Linker histone H1 associates with nucleosomes, facilitating folding and packaging of DNA into higher order chromatin structure. With 11 variants in mammals, histone H1 is the most divergent histone class. Histone H1 variants are differentially expressed during development and cellular differentiation, and regulate specific gene expression in vivo. Ample studies have established the role of linker histone H1 in chromatin compaction and gene expression regulation; however, its role in diseases, such as cancer, remain understudied. In this study, we explore the role of H1 in ovarian cancer, one of the most devastating gynecological cancers due to its poor prognosis and difficulty in early diagnosis. Although mutations of genes responsible for cell proliferation, differentiation and survival have been found in ovarian cancers, ample evidence also suggests an important role of epigenetic changes in the disease occurrence and progression. Because epigenetic changes do not alter DNA sequence and can be reversed or reprogrammed, they offer an attractive avenue for therapeutic intervention in cancer treatment. Using quantitative RT-PCR assays, we systematically examined the expression of 7 H1 genes in 33 human epithelial ovarian tumors. By clustering analysis, we found that ovarian malignant adenocarcinomas and benign adenomas exhibited characteristic expression patterns. We demonstrate that expression profiling of 7 H1 genes in tumor samples discriminates adenocarcinomas vs. adenomas with high accuracy. These findings indicate that the expression of H1 variants is exquisitely regulated and may serve as potential epigenetic biomarkers for ovarian cancer. To further investigate the role of H1 subtypes in ovarian cancer cells, we employ an over-expression approach to test the function of H1 subtypes in an ovarian cancer cell line OVCAR-3. We found that histone H1.3 over-expression significantly suppresses the growth and colony formation of OVCAR-3 cells. Gene expression arrays identified many genes affected by H1.3 over-expression, and oncogene H19 is among the genes most dramatically repressed by H1.3 over-expression. Over-expression of several other H1 subtypes does not lead to significant reduction of H19 expression, suggesting a specific effect by H1.3. Consistently, knockdown of H1.3 increases H19 expression. Furthermore, increased expression of H1.3 leads to accumulation of H1.3 as well as increased DNA methylation at the regulatory regions of H19. Finally we identified a synergistic effect of H1.3 over-expression and H19 knockdown on inhibition of ovarian cancer cell growth. These results establish oncogene H19 as a direct target of histone H1.3, identify a novel role of H1 variants in ovarian cancer mediated through regulating oncogene H19 expression, and may offer new approaches for ovarian cancer therapeutics.

Mechanisms of arsenic toxicity are not yet clear. Arsenite has effects on methylation pathways, by decreasing expression of DNA methylases and depletion of S-adenosylmethionine. Histones are DNA packing proteins that regulate gene expression modulating chromatin accessibility. Methylation at Lysine 9 of Histone H3 (K9H3) is a hallmark of heterochromatin. Dimethyl K9H3 is a mark of facultative heterochromatin and trimethyl K9H3 is present on constitutive heterochromatin. HeLa cells exposed for 24 hrs to 1 uM or 5 uM Sodium Arsenite were fixed and different posttranslational modifications of histones were detected by indirect immunofluorescence. Images were analyzed to assess the change on average methylated species of K9H3 in cell nuclei. Interestingly Arsenite (1 uM and 5 uM) treated cells had a significant increase in the trimethylated and dimethylated of K9H3, evaluated throught the comparison of average nuclei brightness and pixel value analysis between treatments.

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Universiẗat, Diss., 2007--Giessen.

Thèse (Ph. D.)--Université Laval, 2005. / Titre de l'écran-titre (visionné le 13 févr. 2008). Bibliogr.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 135-159). Also available in electronic version. Access restricted to campus users.

Lysine methylation modulates diverse biological processes and is catalyzed by SET domain methyltransferases such as the SMYDs (SMYD1-5), which possess a SET domain split by a MYND motif. Through association with NCoR, the H4 Lys20 methyltransferase activity of SMYD5 represses inflammation by restricting TLR-4 mediated expression in macrophages, yet biochemical and structural features required for SMYD5 methylation activity remain elusive. To determine how SMYD5 catalyses methylation, crystallization screens were conducted with SMYD5 in complex with the co-factor AdoMet and histone H4. Screens yielded lead conditions but no crystals. To determine the motif recognized by SMYD5 and decipher its methylome, peptide arrays were conducted to produce a methylation motif used to identify putative substrates. Surprisingly, arrays revealed that substitution of Lys16, not Lys20, is detrimental to SMYD5 activity. Further enzymatic assays are required to determine if SMYD5 methylates residues other than Lys20 on the H4 tail, or if structural determinants or interacting partners restrict methylation of target lysines.

The histone variant H2A.Z is a key player in transcription regulation in eukaryotes. Histone acetylations by the NuA4/TIP60 complex are required to enable proper incorporation of the histone variant and to promote the recruitment of other complexes and proteins required for transcription initiation. The second key player in H2A.Z-mediated transcription is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant. By the time this project started little was known about H2A.Z in the unicellular parasite Trypanosoma brucei. Like in other eukaryotes H2A.Z was exclusively found in the transcription start sites of the polycistronic transcription units where it keeps the chromatin in an open conformation to enable RNA-polymerase II-mediated transcription. Previous studies showed the variant colocalizing with an acetylation of lysine on histone H4 and a methylation of lysine 4 on histone H3. Data indicated that HAT2 is linked to H2A.Z since it is required for acetylation of lyinse 10 on histone H4. A SWR1-like complex and a complex homologous to the NuA4/TIP60 could not be identified yet. This study aimed at identifying a SWR1-like remodelling complex in T. brucei and at identifying a protein complex orthologous to NuA4/TIP60 as well as at answering the question whether HAT2 is part of this complex or not. To this end, I performed multiple mass spectrometry-coupled co-Immunoprecipitation assays with potential subunits of a SWR1 complex, HAT2 and a putative homolog of a NuA4/TIP60 subunit. In the course of these experiments, I was able to identify the TbSWR1 complex. Subsequent cell fractionation and chromatin immunoprecipitation-coupled sequencing analysis experiments confirmed, that this complex is responsible for the incorporation of the histone variant H2A.Z in T. brucei. In addition to this chromatin remodelling complex, I was also able to identify two histone acetyltransferase complexes assembled around HAT1 and HAT2. In the course of my study data were published by the research group of Nicolai Siegel that identified the histone acetyltransferase HAT2 as being responsible for histone H4 acetylation, in preparation to promote H2A.Z incorporation. The data also indicated that HAT1 is responsible for acetylation of H2A.Z. According to the literature, this acetylation is required for proper transcription initiation. Experimental data generated in this study indicated, that H2A.Z and therefore TbSWR1 is involved in the DNA double strand break response of T. brucei. The identification of the specific complex composition of all three complexes provided some hints about how they could interact with each other in the course of transcription regulation and the DNA double strand break response. A proximity labelling approach performed with one of the subunits of the TbSWR1 complex identified multiple transcription factors, PTM writers and proteins potentially involved in chromatin maintenance. Overall, this work will provide some interesting insights about the composition of the complexes involved in H2A.Z incorporation in T. brucei. Furthermore, it is providing valuable information to set up experiments that could shed some light on RNA-polymerase II-mediated transcription and chromatin remodelling in T. brucei in particular and Kinetoplastids in general. / Die Histonvariante H2A.Z ist ein Schlüsselelement bei der Transkriptionsregulation in Eukaryoten. Histonacetylierungen die vom NuA4/Tip60 Komplex prozessiert werden, sind für den korrekten Einbau der Variante unerlässlich. Darüber hinaus erlauben diese posttranslationellen Modifikationen die Rekrutierung weiterer Proteine und Komplexe die für die Transkription notwendig sind. Ein weiteres Schlüsselelement der mittels H2A.Z regulierten Transkription ist der Komplex zur Umstrukturierung des Chromatins SWR1, welcher das kanonische Histon H2A gegen seine Variante austauscht. Zu Beginn dieses Projektes war der Wissenstand bezüglich der Histonvariante H2A.Z in dem einzelligen Parasiten Trypanosoma brucei limitiert. Wie in anderen eukaryotischen Organismen wurde die Variante ausschließlich an den Startpunkten der polyzistronischen Transkriptionseinheiten gefunden, an denen es für die Öffnung des Chromatins verantwortlich ist und so die Transkription mittels RNAPolymerase II ermöglicht. Vorangegangene Studien konnten zeigen, dass die Variante mit einer Acetylierung des Lysins 10 im Histon H4 und einer Methylierung des Lysins 4 im Histon H3 co-lokalisiert. Einige Daten lieferten den Hinwies, dass die Histon-Acetyltransferase HAT2 mit H2A.Z in Zusammenhang steht, da diese die Acetylierung des Lysins 10 im Hinston H4 prozessiert. Komplexe die in ihrer Funktion dem SWR1 oder dem NuA4/TIP60 Komplex entsprechen, konnten bisher noch nicht gefunden werden. Die vorliegende Arbeit zielt darauf ab Komplexe zu identifizieren, die in ihrer Funktion dem SWR1 sowie dem NuA4/TIP60 Komplex entsprechen. Zudem soll die Frage geklärt werden ob HAT2 Teil eines möglichen NuA4/TIP60 Komplexes ist. In diesem Zusammenhang habe ich mehrere Massenspektrometrie gekoppelte Co-Immunopräzipitationen mit potenziellen Untereinheiten eines SWR1 Komplexes sowie HAT2 und einem Protein welches otholog zu einer NuA4/TIP60 Untereinheit ist, durchgeführt. Im Verlauf dieser Experimente konnte der SWR1 Komplex in T. brucei (TbSWR1) identifiziert werden. Anschließende Zellfraktionierungen sowie Chromatin Immunopräzipitationen gekoppelte Sequenzanalysen konnten bestätigen, dass der identifizierte Komplex für den Einbau der Histonvariante H2A.Z zuständig ist. Darüber hinaus konnten neben diesem Komplex noch zwei weitere Komplexe identifiziert werden, die jeweils die Histonacetyltransferasen HAT1 und HAT2 als Kernkomponenten enthalten. Im Verlauf meiner Arbeit wurden von der Arbeitsgruppe von Nicolai Siegel Daten publiziert die zeigten, dass die Histonacetyltransferase HAT2, in Vorbereitung auf den Einbau von H2A.Z, für die Acetylierung des Histons H4 verantwortlich ist. Im Gegenzug ist HAT1 für die Acetylierung von H2A.Z notwendig, welche wiederum für die korrekte Initiation der Transkription benötigt wird. Damit entspricht die Funktion der Acetylierung von H2A.Z in T. brucei der in der Literatur beschriebenen Funktion. Experimentelle Daten die im Verlauf dieser Arbeit generiert wurden, lieferten einen Hinweis darauf, dass H2A.Z auch an der Reparatur von DNS Doppelstrangbrüchen beteiligt ist. Die Aufschlüsselung der spezifischen Zusammensetzung aller drei Komplexe gab einige Hinweise darauf, wie sie sowohl während der Transkriptionsregulation als auch der Reparatur von DNS Doppelstrangbrüchen miteinander interagieren. Im Zuge einer molekularen Umgebungskartierung, die mit einer der Untereinheiten des TbSWR1 Komplexes durchgeführt wurde, konnten mehrere Transkriptionsfaktoren und Enzyme zur Histonmodifizierung identifiziert werden. Dabei wurden auch einige Proteine identifiziert, welche möglicherweise mit der Umformung des Chromatins in Zusammenhang stehen. Abschließend ist festzuhalten, dass diese Arbeit einige äußerst interessante Einsichten über die Zusammensetzung der Komplexe, die am H2A.Z Einbau in T. brucei beteiligt sind, liefern konnte. Darüber hinaus stellt sie einige wertvolle Informationen zur Verfügung. Diese könnten zur gezielten Planung von Experimenten genutzt werden, um mehr über RNA-Polymerase II vermittelte Transkription und Chromatin Umstrukturierung in T. brucei im speziellen und in Kinetoplastiden im Allgemeinen zu erfahren.

Condensation of chromatin and alteration of chemical groups in the proteins around which the DNA is wrapped play major role in regulation of transcription. Histones are basic proteins rich in arginine and lysine residues which form the nucleosomal core. Histone modifications like acetylation, methylation, phosphorylation, etc. have broadened the horizon for researchers to study epigenetics more in detail. Histone methyl transferases and histone demethyl transferases are enzymes which add or remove methyl groups on histone lysine and arginine residues respectively. In this study a jumonji domain containing putative histone demethyltransferase has been shown to be responsible in controlling flowering phenotype in Arabidopsis thaliana. The knocked out mutants for this gene (JMJ14) showed an early flowering phenotype along with elevated levels of FT transcript (Flowering locus T, gene responsible for controlling the flowering time in Arabidopsis thaliana). We show that methylation was altered on H3K36 in the FT ene in the mutants using ChIP (chromatin immunoprecipitation experiments). The possible role of SDG8 gene, a histone methyl transferase in ABA signaling was also studied during the research. A SET domain containing Sdg8 (group 8 methyltransferase) mutant was found to be responsible for ABA signaled altered root growth in Arabidopsis thaliana. The cell number and cell size in roots decreased in both meristematic and elongation zones leading to decrease in root size in sdg8 mutants and number of root hairs increased when treated with Abscisic acid, a plant hormone. In this part of study, as part of an interaction between epigenetics and gene regulation, it was observed that a putative histone demethylase gene, JMJ14 was responsible for regulating the flowering time by controlling the expression of FT and SDG8 played a role in altered root growth in response to ABA in Arabidopsis thaliana. Further studies on these genes could lead to generation of commercial crops with phenotypes that would increase the plant productivity and be beneficial agronomically.

No description available.