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Expression of class I histone deacetylases in insect cellsBryan, Erin E. January 2006 (has links)
Thesis (M.S.) -- Worcester Polytechnic Institute. / Keywords: histone deacetylase. Includes bibliographical references (p.35-36).
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The role of histone deacetylase 10 in y-globin gene regulation /Nimer, Sadeieh Abedaljaleel, January 2008 (has links)
Thesis (M.S.)--University of Texas at Dallas, 2008. / Includes vita. Non-Latin script record Includes bibliographical references (leaves 39-40)
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Mécanisme épigénétique impliqué dans la déposition de CENP-A aux centromeres / Epigenetic mechanism of CENP-A loading to centromeresShuaib, Muhammad 08 June 2012 (has links)
La ségrégation fidèle des chromosomes est dirigée par le centromère, un locus chromosomique spécialisé qui est requis pour l’assemblage des kinetochores actifs. Les centromères sont marqués épigénétiquement par la présence d’un nucléosome unique qui contient un variant centromérique de l’histone H3 appelé Centromere protein A (CENP-A). Une question fondamentale est comment CENP-A est spécifiquement déposé aux centromères. L’objectif de ma thèse a été d’identifier les facteurs spécifiques de la déposition de CENP-A. Pour identifier les facteurs spécifiques impliqués dans la déposition de CENP-A aux centromères, j’ai utilisé la méthode de purification TAP-TAG à partir d’une fraction nucléaire soluble de cellules HeLa exprimant stablement une copie ectopique de CENP-A (e-CENP-A). J’ai ainsi pu identifié la protéine Holliday Junction Recognition protein (HJURP). En utilisant un siRNA spécifique de HJURP, j’ai montré que la localisation et la déposition de CENP-A étaient fortement affectées. La protéine recombinante HJURP lie de manière stoechiométrique le tétramère CENP-A/H4 mais il ne lie pas le tétramère H3/H4. La liaison se fait grâce à un petit domaine conservé en position N-terminal de HJURP, dénommé CBD (CENP-A binding domain). De plus, j’ai pu mettre en évidence in vitro que HJURP facilitait la déposition du tétramère CENP-A/H4 sur de l’ADN satellite. L’ensemble de mes résultats démontre très clairement que HJURP est la principale chaperone responsable de la déposition de CENP-A aux centromères. / Centromere is a specialized chromosomal locus, where kinetochore assembles, which is required for correct chromosome segregation during cell division. In higher eukaryotes, centromere specification is independent of the DNA sequence and is determined epigenetically by the presence of a unique nucleosome that contains a histone H3 variant, called CENP-A. A fundamental question in centromere biology is that how CENP-A is specifically delivered to and maintained on centromeres. The aim of my thesis was to identify specific chaperone in human, responsible for CENP-A loading to centromeres, by using biochemical and proteomic strategies. To identify CENP-A deposition machinery, I purified the prenucleosomal CENP-A complex from HeLa cells stably expressing epitope tagged CENP-A. By mass spectrometry analysis of proteins present in CENP-A and H3.1 complex, I found HJURP uniquely in CENP-A prenucleosomal complex. Down regulation of HJURP by specific siRNA strongly diminished centromeric localization of CENP-A. Bacteriallyexpressed HJURP specifically binds to the CATD domain of CENP-A, via a highly conserved Nterminal domain, called CBD. Finally, I showed that HJURP is able to facilitate the efficient deposition of CENP-A/H4 tetramer on naked DNA. Taken together, my data demonstrate that HJURP is a key chaperone responsible for the targeting and deposition of newly synthesized CENPA at centromeres.
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The Snf2 Domain SnAC binds specifically to the Interior of Histone H3Jones, Megan Ashely 01 August 2015 (has links)
Combined with the data showing that the analogous section of BRG1 has histone binding properties, the second proposed model, where the SnAC domain acts as a histone anchor required for remodeling and directly binds to a location on the histone surface, was pursued. This was supported by Fe-BABE targeted cleavage that showed a cleavage site located within the SnAC domain as well as crosslinking data that showed there was a 2-3 fold reduction of Snf2 crosslinking upon deletion of the SnAC domain [62]. In order to find which histones SnAC binds to specifically and the regions of the histone(s) involved, a recombinant SnAC domain protein was generated and used in pull down assays with histones H2A, H3, and H4 in order to show binding and specificity. Finally gH3, a tailless histone H3, was purified to determine if the interaction between the SnAC domain and the histone occurred in either the tail, or core domain of the protein.
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Effect of p300 HAT Activity on Myogenic DifferentiationHamed, Munerah January 2013 (has links)
Skeletal muscle specification and differentiation programs are regulated by the myogenic regulatory factors which include Myf5, MyoD, myogenin and Mrf4. Upstream of the MRFs, the transcription co-activators and other intracellular and extracellular signals play crucial roles in regulating skeletal myogenesis. Histone acetyltransferase activity of p300 is required for Myf5 and MyoD expression. Furthermore, the MyoD core enhancer region is indispensable for MyoD expression. However, the mechanism by which p300 activates MyoD gene expression is to be determined. The histone acetyltransferase activity of p300 can be inhibited by small molecule inhibitors such as curcumin. Thus, using the inhibitor approach on stem cells is useful to investigate the role of p300 in activating MyoD expression during myogenesis. We here show that curcumin was able to inhibit stem cell determination and differentiation into skeletal myocytes. We also show that p300 is present, and histone acetylation is high at the core enhancer region. Therefore, we provide evidence that p300 is directly involved in MyoD gene expression during skeletal myogenesis.
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Chondrosarcome : mécanismes de résistance aux traitements conventionnels et thérapies innovantes / Chondrosarcoma : resistance mechanisms to conventional treatments and innovative therapiesLhuissier, Eva 28 September 2017 (has links)
Les chondrosarcomes sont des tumeurs malignes osseuses, considérés comme radio- et chimio-résistants, du fait de leur environnement hypoxique. Dans ce contexte, cette étude vise à mieux comprendre le rôle de l’hypoxie dans la résistance de ces tumeurs à la chimiothérapie (cisplatine) et à la radiothérapie (rayons X) et à identifier de nouvelles stratégies thérapeutiques permettant de sensibiliser les chondrosarcomes aux traitements, par un ciblage épigénétique de la méthylation de la lysine 27 de l’histone H3 (H3K27).Dans un premier temps, nous avons montré que, contrairement à ce qui est communément admis, l’hypoxie n’a pas d’effet sur la sensibilité au cisplatine ou aux rayons X dans certains chondrosarcomes alors qu’il augmente la résistance au cisplatine et la sensibilité aux rayons X uniquement dans une lignée de chondrosarcome. Dans un second temps, nous avons montré que le 3-deazaneplanocine A (DZNep) induit l’apoptose dans ces tumeurs, par un mécanisme indépendant de la méthylation de H3K27 et de sa méthylase EZH2 et semblerait agir par la voie Rhoβ/EGFR. Cependant, il provoque des effets secondaires sur la fertilité masculine. Par ailleurs, son association avec le cisplatine potentialise ses effets toxiques sur les chondrosarcomes. Le GSK-J4, quant à lui ralentit la croissance cellulaire des chondrosarcomes et son association avec le cisplatine augmente cet effet. Cette étude souligne que les chondrosarcomes possèdent des mécanismes de régulation cellulaires différents, d’où l’importance de mener des études sur plusieurs lignées cellulaires afin de mieux prédire la réponse aux traitements. De plus, ces travaux démontrent les propriétés anti-tumorales du DZNep et du GSK-J4 dans le traitement de ces tumeurs. / Chondrosarcomas are bone malignant tumors, considered as radio- and chemo-resistant, due to their hypoxic environment. In this context, this study aimed to better understand the role of hypoxia in the resistance of these tumors to chemotherapy (cisplatin) and radiotherapy (X-rays) and to identify new therapeutic strategies to re-sensitize chondrosarcomas by epigenetic targeting of H3K27 methylation. First, we showed that, contrary to what is commonly accepted, hypoxia has differential effect on cisplatin or X-ray sensitivity in chondrosarcomas, while it increases cisplatin resistance and X-ray sensitivity only in one cell line. Secondly, 3-deazaneplanocin A (DZNep) induces apoptosis in these tumors by a mechanism independent of H3K27 methylation and its methylase EZH2 and seems to act through the Rhoβ / EGFR pathway. However, it causes side effects on male fertility. In addition, its association with cisplatin potentiates its toxic effects on chondrosarcomas. The GSK-J4, on the other hand, decreases cell growth and its association with cisplatin increases this effect.This study highlights that chondrosarcomas use different cellular regulation mechanisms, showing the importance of conducting studies on several cell lines in order to better predict the response to treatments. In addition, these studies demonstrate the anti-tumoral properties of DZNep and GSK-J4 in the treatment of these tumors.
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The Relationship Between Metabolic Circumstance and Epigenetic Acetylation in Myoblast Fate and FunctionLim, Sean 13 September 2021 (has links)
Muscle tissue is grown and maintained by muscle stem cells termed satellite cells. Activated satellite cells become myoblasts, which must proliferate then differentiate into functional muscle. This process, known as myogenesis, is controlled by a cascade of epigenetic regulatory events. One facet of this regulation is histone acetylation, which can be influenced by the availability of metabolites within a cell. In this study, the ability of glucose, pyruvate, or glutamine to change histone acetylation levels in cultured myoblasts was investigated. Changing concentrations of glucose or pyruvate had no effect but decreasing the availability of glutamine in cell culture from 2mM to 0.2mM resulted in proliferating myoblasts accruing a hyperacetylated histone phenotype. However, when the same concentration of glutamine was used on differentiating myoblasts the hyperacetylated phenotype was lost and no change to differentiation was observed. This study demonstrates the potentials and limitations of altering epigenetic acetylation with metabolic circumstance. -- Le développement du tissu musculaire est soutenu par les cellules souches musculaires, communément appelées cellules satellites. Les cellules satellites activées se transforment en myoblastes qui doivent ensuite proliférer et se différencier en muscle fonctionnel. Ce processus, connu comme myogenèse, est contrôlé par une cascade de régulation épigénétique. Un aspect de ce processus est l’acétylation d’histones, qui peut être influencée par la disponibilité de métabolites dans la cellule. Dans cette étude de cas, la capacité du glucose, pyruvate, ou glutamine à changer les niveaux d’acétylation d’histones a été examinée. Le changement des concentrations de glucose ou de pyruvate n’a généré aucun effet, mais la diminution de la disponibilité de la glutamine dans la culture cellulaire de 2mM à 0.2mM a eu pour résultat une prolifération de myoblastes présentant un phénotype d’histones hyper-acétylées. Pourtant, quand la même concentration de glutamine a été utilisée pour différencier les myoblastes, le phénotype hyper-acétylé n’a pas été observé et aucun changement de différenciation n’a pu être détecté. Cette étude démontre le potentiel et les limites des modifications de l’acétylation épigénétique selon les circonstances métaboliques.
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Histone methyltransferases regulate responses to biotic and abiotic factors in tomatoCarol N Bvindi (8500842) 12 October 2021 (has links)
<div><br></div><div>Plants are constantly exposed to biotic and abiotic factors throughout their developmental stages which threaten their growth and productivity. Environmental stresses limit crop productivity and are likely to increase in severity due to the drastic and rapid changes in global climate. In this project, we studied the genetic factors that contribute to plant adaption to pathogens and other environmental factors in tomato. The results of these are presented in chapters 2-4 of this thesis. Chapter 1 covers background information and the review of the current literature in plant responses to biotic and abiotic stress. Chapter 2 deals with functional analysis of tomato histone methyltransferases SDG33 and SDG34 and their role in plant defense and stress tolerance. Chapter 3 focuses on the role of SDG33 and SDG34 on plant responses to Nitrogen. Finally, Chapter 4 summarizes the results from a reverse genetic screen using CRISPR cas9 genome editing to identify Receptor Like Cytoplasmic Kinases (RLCKs) required for plant resistance to fungal pathogens. </div><div>Plant responses to environmental cues are underpinned by rapid and extensive transcriptional reprogramming. Post translational modification of histones orchestrate these reprogramming and cellular responses by altering chromatin structure and establishing permissive or repressive states. Histone lysine methylation (HLM) is a principal modification of chromatin that affects various cellular processes. HLM is mediated by histone methyltransferases (HMTs) that deposit methyl groups to specific lysine residues on n-terminal histones tails. Although it is known that chromatin modifications occur in response to environmental cues, the mechanisms by which this is achieved, and the biological functions of HMTs are poorly understood. The function of tomato histone methyltransferases Set Domain Group (SDG)33 and SDG34 in biotic and abiotic stress responses were studied using tomato mutants generated through CRISPR/cas9 genome editing. </div><div>SDG33 and SDG34 genes were induced by pathogens, drought stress, the plant hormones methyl jasmonate, salicylate and abscisic acid. The sdg33 and sdg34 mutants display altered global HLMs. SDG34 is required for global H3K36 and H3K4 mono, di- and tri-methylation while SDG33 is primarily responsible for di- and tri- H3K36 and H3K4 methylation. Tomato SDG33 and SDG34 are orthologues of the Arabidopsis SDG8, an H3K4 and H3K36 methyl transferase previously implicated in plant immunity and plant growth through epigenetic control of Carotenoid Isomerase (CCR2) and other target genes. However, the tomato sdg33 or sdg34 single mutants showed no altered responses to fungal and bacterial pathogens likely due to functional redundancy of the tomato SDG33 and SDG34 genes consistent with their overlapping biochemical activities. Interestingly, tomato SDG33 or SDG34 genes rescued the disease susceptibility and early flowering phenotypes of Arabidopsis sdg8 mutant. Expression of CCR2 gene is completely inhibited in Arabidopsis sdg8 mutant attributed to loss of H3K36 di- and tri methylation at CCR2 chromatin. CCR2 gene expression was partially restored by transgenic expression of tomato SDG33 or SDG34 genes in Arabidopsis sdg8. In tomato, the single CCR2 gene is expressed independent of SDG33 or SDG33 genes suggesting that the genomic targets of the tomato HMTs are different. Unexpectedly, sdg33 and sdg34 plants were more tolerant to osmotic stress, maintain a higher water status during drought which translated to better survival after drought. Tolerance of sdg33 and sdg34 to drought stress is accompanied by higher expression of drought responsive genes. Collectively, our data demonstrate the critical role of tomato HLM in pathogen and stress tolerance likely through the regulation of gene expression.</div><div>In parallel, we characterized the role of SDGs in mediating nitrogen responses in tomato. The results are described in Chapter 2. Few studies have focused on the role of histone lysine methylation in regulating changes to nutrient availability. Transcriptome analysis in the shoot and roots showed that SDG33 and SDG34 have both overlapping and distinct regulated targets in tomato. In response to nitrogen, 509 and 245 genes are regulated by both SDG33 and SDG34 in response to nitrogen states in the roots and shoot respectively. In the roots these genes were enriched with GO terms such as ‘regulation of gene expression’, regulation of N metabolism’ and ‘regulation of hormone stimuli’. ‘Response to stimulus’, ‘photosynthesis’ and ‘N assimilation’ were the biological processes significantly enriched in the shoots. Overall, we show that SDG33 and SDG34 are involved in regulating nitrogen responsive gene expression and hence physiological nitrogen responses in the roots and shoots. </div><div>We also studied the Set Domain Group 20 (SlSDG20) an orthologue of Arabidopsis SDG25 in tomato. The details of our observations are presented in Chapter 3. SlSDG20 belongs to class III HMTs, it has the SET, Post-SET domain and GYF domain important for proline-rich sequence recognition. SlSDG20 is highly induced by B. cinerea, Methyl Jasmonate and Ethylene. To further understand the functions of SlSDG20 in tomato physiological development and plant immunity we generated slsdg20 knockout mutants through CRSIPR/Cas9. We identified one homozygous slsdg20 mutant with 151bp deletion in an exon immediately before the SET domain. Global methylation assay on the slsdg20 mutant confirmed that SlSDG20 is an H3K4 methyltransferase. The slsdg20 mutant is shorter than the wild type, produce more adventitious shoots causing prolific branching, and produce narrow leaves. Further, the mutant produces abnormal fruit and few seeds that hardly germinate. The slsdg20 mutant is highly susceptible to B. cinerea compared to the wild type. In response to Pst DC3000, slsdg20 mutant plants are comparable of the wild type. Resistance to hrcC strain of Pst DC3000 was impaired in the slsdg20 mutant, suggesting a possible role of SlSDG20 in PTI. In sum, tomato SDG20 is regulates plant immunity and plant growth including fertility.</div><div>The final chapter focuses on tomato Receptor like cytoplasmic kinases (RLCKs). Plants perceive the presence of pathogens through Pattern Recognition Receptors (PRR) which are predominantly RLKs, and subsequently recruit RLCKs to signal to downstream regulators of defense responses. Many RLCKs were characterized from Arabidopsis for their role in signalling of responses to bacterial infection. An example of RLCKs is Arabidopsis BIK1 which is implicated in signal transmission of pathogen recognition event at the cell surface. The tomato genome encodes 647 RLK/RLCKs comprising about 2% of its predicted genes. The functions of most of these predicted tomato RLCKs and RLKs have not been determined. Previously, our lab characterized the Arabidopsis BIK1 and tomato TPK1b RLCKs for fungal resistance. Here, we conducted a reverse genetic screen focused on BIK1 and TPK1b related tomato RLCKs to identify a subset with defense functions. Virus induced gene silencing and pathogen assays conducted on 15 RLCKs identified four RLCK genes with potential role in plant immunity. Then, tomato knock out mutants were generated for four RLCK genes through CRISPR/cas9 genome editing to validate the VIGS data. Subsequently, we demonstrated that TPK07, TPK09, TPK011 and TRK04 are required for resistance to B. cinerea. The data are supported by the pathogen induced expression of these genes. Furthermore, trk04 seedlings are impaired in seedling growth responses to Jasmonic acid. Our study establishes that tomato TPK07, TPK09, TPK011 and TRK04 contribute to defense against B. cinerea but their mechanism of function needs to be elucidated in future studies</div><div><br></div>
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Characterizing the Function of Histone Variant H3.3 in Adult Skeletal Muscle RegenerationLi, Yuefeng 27 April 2022 (has links)
No description available.
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A Study of Abscisic Acid Regulated Enzymes and Histone Binding Proteins in PlantsKolli, Udhghatri 14 August 2015 (has links)
Plant hormone abscisic acid (ABA) plays a main role in coordinating various stress signals in plants. ABA regulates the expression of genes and activities of enzymes in response to various stress conditions. In the following studies we were able to study the ABA mediated regulation of enzymes in plants. Using in-gel activity analysis we identified that ABA regulates the activity of aspartate aminotransferase (AAT), an enzyme involved in nitrogen assimilation and carbohydrate metabolism. Our results indicate that phosphorylation of AAT by SnRK2.2 and 2.3 kinases results in down regulation of AAT2 and AAT3 isozyme activities in Arabidopsis. AAT was identified as a negative regulator of drought stress and aat mutant plants showed improved survival following drought conditions. Using in-gel staining method we were able to visualize sugar phosphatases like fructose 1-6 bisphosphatase family, sedoheptulase-1,7-bisphosphatase, inositol mono phosphatases; protein serine/threonine phosphatases, protein tyrosine phosphatases and studied their response to ABA and drought stress. Fructose-1-6 bisphosphatase family of phosphatases were identified to be induced by ABA in Arabidopsis and rice. N-acetylglucosamine (GlcNAc) is present on glycoproteins and as post translational modification (PTM) in cytoplasmic and nuclear proteins. N-acetylglucosamine is removed from target proteins by hexosaminidases. Little is known about the hexosaminidases in plants. Using in-gel activity analysis we were able to identify an ABA induced Beta-hexosaminidase with a neutral pH optimum in soybean. The nuclear DNA in chromatin is associated with basic proteins called histones. The N-terminal tails of histones contain different PTMs including methylation, phosphorylation, ubiquitination, acetylation, ADP-ribosylation and glycosylation. The histone lysine methylation can serve as a binding site or repel/disrupt the histone binding proteins. The effector/reader proteins specifically recognize the post translational modifications and responsible for the downstream process. Many histone methyl modification effector proteins have been characterized but very few proteins whose binding was disrupted by the presence of a PTM were identified. Using peptide pulldown analysis, far western analyses we identified a WD-40 domain containing histone binding (HB01) protein as direct interactor of unmodified histone. The presence of post translational modifications disrupts HB01 binding to histone H3.
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