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Jansen, Hailey Janice. "Characterization of chromatin assembly in murine embryos." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44768.
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During differentiation, changes in chromatin proteins lead to the establishment and maintenance of gene expression patterns. Histone H3 trimethylated at lysine 4 (H3K4me3) by the trithorax group (trxG) gene family Mixed lineage leukemia (MLL) is associated with active genes. H3K27me3 is trimethylated by the Polycomb group (PcG) Enhancer of Zeste (EZH2) at repressed genes. In Drosophila embryos, trxG and PcG proteins but not H3K4me3 or H3K27me3 are stable to DNA replication. In contrast, methylated histones are detected on nascent DNA in Drosophila and murine cell lines. Therefore some aspect of chromatin assembly or histone trimethylation must differ in different cells. My first aim was to determine if there is a change in the abundance of methylated histones at the replication fork in undifferentiated versus differentiated murine ES cells using two novel in vivo assays. Most undifferentiated ES cells lack early H3K4me3 and H3K27me3, but after 4 days of differentiation, most cells have early trimethylation of H3K4 and H3K27. I propose that the change in kinetics of histone methylation correlates with differentiation. To test this hypothesis, I carried out similar experiments on cells dissociated from day 9.5 (E9.5) and 14.5 (E14.5) murine embryos. In E9.5 cells there are two populations of cells, one that lacks methylated histones and the other contains methylated histones on nascent DNA. By E14.5 most cells exhibit H3K4me3 and H3K27me3 on nascent DNA. To determine if the presence of histone methyltransferases could account for the changes in histone methylation, I tested MLL1 and a subunit of the EZH2 complex, Su(z)12. Both are present continuously on nascent DNA, suggesting that their activity is regulated. Methylation and acetylation antagonize each other at the same residue. However I showed that the presence of acetylated H3K27 is not anticorrelated with H3K27me3 in most murine embryos cells. My results using inhibitors of the appropriate histone acetyltransferase were not conclusive owing to toxicity of the inhibitors. Overall, my results support the hypothesis that trimethylation of H3K4 and H3K27 on nascent DNA is developmentally regulated.
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Dunleavy, Elaine. "Assembly of centromeric chromatin in fission yeast." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/13739.
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Fission yeast centromeres are composed of a central domain surrounded on both sides by outer repeat heterochromatin. Marker genes inserted within the central domain or the outer repeats are transcriptionally silenced. A screen performed to identify mutants that specifically alleviate silencing at the central domain, identified the sim (silencing in the middle of the centromere) mutants. The sim6 mutant was unusual in that it was found to alleviate both central domain and outer repeat silencing and suggests that there may be cross talk between kinetochore assembly and the integrity of neighbouring heterochromatin. To identify other factors with a similar phenotype a screen was performed in which the cos (central core and outer repeat silencing) mutants were isolated. Several mutants allelic to sim6+ (renamed cos1+) were isolated. Cos1+ is allelic to fission yeast mcl1+ (mini-chromosome loss 1) and may function to ensure that features of silent chromatin and sister chromatin cohesion are properly maintained after replication. sim3 mutants were found to specifically alleviate silencing at the central domain. Sim3 is homologous to the histone binding proteins mammalian NASP (nuclear autoantigenic sperm protein) and Xenopus laevis N1/N2. Cells with defective Sim3 have reduced levels CENP-ACnp1 at centromeres and subsequently display defects in mititotic chromosome segregation. Sim3 may be acting as a CENP-ACnp1 ‘escort’, assisting in the replication independent assembly of CENP-ACnp1 chromatin at centromeres, however Sim3 may also contribute to the loading of Sim3 at other stages of the cell cycle. It remains to be determined whether NASP/N1/N2 plays a similar role in metazoa.
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Kats, Ellen Simona. "De-caf-einated life without chromatin assembly factors /." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3221182.
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Thesis (Ph. D.)--University of California, San Diego, 2006.Title from first page of PDF file (viewed September 8, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Royle, Nikki. "Expression, purification and characterisation of recombinant chromatin assembly factor 1." Thesis, University of Cambridge, 2013. https://www.repository.cam.ac.uk/handle/1810/244243.
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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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Rasala, Beth A. "Defining the early steps in nuclear pore assembly chromatin-associated ELYS initiates pore assembly /." Diss., View abstract only; access to full text of dissertation for UC IP will be available after 1/1/2011, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p3296834.
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Thesis (Ph. D.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed June 3, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Pietrobon, Violena. "Chromatin assembly by CAF-1 during homologous recombination : a novel step of regulation." Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00977568.
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The replication of chromosomes can be challenged by endogenous and environmental factors, interfering with the progression of replication forks. Therefore, cells have to coordinate DNA synthesis with mechanisms ensuring the stability and the recovery of halted forks. Homologous recombination (HR) is a universal mechanism that supports DNA repair and the robustness of DNA replication. Nonetheless, mechanisms regulating HR pathways, such as ectopic versus allelic recombination, remain poorly understood. Another essential pathway for genome stability is the wrapping of newly replicated DNA around nucleosomes, leading to the constitution of a chromatin fibre, which allows the structural organization of the genetic material. In Saccharomyces cerevisiae, deficiencies in chromatin assembly pathways lead to replication forks instability and consequent increase in the rate of HR. Histone chaperones play a crucial role during chromatin assembly, thus I decided to focus on the H3-H4 histone chaperone Chromatin Assembly Factor 1 (CAF-1), to study its role in HR processes in Schizosaccharomyces pombe. Indeed, HR includes a DNA synthesis step and little is known about the associated chromatin assembly. My data excluded a role for CAF-1 in allelic recombination and in the maintenance of forks stability. However, CAF-1 was found to play an important role during ectopic recombination, in promoting chromosomal rearrangements induced by halted replication forks. My data support a model according to which CAF-1 allows the stabilization of early recombination intermediates (D-loop), via nucleosome deposition during the elongation of these intermediates. Doing so, CAF-1 counteracts the dissociation of early recombination intermediates by the helicase Rqh1. Therefore, CAF-1 appears to be part of an equilibrium that regulates stability/dissociation of early steps of recombination events. Importantly, I found that the role of CAF-1 in this equilibrium is of particular importance during non-allelic recombination, revealing a novel regulation level of HR mechanisms and outcomes by chromatin assembly.
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Nabatiyan, Arman. "The role of chromatin assembly factor-1 in human cells." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615283.
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Wong, Hiu-ting. "A role of TSPYL2, a novel nucleosome assembly protein, in transcriptional regulation." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43085726.
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Huanyu, Wang. "Characterization of N1/N2 Family Histone Chaperones: Hif1p and NASP." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1279815431.
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Hunt, Spencer Philip. "Whole-Genome Assembly of Atriplex hortensis L. Using OxfordNanopore Technology with Chromatin-Contact Mapping." BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/8580.
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Atriplex hortensis (2n = 2x = 18, 1C genome size ~1.1 gigabases), also known as garden orach, is a highly nutritious, broadleaf annual of the Amaranthaceae-Chenopodiaceae family that has spread from its native Eurasia to other temperate and subtropical environments worldwide. Atriplex is a highly complex and polyphyletic genus of generally halophytic and/or xerophytic plants, some of which have been used as food sources for humans and animals alike. Although there is some literature describing the taxonomy and ecology of orach, there is a lack of genetic and genomic data that would otherwise help elucidate the genetic variation, phylogenetic position, and future potential of this species. Here, we report the assembly of the first highquality, chromosome-scale reference genome for orach cv. ‘Golden’. Sequence data was produced using Oxford Nanopore’s MinION sequencing technology in conjunction with Illumina short-reads and chromatin-contact mapping. Genome assembly was accomplished using the high-noise, single-molecule sequencing assembler, Canu. The genome is enriched for highly repetitive DNA (68%). The Canu assembly combined with the Hi-C chromatin-proximity data yielded a final assembly containing 1,325 scaffolds with a contig N50 of 98.9 Mb and with 94.7% of the assembly represented in the nine largest, chromosome-scale scaffolds. Sixty-eight percent of the genome was classified as highly repetitive DNA, with the most common repetitive elements being Gypsy and Copia-like LTRs. The annotation was completed using MAKER which identified 31,010 gene models and 2,555 tRNA genes. Completeness of the genome was assessed using the Benchmarking Universal Single Copy Orthologs (BUSCO) platform, which quantifies functional gene content using a large core set of highly conserved orthologous genes (COGs). Of the 1,375 plant-specific COGs in the Embryophyta database, 1,330 (96.7%) were identified in the Atriplex assembly. We also report the results of a resequencing panel consisting of 21 accessions which illustrates a high degree of genetic similarity among cultivars and wild material from various locations in North America and Europe. These genome resources provide vital information to better understand orach and facilitate future study and comparison.
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Wong, Hiu-ting, and 王曉婷. "A role of TSPYL2, a novel nucleosome assembly protein, in transcriptional regulation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43085726.
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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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Haddad, John. "Structural and Biochemical Insights into the Assembly of the DPY-30/Ash2L Heterotrimer." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36616.
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In eukaryotes, the SET1 family of methyltransferases carry out the methylation of Lysine 4 on Histone H3. Alone, these enzymes exhibit low enzymatic activity and require the presence of additional regulatory proteins, which include RbBP5, Ash2L, WDR5 and DPY-30, to stimulate their catalytic activity. While previous structural studies established the structural basis underlying the interaction between RbBP5, Ash2L and WDR5, the formation of the Ash2L/DPY-30 complex remains elusive. Here we report the crystal structure of the Ash2L/DPY-30 complex solved at 2.2Å. Our results show that a Cterminal amphipathic α-helix on Ash2L makes several hydrophobic interactions with the DPY-30 homodimer. Moreover, the structure reveals that a tryptophan residue on Ash2L, which directly precedes its C-terminal amphipathic α-helix, makes key interactions with one of DPY-30 α-helix. Finally, biochemical studies of Ash2L revealed a hitherto unknown ability of this protein to bind anionic lipids.
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Ai, Xi. "Biochemical characterization of a hat1p-containing histone acetyltransferase complex." Columbus, Ohio : Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1085509452.
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Thesis (Ph. D.)--Ohio State University, 2004.Title from first page of PDF file. Document formatted into pages; contains xv1, 151 p.; also includes graphics. Includes abstract and vita. Advisor: Mark Parthun, Dept. of Biochemistry. Includes bibliographical references (p. 138-151).
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English, Christine Marie. "Insights into chromatin assembly through the characterization of the histone chaperone ASF1 bound to histones H3-H4 /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2006.
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Thesis (Ph.D. in Biochemistry & Molecular Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006.Typescript. Includes bibliographical references (leaves 169-185). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
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Aguilar, Gurrieri Carmen. "Etudes structurales sur l'assemblage du nucléosome." Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENV017/document.
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Au sein du noyau, l'ADN est organise en chromatine dont l'unité de base est le nucléosome. La structure de la chromatine est très dynamique, ce qui est nécessaire pour la plupart des opérations qui se produisent dans l'ADN telles que la réplication, la transcription, la réparation et la recombinaison. Le nucléosome est constitué de deux dimères H2A/H2B et deux dimères H3/H4 associés avec 147 paires de bases d'ADN. La protéine Nap1 est un chaperon d'histone H2A/H2B impliquée dans l'assemblage et démontage des nucléosomes. Nap1 protège les interactions non spécifiques entre l'ADN chargé négativement et les dimères H2A/H2B chargés positivement, afin de permettre la formation de la structure ordonnée des nucléosomes. Lors de l'assemblage des nucléosomes, les dimères d'histones H3/H4 sont déposés en premier lieu, suivi par le dépôt de dimères H2A/H2B. Lors du démontage du nucléosome, les dimères H2A/H2B sont retirés avant le retrait des dimères H3/H4. La determination de la structure du complexe Nap1-H2A/H2B pourra permettre une meilleure compréhension du processus d'assemblage du nucléosome. Dans cette étude, nous voulons comprendre comment le chaperon Nap1 cible spécifiquement les dimères d'histones H2A/H2B pour l'assemblage des nucléosomes. Notre objectif est de caractériser la structure et la fonction du complexe de Nap1-H2A/H2B. Ainsi nous nous sommes tout d'abord intéresse à la stoechiometrie de ce complexe. Nous avons trouvé qu'un dimère de Nap1 s'associe à un dimère H2A/H2B (Nap1_2-H2A/H2B). D'autre part, l'analyse par spectrométrie de masse non-dénaturante a montré que ce complexe de base peut s'oligomériser et contenir jusqu'à 6 copies de Nap1_2-H2A/H2B. L'analyse de ce complexe par spectrométrie de masse non-dénaturant a montré que ce complexe peu oligomériser dans un grand complexe contenant jusqu'à 6 copies de Nap1_2-H2A/H2B. Nous avons également obtenu la première structure cristalline à basse résolution de ce complexe. L'analyse du même complexe par microscopie électronique à coloration négative a révélé la présence en solution du même oligomère que dans l'unité asymétrique du cristal, qui contient aussi 6 copies de Nap1_2-H2A/H2B. Ainsi, nous avons pu mettre en évidence de nouvelles interfaces d'interaction entre les différents composants de ce complexe qui nous permettent de mieux comprendre le processus d'assemblage des nucléosomes. Le remodelage de la chromatine permet l'expression des gènes eucaryotes. Ce remodelage nécessite des enzymes telles que des histone acétyltransférases (HAT) et les chaperons d'histones. Les HATs acétylent les chaînes latérales des lysines. Il a été proposé que les HATs et les histones chaperons agissent en synergie pour moduler la structure de la chromatine pendant la transcription. La HAT p300 a été proposé d'interagir avec l'histone chaperon Nap1. Nous avons entrepris de caractériser cette interaction. Malheureusement, nos expériences n'ont pas pu détecter d'interaction directe entre ces protéinesAssembly of chromatin is an essential process that concerns most DNA transactions in eukaryotic cells. The basic repeating unit of chromatin are nucleosomes, macromolecular complexes that consist of a histone octamer that organizes 147 bp of DNA in two superhelical turns. Although, the structures of nucleosomes are known in detail, their assembly is poorly understood. In vivo, nucleosome assembly is orchestrated by ATP-dependent remodelling enzymes, histone-modifying enzymes and a number of at least partially redundant histone chaperones. Histone chaperons are a structurally diverse class of proteins that direct the productive assembly and disassembly of nucleosomes by facilitating histone deposition and exchange. The currently accepted model is that nucleosome assembly is a sequential process that begins with the interaction of H3/H4 with DNA to form a (H3/H4)2 tetramer-DNA complex. The addition of two H2A/H2B dimers completes a canonical nucleosome. High-resolution structures of histone chaperons in complex with H3/H4 histones have resulted in detailed insights into the process of nucleosome assembly. However, our understanding of the mechanism of nucleosome assembly has been hampered by the as yet limited number of co-crystal structures of histone–chaperone complexes. In particular it remains unclear how histone chaperons mediate H2A/H2B deposition to complete nucleosome assembly. In this work, we have investigated the role of the H2A/H2B chaperon Nap1 (Nucleosome assembly protein 1) in nucleosome assembly. We have determined the crystal structure of the complex between Nap1 and H2A/H2B and analysed the assembly by various biophysical methods. The structure shows that a Nap1 dimer binds to one copy of H2A/H2B (Nap1_2-H2A/H2B). A large ~550 kDa macromolecular assembly containing 6 copies of the Nap12-H2A/H2B complex is seen in the asymmetric crystallographic unit. We confirmed by both non-denaturing mass spectroscopy and negative stain electron microscopy studies that this assembly is the predominant form of the Nap1_2-H2A/H2B complex in solution. We further investigated the potential interplay between p300-mediated histone acetylation and nucleosome assembly. Together, the structure and associated functional analysis provide a detailed mechanism for the Nap1 chaperon activity, its role in H2A/H2B deposition and in nucleosome assembly
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Keenen, Bridget A. "The role of SWI/SNF chromatin remodeling enzymes in melanoma." Toledo, Ohio : University of Toledo, 2010. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1271819328.
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Dissertation (Ph.D.)--University of Toledo, 2010."Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Sciences." Title from title page of PDF document. "A Dissertation entitled"--at head of title. Bibliography: p. 63-71, 126-140.
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Rodriges, Blanko Elena V. "ANALYSIS OF HUMAN DNA MISMATCH REPAIR IN THE CHROMATIN ENVIRONMENT." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/dissertations/967.
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Mismatch repair corrects errors made during DNA replication and inactive mismatch repair is associated with Lynch Syndrome and sporadic cancer. Genome replication in eukaryotes is accompanied by chromatin formation. The first step in chromatin establishment is nucleosome assembly, that starts with histone tetramer deposition. It is not clear how three important cellular processes: genome replication, mismatch repair and nucleosome assembly are coordinated. Here we analyzed human mismatch repair in the presence of histone deposition in a reconstituted system. We showed that mismatch repair factor inhibits nucleosome assembly on the DNA region with the replicative error. Such a mechanism is important, since in this way DNA with errors remains accessible for mismatch repair system to perform the repair. The DNA synthesis step in mismatch repair is performed by DNA polymerase. Eukaryotes possess two major replicative DNA Polymerases: DNA Polymerase delta and DNA Polymerase epsilon. DNA polymerase delta is involved in mismatch repair. However, it was unknown whether DNA polymerase epsilon can also work in mismatch repair. Here we analyzed human mismatch repair with DNA Polymerase delta and DNA Polymerase epsilon in the environment of histone deposition. Our results indicated that repair activity with both polymerases was activated by histone deposition. Here it was first shown that human DNA Polymerase epsilon performs DNA synthesis during mismatch repair in vitro. Importantly, recent studies have revealed association of Polymerase epsilon mutations with cancer. Since our data showed activity of DNA Polymerase epsilon in mismatch repair, a possible tumor development mechanism may involve inactivation of mismatch repair due to Polymerase epsilon mutations. Overall, our study expanded the understanding of the mechanism of human mismatch repair in the chromatin environment.
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Walfridsson, Julian. "The CHD chromatin remodeling factors in schizosaccharomyces pombe /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-106-7/.
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Bennett, Gwendolyn M. "Chromatin Regulators and DNA Repair: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/742.
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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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Bennett, Gwendolyn M. "Chromatin Regulators and DNA Repair: A Dissertation." eScholarship@UMMS, 2012. http://escholarship.umassmed.edu/gsbs_diss/742.
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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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Ge, Zhongqi. "Role of Nuclear Hat1p Complex and Acetylation of Newly Synthesized Histone H4 in Chromatin Assembly." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1356622980.
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Agudelo, Garcia Paula A. "Identification of New Roles for Histone Acetyltransferase 1." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492599746298382.
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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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Liu, Jessica Chishow. "Biochemical Characterization of the Domain Architecture of Chromatin Assembly Motor Proteins Human CHD1 and CHD2." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226060.
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The sites where the basic unit of chromatin, the nucleosome, is assembled greatly affects the dynamic compaction/decompaction of eukaryotic genetic material and how the DNA is accessed, read, and interpreted. The nucleosome, which consists of ~147 base pairs of DNA wrapped in a left-handed superhelix around an octameric core made up of histone proteins, is the targeted substrate for ATP-dependent protein machineries called chromatin remodelers. Remodelers are essential regulators of DNA accessibility and are often grouped into four families: SWI/SNF, INO80/SWR1, ISWI, and CHD. Though remodelers can act as large multi-subunit complexes, all have a unique core SNF2-like ATPase that utilizes the energy from ATP hydrolysis to translocate along DNA. This DNA translocase activity of the catalytic ATPase domain acts in coordination with auxiliary domains or accessory subunits to disrupt histone-DNA contacts, resulting in distinct remodeling outcomes. Furthermore, the assembly of DNA into nucleosomal arrays is a specialized activity catalyzed by a subset of remodelers. Identifying remodeler proteins responsible for nucleosome assembly and delineating the mechanisms through which remodelers assemble and remodel nucleosomes are key goals in the field of chromatin biology.
CHD proteins have important roles in regulating gene expression through their remodeling activities. While yeast cells only have one CHD protein (CHD1), mammalians possess nine proteins (CHD1-9) that are further categorized into subfamilies on the basis of additional sequences flanking the central ATPase domain. CHD2 is in the same subfamily as CHD1 and has been linked to developmental regulation but the enzymatic activity of CHD2 has not been well characterized. Given the homology between human CHD2 and CHD1, which is an important assembly protein in other species (S. cerevisiae and D. melanogaster), we set out to delineate the biochemical properties of human CHD2 and the CHD1 human counterpart.
In this dissertation work, we examined the biochemical activities of recombinant human CHD1 and CHD2. We used in vitro chromatin assembly and remodeling assays and showed CHD2 assembles nucleosomal arrays and remodels nucleosomes while CHD1 exhibits less robust activity by comparison. We used radiometric ATPase and electrophoretic mobility gel shift assays to measure the ATPase and DNA-binding activities of human CHD1 and CHD2 and assessed the contribution from conserved accessory domains using systematic protein truncations. We found the N-terminal chromodomains are inhibitory for the ATPase and DNA-binding activities of both CHD1 and CHD2 while providing substrate specificity for the latter. Moreover, we showed the DNA-binding domain of CHD2 enhances its ATPase and remodeling activities. The distinct in vitro activities exhibited by human CHD1 and CHD2 suggest they have non-redundant roles in vivo with important mechanistic implications for remodeling by CHD proteins. In a broader sense, our findings have added to the number of known assembly motor proteins and aids in our understanding of how remodelers have evolved auxiliary domains to carry out specific functions such as chromatin assembly.
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Klapholz, Benjamin. "Le facteur d'assemblage de la chromatine CAF-1 : analyse fonctionnelle chez la drosophile." Paris 6, 2008. http://www.theses.fr/2008PA066322.
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La chromatine joue un rôle essentiel dans la stabilité du génome et lors de la différentiation. Mon travail a principalement consisté à caractériser les phénotypes d’un mutant pour la grande sous-unité du facteur d’assemblage de la chromatine CAF-1 chez la drosophile. Les larves mutantes meurent à cause de défauts s’accumulant dans leurs cellules endocyclantes, alors que la progression de leur cycle cellulaire est normale. Ces cellules présentent des défauts d’organisation des nucléosomes, d’efficacité de la réplication des régions d’euchromatine et d’intégrité du génome. Cette étude a permis de corréler l’assemblage des nucléosomes avec l’efficacité du processus de réplication in vivo. Des résultats préliminaires indiquent aussi que les cellules mitotiques pourraient présenter des besoins variables en CAF-1, selon leur différentiation ou la nature de leur division. Enfin, j’ai participé à la caractérisation de la fonction de l’interaction entre CAF-1 et la protéine HP1a in vivo.
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Pugieux, Céline. "Meiotic spindle assembly on chromatin micropatterns : investigating the roles of Augmin, Kinesin-10 and Kinesin-4." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAJ011.
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La division cellulaire est essentielle pour la survie de chaque être vivant. Au cours de ce processus, les chromosomes de la cellule en division sont transmis aux deux cellules filles. La répartition des chromosomes est orchestrée par une structure cellulaire transitoire appelée fuseau mitotique (ou fuseau méiotique dans les cellules reproductrices). Le fuseau est composé de microtubules, de nombreuses protéines et de moteurs moléculaires, qui interagissent de manière complexe et précise aboutissant à l’organisation d’une structure bipolaire dynamique. Comme certains mécanismes moléculaires restent mal compris, nous avons choisi d'aborder la question de l'assemblage du fuseau méiotique dans des extraits d'oeufs de grenouille. Xenopus laevis est un organisme modèle car il est proche, d’un aspect phylogénétique, de l'homme, et il est particulièrement adapté à l’étude de la division cellulaire. Nous avons également utilisé une méthode in vitro (appelée spindle array ou puce à fuseaux) qui a été développée au sein du groupe de recherche auparavant, et qui offre certains avantages par rapport aux approches existantes. Une puce à fuseaux est composée de billes recouvertes de chromatine immobilisées selon des micro-motifs géométriques obtenus selon une technique d’impression par microcontact. L'assemblage des fuseaux méiotiques a été visualisé par microscopie confocale à fluorescence. Grâce à ces outils, nous avons, lors d’un premier projet, abordé le rôle de l’Augmin dans l'assemblage des fuseaux. L’Augmin est un complexe protéique récemment identifié grâce à son hypothétique rôle dans la nucléation de microtubules à partir de microtubules existants. Après déplétion de l’Augmin, nous avons constaté que la nucléation des microtubules était réduite et que les fuseaux avaient une morphologie anormale. De plus, ces derniers qui étaient essentiellement multipolaires sont progressivement devenus bipolaires grâce à une voie de nucléation des microtubules, découverte lors de notre étude, émanant des pôles acentrosomaux et qui est indépendante de l’Augmin. Nos résultats révèlent que l’Augmin est essentiel pour l’assemblage et la bipolarité du fuseau acentrosomal. Au cours d’un second projet, nous avons étudié les fonctions des chromokinésines kinésine-4 (Xklp1) et kinésine-10 (Xkid) dans l'assemblage des fuseaux et leurs mouvements. Xkid participe à la force d’éjection polaire nécessaire à la congression des chromosomes alors que Xklp1 contribue principalement à la régulation de la dynamique des microtubules. En étudiant l'assemblage de fuseaux dans des extraits après déplétion de Xkid, Xklp1 ou les deux, nous avons démontré que Xkid limite la dynamique des mouvements longitudinaux des fuseaux, contribue à la mise en place de la bipolarité et régule la longueur des fuseaux. Nous avons également quantifié la cinétique de nucléation des microtubules et confirmé le rôle de Xklp1 dans la régulation de la dynamique des microtubules. L’ensemble de nos travaux contribuent à une meilleure compréhension des mécanismes d’assemblage du fuseau méiotique et confirme la pertinence de notre méthode pour l'étude de sa morphogenèseCell division is essential for the survival of every living organism. During this process, the chromosomes of the dividing cell are transmitted to the two daughter cells. The partition of the chromosomes is orchestrated by a transient sub-cellular structure called the mitotic spindle (or meiotic spindle in gamete cells). The spindle is composed of microtubules, numerous proteins and molecular motors, which interact in an intricate and yet precise manner leading to a highly dynamic and complexstructure. As some molecular mechanisms remain elusive, we have chosen to address the question of meiotic spindle assembly in Xenopus egg extracts. Xenopus laevis is a model system that is evolutionary close to human, and suitable for cell division studies. We have combined this with an in vitro assay - spindle array - which we developed prior to this work, and which provides advantages over existing approaches. A spindle array is composed of chromatin-coated beads that are immobilized according to geometrical patterns obtained by microcontact printing. The assembly of meiotic spindles wasvisualized by time-lapse fluorescence confocal microscopy. Using these tools, we first addressed the role of augmin in the assembly of meiotic spindles. Augmin is a recently identified protein complex that has been hypothesized to induce microtubule nucleation from the side of preexisting microtubules. By depleting augmin, we found that microtubule nucleationwas reduced and that spindles were morphologically impaired. Spindles were predominantly multipolar but finally reached bipolarity as a result of a newly uncovered augmin-independent microtubule nucleation pathway from acentrosomal poles. Our results thus reveal that augmin is essential for the proper establishment of the microtubule scaffolding and the bipolarity ofacentrosomal spindles. Secondly, we investigated the functions of the chromokinesins kinesin-4 (Xklp1) and kinesin-10 (Xkid)in acentrosomal spindle architecture and motions. Xkid plays a major role in the polar ejection forces leading chromosome movements during congression while the main function of XKlp1 is to regulate microtubule dynamics. We studied spindle assembly in depleted extracts and we report that Xkid limits the dynamics of spindle longitudinal movements, contributes to spindle bipolarity and affects spindle length while XKlp1 controls the spindle microtubule mass. Altogether these findings contribute to a better understanding of meiotic spindle assembly and confirm the pertinence of our method to study spindle morphogenesis
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Garinther, Wendy Irene. "Studies of the topoisomerase and cofactor requirements for nucleosome reconstitution in a yeast chromatin assembly system." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/mq22598.pdf.
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Mocavini, Ivano 1991. "Variations of Polycomb assembly in mouse embryonic stem cells and early differentiation." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2022. http://hdl.handle.net/10803/673953.
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Polycomb repressive complex (PRC)1 and 2 are major players in gene regulation, devoted to maintenance of the epigenetic memory of gene silencing. Mouse embryonic stem cells (mESCs) have been extensively exploited as a model system to study Polycomb complexes composition and function, leading to the identification of many accessory factors. However, the mechanisms regulating PRC1/2 composition in mESCs are still poorly understood. Moreover, several reports have shown that changes in composition occur upon cell differentiation, although little is known about the functional relevance of these changes. In this doctoral thesis I aim to address these questions by focusing on two examples of these aspects: first, I characterize the PRC1 interactome upon mESCs differentiation to primitive endoderm (PrE) and try to assess its role in this cell fate transition. Secondly, I report the identification of a novel splicing isoform of PRC2 core component Suz12, with implications in PRC2 composition and activity on chromatinLos complejos de represión Polycomb (PRC)1 y 2 juegan un papel central en la regulación de la expresión génica, preservando la memoria epigenética del estado de silenciamiento. El uso de células madre embrionarias de ratón (mESCs) como modelo para el estudio de los complejos Polycomb, ha permitido la identificación de muchos factores accesorios. Sin embargo, no se conocen los mecanismos que regulan la composición de PRC1/2 en las mESCs. Además, varios informes han demostrado que, tras la diferenciación celular, ocurren ciertos cambios en la composición, aunque se sabe poco sobre la relevancia funcional de estos. En esta tesis doctoral trato de abordar estas cuestiones centrándome en dos ejemplos concretos: primero, caracterizo la red de proteínas asociadas a PRC1 tras la diferenciación de mESCs al endodermo primitivo (PrE), e intento evaluar su rol durante esta transición de identidad celular. En segundo lugar, informo del hallazgo de una nueva isoforma de splicing de Suz12, un componente central de PRC2, con implicaciones para la composición y la actividad de PRC2 en la cromatina.
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Swygert, Sarah G. "The Shape of Silence: The Solution-State Conformation of Sir Heterochromatin: A Dissertation." eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/790.
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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.
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Tacheva, Silvia K. "Post-translational Modifications of Newly Synthesized Histones H3 and the Role of H3 K56 Acetylation on Chromatin Assembly in Mammalian Cells." Thesis, Boston College, 2010. http://hdl.handle.net/2345/1745.
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Thesis advisor: Anthony T. AnnunziatoThe project I am presenting aimed to: 1. Elucidate the pattern of post- translational modification on the different variants of newly synthesized histones H3 in mammalian cells; 2. Reveal whether the acetylation of residue K56 on newly synthesized H3 histones plays a role in the incorporation of the histone into chromatin in mammalian cells; and 3. Determine whether the acetylation of residue K56 on newly synthesized H3 histones plays a role in the incorporation of the histone specifically in replicating chromatin in mammalian cells. The experiments to answer these questions were performed using HEK293 cells with inducible expression of FLAG-histones, enabling us to control the synthesis of new histones of interest and to detect and analyze their presence and relative levels in the cells. The results suggest that the acetylation of lysine 56 on histone H3 may play a positive role in the incorporation of the histone into new chromatin, and lack of acetylation may be reducing the efficiency of incorporation compared to acetylated histones
Thesis (MS) — Boston College, 2010
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology
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Harding, Katherine. "Silencing Proteins Sir3 and Sir4 have Distinct Roles in the Assembly of Silent Chromatin in Budding Yeast." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31578.
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The Silent Information Regulator (SIR) complex is responsible for the formation of silent chromatin domains in Saccharomyces cerevisiae, and consists of the NAD-dependent histone deacetylase Sir2, and histone binding proteins Sir3 and Sir4. The current model of silent chromatin assembly proposes that histone deacetylation by Sir2 is required to promote recruitment of Sir3 and Sir4, and assembly of full SIR complexes on chromatin. However, recent work has suggested unique roles for the histone binding proteins Sir3 and Sir4 in this process. Here we present data suggesting that Sir3 is primarily responsible for mediating the spreading of silent chromatin from sites of nucleation, while regulation of Sir4 abundance controls the rate of silencing establishment. We have also investigated a potential novel dimerization domain in Sir3, which may represent a conserved function in vertebrates. Investigations into the regulation of silent chromatin assembly in budding yeast will facilitate our understanding of the mechanisms that control heterochromatin-mediated gene repression in higher organisms.
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Lu, Chenning. "Cooperative Binding of Sir Proteins to Nucleosomes and Its Implications for Silent Chromatin Assembly in Saccharomyces Cerevisiae." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467201.
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Silent chromatin, or heterochromatin, refers to regions of the genome in which genes are constitutively repressed. These regions are important for regulating developmental genes and for maintaining genome stability, and are epigenetically inherited. In Saccharomyces cerevisiae, subtelomeres and silent mating type loci are assembled into silent chromatin by the Silent Information Regulator (SIR) complex, composed of Sir2, Sir3 and Sir4, which deacetylates histones and spreads along chromatin. Many questions remain regarding the mechanism of Sir protein spreading along chromatin and the mechanism of epigenetic inheritance of silent chromatin domains. It has been hypothesized that the lateral Sir-Sir protein interactions together with Sir-nucleosome interactions cooperatively recruit Sir proteins to spread along chromatin.
In my thesis project, I set out to test this cooperativity hypothesis by examining the interaction of Sir proteins with well-defined in vitro reconstituted mono- and di-nucleosomes. Using electrophoretic mobility shift assay (EMSA), I find that Sir3, the main nucleosome-binding component of the SIR complex, associates with nucleosomes cooperatively, involving the dimerization of Sir3 bound to neighboring nucleosomes. I demonstrate that this inter-nucleosomal cooperativity is mediated by the Sir3 C-terminal winged helix (wH) dimerization domain and is further stabilized by the Sir4 coiled-coil (CC) domain, which mediates both Sir4 homodimerization and Sir3-Sir4 interactions. There is functional redundancy from the two domains in mediating binding cooperativity, as suggested by the measurement of cooperativity free energy. Surprisingly, my binding measurements suggest that there are no Sir-Sir protein interactions on the same nucleosome. Moreover, by using an in vitro bridging assay, I show that Sir3 effectively bridges free nucleosomes in solution and that its wH domain is required for its bridging activity. My in vitro results are corroborated by in vivo ChIP-seq results showing that either the Sir3 wH domain or the Sir4 CC domain alone could mediate weak spreading of Sir3 protein, away from recruitment sites, under Sir3 overexpression conditions. However, mutations in both domains abolish the spreading completely.
Both histone H4 lysine 16 (H4K16) acetylation and histone H3 lysine 79 (H3K79) methylation are hallmarks of euchromatin in S. cerevisiae. I quantify the effect of either modification alone and both modifications in combination on Sir3-nucleosome binding affinity. This shows that either modification alone decreases Sir3 binding affinity towards nucleosomes by 3-4 fold, and that the two modifications work together to reduce the binding affinity even further. Statistical mechanical modeling of the nucleosome binding results indicate that the combined effect of H4K16 acetylation and H3K79 methylation can account for partitioning of Sir3 between silent and active chromatin regions in vivo.
Our findings and their quantitative analysis suggest that SIR complexes spread along chromatin discontinuously, arguing against the stepwise polymerization model for silent chromatin assembly.Medical Sciences
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Levenstein, Mark E. "Development and characterization of a highly defined, fully recombinant in vitro chromatin assembly system with Drosophila factors /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2002. http://wwwlib.umi.com/cr/ucsd/fullcit?p3045785.
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Manning, Benjamin J. "ATP-Dependent Heterochromatin Remodeling: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/795.
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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.
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Manning, Benjamin J. "ATP-Dependent Heterochromatin Remodeling: A Dissertation." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/795.
Full textAbstract:
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.
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Gao, Min. "Structural and Dynamic Studies of Supramolecular Assemblies by Solid State NMR Spectroscopy." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1385135235.
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Yang, Xiaofang. "Functional and Structural Dissection of the SWI/SNF Chromatin Remodeling Complex: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/330.
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The yeast SWI/SNF complex is the prototype of a subfamily of ATP-dependent chromatin remodeling complexes. It consists of eleven stoichiometric subunits including Swi2p/Snf2p, Swi1p, Snf5p, Swi3p, Swp82p, Swp73p, Arp7p, Arp9p, Snf6p, Snf11p, and Swp29p, with a molecular weight of 1.14 mega Daltons. Swi2p/Snf2p, the catalytic subunit of SWI/SNF, is evolutionally conserved from yeast to human cells. Genetic evidence suggests that SWI/SNF is required for the transcriptional regulation of a subset of genes, especially inducible genes. SWI/SNF can be recruited to target promotors by gene specific activators, and in some cases, SWI/SNF facilitates activator binding. Biochemical studies have demonstrated that purified SWI/SNF complex can hydrolyze ATP, and it can use the energy from ATP hydrolysis to generate superhelical torsion, mobilize mononucleosomes, enhance the accessibility of endonucleases to nucleosomal DNA, displace H2A/H2B dimers, induce dinucleosome and altosome formation, or evict nucleosomes. A human homolog of Swi2p/Snf2p, BRG1, is the catalytic subunit of the human SWI/SNF complex. Interestingly, isolated BRG1 alone is able to remodel a mononucleosome substrate. Importantly, mutations in mammalian SWI/SNF core subunits are implicated in tumorigenesis. Therefore, it remains interesting to characterize the role(s) of each subunit for SWI/SNF function. In this thesis project, I dissected SWI/SNF chromatin remodeling function by investigating the role of the SANT domain of the Swi3p subunit. Swi3p is one of the core components of SWI/SNF complex, and it contains an uncharacterized SANT domain that has been found in many chromatin regulatory proteins. Earlier studies suggested that the SANT domain of Ada2p may serve as the histone tail recognition module. For Swi3p, a small deletion of eleven amino acids from the SANT domain caused a growth phenotype similar to that of other swi/snf mutants.
In chapter I, I have reviewed recent findings in the function of chromatin remodeling complexes and discuss the molecular mechanism of their action.
In chapter II, I characterized the role of the SANT domain of Swi3p. I found that deletion of the SANT domain caused a defect in a genome-wide transcriptional profile, SWI/SNF recruitment, and more interestingly impairment of the SANT domain caused the dissociation of SWI/SNF into several subcomplexes: 1) Swi2p/Arp7p/Arp9p, 2) Swi3p/Swp73p/Snf6p, 3) Snf5p, and 4) Swi1p. Artificial tethering of SWI/SNF onto a LacZ reporter promoter failed to activate the reporter gene in the absence of the SANT domain, although Swi2p can be recruited to the LacZ promoter. We thus demonstrated that the Swi3p SANT domain is critical for Swi3p function and serves as a protein scaffold to integrate these subcomplexes into an intact SWI/SNF complex.
In Chapter III, I first characterized the enzymatic activity of the subcomplexes, especially the minimal complex of Swi2p/Arp7p/Arp9p. We found that this minimal subcomplex is fully functional for chromatin remodeling in assays including cruciform formation, restriction enzyme accessibility in mononucleosomal and nucleosomal array substrates, and mononucleosome mobility shift. However, it is defective in ATP-dependent removal of H2A/H2B dimers. Moreover, we found that Swi3p and the N-terminal acidic domain of Swi3p strongly interact with GST-H2A and H2B but not GST-H3 or H4 tails. We purified a SWI/SNF mutant (SWI/SNF-Δ2N) that lacks 200 amino acids within the N-terminal acidic domain of Swi3p. Intriguingly, SWI/SNF-Δ2N failed to catalyze ATP-dependent dimer loss, although this mutant SWI/SNF contains all the subunits and has intact ATP-dependent activity in enhancing restriction enzyme accessibility. These data help to further understand the molecular mechanism of SWI/SNF, and show that H2A/H2B dimer loss is not an obligatory consequence of ATP-dependent DNA translocation, but requires the histone chaperone function of the Swi3p subunit. Based on these findings, we proposed a new model of the structural and functional organization of the SWI/SNF chromatin remodeling machinery: SWI/SNF contains at least four distinct modules that function at distinct stages of the chromatin remodeling process. 1) Swi1p and Snf5p modules directly interact with gene specific activators and function as the recruiter; 2) Swi2p/Arp7p/Arp9p generates energy from ATP hydrolysis and disrupts histone/DNA interactions; and 3) Swi3p/Swp73p/Snf6p may play dual roles by integrating each module into a large remodeling complex, as well as functioning as a histone H2A/H2B chaperone to remove dimers from remodeled nucleosomes.
Chapter IV is a perspective from current work in this project. I first discuss the interest in further characterizing the essential role of Snf6p, based on its activation of LacZ reporter on its own. Using in vitro translated protein and co-IP studies, I tried to pinpoint the requirement of the SANT domain for SWI/SNF assembly. I found that Swi3p directly interacts with Swp73p, but not with other subunits. When Swi3p is first incubated with Swp73p, Swi3p also interacts with Snf6p, indicating that Swi3p indirectly interacts with Snf6p, therefore forming a subcomplex of Swi3p/Swp73p/Snf6p. This subcomplex can also be reconstituted using in vitro co-translation. Consistent with the TAP preparation of this subcomplex, partial deletion of the SANT domain of Swi3p does not affect the assembly of Swi3p/Swp73p/Snf6p in vitro. However, the assembly of SWI/SNF complex was not detected in the presence of eight essential in vitro translated subunits or from co-translation of all the subunits. I have discussed the interest in further characterizing the histone chaperone role of the Swi3p N-terminal acidic domain and the role of other core subunits of SWI/SNF such as Snf6p for transcriptional regulation.
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Bryant, Victoria. "CMG Helicase Assembly and Activation: Regulation by c-Myc through Chromatin Decondensation and Novel Therapeutic Avenues for Cancer Treatment." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6191.
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The CMG (Cdc45, MCM, GINS) helicase is required for cellular proliferation and functions to unwind double-stranded DNA to allow the replication machinery to duplicate the genome. Cancer cells mismanage helicase activation through a variety of mechanisms, leading to the potential for the development of novel anti-cancer treatments. Mammalian cells load an excess of MCM complexes that act as reserves for new replication origins to be created when replication forks stall due to stress conditions, such as drug treatment. Targeting the helicase through inhibition of the MCM complex has sensitized cancer cells to drugs that inhibit DNA replication, such as aphidicolin and hydroxyurea. However, these drugs are not used in the clinical management of cancer. We hypothesized that the effectiveness of the clinically relevant drugs gemcitabine and 5-FU against pancreatic cancer cells, and oxaliplatin and etoposide against colorectal cells, could be increased through co-suppression of the MCM complex. The oncogene c-Myc also leads to the mismanagement of CMG helicases in part due to a non-transcriptional role in overactivating replication origins and causing DNA damage. We sought to elucidate the mechanism by which Myc causes overactivation of CMG helicases.
Herein we demonstrate that co-suppression of reserve MCM complexes in pancreatic or colorectal cancer cell lines treated with clinically applicable chemotherapeutic compounds causes significant loss of proliferative capacity compared with cells containing the full complement of reserve MCMs. This is in part due to an inability to recover DNA replication following drug exposure, leading to an increase in apoptosis. Targeting of Myc to genomic sites induced large-scale decondensation of higher order chromatin that was required for CMG helicase assembly and activation at reserve MCM complexes. The physiological mediators of Myc, GCN5 and Tip60, are required for the chromatin unfolding and Cdc45 recruitment.
We conclude that depletion of the reserve MCM complexes causes chemosensitization of multiple human tumor cell types to several chemotherapeutic drugs used in the clinical management of human cancer. This argues for the development and use of anti-MCM drugs in combination with chemotherapeutic compounds, which has the potential to increase the therapeutic index of existing clinical compounds. We have also identified a previously unknown role for Myc in normal cell cycle progression whereby DNA replication initiation is regulated through the assembly and activation of CMG helicases on Myc-mediated open chromatin regions. Our results also provide new mechanistic insight into Myc oncogenic transformation in which overstimulation of DNA replication could result in genomic instability and provide an explanation for Myc driven oncogenic transformation.
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Hughes, Amanda L. "Dissecting cis and trans Determinants of Nucleosome Positioning: A Dissertation." eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/743.
Full textAbstract:
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.
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Hughes, Amanda L. "Dissecting cis and trans Determinants of Nucleosome Positioning: A Dissertation." eScholarship@UMMS, 2011. http://escholarship.umassmed.edu/gsbs_diss/743.
Full textAbstract:
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.
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Alvarez-Saavedra, Matias A. "The Snf2h and Snf2l Nucleosome Remodeling Proteins Co-modulate Gene Expression and Chromatin Organization to Control Brain Development, Neural Circuitry Assembly and Cognitive Functions." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/30304.
Full textAbstract:
Chromatin remodeling enzymes are instrumental for neural development as evidenced by their identification as disease genes underlying human disorders characterized by intellectual-disability. In this regard, the murine Snf2h and Snf2l genes show differential expression patterns during embryonic development, with a unique pattern in the brain where Snf2h is predominant in neural progenitors, while Snf2l expression peaks at the onset of differentiation. These observations led me to investigate the role of Snf2h and Snf2l in brain development by using conditionally targeted Snf2h and Snf2l mice.
I selectively ablated Snf2h expression in cortical progenitors, cerebellar progenitors, or postmitotic Purkinje neurons of the cerebellum, while Snf2l was deleted in the germline. I found that Snf2h plays diverse roles in neural progenitor expansion and postmitotic gene expression control, while Snf2l is involved in the precise timing of neural differentiation onset. Gene expression studies revealed that Snf2h and Snf2l co-modulate the FoxG1 and En1 transcription factors during cortical and cerebellar neurogenesis, respectively, to precisely control the transition from a progenitor to a differentiated neuron. Moreover, Snf2h is essential for the postmitotic neural activation of the clustered protocadherin genes, and does so by functionally interacting with the matrix-attachment region protein Satb2. My neurobehavioral studies also provided insight into how Snf2h loss in cerebellar progenitors results in cerebellar ataxia, while Snf2h loss in cortical progenitors, or in postmitotic Purkinje neurons of the cerebellum, resulted in learning and memory deficits, and hyperactive-like behavior.
Molecularly, Snf2h plays an important role in linker histone H1e dynamics and higher order chromatin packaging, as evidenced by loss of chromatin ultrastructure upon Snf2h deletion in progenitor and postmitotic neurons. I further demonstrated that Snf2h loss in a neuronal cell culture model results in reduced H1e deposition, and that overexpression of human SNF2H or SNF2L upon Snf2h knockdown rescues this biochemical dysfunction. My experiments suggest that Snf2h and Snf2l are regulatory nucleosome remodeling engines that co-modulate the gene expression programs necessary for proper brain development, maturation and function.
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Marfella, Concetta G. A. "The Role of CHD2 in Mammalian Development and Disease: a Dissertation." eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/334.
Full textAbstract:
Chromatin structure is intricately involved in the mechanisms of eukaryotic gene regulation. In general, the compact nature of chromatin blocks DNA accessibility such that components of the transcriptional machinery are unable to access regulatory sequences and gene activation is repressed. These repressive effects can be overcome or augmented by the actions of chromatin remodeling enzymes. Numerous studies highlight two classes of these enzymes: those that covalently modify nucleosomal histones and those that utilize energy derived from ATP hydrolysis to destabilize the histone-DNA contacts within the nucleosome (13, 14, 92). Members of each of these groups of chromatin remodeling enzymes play pivotal roles in modulating chromatin structure and in facilitating or blocking the binding of transcription factors. Mutations in genes encoding these enzymes can result in transcriptional deregulation and improper protein expression. Therefore, the regulation of chromatin structure is critical for precise regulation of almost all aspects of gene expression. Consequently, enzymes regulating chromatin structure are important modulators of cellular processes such as cell viability, growth, and differentiation. There remain many uncharacterized members of the ATP-dependent class of remodeling enzymes; characterization of these proteins will further elucidate the cellular functions these enzymes control.
Here, we focus primarily on the ATP-dependent remodeling complexes, specifically the chromodomain helicase DNA-binding (CHD) family. The CHD proteins are distinguished from other ATP-dependent complexes by the presence of two N-terminal chromodomains that function as interaction surfaces for a variety of chromatin components. These proteins also contain a SNF2-like ATPase motif and are further classified based on the presence or absence of additional domains. Genetic, biochemical, and structural studies demonstrate that CHD proteins are important regulators of transcription and play critical roles during developmental processes. Numerous CHD proteins have also been implicated in human disease.
The first CHD family member, mChd1, was identified in 1993 in a search for DNA-binding proteins with an affinity for immunoglobin promoters. Since then, additional CHD genes have been identified based on sequence and structural homology to mChd1. Despite an increase in the number of studies relating to CHD proteins, the function of most remains unknown or poorly characterized. Using embryonic stem (ES) cells containing an insertional mutation in the murine Chd2 locus, we generated a Chd2-mutant mouse model to address the biological effects of Chd2 in development and disease. The targeted Chd2 allele resulted in a stable Chd2-βgeo fusion protein that contained the tandem chromodomains, the SNF2-like ATPase motif, but lacked the C-terminal portion of the DNA-binding domain.
We demonstrated that the mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis as well as perinatal lethality. Similarly, heterozygous mice showed a decreased neonatal viability. Moreover, the surviving heterozygous mice showed a general growth delay during the neonatal period and increased susceptibility to non-neoplastic lesions affecting multiple organs, most notably the kidneys.
We further examined the connection between Chd2 and kidney disease in this murine model. Our findings revealed that the kidney phenotype observed in Chd2 mutant mice led to the development of membranous glomerulopathy, proteinuria, and ultimately to impaired kidney function. Additionally, serum analysis revealed decreased hematocrit levels in the Chd2-mutant mice, suggesting that the membranous glomerulopathy observed in these mice is associated with anemia.
Lastly, we investigated whether the type of anemia observed in the Chd2-mutant mice. Red blood cell (RBC) indices and morphological examination of the RBCs indicated that the anemia seen in the Chd2-mutant mice can be classified as normocytic and normochromic. Further analyses have been initiated to determine if the anemia is due to an intrinsic effect in erythropoiesis or a secondary consequence of the glomerular disease.
In summary, our findings have contributed to our understanding of the putative chromatin remodeling enzyme Chd2. Although much remains to be studied, these findings demonstrate a role for Chd2 in mammalian development and have revealed a link between Chd2 and disease.
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Kolpa, Heather J. "XIST and CoT-1 Repeat RNAs are Integral Components of a Complex Nuclear Scaffold Required to Maintain SAF-A and Modify Chromosome Architecture: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/825.
Full textAbstract:
XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.
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Kolpa, Heather J. "XIST and CoT-1 Repeat RNAs are Integral Components of a Complex Nuclear Scaffold Required to Maintain SAF-A and Modify Chromosome Architecture: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/825.
Full textAbstract:
XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.
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Salma, Nunciada. "Transcriptional Regulation During Adipocyte Differentiation: A Role for SWI/SNF Chromatin Remodeling Enzymes: A Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/50.
Full textAbstract:
Chromatin has a compact organization in which most DNA sequences are structurally inaccessible and functionally inactive. Reconfiguration of thechromatir required to activate transcription. This reconfiguration is achieved by the action of enzymes that covalently modify nucleosomal core histones, and by enzymes that disrupt histone-DNA interactions via ATP hydrolysis.
TheSWI/SNF family of ATP-dependent chromatin remodeling enzymes has been implicated not only in gene activation but also in numerous cellular processes including differentiation, gene repression, cell cycle control, recombination and DNA repair. PPARγ, C/EBPα and C/EBPβ are transcription factors with well established roles in adipogenesis. Ectopical expression of each of these factors in non-adipogenic cells is sufficient to convert them to adipocyte-like cells.
To determine the requirements of SWI/SNF enzymes in adipocyte differentiation, we introduced PPARγ, C/EBPα or C/EBPβ into fibroblasts that inducibly express dominant-negative versions of the Brahma-Related Gene 1 (BRG1) or human Brahma (BRM), which are the ATPase subunits of the SWI/SNF enzymes. We found that adipogenesis and expression of adipocyte genes were inhibited in the presence of mutant SWI/SNF enzymes. Additionally, in cells expressing C/EBPα or C/EBPβ, PPARγ expression was SWI/SNF dependent. These data indicate the importance of these remodeling enzymes in both early and late gene activation events.
Subsequently, we examined by chromatin immunoprecipitation (ChIP) assay the functional role of SWI/SNF enzymes in the activation of PPARγ2, the master regulator of adipogenesis. Temporal analysis of factors binding to the PPARγ2 promoter showed that SWI/SNF enzymes are required to promote preinitiation complex assembly and function.
Additionally, our studies concentrated on the role of C/EBP family members in the activation of early and late genes during adipocyte differentiation. During adipogenesis, C/EBPβ and δ are rapidly and transiently expressed and are involved in the expression of PPARγ and C/EBPα, which together activate the majority of the adipocyte genes. Our studies determined the temporal recruitment of the C/EBP family at the promoters of early and late genes by ChIP assay during adipocyte differentiation. We found that all of the C/EBP members evaluated are present at the promoters of early and late genes, and the binding correlated with the kinetics of the C/EBPs expression. Binding of C/EBPβ and δ is transient, subsequently being replaced by C/EBPα. These studies demonstrated that C/EBPβ and δ are not only involved in the regulation of PPARγ and C/EBPα, but also in the activation of late expressed adipocyte genes.
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Silveira, Alexandra C. "Characterization of SUDS3 as a BRMS1 family member in breast cancer." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008p/silveira.pdf.
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Linger, Jeffrey G. "The role of histone chaperones in double-strand DNA repair and replication-independent histone exchange /." Connect to full text via ProQuest. IP filtered, 2006.
Find full textAbstract:
Thesis (Ph.D. in Biochemistry) -- University of Colorado, 2006.Typescript. Includes bibliographical references (leaves 153-171). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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BERTORA, STEFANIA. "ROLE OF NUCLEAR ENVELOPE PROTEIN MAN1 IN NUCLEAR ORGANISATION AND MAINTENANCE OF GENOME STABILITY." Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/554706.
Full textAbstract:
The eukaryotic cell nucleus is characterized by a defined spatial organization of the chromatin, which relies on the physical tethering of many genomic loci to the inner surface of the nuclear envelope. This interaction is mainly mediated by lamins and lamin-associated proteins, which create a protein network at the nuclear periphery called nuclear lamina. Man1 is a member of a lamin-associated protein family known as LEM-domain proteins, which are characterized by the presence of a highly conserved domain, called LEM, that mediates the interaction with the chromatin. Data obtained with the yeast Man1 homolog Src1 underline the importance of this protein in different processes of the cell cycle, such as chromosome segregation, nuclear pores assembly, gene expression, chromatin organization and maintenance of genome stability, while in animal models, the function of Man1 has been associated to the regulation of developmental signalling pathways during embryogenesis. In this study, truncated recombinant mutants of Man1, containing the LEM domain, were shown to inhibit nuclear assembly and alter nuclear pore formation when added to Xenopus laevis cell-free extracts. Moreover, Xenopus nuclei assembled in the presence of Man1 truncated fragments were characterized by defects in chromatin organization, DNA replication and accumulation of DNA damage and, as a consequence, they failed to progress through mitosis. Furthermore, mouse embryonic stem cells (mESCs) depleted for Man1 showed evident signs of spontaneous differentiation, indicating inability in the maintenance of stem cell features. Intriguingly, preliminary analysis of Man1-knockout mESCs transcriptional profile showed an alteration of gene expression at the level of pericentromeric and telomeric regions, underlining a potential link between Man1 and genomic stability of these particular regions. In conclusion, this study illustrates the importance of Man1 in ensuring the proper chromatin organization necessary to support different cellular and DNA metabolic processes.
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Pennini, Meghan E. "Toll-like Receptor 2-dependent Inhibition of Interferon gamma Signaling by Mycobacterium tuberculosis." Connect to text online, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=case1152115234.
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