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Gocevski, Goran. "Interplay of Mye and Max with Epigenetic Regulator Bmi1." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114264.
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The polycomb group protein Bmi1 is an epigenetic regulator essential for the proliferation of many types of cancers. By impeding the expression of the tumor suppressor p53, Bmi1 is able to prevent apoptosis and senescence. c-Myc, a prominent oncogene, cooperates with Bmi1 to stimulate cellular transformation and tumorigenesis. Further investigation of the basic biological interplay between Bmi1 and c-Myc is crucial for our understanding of their tumorigenic ability. In my project I demonstrated that c-Myc and Bmi1 directly interact with each other and form nuclear foci. Overexpression of Max, a known partner of Myc, disrupts the Bmi1 and c-Myc interaction and prevents the formation of nuclear foci. Similar results were obtained with another member of the Myc family, L-Myc. Additionally, I found that HDAC3 interacts and co-localizes with Myc. HDAC3 also forms nuclear foci with Bmi1 and the addition of Max abrogates this interaction. In addition to the well-established role of Bmi1 as an epigenetic regulator, it has been recently shown that Bmi1 is part of an E3 ubiquitin-ligase complex, known as the Bmi1/RING1A or B complex. This complex controls the stability of many proteins. I showed that Bmi1 induces an L-Myc ubiquitination, which in turn causes the degradation of L-Myc. This data proposes a novel regulatory mechanism for the stability of the Myc oncogenes. The results of this thesis provide new insight into the basic biochemical interplay ofBmi1 with Myc and Max.<br>La protéine de groupe polycomb Bmi1 est essentielle pour la prolifération de nombreux types de cancers. En freinant l'expression du suppresseur de tumeur p53, Bmi1 est capable de prévenir l'apoptose et la sénescence. c-Myc, une autre oncogène, s'associe à Bmi1 pour stimuler la transformation et la tumorigenèse. Une enquête plus approfondie de l'interaction biologique fondamentale entre Bmi1 et c-Myc est crucial pour notre compréhension de leur capacité à promouvoir la tumorigène. Dans mon projet, j'ai démontré que c-Myc et Bmi1 interagissent directement et forment des foyers nucléaires. La surexpression de Max, un partenaire connu de Myc, perturbe l'interaction entre Bmi1 et c-Myc et empêche la formation de foyers nucléaires. Des résultats similaires ont été obtenus avec un autre membre de la famille Myc, L-Myc. En outre, j'ai constaté que HDAC3 interagi et se co-localise avec Myc. HDAC3 forme aussi des foyers nucléaires avec Bmi1 et l'ajout de Max abroge cette interaction. En plus du rôle bien établi de Bmi1 comme un régulateur épigénétique, il a été démontré récemment que Bmi1 fait partie d'une ubiquitine-ligase E3 complexe, connu sous le nom complexe Bmi1/RING1A ou B. Ce complexe contrôle la stabilité de nombreuses protéines. J'ai démontré que Bmi1 induit l'ubiquitination de L-Myc qui à son tour provoque la dégradation de celle-ci. Ces données proposent un nouveau mécanisme de règlementation pour la stabilité des oncogènes Myc. Les résultats de cette thèse fournissent un nouvel éclairage sur l'interaction biochimique de Bmi1 avec Myc et Max.
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Almoflehi, Sakhar. "Cord Blood CD34+ Expansion Using Vitamin-C: An Epigenetic Regulator." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41413.
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Vitamin-C (Vit-C) has been shown to modulate hematopoietic stem cells and leukemia stem cell frequency in-vivo. Herein, Vit-C analogue, L-ascorbic acid 2-phosphate (AA2P), was investigated as a new potential HSC expansion agonist. Cord blood CD34+ cells were expanded in cultures with or without AA2P. AA2P induced a 2-fold increase in the expansion of stem and progenitor subsets including lymphoid-primed multi-potential progenitors (p<0.05, n=3) and functional colony forming progenitors. The functional properties of AA2P grafts was evaluated with a xenotransplant model. Superior platelet levels in the periphery (p<0.05) and human bone marrow engraftment (median 75% hCD45+ cells for AA2P Vs. 48% for PBS control at week-22, n=3, p<0.05) was detected in AA2P cohorts Vs. control. In summary, my results demonstrate that AA2P is a new stem and progenitor expansion agonist with AA2P-expanded stem and progenitor cells capable of increased engraftment and higher platelet recovery. These findings may aid to overcome cord blood limitations; thereby, improving clinical relevance.
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Lu, Yizhen. "Physical interation of parathyroid hormone-related protein with the epigenetic regulator Bmi1." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96929.
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As a cause of malignancy-induced hypercalcemia, PTHrP (parathyroid hormone-related protein) plays an important role in cell growth and differentiation. This peptide is unique in that it not only acts through membrane receptors but also translocates directly to the nucleus. Studies have shown that the repression of target gene expression is achieved through chromatin modifications induced by the PcG complex. As a core protein of the PcG complex, Bmi1 functions as a transcriptional repressor for various genes involved in development and cell proliferation. Recent studies have indicated that the skeletal phenotypes of PTHrP(1-84) knock-in mice are consistent with ones observed in Bmi1-/- mice in vivo. In addition, the down-regulation of Bmi1 expression was detected in PTHrP(1-84) knock-in mice. Both phenotypes indicate that there is correlation between Bmi1 and PTHrP. However, the molecular mechanism involved in correlation of PTHrP and Bmi1 regulation is poorly understood. The aim of this study was to gain insight into the underlying molecular mechanism of the way of PTHrP regulating Bmi1. We focused on the interaction between PTHrP and Bmi1 in vitro and in vivo system and the consequences exerted by this interaction. At first, co-localization of PTHrP and Bmi1 was demonstrated and the N-terminus of PTHrP was found to be responsible for the interaction both in vivo and in vitro. Second, we set up the repression assays in vivo to identify the promoters' activities and cell survival influenced by this direct interaction. As a result, overexpression of PTHrP and Bmi1 in HEK293 cells was shown to have an effect on p19Arf and Gal4 promoter activities in vivo. Thirdly, increased cell proliferation was detected in HEK293 cells and NIH 3T3 cells with overexpressed PTHrP and Bmi1 together. At the same time, I also discovered the elevation of cell survival rate in HEK293 and NIH 3T3 cells when PTHrP and Mel18 were expressed together. This study provides evidence that the hormone PTHrP physically and functionally interacts with Bmi1 and Mel18 to affect the activities of promoters in the nucleus and regulate cell proliferation.<br>La protéine Parathyroid hormone related-protein (PTHrP) joue un rôle très important dans la croissance et la différentiation cellulaire en plus d'être responsable de l'hypercalcémie induite par la malignité. Ce peptide est unique non seulement parce qu'il agit par l'intermediate de récepteurs transmembranaires, mais aussi parce qu'il est transloqué directement au noyau. Bmi-1, un peptide essentiel du PcG complexe, fonctionne comme un répresseur de transcription pour plusieurs gènes importants dans le développement et de l'organisme de la prolifération cellulaire. Cette fonction répressive régule l'expression des gènes cibles en induisant des modifications sur la chromatine (73). Des études publiées récemment démontrent que PTHrP influence l'expression moléculaire de Bmi-1 (95). Cependant, le mécanisme par lequel Bmi-1 contrôle PTHrP n'est pas encore bien documenté. Mon but premier est d'élucider les mécanismes moléculaires de cette interaction ensuite de trouver quelles conséquences fonctionnelles peuvent résulter de cette interaction. Au départ, la colocalisation de PTHrP et Bmi-1 a été démontrée dans le noyau de cellules HEK293. Ensuite, l'interaction entre Bmi-1 et PTHrP a été illustrée in vivo et in vitro. On a trouvé que c'est le N-Terminal qui est responsable des interactions in vivo et in vitro. De plus, la surexpression de PTHrP et Bmi-1 dans les cellules HEK293 provoque des effets minimes sur l'activité transcriptionelle des gènes et de l'expression du gène P19arf. En outre, la surexpression de PTHrP et Bmi-1 cause une augmentation du niveau de prolifération cellulaire dans les cellules HEK293 et NIH 3T3. En parallèle, j'ai découvert une augmentation du taux de survie des cellules HEK 293 et NIH 3T3 suite à surexpression des peptides PTHrP et Mel18. A été noteé ces études démontrent que l'hormone PTHrP interagit physiquement et est attaché fonctionnellement avec Bmi-1.
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Lubitz, Sandra. "Analysis of an epigenetic regulator in mouse embryonic stem cell self-renewal and differentiation." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1139479284063-94996.
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Mammals have two orthologs, Mll and Trx2, for the Drososphila protein Trithorax (TRX), which is the founding member of the trithorax group (TrxG) of epigenetic regulators. TrxG proteins are characterized by an evolutionary conserved SET domain. A major function of all SET domain- containing proteins is to modulate gene activity, but the underlying mechanisms are poorly understood. Apparently TRX, Mll and Trx2 are histone H3 lysine 4 specific methyltransferases. So far all evidence points to roles in expression of specific target genes. However, target genes and function of the epigenetic regulator Trx2 were still unknown. Homozygous trx2 mutant embryos arrest in development because of severe and widespread defects {Glaser, 2005 #296}. Thus mouse embryonic stem (ES) cells carrying a null mutation of trx2 were used as an alternative model system to address the implication of Trx2 in differentiation. This study showed that Trx2 is redundant for ES cell self-renewal. Homozygous trx2 knockout ES cells did not exhibit cell cycle defects. However, loss of Trx2 resulted in reduced proliferation and increased apoptosis rates in trx2-/- ES cells. Due to the fact that differentiation requires an appropriate rate of population growth, trx2-/- cells were affected adversely upon in vitro differentiation. Neurogeneic differentiation of trx2 mutant cells generated fewer mature neurons than wild type cells. Moreover a temporal delay in the developmental progression to differentiation became apparent. Cardiac differentiation of trx2-/- cells confirmed the developmental defect and temporal delay. Notably differentiation of trx2-/- cells was merely delayed or impaired but it was not absent, implying that Trx2 is not required for gene expression programs specific for neurons or cardiac myocytes. We propose that differentiation of trx2-/- ES cells is impaired because apoptosis is disturbing differentiation. Apart from analyzing the phenotype of trx2 mutant cells, this work was focused on the identification of Trx2 target genes. Oligonucleotide expression arrays were used to identify genes whose expression levels were affected by the absence of Trx2. In general, loss of Trx2 function resulted in more genes with decreased than increased expression levels. This is consistent with the hypothesis that Trx2 functions as a transcriptional activator. Comparison of gene expression profiles for constitutive and conditional trx2 mutant cells enabled a distinction between direct and indirect target genes for Trx2. As a result Magoh2 was identified as the key candidate target gene for Trx2. Interaction between Trx2 and Magoh2 suggested a potential regulatory role for Trx2 in alternative splicing. Furthermore this work provided evidence that Trx2 could be involved in the maintenance of CpG island promoter gene expression, thus providing a potent regulatory mechanism for ubiquitously expressed genes.
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Lubitz, Sandra. "Analysis of an epigenetic regulator in mouse embryonic stem cell self-renewal and differentiation." Doctoral thesis, Technische Universität Dresden, 2005. https://tud.qucosa.de/id/qucosa%3A24639.
Full textAbstract:
Mammals have two orthologs, Mll and Trx2, for the Drososphila protein Trithorax (TRX), which is the founding member of the trithorax group (TrxG) of epigenetic regulators. TrxG proteins are characterized by an evolutionary conserved SET domain. A major function of all SET domain- containing proteins is to modulate gene activity, but the underlying mechanisms are poorly understood. Apparently TRX, Mll and Trx2 are histone H3 lysine 4 specific methyltransferases. So far all evidence points to roles in expression of specific target genes. However, target genes and function of the epigenetic regulator Trx2 were still unknown. Homozygous trx2 mutant embryos arrest in development because of severe and widespread defects {Glaser, 2005 #296}. Thus mouse embryonic stem (ES) cells carrying a null mutation of trx2 were used as an alternative model system to address the implication of Trx2 in differentiation. This study showed that Trx2 is redundant for ES cell self-renewal. Homozygous trx2 knockout ES cells did not exhibit cell cycle defects. However, loss of Trx2 resulted in reduced proliferation and increased apoptosis rates in trx2-/- ES cells. Due to the fact that differentiation requires an appropriate rate of population growth, trx2-/- cells were affected adversely upon in vitro differentiation. Neurogeneic differentiation of trx2 mutant cells generated fewer mature neurons than wild type cells. Moreover a temporal delay in the developmental progression to differentiation became apparent. Cardiac differentiation of trx2-/- cells confirmed the developmental defect and temporal delay. Notably differentiation of trx2-/- cells was merely delayed or impaired but it was not absent, implying that Trx2 is not required for gene expression programs specific for neurons or cardiac myocytes. We propose that differentiation of trx2-/- ES cells is impaired because apoptosis is disturbing differentiation. Apart from analyzing the phenotype of trx2 mutant cells, this work was focused on the identification of Trx2 target genes. Oligonucleotide expression arrays were used to identify genes whose expression levels were affected by the absence of Trx2. In general, loss of Trx2 function resulted in more genes with decreased than increased expression levels. This is consistent with the hypothesis that Trx2 functions as a transcriptional activator. Comparison of gene expression profiles for constitutive and conditional trx2 mutant cells enabled a distinction between direct and indirect target genes for Trx2. As a result Magoh2 was identified as the key candidate target gene for Trx2. Interaction between Trx2 and Magoh2 suggested a potential regulatory role for Trx2 in alternative splicing. Furthermore this work provided evidence that Trx2 could be involved in the maintenance of CpG island promoter gene expression, thus providing a potent regulatory mechanism for ubiquitously expressed genes.
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Grinat, Johanna. "The epigenetic regulator Mll1 is required for Wnt-driven intestinal tumorigenesis and cancer stemness." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/22192.
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Genetisch bedingte Veränderungen im Wnt-Signalweg sind in der Tumorigenese des Darms von zentraler Bedeutung. Mutationen des Wnt-Effektormoleküls β-Catenin in den adulten Stammzellen des Darmepithels führen zu unkontrollierter Proliferation und Expansion der Darmstammzellen und initiieren die Tumorentstehung. Auch in fortgeschrittenen Darmtumoren unterstützt die Wnt-Signalgebung maßgeblich das Tumorwachstum und den Erhalt von Tumorstammzellen. Nach erfolgreicher chemotherapeutischer Behandlung treten oftmals Tumorrezidive auf, für deren Entstehung therapieresistente Tumorstammzellen verantwortlich gemacht werden. Trotz intensiver Forschung fehlen in der Darmkrebstherapie nach wie vor Behandlungsansätze zur gezielten Therapie der Tumorstammzellen. Ziel dieser Dissertation ist es, unser Verständnis der molekularen Regulationsmechanismen in Kolonkarzinomen zu erweitern und die Entwicklung rationaler Behandlungsstrategien zu fördern. Ich konnte die Histonmethyltransferase Mll1 als entscheidenden Faktor in der epigenetischen Regulation humaner und muriner Darmkrebsstammzellen und -tumore identifizieren. Humane Kolonkarzinome weisen eine erhöhte Mll1-Expression auf, die mit dem Level an nukleärem β-Catenin korreliert. Im adulten Darmepithel ist Mll1 insbesondere in den Lgr5+ Stammzellen exprimiert und maßgeblich an der Wnt/β-Catenin-induzierten Stammzellexpansion sowie der Tumorentstehung beteiligt. Der konditionelle Verlust von Mll1 im murinen Darmkrebsmodell verhindert die β-Catenin-induzierte Tumorigenese. Mll1 unterstützt die Selbsterneuerungsfähigkeit und Proliferation der Tumorstammzellen, indem es die Expression von essentiellen Stammzellgenen wie dem Wnt-abhängigen Stammzellmarker Lgr5 aufrechterhält. Eine Inhibition der Mll1-Funktion in der Darmkrebstherapie kann eine gezielte Eliminierung der Tumorstammzellen ermöglichen, wodurch das fortschreitende Tumorwachstum unterbunden und die Bildung von Rezidiven verhindert werden kann.<br>Genetic mutations inducing aberrant activity of Wnt signalling are causative for intestinal tumorigenesis. Mutations of the Wnt effector molecule β-catenin in adult stem cells of the intestinal epithelium drive uncontrolled proliferation, expand the stem cell pool and initiate tumor formation. In advanced tumors, aberrant Wnt signalling promotes tumor growth and maintains cancer stem cells. The cancer stem cells are highly resistant to conventional chemotherapy and frequently initiate tumor relapse after completion of treatment. Despite extensive research, we are still lacking efficient therapies for colon cancer that specifically eliminate the cancer stem cells. This dissertation aims to expand our knowledge on molecular gene regulatory mechanisms in colon cancer cells to promote the identification and future development of rational therapies for colon cancer patients. I identified the histone methyltransferase Mll1 as an epigenetic regulator in human and mouse intestinal cancer stem cells and tumors. Human colon carcinomas with nuclear β-catenin exhibit high levels of Mll1. In the adult intestinal epithelium of mice, Mll1 is highly expressed in the Lgr5+ stem cells and is a prerequisite for the oncogenic Wnt/β-catenin-mediated stem cell expansion and tumorigenesis. Conditional knockout of Mll1 in an intestinal mouse tumor model prevents the β-catenin-driven intestinal tumorigenesis. Knockdown of Mll1 impairs the self-renewal and proliferation of colon cancer sphere cultures and halts tumor growth in xenografts. Mechanistically, Mll1 sustains the expression of intestinal stem cell genes including the Wnt/β-catenin target gene Lgr5 by antagonizing gene silencing through polycomb repressive complex 2-mediated H3K27 tri-methylation. Interfering with Mll1 function can efficiently eliminate colon cancer stem cells, and has potential as a rational therapy for colon cancer.
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Punnia-Moorthy, Gayathiri. "Defining the functional roles of X-linked epigenetic regulator lysine demethylase 6A (KDM6A) in Melanoma." Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/28897.
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Melanoma is an aggressive form of skin cancer and Australia has one of highest incidences of melanoma in the world. Current treatments for metastatic melanoma are plagued by the resistance melanomas develop against immunotherapies and targeted therapies. Lysine demethylases (KDMs) are epigenetic enzymes that remove methyl groups from the amino acid lysine (K) on histone proteins, which effects gene expression. One of these KDMs is KDM6A (an X-linked gene also known as UTX) that removes methyl groups from histone 3, lysine number 27 (H3K27me3) inducing activation of gene expression. KDM6A has been reported to play roles in the progression of multiple cancers, however the role of KDM6A in melanoma is yet to be investigated.
In this study, the effects of KDM6A expression on clinical parameters, including survival, and gene expression patterns were investigated in a cohort of 458 melanoma patients obtained from The Cancer Genome Atlas (TCGA). In addition, the effects of a KDM6 inhibitor GSK-J4 and KDM6A knockout using the CRISPR-Cas9 system in melanoma cells was investigated in vitro using a variety of molecular and cell biology assays. RNA sequencing was used to determine which genes and pathways were significantly upregulated and downregulated in drug treated and KDM6A knockout melanoma cells.
Results showed that high KDM6A expression significantly correlated with gender in melanoma patients KDM6A expression was associated with better overall survival in melanoma patients, particularly in females but not in males. High KDM6A expression was associated with upregulation of interferon pathways and downregulation of pro-survival pathways which may prevent melanoma growth. High KDM6A expression was also associated with multiple immune cell infiltration in melanoma patient tumours, especially in females. In addition, KDM6A expression significantly correlated with COMPASS components, an important epigenetic complex in which KDM6A is an essential enzymatic component.
In vitro studies showed that KDM6A knockout in melanoma cells significantly increased proliferation and colony formation, hence promoting melanoma cell growth and supporting the role of KDM6A in tumour suppressive function. RNA-seq analysis in KDM6A depleted cells showed significant upregulation of oncogenic pathways and downregulation of tumour suppressive pathways. Surprisingly, GSK-J4 treatment in melanoma cells showed the opposite effect to KDM6A knockdown with increased apoptosis and decreased viability, colony formation and 3D spheroid formation, but had no effect on cell cycle regardless of basal KDM6A expression. In addition, GSK-J4 also targeted other histone markers which include H3K4me3. RNA-seq analysis in drug treated melanoma cells showed a significant upregulation of pathways involved in DNA regulation and significant downregulation of cell metabolic pathways. These results suggest that KDM6A appears to have a protective effect in melanoma patients, especially in females, indicating a potential tumour suppressive role.
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Grinat, Johanna [Verfasser]. "The epigenetic regulator Mll1 is required for Wnt-driven intestinal tumorigenesis and cancer stemness / Johanna Grinat." Berlin : Humboldt-Universität zu Berlin, 2020. http://d-nb.info/1223452255/34.
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Trippel, Franziska Katharina [Verfasser], and Roland [Akademischer Betreuer] Kappler. "The role of NFE2L2 mutations and the epigenetic regulator UHRF1 in hepatoblastoma / Franziska Katharina Trippel. Betreuer: Roland Kappler." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1096162644/34.
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Elangovan, Venkateswaran Ramamoorthi, Sara M. Camp, Gabriel T. Kelly, et al. "Endotoxin- and Mechanical Stress–Induced Epigenetic Changes in the Regulation of the Nicotinamide Phosphoribosyltransferase Promoter." UNIV CHICAGO PRESS, 2016. http://hdl.handle.net/10150/622492.
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Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.
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Borsari, Beatrice 1992. "Epigenetic regulation of the transcriptome." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2021. http://hdl.handle.net/10803/671429.
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We have monitored the transcriptome and the epigenome of human pre-B cells transdifferentiating into macrophages. Analysis of these data provides a general framework to understand the relationship between gene expression and chromatin. We have observed widespread uncoupling of gene expression and epigenetic features during transdifferentiation, with several genes characterized by unvaried chromatin state throughout the process, irrespective of changes in gene expression. Nevertheless, we report a strong association between transcription and chromatin marking of promoter regions at the time of initial gene activation. We have also analyzed the genomic location of distal regulatory elements in developmental and adult samples, and found that tissue-specific enhancer signatures in the human genome tend to accumulate within introns, while those shared among tissues are more frequently intergenic. By focusing on intronic segments, we have additionally uncovered both constraint and variation in the timing of splicing, with a subset of introns that switch from co-transcriptional to post-transcriptional splicing across distinct cell types.<br>Hemos monitorizado el transcriptoma y el epigenoma de células pre-B durante su transdiferenciación en macrófagos. El análisis de estos datos proporciona un marco general para comprender la relación entre la expresión génica y la cromatina. Observamos un desacoplamiento generalizado entre la expresión génica y las marcas epigenéticas durante la transdiferenciación, con multitud de genes caracterizados por un único estado de la cromatina, independientemente de los cambios en su expresión. No obstante, encontramos una fuerte asociación entre la transcripción y las marcas de la cromatina en los promotores durante la activación inicial de los genes. También hemos estudiado la localización genómica de elementos reguladores distales (enhancers), en muestras obtenidas tanto de tejidos embrionarios como adultos, encontrando que los enhancers específicos de tejido tienden a estar situados en los intrones, mientras que aquellos compartidos entre tejidos son, a menudo, intergénicos. Por último, centrándonos en el estudio de los intrones, hemos identificado tanto conservación como variabilidad en la temporalidad del splicing, con un subconjunto de intrones que cambian de splicing co-transcripcional a post-transcripcional en distintos tipos celulares.
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Mondal, Tanmoy. "Epigenetic Regulation by Noncoding RNA." Doctoral thesis, Uppsala universitet, Genomik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-160326.
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High throughput transcriptomic analyses have realized us with the fact that eukaryotic genome encodes thousands of noncoding RNAs (ncRNAs) with unknown function. In my thesis, I sought to address epigenetic regulation of transcription by ncRNA using the Kcnq1 imprinted cluster as a model system. Genomic imprinting is an epigenetic phenomenon whereby one of the parental alleles is silenced by epigenetic mechanism in a parent of origin-specific manner. A long ncRNA Kcnq1ot1 regulates imprinting of nearly 8 protein coding genes in the Kcnq1 imprinted cluster. Expression of Kcnq1ot1 is restricted to the paternal chromosome while that of protein-coding genes to the maternal chromosome. Kcnq1ot1 is a 91kb long, moderately stable, nuclear localized and RNAPII encoded transcript. We demonstrated that Kcnq1ot1 RNA itself mediates lineage specific silencing on the paternal chromosome by interacting with chromatin and recruiting the repressive chromatin modifiers to the imprinted gene promoters. Previously we identified an 890bp silencing domain (SD) at the 5´end of the Kcnq1ot1 RNA which is responsible for gene silencing. Targeted deletion of the 890SD in mouse resulted in specific loss of silencing of ubiquitously imprinted genes. We have further shown that Kcnq1ot1 interacts with Dnmt1 and recruit Dnmt1 at the somatic DMRs flanking some of the ubiquitously imprinted genes. We next addressed the stability of the Kcnq1ot1 mediated epigenetic silencing using transgenic mouse where we have conditionally deleted the Kcnq1ot1 RNA at different developmental stages and we found that Kcnq1ot1 RNA is required to maintain the silencing of the ubiquitously imprinted genes. In addition, DNA methylation, which controls imprinting of the ubiquitous genes require Kcnq1ot1 for its maintenance. To characterize the ncRNAs that mediate gene regulation through chromatin interaction we have isolated chromatin associated RNAs (CARs) from sucrose gradient fractioned chromatin. High-throughput sequencing of the CARs resulted in the identification of the 141 intronic and 74 intergenic regions harboring CARs. We characterized one of the intergenic CARs which regulate the transcription of the two neighboring genes by modulating the chromatin marks. In summary current thesis has uncovered unprecedented role of ncRNAs in gene expression via chromatin level regulation.
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Hellebrekers, Debby Maria Elisabeth Ida. "Epigenetic regulation of tumor angiogenesis." Maastricht : Maastricht : Universiteit Maastricht ; University Library, Universiteit Maastricht [host], 2006. http://arno.unimaas.nl/show.cgi?fid=5612.
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Johansson, Jennie. "Epigenetic Regulation of Mitochondrial DNA." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-166684.
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This mini-review investigates and compiles the latest knowledge regarding epigenetic changes on the mammalian mitochondrial DNA and its proteins. Methylation of the DNA, acetylation of the proteins and silencing of genes by short non-coding RNAs are the main epigenetic changes known today to affect mitochondrial DNA, mostly leading to repression. Methylation mainly occurs at non-CpG sites in the main non-coding region called the D-loop, with methylation patterns being cell type specific. Acetylation of proteins are mainly controlled by the deacetylase SIRT3, with its function being correlated to longevity. On the other hand, mitochondrial dysfunction is directly associated with a plethora of diseases, such as neurodegenerative disorders and heart disorders. The mitochondrion and nucleus are immensely dependent on each other and exchange vital proteins and RNAs, with epigenetic changes on one potentially affecting the other. Recent research shows that heteroplasmy is a proven cause of mitochondrial malfunction and that paternal inheritance is possible. The mitochondrial haplotype also shows different vulnerability to certain diets and diseases, leading to the conclusion that the mitochondrial haplotype can be used to more than just tracing human origins, such as to predicting and preventing diseases.
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Kotzin, Megan D., and Megan D. Kotzin. "Epigenetic Regulation: A Literature Review." Thesis, The University of Arizona, 2017. http://hdl.handle.net/10150/625025.
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Epigenetic regulation describes the manner in which gene expression is modified without changes to the DNA sequence. It is a complex process that involves the interaction of many biological molecules. The three most well-characterized mechanisms of epigenetic regulation are DNA methylation, histone modifications, and non-coding RNA activity. It is well established that environmental factors can regulate the activity of these mechanisms. This review specifically focuses on the manner by which physical activity and nutrition can alter gene expression through epigenetic regulation, proposing an explanation of why certain eating and exercise behaviors produce particular outcomes. The results of my research indicate that engaging in physical activity positively regulates the activity of signaling pathways that lead to tissue remodeling and an increase in aerobic capacity. Many studies have demonstrated the importance of early-life nutrition and how poor periconceptional nutrition negatively affects offspring. Type II diabetes, is a metabolic condition largely characterized by insulin resistance and the pathways that regulate insulin-mediated glucose uptake are susceptible to epigenetic regulation.
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Hoekenga, Owen Andrew. "Epigenetic regulation of Pl-blotched /." free to MU campus, to others for purchase, 1998. http://wwwlib.umi.com/cr/mo/fullcit?p9901242.
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Hong, Ted. "Alteration of Human Gene Regulatory Networks by Human Virus Transcriptional Regulators." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1593273403439508.
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Morikawa, Hiromasa. "Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation." Kyoto University, 2013. http://hdl.handle.net/2433/180610.
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Sousa, Rute Inês Silva e. 1983. "The Epigenetic regulation of Drosophila telomeres." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/96908.
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Drosophila telomere maintenance depends on the transposition of three specialized retrotransposons – HeT-A, TART and TAHRE (HTT). Controlling the activation and silencing of these elements is crucial to maintain telomere length homeostasis without compromising genomic instability. In this thesis, I have identified the role of different chromosomal proteins involved in creating the correct chromatin environment to achieve telomere length homeostasis and stability. JIL-1, together with HP1a and Z4, act as a boundary at the telomere-subtelomere frontier. The interplay of these proteins leads to an equilibrium in the activation/repression state of the telomere retrotransposons. Additionally, I have contributed to the finding that the HeT-A Gag protein is a key component targeting different protein complexes to the telomeres and guaranteeing genome stability. I have also been able to demonstrate that the Z4 partners DREF, TRF2 and KEN are also involved in the silencing of HTT, probably by mediating chromatin remodeling. Finally, I have identified a special subtelomere domain at the 4R telomere with different chromatin characteristics and demonstrated that SETDB1, HP1a and POF are involved in the regulation of the telomeric retrotransposons in the 4th chromosome. These results provide important insights to better understand how in Drosophila the telomere retrotransposons are orchestrated to achieve a telomere function analogous to telomerase telomeres in other eukaryotes.<br>El manteniment dels telòmers de Drosophila depèn de la transposició especialitzada de tres retrotransposons, HeT-A, TART i TAHRE (HTT). El control de l’activació i la repressió d’aquests elements és crucial a l’hora de mantenir la llargada telomèrica sense comprometre l’estabilitat genòmica. En aquesta tesi jo he pogut identificar el paper de diferents proteïnes cromosòmiques involucrades en crear un estat de la cromatina adient per mantenir la longitud i l’estabilitat telomèrica. JIL-1 juntament amb HP1a i Z4 ajuden a crear el llindar entre la frontera dels domini telomèric i subtelomèric. L’actuació conjunta d’aquestes proteïnes aconsegueix un estat d’equilibri activació/repressió dels retrotransposons telomèrics. A més a més, he contribuït a la descoberta de la implicació de la proteïna HeT-A Gag en el reclutament de diferents complexes proteics als telomèrs de Drosophila per poder garantir l’estabilitat telomèrica. També he pogut demostrar que altres membres dels complexes on participa Z4, com ara: DREF, TRF2 i KEN, estan també implicats en el silenciament dels retrotransposons telomèrics segurament per mitjà de la remodelació de la cromatina. Finalment he pogut demostrar que el domini subtelomèric del telòmer 4R, té una estructura cromatínica diferent a la resta dels dominis subtelomèrics dels altres cromosomes i he pogut demostrar que les proteïnes SETDB1, HP1a i POF estan implicades en la regulació de l’HTT del cromosoma 4. Els resultats d’aquesta tesi ajuden de manera substancial a comprendre com els retrotransposons telomèrics estan orquestrats per tal de poder fer una funció anàloga als telòmers de telomerasa en altres eucariotes.
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Kobow, Katja. "Epigenetic gene regulation in focal epilepsies." kostenfrei, 2009. http://d-nb.info/999752243/34.
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Chan, Yvonne. "Epigenetic regulation of enos gene expression." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0012/MQ40769.pdf.
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Huang, Hsuan-Ting. "Epigenetic Regulation of Hematopoiesis in Zebrafish." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10175.
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The initiation of the hematopoietic program is orchestrated by key transcription factors that recruit chromatin regulators in order to activate or inhibit blood target gene expression. To generate a complete compendium of chromatin factors that establish the genetic code during developmental hematopoiesis, we conducted a large-scale reverse genetic screen targeting 425 chromatin factors in zebrafish and identified over 30 novel chromatin regulators that function at distinct steps of embryonic hematopoiesis. In vertebrates, developmental hematopoiesis occurs in two waves. During the first and primitive wave, mainly erythrocytes are produced, and we identified at least 15 chromatin factors that decrease or increase formation of \(scl^+\), \(gata1^+\), and \(\beta-globin e3^+\) erythroid progenitors. In the definitive wave, HSCs capable of self-renewal and differentiation into multiple lineages are induced, and we identified at least 18 chromatin factors that decrease or increase the formation of \(c-myb^+\) and \(runx1^+\) stem and progenitor cells in the aorta gonad mesonephros (AGM) region, without disruption of vascular development. The majority of the chromatin factors identified from the screen are involved in histone acetylation, histone methylation, and nucleosome remodeling, the same modifications that are hypothesized to have the most functional impact on the transcriptional status of a gene. Moreover, these factors can be mapped to subunits of chromatin complexes that modify these marks, such as HBO/HAT, HDAC/NuRD, SET1A/MLL, ISWI, and SWI/SNF. One of the strongest phenotypes identified from the screen came from knockdown of chromodomain helicase DNA binding domain 7 (chd7). Morpholino knockdown of chd7 resulted in increased primitive and definitive blood production from the induction of stem and progenitor cells to the differentiation of myeloid and erythroid lineages. This expansion of the blood lineage is cell autonomous as determined by blastula transplantation experiments. Though chromatin factors are believed to function broadly and are often expressed ubiquitously, the combined results of the screen and chd7 analysis show that individual factors have very tissue specific functions. These studies implicate chromatin factors as playing a major role in establishing the programs of gene expression for self-renewal and differentiation of hematopoietic cells.
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Wiersma, Maaike. "Epigenetic regulation at MLL1 target genes." Thesis, University of Birmingham, 2015. http://etheses.bham.ac.uk//id/eprint/5813/.
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The mixed-lineage leukaemia 1 protein is a histone methyl-transferase that deposits the gene activating H3K4 trimethyl mark, and is often mutated in leukaemia. MLL1 is normally associated with a cohort of cofactors, but the mechanisms regulating the histone methyl-transferase activity remain unclear. Here I examine the role of Msk1, a downstream kinase of the MAP-kinase pathway, in regulating MLL1 activity. Msk1 is known to deposit the H3S10 phosphorylation mark, which was found to stimulate MLL1’s methylation activity in vitro. Here I demonstrate that MLL1 and Msk1 can be immunoprecipitated and their patterns of genomic binding show an overlap at ~30 of sites, suggesting a direct functional interaction. In transient MLL1 and Msk1 knock-down cells, known MLL1 target genes were down-regulated and at a global level, 30% of all responding genes were regulated in the same manner. Furthermore, key histone modifications at MLL1 target genes change in Msk1 knock-down cells, suggesting that histone cross-talk within the MLL1 complex acts as a means of gene regulation. Finally, cell cycle studies suggest MLL1-Msk1 cross-talk may stimulate MLL1-driven gene expression after mitosis. These findings suggest that MLL1 is regulated by Msk1 and therefore by extracellular signals via the MAP-kinase pathway.
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Hackett, Jamie Alexander. "Epigenetic regulation of germline-specific genes." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/5931.
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In mammals, epigenetic modifications and trans-acting effectors coordinate gene expression during development and impose transcriptional memories that define specific cell lineages and cell-types. Methylation at CpG dinucleotides is an epigenetic mechanism through which transcriptional silencing is established and heritably maintained through development. Functionally, DNA methylation regulates key biological processes such as X-chromosome inactivation, transposon repression and genomic imprinting. However, the extent to which DNA methylation is the primary regulator of single-copy gene expression and the precise mechanism of methylation-dependent silencing remain undetermined. Here, I identify a novel set of germline-specific candidate genes putatively regulated by DNA methylation. Analysis of one candidate gene, Tex19, demonstrates that promoter CpG methylation is the primary and exclusive mechanism for regulating developmental silencing in somatic lineages. Genetic or pharmacological removal of CpG methylation triggers robust de-repression of Tex19 and loss of transcriptional memory. Moreover, Tex19 critically relies on de novo methylation, mediated by Dnmt3b, to impose silencing in differentiating ES cells and somatic cells in vivo from embryonic day (E)7.5. Reporter gene and ChIP analysis demonstrate that Tex19 is strongly activated by general transcription factors and is not marked by repressive histone modifications in somatic lineages, consistent with differential DNA methylation per se being the primary mechanism of regulating expression. Full transcriptional silencing of Tex19 is critically dependent on the methyl-binding protein (MBP) Kaiso, which is only recruited to methylated Tex19 promoter. The reliance on DNA methylation and Kaiso for silencing in somatic cells establishes an epigenetic memory responsible for maintaining expression in germline and pluripotent cell types through successive developmental cycles. This thesis represents the first causal report of lineagespecific promoter DNA methylation directing silencing of an in vivo gene through recruitment of an MBP.
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Scionti, Isabella. "Epigenetic Regulation of Skeletal Muscle Differentiation." Thesis, Lyon, 2017. http://www.theses.fr/2017LYSEN084/document.
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LSD1 et PHF2 sont des déméthylases de lysines capables de déméthyler à la fois les protéines histones qui influencent l’expression génique et les protéines non histones en affectant leurs activités ou stabilités. Des approches fonctionnelles d’inactivation de Lsd1 ou Phf2 chez la souris ont démontré l’implication de ces enzymes dans l'engagement des cellules progénitrices au cours de la différenciation. La myogenèse est l'un des exemples les mieux caractérisés sur la façon dont les cellules progénitrices se multiplient et se différencient pour former un organe fonctionnel. Elle est initiée par une expression temporelle spécifique des gènes régulateurs cibles. Parmi ces facteurs, MYOD est un régulateur clé de l'engagement dans la différenciation des cellules progénitrices musculaires. Bien que l’action de MYOD au cours de la différenciation cellulaire ait été largement étudiée, peu de chose sont connus sur les événements de remodelage de la chromatine associés à l'activation de l'expression de MyoD. Parmi les régions régulatrices de l'expression de MyoD, la région Core Enhancer (CE) qui est transcrite en ARN activateur non codant (CEeRNA) a été démontrée pour contrôler l'initiation de l'expression de MyoD au cours de l'engagement de myoblastes dans la différenciation.Nous avons identifié LSD1 et PHF2 comme des activateurs clés du CE de MyoD. L'invalidation in vitro et in vivo de LSD1 ou l'inhibition de l'activité enzymatique de LSD1 empêche le recrutement de l'ARN PolII sur le CE, empêchant l’expression du CEeRNA. D’après nos résultats, l'expression forcée du CEeRNA restaure efficacement l'expression de MyoD et la fusion myoblastique en l'absence de LSD1. De plus, PHF2 interagit avec LSD1 en régulant sa stabilité protéique.En effet, l'ablation in vitro de PHF2 entraîne une dégradation massive de LSD1 et donc une absence d'expression du CEeRNA. Cependant, toutes les modifications d'histones qui ont lieu dans la région du CE lors de l'activation de la différenciation ne peuvent pas être directement attribuées à l'activité enzymatique de LSD1 ou PHF2. Ces résultats soulèvent la question de l'identité des partenaires de LSD1 et PHF2, qui co-participeraient à l'expression du CEeRNA et donc à l'engagement des myoblastes dans la différenciation cellulaire<br>LSD1 and PHF2 are lysine de-methylases that can de-methylate both histone proteins, influencing gene expression and non-histone proteins, affecting their activity or stability. Functional approaches using Lsd1 or Phf2 inactivation in mouse have demonstrated the involvement of these enzymes in the engagement of progenitor cells into differentiation. One of the best-characterized examples of how progenitor cells multiply and differentiate to form functional organ is myogenesis. It is initiated by the specific timing expression of the specific regulatory genes; among these factors, MYOD is a key regulator of the engagement into differentiation of muscle progenitor cells. Although the action of MYOD during muscle differentiation has been extensively studied, still little is known about the chromatin remodeling events associated with the activation of MyoD expression. Among the regulatory regions of MyoD expression, the Core Enhancer region (CE), which transcribes for a non-coding enhancer RNA (CEeRNA), has been demonstrated to control the initiation of MyoD expression during myoblast commitment. We identified LSD1 and PHF2 as key activators of the MyoD CE. In vitro and in vivo ablation of LSD1 or inhibition of LSD1 enzymatic activity impaired the recruitment of RNA PolII on the CE, resulting in a failed expression of the CEeRNA. According to our results, forced expression of the CEeRNA efficiently rescue MyoD expression and myoblast fusion in the absence of LSD1. Moreover PHF2 interacts with LSD1 regulating its protein stability. Indeed in vitro ablation of PHF2 results in a massive LSD1 degradation and thus absence of CEeRNA expression. However, all the histone modifications occurring on the CE region upon activation cannot be directly attributed to LSD1 or PHF2 enzymatic activity. These results raise the question of the identity of LSD1 and PHF2 partners, which co-participate to CEeRNA expression and thus to the engagement of myoblast cells into differentiation
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Mascheretti, Iride. "regolazione epigenetica del meccanismo autonomo di fioritura in mais (Zea mays)." Doctoral thesis, Università degli studi di Padova, 2014. http://hdl.handle.net/11577/3424659.
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In the B73 maize temperate line, the autonomous flowering pathway controls flowering independently of external signals. In Arabidopsis, the epigenetic mechanisms have been demonstrated to play an important role in the control of floral transition. To understand whether, also in maize, the epigenetic mechanisms are important in the regulation of flowering, we have characterized mutants in epi-regulators that are components of the autonomous flowering pathway. Moreover, we have analyzed mutants in a key regulator of floral transition, for which a role in epigenetic mechanisms has been speculated. During the first approach, we have analyzed lines, which were simultaneously down-regulated in nfc101 and nfc102. The maize Nucleosome remodeling factor complex component101 (nfc101) and nfc102 are putative paralogs encoding WD-repeat proteins with homology to plant and mammalian components of various chromatin modifying complexes. Our results indicate that the NFC101/NFC102 proteins directly bind and repress the Indeterminate1 (Id1) and the Zea mays CENTRORADIALIS8 (ZCN8) genes, two key regulators of the autonomous flowering pathway. In addition, the abolition of NFC101/NFC102 association with repetitive sequences of different transposable elements (TEs) resulted in tissue-specific up-regulation of non-polyadenylated RNAs produced by these regions. All direct NFC101/NFC102 targets showed histone modification patterns linked to active chromatin in nfc101/nfc102 down-regulation lines. However, different mechanisms may be involved because NFC101/NFC102 proteins mediate HDAC recruitment at Id1 and TE repeats but not at ZCN8. These results, along with the pleiotropic effects observed in nfc101/nfc102 down-regulation lines, suggest that NFC101 and NFC102 are components of distinct chromatin modifying complexes, which operate in different pathways and influence diverse aspects of maize development.
In the second strategy, we have analyzed id1 mutants, to understand if Id1 is able to activate ZCN8 and ZCN7 expression through epigenetic mechanisms. We have demonstrated that Id1, which is expressed in the immature leaf, partially contributes to the formation of histone modification patterns linked to active chromatin in the ZCN8 and ZCN7 loci in this tissue. So, in the immature leaf Id1 could be important for the formation of active chromatin at the ZCN8 and ZCN7 loci, which is maintained through mitotic divisions until the formation of mature leaf, where ZCN8 and ZCN7 are expressed. However, our results also indicate that other proteins could play a role in the formation of active chromatin at the ZCN8 and ZCN7 genes, independently from Id1 and could be necessary in the post-transcriptional regulation of ZCN8 and ZCN7.<br>Il meccanismo autonomo di fioritura regola la transizione dalla fase vegetativa alla fase riproduttiva in linee di mais temperato, indipendentemente da segnali ambientali. In Arabidopsis, è stato dimostrato che la regolazione epigenetica svolge un ruolo importante nel meccanismo autonomo di fioritura. Al fine di comprendere se la regolazione epigenetica è importante anche per la fioritura del mais, sono stati caratterizzati mutanti di epi-regolatori, che sono componenti del meccanismo autonomo di fioritura e mutanti di un regolatore chiave della transizione fiorale, per il quale è stata ipotizzata una funzione correlata a meccanismi epigenetici.
Relativamente al primo approccio, sono state analizzate linee che sotto-esprimono i geni Nucleosome remodeling factor complex component 101 (nfc101) e nfc102, due paraloghi codificanti per proteine WD-repeat, componenti di vari complessi che modificano la cromatina. I nostri risultati indicano che le proteine NFC101/NFC102 legano direttamente e reprimono l’espressione dei geni Indeterminate1 (Id1) e Zea mays CENTRORADIALIS8 (ZCN8), regolatori chiave del meccanismo autonomo di fioritura. Le proteine NFC101/NFC102 legano anche le sequenze ripetute di diversi elementi trasponibili (TE), regolando negativamente e in modo tessuto-specifico gli RNA non-poliadenilati da loro prodotti. Nei mutanti nfc101/nfc102, tutti i target diretti di NFC101/NFC102 mostrano un profilo di modifiche istoniche caratterizzanti una cromatina trascrizionalmente attiva. La regolazione mediata da NFC101/NFC102 coinvolge probabilmente meccanismi differenti, poiché esse reclutano la istone deacetilasi di tipo Rpd3 al gene Id1 e ai TE, ma non al gene ZCN8. Nel complesso, questi risultati, insieme agli effetti pleiotropici osservati nei mutanti nfc101/nfc102, indicano che NFC101/NFC102, oltre a modulare l’espressione di regolatori della fioritura mediante modifiche della cromatina, sono coinvolti in molteplici meccanismi che controllano diversi aspetti dello sviluppo del mais.
Nel secondo approccio, è stato utilizzato il mutante nullo id1, per verificare se la sua azione di attivatore del florigeno ZCN8 e del suo paralogo ZCN7 viene svolta mediante regolazione epigenetica. I nostri risultati indicano che la funzione di Id1, espresso nella foglia immatura, correla solo parzialmente con la formazione, in questo tessuto, di un profilo di modifiche istoniche associate a una competenza trascrizionale nei loci dei florigeni, il cui mRNA processato è invece prodotto solo nella foglia matura. Pertanto, Id1 potrebbe effettivamente promuovere nei florigeni la formazione di uno stato epigenetico “istruttivo” per la trascrizione, che è mantenuto durante lo sviluppo della foglia fino all’effettiva sintesi degli mRNA. Tuttavia, le osservazioni fatte suggeriscono che altri fattori sono richiesti per spiegare l’esistenza, nei florigeni, di un profilo di modifiche istoniche indipendente da Id1, così come l’importanza della regolazione post-trascrizionale che caratterizza la regolazione dei florigeni nella foglia matura nel mais.
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Huang, Chieh-Ting. "Epigenetic involvement of GluR2 regulation in Epileptogenesis." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=106297.
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Epilepsy is one of the most common neurological disorders characterized by recurrent seizures. Currently, the underlying mechanisms are not well understood and therapies only serve to relieve the symptoms. A single episode of seizure can trigger epileptogenesis, a process in which the brain undergoes network reorganization including neurodegeneration and sprouting of axons. The mechanisms linking the first seizure to development of epilepsy are currently unknown. Interestingly, changes in neuronal circuitry in epilepsy are accompanied by chronic alterations in the normal brain gene expression profile. Epigenetic mechanisms, including DNA methylation and covalent histone modifications stably program the genome during gestation. However, recent studies suggest also that epigenetic mechanisms might be involved in modifying genome function in response to environmental stimuli. We therefore hypothesize that a single seizure can disrupt normal epigenetic programming in the brain, which results in altered gene expression profiles that drive the network reorganization events. In this study, we used in vitro and in vivo models of temporal lobe epilepsy (TLE) by kainic acid treatment to test whether DNA methylation changes are associated with epileptogenesis. DNA methylation is a covalent modification of DNA by adding a methyl group on the 5' position of cytosine by DNA methyltransferases. We focused our analysis on DNA methylation because of its importance role in gene regulation. Indeed there is an overall inverse correlation between DNA methylation of regulatory regions of genes and gene expression. We closely examined the DNA methylation changes associated with the promoters of the GriA2 gene (codes for glutamate receptor ionotropic AMPA 2 subunit), which has been demonstrated to be down-regulated in epilepsy and to be highly implicated in hyper-excitable neuronal circuitries. We detected rapid hypermethylation in GriA2 after a 2-hour period of epileptiform activity in the in vitro model. Similar changes in GriA2 DNA methylation were also observed in our in vivo model 10 weeks post-kainic acid injection. We also observed a significant positive correlation between the number of seizures recorded by video-EEG and severity assessed by Racine scale and the average GriA2 DNA methylation. Epileptogenic insults induced by kainic acid treatment led to rapid DNA methylation changes in GriA2 gene. This result suggests that alterations in DNA methylation may serve as a molecular memory of the insult, which can lead to the progressive changes in gene expressions, thus contributing to the development of epilepsy as well as the maintenance of an epileptic neuronal circuitry.<br>L'épilepsie est l'une des maladies neurologiques les plus fréquentes, caractérisée par des crises épileptiques répétées et chroniques. Les mécanismes sous-tendant les troubles neurologiques associés à la maladie sont encore mal compris et seuls des traitements symptomatiques sont actuellement disponibles. Une seule crise épileptique peut induire un processus d'épileptogenèse durant lequel une réorganisation des circuits neuronaux s'effectue, incluant une neurodégénérescence et un bourgeonnement anormal des axones. Les mécanismes conduisant au développement de la maladie épileptique en tant que telle à partir d'un premier épisode épileptique sont encore inconnus. De façon intéressante, les réarrangements des circuits neuronaux observés dans l'épilepsie sont accompagnés de changements stables de schémas d'expression de gènes. Les mécanismes épigénétiques, incluant la méthylation de l'ADN ou les modifications covalentes des histones, permettent une régulation stable des schémas d'expression des gènes se mettant en place durant la gestation. Cependant, de récentes études suggèrent que ces mécanismes épigénétiques permettent également une réorganisation des schémas d'expression de gènes en réponse à des stimuli environnementaux. Nous avons alors émis l'hypothèse qu'un seul épisode épileptique peut perturber les profils épigénétiques cérébraux normaux, aboutissant à des schémas d'expression de gènes altérés et aux réorganisations cérébrales caractéristiques de l'épilepsie. Lors de cette étude, nous avons utilisés des modèles in vitro et in vivo de l'épilepsie du lobe temporal (TLE), par traitements au kaïnate, afin de tester si des changements de méthylation de l'ADN sont associés au processus d'épileptogenèse. La méthylation de l'ADN est un processus épigénétique dans lequel les bases cytosines peuvent être modifiées par l'addition d'un groupement méthyle lors d'une réaction catalysée par des ADN méthyltransférases. Nous avons focalisé notre étude sur l'étude des changements de méthylation de l'ADN en raison de son rôle important dans la régulation de l'expression des gènes. En effet, le niveau de méthylation de régions régulatrices de l'ADN telles que les promoteurs est corrélé négativement au niveau d'expression génique. Nous avons en particulier mesuré les modifications des niveaux de méthylation des promoteurs du gène GriA2 (codant pour la sous-unité 2 du récepteur gutamatergique ionotropique AMPA), qui est sous-exprimé dans l'épilepsie et dont la protéine est fortement impliquée dans l'hyperexcitabilité neuronale observée dans les crises épileptiques. Nous avons mesuré une hyperméthylation du gène GriA2 à la suite d'une période de 2 heures d'activité épileptiforme dans le modèle in vitro. Des modifications similaires ont également été observées dans le modèle in vivo, 10 semaines après une injection intracérébrale de kaïnate. Nous avons également observé une corrélation positive significative entre le nombre de crises épileptiques, détectées par Electro-Encéphalogramme Vidéo, la sévérité des crises, évaluée grâce à l'échelle Racine, et le niveau moyen de méthylation du gène GriA2.Les crises épileptiques, induites par un traitement au kaïnate, conduisent à des changements rapides des niveaux de méthylation du gène GriA2. Ce résultat suggère que des modifications des schémas de méthylation de l'ADN pourraient être un mécanisme moléculaire de mémorisation des crises épileptiques, conduisant à des changements progressifs d'expression de gènes et contribuant au développement de l'épilepsie et au maintien de circuits neuronaux anormaux.
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Zhang, Le, and 张乐. "Epigenetic regulation in laminopathy-based premature aging." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46337672.
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Atkinson, Stuart P. "Epigenetic regulation of the telomerase gene promoters." Thesis, University of Glasgow, 2006. http://theses.gla.ac.uk/4035/.
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Epigenetic mechanisms have been implicated in the regulation of telomerase gene expression and here we show that specific modifications within the chromatin environment of the hTR and hTERT promoters correlate with expression of hTR and hTERT in ALT, normal and telomerase-positive tumour cell lines. Lack of expression of hTR and hTERT is associated with repressive histone modification, while, hTR and hTERT expression is associated permissive histone modifications. Methylation of lysine 20 H4 was not linked to gene expression but instead was specific to the hTR and hTERT promoters of ALT cells providing an insight into the differences between ALT and telomerase-positive cells as well as a novel marker for the ALT phenotype. Basal transcription machinery dynamics were also shown to be different between normal and cancer cells at the telomerase gene promoters. Modulation of the chromatin environment was also shown to cause re-expression or increased expression of hTR and hTERT further supporting the role of the chromatin environment in controlling telomerase gene expression. Epigenetic mechanisms are also shown to be involved in the repression of hTERT transcription in human mesenchymal stem cell (hMSCs) and modulation of the chromatin environment is shown to allow re-expression of hTERT expression, while the disruption of telomerase gene expression in human haematopoietic stem cells (hHSCs) in chronic myeloid leukaemia (CML) and the role of the chromatin environment was also studied. These data establishes how epigenetic mechanisms can contribute to transcriptional regulation of telomerase and also highlights the potential importance of epigenetics in senescence and tumourigenicity.
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Rosselló, Tortella Margalida. "Epigenetic Regulation of tRNA Biology in Cancer." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/673026.
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Transfer RNAs (tRNAs) are essential molecules that allow the translation of the genetic code into amino acids. Extensive research during the last 50 years have revealed that, despite their apparently simple structure and function, tRNAs are more than simple adaptors in protein synthesis –they are of high importance in normal cell functions. Reinforcing this, tRNA levels are tightly regulated to match the codon usage patterns of a given cell type or cellular status to meet the cellular specific needs and adapt to stress. Moreover, tRNA nucleoside modifications are critical for their function at multiple levels, such as translation efficiency and fidelity, wobbling and fragmentation.
The relevance of tRNA regulation in cell physiology is emphasized by the recent discovery that these molecules and their derived fragments are deregulated in cancer. Not only tRNA biology imbalance is associated to malignant transformation, but it also actively participates in it. These alterations occur at multiple levels of tRNA biology, such as expression, nucleoside modification and fragmentation, but many open questions remain unanswered. Cancer- specific tRNA deregulation is a very new and still unexplored discipline, and further studies are required to fully understand the molecular mechanisms that account for these alterations and their relevance in tumor biology.
Because alterations in DNA methylation constitute a frequent mechanism by which transformed cells acquire their malignant characteristics, the cornerstone of this thesis is the description of epigenetic lesions that support the cancer-associated tRNA deregulation. To this end, we have designed and performed two independent studies to unveil the epigenetic regulation of tRNA biology in cancer. In the first study, we highlighted the tumor-specific epigenetic silencing of TYW2 as a mechanism to induce tRNAPhe hypomodification at position 37, a phenomenon that was observed for the first time more than forty years ago but whose cause and consequences have remained obscure. Our results established the connection between this epigenetic defect and a phenotype that enhances -1 ribosome frameshifting events to ultimately confer increased migratory capacities and mesenchymal features to the transformed colon cells. In the second study, we established a founded connection between cancer-associated DNA methylation defects with alterations in the expression of specific tRNAs. Our analyses also revealed that the oncogenic tRNA-Arg-TCT-4-1 overexpression in endometrial cancer was guided by DNA hypomethylation. Most importantly from the clinical perspective, the epigenetic alterations identified in both studies can anticipate the patients’ outcome, for which they may serve as biomarkers to allow the identification of high-risk patients that may benefit from a more comprehensive surveillance or complementary therapeutic strategies.<br>Els ARN de transferència (tRNAs) són d’una importància clau en la regulació de la síntesi proteica i l’expressió gènica. La seva rellevància en la fisiologia cel·lular es veu reforçada pel descobriment que aquestes molècules i els seus derivats estan alterats en patologies com el càncer, on contribueixen activament. Les alteracions dels tRNAs en càncer suposen una nova disciplina d’estudi on encara moltes preguntes romanen obertes per tal d’arribar a comprendre quines són les causes d’aquestes defectes i quin impacte tenen sobre la malaltia. Aquesta tesi té com objectiu identificar i caracteritzar alteracions en la metilació de l’ADN subjacents als desequilibris en la biologia dels tRNAs de les cèl·lules tumorals. En el primer estudi, hem descobert el silenciament epigenètic de l’enzim TYW2 en càncer colorectal com a causa de la hipomodificació del tRNAPhe, un fenomen que va ser descrit per primer cop fa més de quaranta anys però les causes i conseqüències del qual no van ser mai estudiades. Els nostres resultats estableixen una clara connexió entre aquest defecte epigenètic i un fenotip que és propens a potencial el frameshift dels ribosomes, cosa que augmenta la capacitat migratòria de les cèl·lules de càncer de colon. El segon estudi ha servit per caracteritzar la relació entre els canvis en la metilació de l’ADN i les alteracions en l’expressió dels tRNAs en càncer. Els nostres resultats han revelat que l’expressió de tRNA-Arg-TCT-4-1 augmenta en càncer d’endometri arrel de la hipometilació del seu gen. Més enllà d’aquests dos mecanismes epigenètics per modular la biologia dels tRNAs, els nostres estudis estableixen una connexió entre aquestes lesions epigenètiques i la prognosi dels pacients amb certs tipus de tumor, per la qual cosa podrien proposar-se com biomarcadors per identificar pacients de risc.
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Sun, Bin. "Epigenetic regulation of postnatal subventricular zone development." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:e9ee95c1-b6cb-43c5-aef8-780e3fd50422.
Full textAbstract:
The postnatal/adult subventricular zone (SVZ) harbours neural stem cells (NSC), which produce neurons that migrate to the olfactory bulbs. SVZ NSC share several biological features with glia, especially reactive astrocytes. However, it is not clear how SVZ NSC simultaneously maintain self-renewal stem cell properties and the potential for generating daughter cells that differentiate into neurons. Multiple cyclin-dependent kinase inhibitors (CDKIs), including p16, p19 and p21 have been identified as indispensable for maintaining stem cell potential, in both cyclin dependent and independent manners. However, how these CDKIs are coordinated remains poorly defined. One possible regulator of CDKIs is the canonical Polycomb Repressive Complex 2 (PRC2) that consists of Eed, Suz12, and Ezh2. Ezh2 functions to methylate lysine 27 of histone 3 (H3K27me3) and consequently suppresses target gene expression. Whereas PRC2 serves to balance self-renewal versus differentiation and neuron versus glial fate choices in early and late embryogenesis, respectively, our understanding of its role in the neonatal and adult SVZ is incomplete. In this thesis, I discovered that the PRC2 core subunit Eed, but not Ezh2, was expressed in SVZ NSC. Eed directly repressed p16 and p19, but indirectly fine-tuned p21 expression in SVZ NSC. Conditional deletion or knockdown of Eed in vivo led to loss of constitutive SVZ stemness and blocked NSC activation. This was partly due to selective activation of the PRC2 targets, Cdkn2a and Gata6; in contrast Ezh2 loss of function only activated Cdkn2a but not Gata6. In the SVZ, Gata6 overexpression was sufficient to limit the neurogenic ability of NSC and also inhibited p21 post-transcriptional expression. I also showed that although reactive astrocytes in the cerebral cortex can acquire stem cell properties in response to brain injury, Eed was not involved in this process. Taken together, I identified novel and divergent regulation of SVZ CDKIs by separate subcomponents of PRC2, and showed that these are essential for SVZ NSC maintenance. Whilst this regulatory pathway was specific in the neurogenic niche it had little influence on parenchymal astrocytes. In a relatively small side project, I screened and identified several long non- coding RNAs (lncRNA) that were highly expressed in the adult rodent SVZ. The lncRNA Paupar, which is transcribed upstream from the Pax6 antisense strand, was enriched in the postnatal SVZ and regulated Pax6 and Ezh2 expression. In vivo, Paupar knockdown showed it is necessary for stem cell maintenance and thus regulates postnatal neurogenesis. To conclude, I discovered two interacting epigenetic regulators that control postnatal SVZ NSC and neurogenesis.
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Zhang, Qunshu. "Epigenetic Regulation of Apoptosis in Prostate Cancer." Diss., North Dakota State University, 2015. https://hdl.handle.net/10365/27614.
Full textAbstract:
Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive
complex 2 and suppresses gene expression by catalyzing histone H3 methylation on lysine 27.
EZH2 is overexpressed in metastatic prostate cancer and has been shown to promote cell
proliferation and metastasis. Here we show that EZH2 also suppresses prostate cancer apoptosis
by coordinating the epigenetic silencing of two pro-apoptotic microRNAs, miR-205 and miR-31.
We previously reported that miR-205 is silenced in prostate cancer through promoter
methylation. In this study, we found that EZH2 suppresses miR-31 expression by trimethylation
of H3K27 on the miR-31 promoter. SiRNA knockdown of EZH2 increased miR-31 expression
and decreased the anti-apoptotic protein E2F6 (a target of miR-31), resulting in the sensitization
of prostate cancer cells to docetaxel-induced apoptosis and vice versa. We further demonstrated
that miR-205 silencing is linked to miR-31 silencing through EZH2. Suppression of miR-205
caused an increase of EZH2 protein, which in turn inhibited miR-31 expression and vice versa.
Thus, EZH2 integrates the epigenetic silencing of miR-205 and miR-31 to confer resistance to
chemotherapy-induced apoptosis.
Besides the histone modification by histone methyltransferases (HMTs) such as EZH2,
histone deacetylases (HDACs) offer another mechanism to epigenetically regulate gene
expressions in cancer. The class I selective inhibitor of HDACs, mocetinostat, has promising
antitumor activities in both preclinical studies and the clinical trials. To understand how
mocetinostat induces apoptosis in prostate cancer cells, we examined the effects of mocetinostat
on miR-31. We found that miR-31 was significantly upregulated by mocetinostat in prostate
cancer cells. E2F6 was decreased by mocetinostat treatment. Mocetinostat also increased the
expression of pro-apoptotic protein Bad and activated caspase-3 and caspase-9. SiRNA
iv
knockdown of E2F6 sensitized cancer cells to mocetinostat-induced apoptosis. Importantly, we
found the same results in the primary prostate cancer stem cells. Thus, activation of miR-31 and
downregulation of E2F6 contribute to mocetinostat-induced apoptosis in prostate cancer.
In summary, the epigenetic silencing of miR-31 confers a resistance mechanism for
chemotherapy-induced apoptosis in prostate cancer cells. Using mocetinostat to activate miR-31
expression is a novel strategy to overcome resistance to apoptosis and improve response to
therapy.
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Maleszewska, Marta. "Epigenetic regulation of haematopoietic stem cell differentiation." Paris 7, 2009. http://www.theses.fr/2009PA077097.
Full textAbstract:
Le rôle des événements épigénétiques dans le maintien de la multipotentialité et la détermination cellulaire des cellules souches hématopoïétiques (CSH) reste mal connu. L'objet de ma thèse a été de définir les éléments épigénétiques qui sous-tendent la multipotentialité des CSH. Les modifications des histones, la méthylation de l'ADN et la localisation sub-nucléaire de loci spécifiques des lignées hématopoïétiques ont été étudiées dans les CSH CD34+CD381o et dans des précurseurs hématopoïétiques. Nous montrons que dans les CSH les gènes hématopoïétiques ont une structure chromatinienne particulière, avec de hauts niveaux d'acétylation de l'histone H4 et de H3K4me2, en l'absence des marqueurs actif H3K4me3 et répressifs H3K9me3 et H3K27me3. La différenciation des cellules souches est associée à une inhibition épigénétique des loci non spécifiques de la lignée (diminution des marqueurs d'activation et augmentation des marqueurs d'inhibition) et à un enrichissement des gènes spécifiques de la lignée en marqueurs d'activation, notamment acétylation de l'histone H3 et H3K4me3. Les modifications des histones aux loci D -globine et Ig précèdent leur changement de localisation sub-nucléaire aux cours de l'hématopoïèse. Nous montrons que la région JH du locus IgH est méthylée de façon hétérogène en tout-ou-rien dans les cellules CD34+CD381o, du fait soit de différences spécifiques d'allèle dans la méthylation de l'ADN soit d'une hétérogénéité au sein des cellules CD34+CD381o. Au total, les CSH présentent des caractéristiques épigénétiques qui pourraient contribuer à établir la multipotentialité de ces cellules souches<br>The role of chromatin in haematopoietic stem cell (HSC) cell fate decisions is poorly understood. My PhD studies were aimed at defining epigenetic correlates that underlie the multilineage potential of HSC. To this end, the level of histone modifications, DNA methylation and subnuclear localisation of lineage-specific haematopoietic loci were analysed in CD34+CD381o HSC and lineage committed precursors. This study shows that HSC maintain haematopoietic genes in a distinct chromatin conformation, characterised by the presence of histone H4 acetylation and H3K4me2 in the absence of active H3K4me3 or repressive H3K27me3 or H3K9me3 histone marks. This chromatin structure appears to describe a transcriptionally competent "ground state" for these genes keeping them silent but poised for expression at later stages of HSC differentiation. Progressive lineage restriction and differentiation of HSC was accompanied by epigenetic silencing of lineage-inappropriate genes associated with loss of active and addition of repressive histone marks, while lineage-specific genes were further enriched for active histone H3 acetylation and H3K4me3 marks. Furthermore, we found that changes in histone modifications at the β-globin and Ig loci precede changes in subnuclear localisation during HSC differentiation. DNA methylation analysis indicted that the IgH JH region is heterogeneously methylated in CD34+CD381o progenitors, perhaps reflecting allele-specific differences in HSC or heterogeneity within the CD34+CD381o population. All together, the results of this study have identified epigenetic features that may serve to establish and maintain the multilineage potential of HSC
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Pliuskys, Laurynas. "Epigenetic regulation of the myeloid cell lineage." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:f4ee6659-ce0b-4730-ae5b-95c141f82e10.
Full textAbstract:
The myeloid cell lineage is a fundamental element of the immune system and it can give rise to a diverse set of terminally differentiated cells, such as macrophages or osteoclasts among many others. Mutations or misregulation of gene expression may lead to severe clinical conditions, such as arthritis, osteoporosis or cancers. Epigenetics, the regulation of gene expression and chromatin remodelling, is implicated in cell differentiation, function and disease, and hence it is a promising new area to explore in order to explain underlying cellular mechanisms. Firstly, human macrophage subtypes were studied. Chemokine (C-C motif) ligand (CCL) 1 and mannose receptor were validated to be granulocyte macrophage (GM) colony stimulating factor (CSF) induced macrophage markers, while CCL<sub>2</sub> was specifically expressed in macrophage CSF (MCSF) macrophage population. By utilising publicly available high-throughput sequencing data, new biomarkers dehydrogenase/reductase (SDR family) member 2 and CCL<sub>26</sub> were discovered to be MCSF-macrophage specific while guanylate binding protein 5 and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A were highly up-regulated in GMCSF cells. Secondly, a range of gene knock-down techniques for the myeloid cell lineage were optimised and established. Lentiviral short-hairpin RNA (shRNA) delivery methods were shown to induce an undesirable pro-inflammatory response in macrophages. Furthermore, the frequently utilised cytomegalovirus promoter for gene expression was shown to be completely silenced in macrophage populations. Locked nucleic acids were selected as a suitable alternative to shRNA knock-down and by employing this new tool it was shown that a histone demethylase lysine (K)-specific demethylase (KDM) 6B is fundamental for macrophage differentiation. Finally, a small molecule GSK-J<sub>4</sub>, a potent inhibitor of histone demethylases KDM6A, KDM6B and KDM<sub>5</sub>B specific for H<sub>3</sub>K<sub>27me3</sub> and H<sub>3</sub>K<sub>4me3</sub>, respectively, was used to dissect epigenetic signalling in osteoclasts and multiple myeloma. In osteoclasts it was shown to act mainly by inhibiting transcriptional changes required for osteoclastogenesis when MCSF-macrophages are stimulated with Receptor Activator Of Nuclear Factor Kappa-B Ligand (RANKL), as indicated by the differential increase in H<sub>3</sub>K<sub>27me3</sub> marks, leading to inhibition of c-Jun and potentially abolition of transcription factor AP-1, required for the transcriptional initiation of nuclear factor of activated T-cells 1 (NFATc1). In multiple myeloma cells, GSK-J<sub>4</sub> causes a dramatic increase in expression, further supported by the build-up of global H<sub>3</sub>K<sub>4me3</sub> marks, which results in the upregulation of the unfolded protein response pathway. In both cell systems, there is an early upregulation of metallothionein genes, which in multiple myeloma was shown to increase potentially due to rapid influx of zinc ions within the first 30 minutes, and as such may cause induction of apoptosis in multiple myeloma and may inhibit differentiation of osteoclasts.
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Corbett, Laura. "Morphogen and epigenetic regulation of wound healing." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2935.
Full textAbstract:
Events during wound repair are reminiscent of developmental events such as cell migration, redifferentiation and proliferation. Factors controlling these processes in the early embryo may therefore be important regulators in adult wound healing. Fibrosis is a disease of dysregulated wound healing with fibroproliferative disorders accounting for 45% of deaths in developed nations. Despite this, effective antifibrotic therapy is limited. Understanding factors regulating wound repair process will aid identification of potential therapeutic targets. This project first explored how activation of developmental signalling pathways influences regulation of myofibroblast transdifferentiation and behaviour in liver fibrosis, focusing on the Wnt signalling pathway. Wnt signalling was upregulated in activated myofibroblasts, with significantly increased expression of non-canonical ligands. Wnt stimulus did not provoke a canonical/β-Catenin mediated response, with myofibroblasts responding through non-canonical associated signalling instead. Inhibition of Wnt signalling reduced classic markers of fibrosis, suggesting a profibrotic role for Wnt signalling during liver fibrosis. Stimulus with the non-canonical ligand Wnt5a did not directly affect expression of fibrotic markers in myofibroblasts. Instead it appeared to act as a prosurvival factor and increased expression of profibrotic cytokines in resident liver macrophages. Dynamic changes in DNA methylation pattern also regulate embryonic development. This project next explored whether inheritance of epigenetic marks could alter early DNA methylation patterns and affect response to injury in adult, focusing on the histone variant H2A.Z. H2A.Z is thought anti-correlative to DNA methylation and has been shown to be enriched in the sperm of injured animals in a model of transgenerational adaptation to wound healing. H2A.Z expression was depleted via morpholino injection in early zebrafish embryos, resulting in significant mortality and phenotypic abnormality. Injected animals displayed significant hypermethylation of DNA during early embryonic periods. This suggests that alteration of epigenetic marks can influence methylation status of DNA.
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Wu, Hao. "Epigenetic regulation of neural stem cell differentiation." Diss., Restricted to subscribing institutions, 2009. http://proquest.umi.com/pqdweb?did=1835827841&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Albadrani, Ghadeer Mohsen. "Epigenetic regulation of Ewing's sarcoma stem cells." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/20321/.
Full textAbstract:
Emerging evidence suggests that cancer stem-like (CS-like) cells are responsible for cancer progression and relapse. The identification and characterisation of CS-like cells is therefore important to reveal potential targets that could be used to design more effective personalised treatment to improve outcomes. The cell surface marker prominin-1 (CD133) has been used to identify Ewing sarcoma (ES) CS-like cells (ES-CS-like), however some primary ES cells are devoid of CD133 and may be down-regulated by the microenvironment in cell culture. Therefore, alternative approaches are required to identify ES-CS-like cells. Two approaches were compared to enrich for putative ES-CSCs from ES cell lines; isolation of ES-CS-like cells by CD133 expression and using a functional single cell self-renewal assay. The second approach was more reliable for studying the miRNA and mRNA expression profiles in ES-CS-like spheroids compared to ES cells grown as monolayers. In ES-CS-like spheroids, the expression of the stem cell marker EBAF (q < 0.03), miR-210-3p (q < 0.12) and the ABC transporter protein ABCG1 (q < 0.14) were all significantly increased. The phenotypic significance of ABCG1 was investigated using knock-in and knock-out experiments. Overexpressing ABCG1 protein using a lentiviral vector was unachievable as a result of the increased expression of the E3 ligase; NEDD4-1. This ligase prevented the post translational overexpression of ABCG1 mRNA. Interestingly, knock-down of ABCG1 mRNA appeared to stabilise ABCG1 protein and decrease viable number of ES cell line (SK-N-MC), suggesting ABCG1 may have a cell cycle or survival function. In conclusion, growth of ES spheroids from single cells enriches for cells which express stem cell markers, suggesting that the single cell self-renewal assay can be used to enrich for ES-CS-like cells. Furthermore, ABCG1 and miR-210-3p may be drivers of the ES-CS-like phenotype and might be used to select patients at greatest risk and inform design of targeted treatments. These observations require validation using functional assays and confirmation in patient derived cells and tumours. Whether CDKN1A-interacting zinc finger protein 1 (CIZ1) plays a role in the ES-CS-like phenotype is yet to be investigated.
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Islam, Abul 1978. "Delineating epigenetic regulatory mechanisms of cell profileration and differentiation." Doctoral thesis, Universitat Pompeu Fabra, 2012. http://hdl.handle.net/10803/85721.
Full textAbstract:
Recent advances in high throughput technology have opened the door to systematic studies of epigenetic mechanisms. One of the key components in the regulation of the cell cycle and differentiation is the retinoblastoma protein (pRB), a component of the RB/E2F tumor suppressor pathway that is frequently deregulated in cancer. The RBP2/KDM5A histone demethylase was shown to interact with pRB and regulate pRB function during differentiation. However, how precisely differentiation is coupled with halted cell cycle progression and whether an epigenetic mechanism is involved remain unknown. In the present study, I analyzed gene expression levels of human histone methyltransferases (HMT) and demethylases (HDM), as well as their targets in human cancers; and focused on RB/KDM5A connection in control of cell cycle and differentiation. In particular, I used Drosophila as a model to describe a novel mechanism where the RB/E2F pathway interacts with the Hippo tumor suppressor pathway to synergistically control cell cycle exit upon differentiation. Studying the role of miR-11, I found that the inhibition of dE2F1-induced cell death is its highly specialized function. Furthermore, I studied the induction of differentiation and apoptosis as the consequences of KDM5A deletion in cells derived from Rb knockout mice. I concluded that during differentiation, KDM5A plays a critical role at the enhancers of cell type-specific genes and at the promoters of E2F targets; in cooperation with other repressor complexes, it silences cell cycle genes. I found that KDM5A binds to transcription start sites of the majority of genes with H3K4 methylation. These are highly expressed genes, involved in certain biological processes, and occupied by KDM5A in an isoform-specific manner. KDM5A plays a unique and non-redundant role in histone demethylation and its promoter binding pattern highly overlaps with the opposing enzyme, MLL1. Finally, I found that HMT and HDM enzymes exhibit a distinct co-expression pattern in different cancer types, and this determines the level of expression of their target genes.<br>Los avances recientes en las tecnologías de alto flujo han abierto el camino a los estudios sistemáticos de los mecanismos epigenéticos. La proteína retinoblastoma (pRB), uno de los elementos de la ruta de supresión de tumores RB/E2F que se encuentra desregulado con frecuencia en el cáncer, es uno de los componentes esenciales de la regulación del ciclo celular y la diferenciación. Sin embargo, aún no se conoce de qué manera precisa la diferenciación se acopla a la detención del avance del ciclo celular y si hay algún mecanismo epigenético vinculado a este proceso. En este estudio, he analizado los niveles de expresión de histona metiltransferasas (HMT) y desmetilasas humanas (HDM), así como sus dianas en cánceres humanos, y me he centrado en la conexión de RB/KDM5A en el control del ciclo celular y la diferenciación. Específicamente, utilicé Drosophila como modelo para describir un mecanismo nuevo mediante el cual RB/E2F interactúa con la ruta Hippo de supresión de tumores para controlar de manera sinérgica la detención del ciclo celular relacionada con la diferenciación. Mediante la investigación del papel de miR-11, determiné que su función altamente especializada es la inhibición de la muerte celular inducida por dE2F1. Además, estudié la inducción de la diferenciación y la apoptosis como consecuencia de la pérdida de KDMA5 en células obtenidas a partir de ratones sin Rb. Extraje como conclusión que, durante la diferenciación, KDMA5 desempeña un papel esencial sobre los estimuladores de los genes específicos de los tipos celulares, así como en los promotores de las dianas de E2F; en cooperación con otros complejos represores silencia a los genes del ciclo celular. Investigué el mecanismo de reclutamiento de KDM5A y encontré que se une al sitio de inicio de la transcripción de la mayoría de los genes que poseen metilación en H3K4. Estos genes tienen elevados niveles de expresión, están involucrados en determinados procesos biológicos y están ocupados por diferentes isoformas de KDM5A. KDM5A desempeña un papel único y no redundante en la desmetilación de las histonas y que en gran medida se solapa con la enzima con la función opuesta, MLL1. Para terminar, encontré que las enzimas HMT y HDM muestran patrones de co-expresión distintos en diferentes tipos de cáncer, y que este hecho determina los niveles de expresión de sus genes diana.
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Jubierre, Zapater Luz. "Epigenetic regulators in neuroblastoma: BRG1, a future therapeutic target." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/457983.
Full textAbstract:
El Neuroblastoma (NB) es un cáncer derivado del sistema nervioso simpático, y es el tumor sólido más común en los niños de cero a dos años. Los NB son muy heterogéneos, tienen un curso clínico que puede variar desde la regresión espontánea hasta la resistencia a toda forma de tratamiento existente. Los pacientes de alto riesgo necesitan altas dosis de quimioterapia y pese a ello solo el 30-40% se curan. Los tumores recidivantes o metastáticos adquieren resistencia a las terapias, poniendo de manifiesto la necesidad del desarrollo de nuevos tratamientos. Debido a los diversos mecanismos responsables de la quimioresistencia del NB, proponemos usar como diana terapéutica a los factores epigenéticos que controlan varias vías celulares para evitar la resistencia a terapia. Hemos encontrado que el gen SMARCA4/BRG1 (de sus siglas en inglés SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4) está consistentemente sobreexpresado en estadios avanzados de NB, y que los altos niveles de BRG1 son indicativos de mal pronóstico. Los experimentos de pérdida de función tanto in vitro como in vivo demostraron que BRG1 es esencial para la proliferación de las células de NB. Además, un análisis transcriptómico global mostró que BRG1 controla la expresión de elementos claves en vías oncogénicas como las de PI3K/AKT o BCL-2, lo que ofrece una nueva y prometedora combinación de terapia para los NB de alto riesgo.<br>Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system, and is the most common solid tumor of infancy. NBs are very heterogeneous, with a clinical course ranging from spontaneous regression to resistance to all current forms of treatment. High-risk patients need intense chemotherapy, and only 30-40% will be cured. Relapsed or metastatic tumors acquire multi-drug resistance, raising the need for alternative treatments. Owing to the diverse mechanisms that are responsible of NB chemoresistance, we aimed to target epigenetic factors that control multiple pathways to bypass therapy resistance. We found that the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4/BRG1) was consistently upregulated in advanced stages of NB, with high BRG1 levels being indicative of poor outcome. Loss-of-function experiments in vitro and in vivo showed that BRG1 is essential for the proliferation of NB cells. Furthermore, whole-genome transcriptome analysis revealed that BRG1 controls the expression of key elements of oncogenic pathways such as PI3K/AKT and BCL2, which offers a promising new combination therapy for high-risk NB.
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BASON, RAMONA. "EPIGENETIC CHARACTERIZATION OF TUMOR INFILTRATING CD4+ T REGULATORY CELLS." Doctoral thesis, Università degli Studi di Milano, 2022. http://hdl.handle.net/2434/917092.
Full textAbstract:
In recent years, the role of CD4+ regulatory T cells (Treg cells) in inhibiting the anti cancer activity of effector T cells has become increasingly evident and they are therefore currently considered promising targets for cancer immunotherapy. Despite Treg cell depletion has been reported to increase anti-tumor specific immune responses and to reduce tumor burden, some relevant issues still remain to be addressed, for a safer, more effective clinical application of these therapies.
Previous findings in our lab identified unique transcriptional profiles of human Treg cells infiltrating colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) supporting the existence of an underlying regulatory hubs that specifically shape tumor Treg identity and represent potential target for their functional modulation specificially in the tumor microenvironment.
Treg cell transcriptome represents just a layer of the machinery that drive the acquisition of their tumor cell state. Ultimately, a comprehensive understanding of the regulatory networks that govern tumor Treg gene expression programs can provide insights on how a more selective inhibition can be achieved by interfering with the hubs that translate the cues coming from the tumor microenvironment.
To define the epigenetic blueprints specific for tumor infiltrating Treg cells we have integrated distinct histone marks (H3K4me3, H3K4me1, H3K36me3, H3K27ac, H3K27me3) with ATAC-seq derived chromatin accessibility data we generated in Treg cells isolated from peripheral blood, normal and tumor tissues. To infer genome chromatin states we have employed ChromHMM a machine learning based approach that predicts the molecular structure of promoters and enhancers based on data from chromatin accessibility assays and a set of histone modification. We focused in particular on active enhancer regions, characterized by the co-presence of H3K27Ac and H3K4me1. Transcription factor footprinting analysis on the identified regulatory regions showed TF groups with distinct binding activity profile across Treg populations that are clearly lost or gained specifically in tumor Treg cells. Based on our findings we are implementing CRISPR based epigenetic modulation of selected enhancers that represent a novel strategy to reverse Treg mediated immunosuppression in the tumor microenvironment.
The new knowledge coming from this project cues will lead to a better understanding of tiTreg plasticity and to the identification of novel potential targets that can guide the rational development of tiTreg reprograming and innovative therapeutics for cancer with increased efficacy and reduced adverse effects.
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Bergström, Rosita. "Epigenetic Regulation of Replication Timing and Signal Transduction." Doctoral thesis, Uppsala universitet, Molekylär cellbiologi, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8413.
Full textAbstract:
Upon fertilization the paternal and maternal genomes unite, giving rise to the embryo, with its unique genetic code. All cells in the human body are derived from the fertilized ovum: hence they all contain (with a few exceptions) the same genetic composition. However, by selective processes, genes are turned on and off in an adaptable, and cell type-specific, manner. The aim of this thesis is to investigate how signals coming from outside the cell and epigenetic factors residing in the cell nucleus, cooperate to control gene expression. The transforming growth factor-β (TGF-β) superfamily consists of around 30 cytokines, which are essential for accurate gene regulation during embryonic development and adult life. Among these are the ligands TGF-β1 and bone morphogenetic (BMP) -7, which interact with diverse plasma membrane receptors, but signal via partly the same Smad proteins. Smad4 is essential to achieve TGF-β-dependent responses. We observed that by regulating transcription factors such as Id2 and Id3 in a specific manner, TGF-β1 and BMP-7 achieve distinct physiological responses. Moreover, we demonstrate that CTCF, an insulator protein regulating higher order chromatin conformation, is able to direct transcription by recruiting RNA polymerase II to its target sites. This is the first mechanistic explanation of how an insulator protein can direct transcription, and reveals a link between epigenetic modifications and classical regulators of transcription. We also detected that DNA loci occupied by CTCF replicate late. The timing of replication is a crucial determinant of gene activity. Genes replicating early tend to be active, whereas genes replicating late often are silenced. Thus, CTCF can regulate transcription at several levels. Finally, we detected a substantial cross-talk between CTCF and TGF-β signaling. This is the first time that a direct interplay between a signal transduction pathway and the chromatin insulator CTCF is demonstrated.
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Orcutt, Timothy Michael. "Dissecting the epigenetic regulation of V[beta] recombination." NCSU, 2007. http://www.lib.ncsu.edu/theses/available/etd-07232007-100353/.
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V(D)J recombination in developing lymphocytes is essential for producing a diverse repertoire of antigen receptors (TCR and Ig). During recombination, the proteins encoded by the recombinase activating genes (RAG1 and RAG2) bind specific DNA sequences flanking individual V, D, and J coding segments within each antigen receptor gene, and introduce double strand DNA breaks at the coding sequence/targeting sequence boundaries. These double strand breaks are then repaired by ubiquitous DNA repair machinery to generate novel coding segment joints. The ability of each developing lymphocyte to independently assemble unique V(D)J joints results in the enormous diversity of antigen receptors expressed by our immune system. Despite a conserved enzymatic activity in both B and T lymphocytes, the assembly of T cell receptors (TCRs) and Immunoglobulins (Igs) in T and B cells respectively follows a highly orchestrated program in which the accessibility of individual targeting sequences varies during lymphocyte development. For example, when the TCRb locus is rearranged, it initially assembles joints between D and J elements. Only after DJ joining do upstream V sequences become accessible and rearrange with the preassembled DJ?s. We have previously shown that DJ rearrangement requires modification of the chromatin structure surrounding individual D and J segments via the coordinated actions of D-associated promoters and a single downstream enhancer. Like the D elements, each V element in TCRb is associated with a transcriptional promoter. But the role these V promoters play in V-to-DJ recombination remains unknown. Similarly, because enhancer deletion ablates D-to-J assembly, the potential role of enhancer activity in V recombination has not been directly tested. We hypothesize that V-to-DJ rearrangement requires both enhancer and promoter-dependent changes in the chromatin surrounding the V RAG binding site, as well as that surrounding the D 5? binding site. To test this hypothesis, I have constructed a panel of recombination substrates which harbor unrearranged or prerearranged DJ elements downstream from a single V element. These ?miniloci? were stably transfected into the chromatin of a recombinase-inducible T cell line, and the chromatin status, expression and recombination potential of each was assessed.
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Bergström, Rosita. "Epigenetic regulation of replication timing and signal transduction /." Uppsala : Acta Universitatis Upsaliensis, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8413.
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Tan, E.-Jean. "Transcriptional and Epigenetic Regulation of Epithelial-Mesenchymal Transition." Doctoral thesis, Uppsala universitet, Ludwiginstitutet för cancerforskning, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-206120.
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The transforming growth factor beta (TGFβ) is a cytokine that regulates a plethora of cellular processes such as cell proliferation, differentiation, migration and apoptosis. TGFβ signals via serine/threonine kinase receptors and activates the Smads to regulate gene expression. Enigmatically, TGFβ has a dichotomous role as a tumor suppressor and a tumor promoter in cancer. At early stages of tumorigenesis, TGFβ acts as a tumor suppressor by exerting growth inhibitory effects and inducing apoptosis. However, at advanced stages, TGFβ contributes to tumor malignancy by promoting invasion and metastasis. The pro-tumorigenic TGFβ potently triggers an embryonic program known as epithelial-mesenchymal transition (EMT). EMT is a dynamic process whereby polarized epithelial cells adapt a mesenchymal morphology, thereby facilitating migration and invasion. Downregulation of cell-cell adhesion molecules, such as E-cadherin and ZO-1, is an eminent feature of EMT. TGFβ induces EMT by upregulating a non-histone chromatin factor, high mobility group A2 (HMGA2). This thesis focuses on elucidating the molecular mechanisms by which HMGA2 elicits EMT. We found that HMGA2 regulates a network of EMT transcription factors (EMT-TFs), such as members of the Snail, ZEB and Twist families, during TGFβ-induced EMT. HMGA2 can interact with Smad complexes to synergistically induce Snail expression. HMGA2 also directly binds and activates the Twist promoter. We used mouse mammary epithelial cells overexpressing HMGA2, which are mesenchymal in morphology and highly invasive, as a constitutive EMT model. Snail and Twist have complementary roles in HMGA2-mesenchymal cells during EMT, and tight junctions were restored upon silencing of both Snail and Twist in these cells. Finally, we also demonstrate that HMGA2 can epigenetically silence the E-cadherin gene. In summary, HMGA2 modulates multiple reprogramming events to promote EMT and invasion.
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McEwen, Kirsten Rose. "Epigenetic regulation of imprinted loci in the mouse." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609297.
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Bruton, Peter Christopher. "Epigenetic regulation of heterochromatin structure and tumour progression." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33232.
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Since the discovery of DNA packaging into chromatin, and McClintock's (1951) work on position-effect variegation providing evidence of non-mendelian inheritance, the principal of a genome maintaining 'on' and 'off' states has been widely adopted. However, the underlying mechanisms that regulate these dynamic chromatin states and their effect on disease are still poorly understood. DNA methylation and histone trimethylation at H3K9 and H4K20 are the core hallmarks of the heterochromatic constitutively 'off' state. Constitutive heterochromatin is predominantly comprised of repetitive satellite containing pericentromeric regions and telomeres and in mouse heterochromatin clusters into large chromocenters. These regions are cytologically more compact and generally transcriptionally silent across embryonic and differentiated mouse cell types. However, in addition to increased genomic instability, mouse tumour cells sustain increased satellite expression suggesting constitutive heterochromatin is disrupted. Therefore how constitutive heterochromatin is maintained has important implications for genome regulation and disease, and remains poorly understood. While satellite DNA sequences are not evolutionarily conserved, pericentromeric and telomeric heterochromatin occurs across species. Heterochromatin formation is therefore independent of the underlying DNA sequence, supporting the hypothesis that epigenetic components can regulate chromatin structure. DNA methylation is generally thought to be associated with transcriptional silencing and chromatin compaction. However, Gilbert et al (2007) showed that the complete loss of DNA methylation did not affect the compaction at heterochromatin or global genome compaction. The role of H3K9me3 in regulating heterochromatin has also been an area of keen interest. H3K9me3 patterns are established by suppressor of variegation 3-9 homologues and provide the binding site for heterochromatic protein 1 [HP1] which can in turn recruit Suv39h1. This Suv3-9h-HP1-H3K9 axis enables its propagation throughout heterochromatin. Peters et al (2001) demonstrated that in mice loss of suv39 homologues 1 and 2 caused a loss of H3K9me3 at constitutive heterochromatic domains. These Suv39h null mice demonstrated decreased genome stability, and an increased prevalence of oncogenesis. However cytological chromocenters are still present in the absence of H3K9me3. Therefore the function of H3K9me3 as a causative agent in heterochromatin formation is still debated. Broadly the aim was to investigate the phenotypic role of heterochromatic epigenetic components in cancer progression, and address whether H3K9me3 effects large scale chromatin structure. To identify heterochromatic gene silencing components, an inhibitor screen was performed in an artificial silenced reporter system. The reporter fluorophore was silenced by the presence of centromeric arrays from yeast/bacterial artificial chromosomes and human alpha satellite repeats enriched for H3K9me3. To address the function of the de-silencing components identified in cancer, the fitness of colon cancer cells [HCT116] was investigated before and after the development of resistance to the MEK inhibitor trametinib. The most intriguing result was that BET protein inhibition resulted in derepression of the reporter construct and trametinib resistant HCT116 cells were more sensitive to BET inhibitors, while subsequent investigation showed HP1 protein levels were altered. Analysis of publically available datasets of tumour drug resistance, showed elevated BET protein binding at HP1 promoters in resistant cell lines suggesting an indirect role in gene silencing. To investigate the consequence of H3K9me3 loss on chromatin structure, mouse embryonic stem cells that lacked both Suv39 homologues were used. Microccocal nuclease digestion and sucrose sedimentation demonstrated a global decompaction of large-scale chromatin fibres whilst re-expression of suv39h1 rescued H3K9me3 at chromocenters and global chromatin decompaction. Loss of Suv39h also increased chromatin associated RNA levels that were also rescued by Suv39h1 re-expression. This suggests that H3K9me3 has a role chromatin fibre compaction globally as well as at constitutive heterochromatin, potentially mediated by chromatin associated RNA. To conclude, multiple components were identified that are involved in transcriptional silencing. Evaluating their function in tumour progression demonstrated a possible role of BET proteins in the development of MEKi resistance that may be mediated through HP1 proteins. H3K9me3 and its binding partner HP1 affect global chromatin compaction. The global decompaction after Suv39h loss correlates with an increase in chromatin associated RNA, suggesting a possible mechanism for changes in chromatin compaction beyond H3K9me3.
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Amar, Sabrina. "Histone modification and its role in epigenetic regulation." Thesis, University of Portsmouth, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516863.
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The development of genome-wide histone modification mapping technologies has provided a significant body of evidence implicating histone modifications in gene regulation, cell specialisation and differentiation processes. Monomethylation of lysine 4 of histone H3 (H3K4Mel) and the presence of the histone variant H2A.Z have previously been shown as a feature of enhancers, defined simply as points enriched in the HAT p300, but it was unclear whether these potential enhancers were linked to inactive, active or poised genes or whether developmental changes were reflected in the occupancy of H3K4Mel. The aim of this study was to document the spatio-temporal distribution of H3K4MeI at developmentally regulated genes and investigate proteins specifically binding to this modification so as to unravel possible control mechanisms. H3K4Mel distributions were determined by native chromatin immunoprecipitation (nChlP) experiments, analysed by real-time PCR, using chicken haematopoietic cell lines representing different erythroid and myeloid differentiation stages. Mapping at the lysozyme locus showed enrichments of H3K4Mel at the gene enhancers in myeloid cells independently of expression status, even in multipotent myeloid progenitor cells where the gene is not expressed. In contrast, in none of the erythroid cells studied - where the gene is never expressed - is there any H3K4Mel found at the lysozyme locus. At the β-globin locus, the β-adult enhancer is strongly enriched in H3K4mel in early erythroid progenitor cells where the globin genes are not expressed but the modification is lost from this enhancer in IS-day embryonic erythrocytes - which strongly express the adult and hatching globin genes - despite a general rise in H3K4Mel levels elsewhere in the locus, including the inactive embryonic globin genes. In none of the myeloid cells studied - where globin genes are never expressed - is there any high H3K4Mel enrichment found at the β-globin locus. At the folate receptor gene, the promoter carries a high level of H3K4Mel when the gene is inactive but this decreases when the gene becomes active. Taken together, the data suggest that H3K4Mel is a pioneer modification that participates in establishing the enhancers of genes poised for future activation. Pull-down experiments using immobilised peptides or reconstituted nucleosomes containing H3K4Mei and extracts from HeLa cell nuclei, followed by tryptic mass spectroscopy of bound proteins, were used to identify possible binding partners. Five potential binding partners were defined and have been shown to associate with chromatin.
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Addicks, Gregory Charles. "Epigenetic Regulation of Muscle Stem and Progenitor Cells." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37112.
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Epigenetic mechanisms are of fundamental importance for resolving and maintaining cellular identity. The mechanisms regulating muscle stem and progenitor cell identity have ramifications for understanding all aspects of myogenesis. The epigenetic mechanisms regulating muscle stem cells are therefore important aspects for understanding the regulation of muscle regeneration and maintenance.
Important roles for the trithorax H3K4 histone methyltransferase (HMT) MLL1 have been established for early embryogenesis, and for hematopoietic and neural identity. Here, using a conditional Mll1 knockout (KO), we find that in vivo, MLL1 is necessary for efficient muscle regeneration, and for maintenance and proliferation of muscle stem and progenitor cells. Loss of Mll1 in cultured myoblasts reveals an essential role for expression of the myogenic specification gene Pax7.
Mll1 KO results in a minor decrease in Pax7 mRNA and a strong decrease of Pax7 protein. While MLL1 was found to bind the Pax7 promoter, Mll1 KO results in a minor decrease of H3K4me3 at Pax7, supporting a recognized non-HMT role for Mll1 at Pax7. Microarray analysis of mRNA expression in Mll1 KO myoblasts finds that Myf5 is the most strongly downregulated of all genes, unexpectedly, mRNA expression of previously identified MLL1 targets are unaffected by loss of MLL1 in myoblasts. Pax7 activates Myf5 expression through recruitment of a H3K4 HMT, and in Mll1 KO myoblasts expression of, and H3K4me3 at Myf5 is lost. Exogenous Pax7 rescues Myf5 expression and H3K4me3 at Myf5 in the absence of MLL1, indicating that Myf5 expression is conditional on Pax7, but not MLL1.
We also show that Myf5 DNA is methylated in non-myogenic cells, and in satellite stem cells that have never expressed Myf5, but is not methylated in satellite cells that are committed to the myogenic lineage, indicating that demethylation of Myf5 may be a fundamental step in myogenic commitment. Intriguingly, Myf5 promoter DNA becomes remethylated in Mll1 KO myoblasts.
This work finds that Pax7 expression and myogenic identity is partly dependent on MLL1 expression. Further, evidence is uncovered that myogenic commitment is initiated by demethylation of Myf5. These findings add to the understanding of the epigenetic mechanisms that regulate and define muscle stem cells.
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SIDDIQUI, HASAN. "RB-MEDIATED REGULATION OF TRANSCRIPTION AND EPIGENETIC MODIFICATIONS." University of Cincinnati / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1148053497.
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Singh, Rajbir. "Histone Isoforms: From Epigenetic Regulation To Cancer Screening." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1384430389.
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