where we monitor DNA movement. Next, we studied the mechanism of nucleosome disassembly by RSC in the presence of Nap1. We found that Nap1 promotes the disassembly of the distal nucleosome that RSC collides with rather than the proximal nucleosomes it mobilizes. SWI/SNF tops the list of the frequently mutated epigenetic factor in cancer with its subunits mutated in more than 20% of all cancers. Loss of hSnf5 is a driver mutation in pediatric rhabdoid tumors. Our lab has previously identified that the deletion of Snf5 causes yeast SWI/SNF to lose an entire module comprised of Snf5, Swp82, and Taf14. In this study, we establish the properties of aberrant SWI/SNF complex formed in the absence of Snf5 module. The deletion resulted in lower ATP hydrolysis and nucleosome mobilization activities of the mutant SWI/SNF. We found that Snf5 module is necessary to couple ATP hydrolysis with DNA translocation. We studied the role of accessory domain AT-hooks in the ATPase subunit of SWI/SNF and found similar results. Interestingly, AT hook and SnAC domains, and Snf5 subunit were found to communicate with the same region in ATPase domain physically. These studies provide valuable mechanistic insights into chromatin structure and function and highlight how different chromatin remodelers catalyze different chromatin remodeling outcomes. We also provide new insights on how the activity of the core ATPase motor is regulated either by accessory domains on the same subunit or by accessory subunits as a part of the larger complex.
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North, Justin A. "Regulation of Nucleosome Dynamics: Mechanisms for Chromatin Accessibility and Metabolism." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354737862.
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Luo, Yi. "Nucleosome Regulation of Transcription Factor Binding Dynamics: a Single-molecule Study." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449093157.
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Jessen, Walter Joseph. "Chromatin dynamics at the Saccharomyces cerevisiae PHO5 promoter." Diss., Texas A&M University, 2004. http://hdl.handle.net/1969.1/3306.
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In eukaryotes, the organization of DNA into chromatin is a primary determinant of gene expression. Positioned nucleosomes in promoter regions are frequently found to regulate gene expression by obstructing the accessibility of cis-regulatory elements in DNA to trans-factors. This dissertation focuses on the chromatin structure and remodeling program at the S. cerevisiae PHO5 promoter, extending the use of DNA methyltransferases as in vivo probes of chromatin structure. Our studies address the diversity of histone-DNA interactions in vivo by examining nucleosome conformational stabilities at the PHO5 promoter. We present high-resolution chromatin structural mapping of the promoter, required to relate in vivo site accessibility to nucleosome stability and show that the PHO5 promoter nucleosomes have different accessibilities. We show a correlation between DNA curvature and nucleosome positioning, which is consistent with the observed differences in accessibility/stability. Kinetic analyses of the chromatin remodeling program at PHO5 show that nucleosomes proximal to the enhancer are disrupted preferentially and prior to those more distal, demonstrating bidirectional and finite propagation of chromatin remodeling from bound activators and providing a novel mechanism by which transactivation at a distance occurs.
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Topal, Salih. "Chromatin Dynamics Regulate Transcriptional Homeostasis." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1062.
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Eukaryotic promoters are inherently bidirectional and allow RNA Polymerase II to transcribe both coding and noncoding RNAs. Dynamic disassembly and reassembly is a prominent feature of nucleosomes around eukaryotic promoters. While H3K56 acetylation (H3K56Ac) enhances turnover events of these promoter-proximal nucleosomes, the chromatin remodeler INO80C ensures their proper positioning. In my dissertation, I explore how chromatin dynamics regulate transcriptional homeostasis. In the first part, I investigate the role of H3K56Ac on the nascent transcriptome throughout the eukaryotic cell cycle. I find that H3K56Ac is a global, positive regulator for coding and noncoding transcription by promoting both initiation and elongation/termination. On the contrary, I find that H3K56Ac represses promiscuous transcription following replication fork passage by ensuring efficient nucleosome assembly during S-phase. In addition, I show that there is a stepwise increase in transcription in the S-G2 transition, and this response to gene dosage imbalance does not require H3K56Ac. This study clearly shows that a single histone modification, H3K56Ac can exert both positive and negative effects on transcription at different cell cycle stages. In the second part, I investigate the role of the chromatin remodeler INO80C on the nascent transcription around replication origins. I show that INO80C, together with the transcription factor Mot1, prevents cryptic transcription around yeast replication origins, and the loss of these proteins lead to an increase in DNA double strand breaks. I hypothesize that recruitment of INO80C ensures proper positioning of nucleosomes around origins and the exclusion of RNA Pol II to prevent cryptic initiation. Together these findings indicate that H3K56Ac regulates transcription globally by enhancing nucleosome turnover, and it prevents cryptic transcription and reinforces transcriptional fidelity by promoting efficient nucleosome assembly in the S-phase. In addition, INO80C maintains genome stability by preventing cryptic transcription around the origins.
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Le, Jenny Vi Le. "Tunable Nanocalipers to Probe Structure and Dynamics in Chromatin." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543163132011865.
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Font, Mateu Jofre 1977. "Dynamics of progesterone receptor interactors in breast cancer cells upon hormone exposure." Doctoral thesis, Universitat Pompeu Fabra, 2016. http://hdl.handle.net/10803/511363.
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El receptor de la progesterona és un regulador clau per la proliferació de les cèl·lules de càncer de mama dependents d’hormona. El mecanisme d’acció del PR ha tingut un paper important en la resolució del mecanisme molecular d’activació de la transcripció. No obstant això, no hi ha hagut un estudi a fons de les seves interaccions en resposta a hormona. En aquest treball s'han identificat per RIME (immunoprecipitació ràpida per l'espectrometria de masses de proteïnes endògenes) 315 interactors d’alta confiança del PR en cèl·lules de càncer de mama exposades a la potent agonista de la progesterona R5020 durant 0, 1, 5, 15, 30 i 60 minuts. Hem identificat 20 interactors coneguts del PR i 295 de nous. Els interactors del PR trobats formen 4 grups dinàmics; El grup basal, 66 proteïnes presents en nivells similars en tots els temps; grup 1, 41 proteïnes que disminueixen la seva interacció després de l'hormona; grup 2, 115 proteïnes que augmenten la seva interacció ràpidament després de l'hormona; i el grup 3, 91 proteïnes que tenen un augment de la seva interacció constant amb el temps. Els interactors del PR formen complexes funcionals que intervenen en la regulació transcripcional, remodelació de la cromatina, el processament del ARNm, reparació de l’ADN danyat, la degradació proteosomal, proteïnes estabilitzadores i proteïnes de l’estructura nuclear. L'exposició de cèl·lules a l’antagonista parcial de la progesterona RU486 manté la majoria dels interactors del PR, però perd els relacionats amb la regulació de la transcripció. Aquest estudi estableix les bases per a l'anàlisi de les noves funcions dels receptors de progesterona en cèl·lules de càncer de mama.Progesterone receptor is a key regulatory element in hormone-dependent breast cancer cells proliferation. The mechanism of action of PR has played an important role in solving the molecular mechanism of transcription regulation. However, it has not been a thorough study of its interactors in response to hormone. In this work we have identified by RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) 315 high confidence PR interactors in breast cancer cells exposed to the potent progesterone agonist R5020 for 0, 1, 5, 15, 30 and 60 minutes. We have identified 20 known PR interactors and 295 new ones. The found PR interactors form 4 dynamic clusters; Basal cluster, 66 proteins present at similar level at all time points; Cluster 1, 41 proteins decreasing their interaction after hormone; cluster 2, 115 proteins increasing their interaction rapidly after hormone; and cluster 3, 93 proteins increasing their interaction steadily over time. PR interactors form functional complexes involved in transcriptional regulation, chromatin remodelling, mRNA processing, DNA damage repair, proteosomal degradation, protein stability and nuclear structural proteins. Exposure of cells to progesterone partial antagonist RU486 maintain the majority of PR interactors, but loses the interactors related to transcription regulation. This study set the bases for analyses of new functions of progesterone receptor in breast cancer cells.
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Ruscio, Jory Zmuda. "Molecular Modeling: Elucidation of Structure/Function Relationships of Proteins and DNA at the Atomic Resolution." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/27219.
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While experiments provide valuable information about biological molecules, current technology cannot yet monitor atomic fluctuations at relevant time scales. Theoretical computational simulations are able to model the appropriate interactions at atomic resolution. Computational techniques have become widely used for identifying interactions in biological systems. Such methods have proven quite accurate in their ability to reproduce experimental data and also in screening and predicting pertinent activities. Molecular modeling employs theoretical and computational techniques to elucidate biologically relevant information from macromolecular structures. Three biological systems, the nucleosome core particle, myoglobin and glycosyl hydrolase family 1 beta-glucosidases will be examined with molecular modeling methods. Results of our analyses provide information about DNA flexibility and packaging, internal migration of ligands in a small protein, and substrate specificity of an enzyme system.Ph. D.
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Terakawa, Tsuyoshi. "p53 search and recognition dynamics on DNA studied by multi-scale simulations." 京都大学 (Kyoto University), 2014. http://hdl.handle.net/2433/188528.
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Nawaz, Kashif [Verfasser], George [Gutachter] Coupland, and Stanislav [Gutachter] Kopriva. "Genome-wide nucleosome dynamics under heat stress in Arabidopsis thaliana / Kashif Nawaz ; Gutachter: George Coupland, Stanislav Kopriva." Köln : Universitäts- und Stadtbibliothek Köln, 2018. http://d-nb.info/1191895750/34.
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Elbahnsi, Ahmad. "Dynamique et stabilité du nucléosome." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLN002/document.
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Le nucléosome est l’unité élémentaire de la compaction de l’ADN dans les cellules eucaryotes. C’est un complexe composé par un long segment d’ADN enroulé 1.7 fois en super-hélice autour d’un cœur de huit protéines histones. Les nucléosomes contrôlent l’accessibilité de l'ADN en s'associant et se dissociant le long des génomes et, ce faisant, sont directement impliqués dans la plupart des processus nucléaires. Le but principal de ce travail a été de décrire l'interface ADN-histones en solution pour mieux comprendre la stabilité du nucléosome. Nous avons voulu savoir en particulier comment l'ADN est maintenu enroulé autour du cœur d'histone et comment la séquence de l'ADN pourrait éventuellement affecter l'interface ADN-histones. Plusieurs nucléosomes ont été étudiés par dynamique moléculaire en solvant explicite ; ils diffèrent par la taille des queues d'histone et par les séquences d'ADN qui les forment. Pour garantir une analyse objective de la topologie de l’interface ADN-histones, une méthode basée sur les pavages de Delaunay-Laguerre originellement dédiée aux protéines a été adaptée aux acides nucléiques. Nous montrons ainsi que l'interface ADN-histones est constituée d'un réseau d'interaction très dense, caractérisé par des aires de contact électrostatique et hydrophobe équivalentes. Les queues d'histone renforcent significativement l'interface. Le comportement dynamique des arginines des cœurs structurés et des queues d'histone qui interagissent avec les petits sillons de l'ADN a été examiné en détail. Les cations écrantent les répulsions entre les hélices d'ADN juxtaposées l'une au dessus de l'autre du fait de l'enroulement en super-hélice. Enfin, l’interface ADN-histones est globalement retrouvée dans les nucléosomes formés avec des séquences d’ADN défavorables au nucléosome. Ceci suggère qu'une fois le nucléosome formé, il n'y a pas d'effet décisif de la séquence de l'ADN sur l'interfaceThe nucleosome is the fundamental unit of DNA compaction in eukaryotic cells. It consists in a long DNA segment (145-147 bp) wrapped in 1.7 left-handed superhelix turns around a histone octamer. Nucleosomes control the DNA accessibility by assembling and disassembling along the genomes and are therefore involved in most nuclear processes.The main aim of the thesis was to describe the DNA-histone interface in solution to better understand the nucleosome stability. We examined in particular how the DNA is maintained wrapped around the histone and how its sequence affects the DNA-histone interface. Several nucleosomes were studied using molecular dynamics in explicit solvent ; they differed by the tail length and the DNA sequences. To ensure an objective analysis of the topology of the DNA-histone interface, a method based on Delaunay-Laguerre tessellations, originally developed for proteins, was adapted to nucleic acids.Our results show that the DNA-histone interface is composed by a dense network of interactions, characterized by equivalent electrostatic and hydrophobic contact area. The histone tails significantly reinforce the interface. The behavior of arginines belonging to the histone structured cores or tails and that interact with the DNA minor groove was scrutinized in detail. Cations shield the repulsive interactions between the two DNA gyres, closely juxtaposed one above the other because of the superhelix wrapping. Finally, the DNA-histone interface is globally not affected in nucleosomes containing DNA sequences known to disfavor nucleosomes. This suggests that, once the nucleosome established, there is no significant effect of the DNA sequence on the interface
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Nguyen, Vu Quang. "Structural insights into the assembly and dynamics of the ATP-dependent chromatin-remodeling complex SWR1." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11606.
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The ATP-dependent chromatin remodeling complex SWR1 exchanges a variant histone H2A.Z-H2B dimer for a canonical H2A-H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 Mega-Dalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single hetero-hexameric Rvb1/2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multi-subunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvb1/2 ring and a distinct substrate-handling mode by SWR1, thereby providing the first structural framework for understanding the complex dimer-exchange reaction.
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Hahn, Liljan [Verfasser], Heinz [Akademischer Betreuer] Neumann, Ulf [Akademischer Betreuer] Diederichsen, and Ralf [Akademischer Betreuer] Ficner. "Investigation of Nucleosome Dynamics by Genetic Code Expansion / Liljan Hahn. Betreuer: Heinz Neumann. Gutachter: Heinz Neumann ; Ulf Diederichsen ; Ralf Ficner." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1078150818/34.
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Riedmann, Caitlyn M. "THE DYNAMIC NATURE OF CHROMATIN." UKnowledge, 2017. http://uknowledge.uky.edu/biochem_etds/31.
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Eukaryotic organisms contain their entire genome in the nucleus of their cells. In order to fit within the nucleus, genomic DNA wraps into nucleosomes, the basic, repeating unit of chromatin. Nucleosomes wrap around each other to form higher order chromatin structures. Here we study many factors that affect, or are effected by, chromatin structure including: (1) how low-dose inorganic arsenic (iAs) changes chromatin structures and their relation to global transcription and splicing patterns, and (2) how chromatin architectural proteins (CAPs) bind to and change nucleosome dynamics and DNA target site accessibility.
Despite iAs’s non-mutagenic nature, chronic exposure to low doses of iAs is associated with a higher risk of skin, lung, and bladder cancers. We sought to identify the genome-wide changes to chromatin structure and splicing profiles behind the cell’s adaptive response to iAs and its removal. Furthermore, we extended our investigation into cells that had the iAs insult removed. Our results show that the iAs-induced epithelial to mesenchymal transition and changes to the transcriptome are coupled with changes to the higher order chromatin structure and CAP binding patterns. We hypothesize that CAPs, which bind the entry/exit and linker DNA of nucleosomes, regulate DNA target site accessibility by altering of the rate of spontaneous dissociation of DNA from nucleosome.
Therefore, we investigated the effects of the repressive CAP histone H1, the activating CAP high mobility group D1 (HMGD1), and the neural CAP methyl CpG binding protein 2 (MeCP2) on the dynamics of short chromatin arrays and mononucleosomes and their effect on nucleosomal DNA accessibility. Using biochemical and biophysical analyses we show that all CAP-chromatin structures tested were susceptible to chromatin remodeling by ISWI and created more stable higher order structures than if CAPs were absent. Additionally, histone H1 and MeCP2 hinder model transcription factor Gal4 from binding its cognate DNA site within nucleosomal DNA.
Overall, we show that chromatin structure is dynamic and changes in response to environmental signals and that CAPs change nucleosome dynamics that help to regulate chromatin structures and impact transcriptional profiles.
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Retureau, Romain. "Interactions acides nucléiques/protéines non spécifiques : le nucléosome et les complexes de la NCp7." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLN057/document.
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Les protéines régulent et exécutent l'ensemble des fonctions vitales des organismes en interagissant notamment avec les acides nucléiques (AN), dont l’ADN, support de l’information génétique. Appréhender la nature de ces types d’interactions est central en biologie. Le nucléosome, qui est l’unité élémentaire de la compaction de l’ADN chez les cellules eucaryotes, est formé d’un d’ADN enroulé autour d’un cœur protéique d’histone ; il contrôle l’accessibilité de l’ADN en se formant et en se dissociant le long des génomes. Ici, le nucléosome a été modélisé par dynamique moléculaire en solution. L’ analyse de l’interface ADN-histone par une méthode géométrique innovante a permis de comprendre comment la forte cohésion de ce complexe était assurée. La description de l’interface a aussi servi à interpréter des expériences d’assemblage et de désassemblage du nucléosome qui ont par ailleurs démontré l’effet de la séquence d’ADN sur ces processus. Enfin, j’ai comparé les dynamiques de l’ADN nucléosomal et de l’ADN nu, et montré quelles propriétés structurales étaient conservées au sein du nucléosome et comment elles sont utilisées pour moduler l’efficacité de l’association ADN-histones. Une stratégie semblable a été appliquée à des structures expérimentales de complexes entre ADN ou ARN et NCp7, une protéine du VIH-1 chaperon des AN. Cette dernière étude propose un mécanisme d’association entre les partenaires sur des bases rationnelles. Dans ces deux études, je mets en évidence des mécanismes de formation des complexes en plusieurs étapes et j’illustre les préférences de structure et de séquence des AN chez des protéines dites non-spécifiquesProteins regulate and perform the vital functions of organisms, in particular by interacting with nucleic acids (NA), including DNA which carries the genetic information. Understanding the nature of these interactions is central in biology. The nucleosome is the basic unit of DNA compaction in eukaryotes. Composed of a DNA wrapped around a histone core, this complex regulates the DNA accessibility by assembling and disassembling along the genome. Here, we carried out molecular dynamic simulations of the nucleosome in solution. The analysis of the DNA-histone interface with an innovative geometrical method highlighted the strong cohesion of the complex. Such an in-depth description of the interface was also used to interpret nucleosome assembly and disassembly experiments. Those experiments emphasized in particular the DNA sequence effect in both assembly and disassembly processes. Finally, the comparison between nucleosomal and free DNA dynamics showed which structural properties were conserved in the complex and how they contributed to the DNA-histone assembly efficiency. A similar strategy was used on experimental structures of NCp7, a HIV-1 NA chaperone protein, complexed with either DNA or RNA. The latter analysis suggested a rational basis to describe the mechanism of partner assembly. In both studies, I evidenced stepwise mechanisms of complex assemblies and I illustrated NA structure and sequence preferences of some so-called non-specific proteins
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Cannan, Wendy J. "Mechanisms and Dynamics of Oxidative DNA Damage Repair in Nucleosomes." ScholarWorks @ UVM, 2016. http://scholarworks.uvm.edu/graddis/628.
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DNA provides the blueprint for cell function and growth, as well as ensuring continuity from one cell generation to the next. In order to compact, protect, and regulate this vital information, DNA is packaged by histone proteins into nucleosomes, which are the fundamental subunits of chromatin. Reactive oxygen species, generated by both endogenous and exogenous agents, can react with DNA, altering base chemistry and generating DNA strand breaks. Left unrepaired, these oxidation products can result in mutations and/or cell death. The Base Excision Repair (BER) pathway exists to deal with damaged bases and single-stranded DNA breaks. However, the packaging of DNA into chromatin provides roadblocks to repair. Damaged DNA bases may be buried within nucleosomes, where they are inaccessible to repair enzymes and other DNA binding proteins. Previous in vitro studies by our lab have demonstrated that BER enzymes can function within this challenging environment, albeit in a reduced capacity.
Exposure to ionizing radiation often results in multiple, clustered oxidative lesions. Near-simultaneous BER of two lesions located on opposing strands within a single helical turn of DNA of one another creates multiple DNA single-strand break intermediates. This, in turn, may create a potentially lethal double-strand break (DSB) that can no longer be repaired by BER. To determine if chromatin offers protection from this phenomenon, we incubated DNA glycosylases with nucleosomes containing clustered damages in an attempt to generate DSBs. We discovered that nucleosomes offer substantial protection from inadvertent DSB formation. Steric hindrance by the histone core in the nucleosome was a major factor in restricting DSB formation. As well, lesions positioned very close to one another were refractory to processing, with one lesion blocking or disrupting access to the second site. The nucleosome itself appears to remain intact during DSB formation, and in some cases, no DNA is released from the histones. Taken together, these results suggest that in vivo, DSBs generated by BER occur primarily in regions of the genome associated with elevated rates of nucleosome turnover or remodeling, and in the short linker DNA segments that lie between adjacent nucleosomes.
DNA ligase IIIα (LigIIIα) catalyzes the final step in BER. In order to facilitate repair, DNA ligase must completely encircle the DNA helix. Thus, DNA ligase must at least transiently disrupt histone-DNA contacts. To determine how LigIIIα functions in nucleosomes, given this restraint, we incubated the enzyme with nick-containing nucleosomes. We found that a nick located further within the nucleosome was ligated at a lower rate than one located closer to the edge. This indicated that LigIIIα must wait for DNA to spontaneously, transiently unwrap from the histone octamer to expose the nick for recognition. Remarkably, the disruption that must occur for ligation is both limited and transient: the nucleosome remains resistant to enzymatic digest before and during ligation, and reforms completely once LigIIIα dissociates.
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Howell, Steven C. "Dynamic Conformations of Nucleosome Arrays in Solution from Small-Angle X-ray Scattering." Thesis, The George Washington University, 2015. http://pqdtopen.proquest.com/#viewpdf?dispub=3738561.
Full textAbstract:
Chromatin conformation and dynamics remains unsolved despite the critical role of the chromatin in fundamental genetic functions such as transcription, replication, and repair. At the molecular level, chromatin can be viewed as a linear array of nucleosomes, each consisting of 147 base pairs (bp) of double-stranded DNA (dsDNA) wrapped around a protein core and connected by 10 to 90 bp of linker dsDNA.
Using small-angle X-ray scattering (SAXS), we investigated how the conformations of model nucleosome arrays in solution are modulated by ionic condition as well as the effect of linker histone proteins. To facilitate ensemble modeling of these SAXS measurements, we developed a simulation method that treats coarse-grained DNA as a Markov chain, then explores possible DNA conformations using Metropolis Monte Carlo (MC) sampling. This algorithm extends the functionality of SASSIE, a program used to model intrinsically disordered biological molecules, adding to the previous methods for simulating protein, carbohydrates, and single-stranded DNA. Our SAXS measurements of various nucleosome arrays together with the MC generated models provide valuable solution structure information identifying specific differences from the structure of crystallized arrays.
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Simon, Marek. "Chromatin Modified! Dynamics, Mechanics, Structure, and HIV Integration." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1336752099.
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Juha, Mikael Lintuluoto. "Self-organization and Dynamic Molecular Recognition of Nucleoside Derivatives through Hydrogen Bonding." Kyoto University, 1997. http://hdl.handle.net/2433/202325.
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Knoch, Tobias A. "Approaching the three-dimensional organization of the human genome." [S.l. : s.n.], 2003. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB10358902.
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Conroy, Daniel William. "Structural Studies of Biomolecules by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1555428362333615.
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Hahn, Liljan. "Investigation of Nucleosome Dynamics by Genetic Code Expansion." Doctoral thesis, 2015. http://hdl.handle.net/11858/00-1735-0000-0023-9661-9.
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"Analysis of Nucleosome Dynamics by Fluorescence Correlation Spectroscopy." Doctoral diss., 2011. http://hdl.handle.net/2286/R.I.9152.
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abstract: Nucleosomes are the basic repetitive unit of eukaryotic chromatin and are responsible for packing DNA inside the nucleus of the cell. They consist of a complex of eight histone proteins (two copies of four proteins H2A, H2B, H3 and H4) around which 147 base pairs of DNA are wrapped in ~1.67 superhelical turns. Although the nucleosomes are stable protein-DNA complexes, they undergo spontaneous conformational changes that occur in an asynchronous fashion. This conformational dynamics, defined by the "site-exposure" model, involves the DNA unwrapping from the protein core and exposing itself transiently before wrapping back. Physiologically, this allows regulatory proteins to bind to their target DNA sites during cellular processes like replication, DNA repair and transcription. Traditional biochemical assays have stablished the equilibrium constants for the accessibility to various sites along the length of the nucleosomal DNA, from its end to the middle of the dyad axis. Using fluorescence correlation spectroscopy (FCS), we have established the position dependent rewrapping rates for nucleosomes. We have also used Monte Carlo simulation methods to analyze the applicability of FRET fluctuation spectroscopy towards conformational dynamics, specifically motivated by nucleosome dynamics. Another important conformational change that is involved in cellular processes is the disassembly of nucleosome into its constituent particles. The exact pathway adopted by nucleosomes is still not clear. We used dual color fluorescence correlation spectroscopy to study the intermediates during nucleosome disassembly induced by changing ionic strength. Studying the nature of nucleosome conformational change and the kinetics is very important in understanding gene expression. The results from this thesis give a quantitative description to the basic unit of the chromatin.Dissertation/Thesis
Ph.D. Chemistry 2011
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Neher, Richard [Verfasser]. "Dynamic aspects of DNA : DNA-slippage and nucleosome dynamics / vorgelegt von Richard Neher." 2007. http://d-nb.info/984536973/34.
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Ettig, Ramona [Verfasser]. "From nucleosomes to chromatin fibers : molecular dynamics and Monte-Carlo simulations of nucleosome organization and interactions / presented by Ramona Ettig." 2010. http://d-nb.info/1008255661/34.
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"The dynamics of nucleosome systems and the posttranslational modification effects: Applications of molecular dynamics simulation in structural biology." UNIVERSITY OF CALIFORNIA, DAVIS, 2009. http://pqdtopen.proquest.com/#viewpdf?dispub=3358973.
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Möbius, Wolfram [Verfasser]. "Physical aspects of chromatin constituents : DNA dynamics and nucleosome positioning / vorgelegt von Wolfram Möbius." 2010. http://d-nb.info/1006683054/34.
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Wocjan, Tomasz [Verfasser]. "Dynamics of DNA in nucleosomes and plasmids studied by Brownian dynamics / presented by Tomasz Wocjan." 2010. http://d-nb.info/1000122883/34.
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Padeganeh, Abbas. "Investigation of the structure and dynamics of the centromeric epigenetic mark." Thèse, 2014. http://hdl.handle.net/1866/11321.
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Le centromère est le site chromosomal où le kinetochore se forme, afin d’assurer une ségrégation fidèles des chromosomes et ainsi maintenir la ploïdie appropriée lors de la mitose. L’identité du centromere est héritée par un mécanisme épigénétique impliquant une variante de l’histone H3 nommée centromere protein-A (CENP-A), qui remplace l’histone H3 au niveau de la chromatine du centromère. Des erreurs de propagation de la chromatine du centromère peuvent mener à des problèmes de ségrégation des chromosomes, pouvant entraîner l’aneuploïdie, un phénomène fréquemment observé dans le cancer. De plus, une expression non-régulée de CENP-A a aussi été rapportée dans différentes tumeurs humaines. Ainsi, plusieurs études ont cherchées à élucider la structure et le rôle de la chromatine contenant CENP-A dans des cellules en prolifération. Toutefois, la nature moléculaire de CENP-A en tant que marqueur épigénétique ainsi que ces dynamiques à l'extérieur du cycle cellulaire demeurent des sujets débat.
Dans cette thèse, une nouvelle méthode de comptage de molécules uniques à l'aide de la microscopie à réflexion totale interne de la fluorescence (TIRF) sera décrite, puis exploitée afin d'élucider la composition moléculaire des nucléosomes contenant CENP-A, extraits de cellules en prolifération. Nous démontrons que les nucléosomes contenant CENP-A marquent les centromères humains de façon épigénétique à travers le cycle cellulaire. De plus, nos données démontrent que la forme prénucléosomale de CENP-A, en association avec la protéine chaperon HJURP existe sous forme de monomère et de dimère, ce qui reflète une étape intermédiaire de l'assemblage de nucléosomes contenant CENP-A.
Ensuite, des analyses quantitatives de centromères lors de différenciation myogénique, et dans différents tissus adultes révèlent des changements globaux qui maintiennent la marque épigénétique dans une forme inactive suite à la différentiation terminale. Ces changements incluent une réduction du nombre de points focaux de CENP-A, un réarrangement des points dans le noyau, ainsi qu'une réduction importante de la quantité de CENP-A. De plus, nous démontrons que lorsqu'une dédifférenciation cellulaire est induite puis le cycle cellulaire ré-entamé, le phénotype "différencié" décrit ci-haut est récupéré, et les centromères reprennent leur phénotype "prolifératif".
En somme, cet oeuvre décrit la composition structurale sous-jacente à l'identité épigénétique des centromères de cellules humaines lors du cycle cellulaire, et met en lumière le rôle de CENP-A à l'extérieur du cycle cellulaire.The centromere is a unique chromosomal locus where the kinetochore is formed to mediate faithful chromosome partitioning, thus maintaining ploidy during cell division. Centromere identity is inherited via an epigenetic mechanism involving a histone H3 variant, called centromere protein-A (CENP-A) which replaces histone H3 in centromeric chromatin. Defects in the centromeric chromatin can lead to missegregation of chromosomes resulting in aneuploidy, a ¬¬frequently observed phenomenon in cancer. Moreover, deregulated CENP-A expression has also been documented in a number of human malignancies. Therefore, much effort has been devoted to uncover the structure and role of CENP-A-containing chromatin in proliferating cells. However, the molecular nature of this epigenetic mark and its potential dynamics during and outside the cell cycle remains controversial. In this thesis, the development of a novel single-molecule imaging approach based on total internal reflection fluorescence and the use of this assay to gain quantitative information about the molecular composition of CENP-A-containing nucleosomes extracted from proliferating cells throughout the cell cycle as well as the dynamics and cellular fate of CENP-A chromatin in terminal differentiation are described. Here, we show that octameric CENP-A nucleosomes containing core Histones H2B and H4 epigenetically mark human centromeres throughout the cell cycle. Moreover, our data demonstrate that the prenucleosomal form of CENP-A bound by the chaperone HJURP transits between monomeric and dimeric forms likely reflecting intermediate steps in CENP-A nucleosomal assembly. Moreover, quantitative analyses of centromeres in myogenic differentiation and adult mouse tissue sections revealed that centromeres undergo global changes in order to retain a minimal CENP-A epigenetic code in an inactive state, upon induction of terminal differentiation. These include a robust decrease in the number of centromeric foci, subnuclear rearrangement as well as extensive loss of CENP-A protein. Interestingly, we show that forced dedifferentiation under cell cycle reentry permissive conditions, rescued the above-mentioned phenotype concomitantly with the restoration of cell division. Altogether, this work delineates the structural basis for the epigenetic specification of human centromeres during the cell cycle and sheds light on the cellular fate of the CENP-A epigenetic code outside the cell cycle.