La chromatine, dont l’unité de base est le nucléosome, est une structure nucléoprotéique dynamique qui nécessite un remodelage au cours de divers processus nucléaires ayant besoin d’un accès direct à l’ADN tels que la réplication, la transcription ou la réparation des lésions. Plusieurs facteurs capables de moduler la structure de la chromatine ont été caractérisés et regroupent les chaperons d’histones, les complexes de remodelage ATP-dépendants, les variants d’histone et les enzymes modifiant les histones de façon post-traductionnelle. Le complexe acéyltransférase NuA4 responsable de l’acétylation des histones H4 et H2A participe activement à la dynamique de la chromatine au cours de ces processus. Il favorise sa relaxation lors de l’activation de la transcription et de la réparation de l’ADN. Il est le seul complexe acétyltransférase dont l’activité est essentielle chez la levure Saccharomyces cerevisiae. Le but de mon projet de doctorat était de mieux comprendre les mécanismes capables de réguler son activité. Nous avons mis en évidence trois niveaux de régulation de son activité, via (i) son auto-acétylation, (ii) son ciblage à la chromatine de façon spécifique et (iii) directement par son substrat histone. Alors que l’acétylation de la sous-unité Yng2 de NuA4 par le complexe lui-même est importante pour le maintien de l’intégrité du complexe et de son activité, nous avons découvert que NuA4 est capable de s’auto-acétyler in vivo et in vitro sur plusieurs de ces sous-unités et que l’acétylation d’un seule lysine dans le domaine MYST de sa sous-unité catalytique Esa1 est essentielle à l’activité du complexe, sans influencer son intégrité. D’autre part, nous avons caractérisé par des techniques de purification un sous-module de NuA4 composé des facteurs Eaf5, Eaf7 et de la protéine à chromodomaine Eaf3 capable de reconnaître les lysines méthylées, qui serait impliqué dans le ciblage de NuA4 à la chromatine. Ce complexe trimérique est également présent indépendant de NuA4 dans la cellule, et serait localisé de façon préférentielle sur la région codante des gènes. Enfin, les travaux du laboratoire ont montré que les modifications post-traductionnelles présentes sur la queue N-ter de H4 peuvent réguler l’activité de NuA4. La phosphorylation de H4 sur sa sérine 1 inhibe son acétylation sur les lysines adjacentes par NuA4. Alors que nous montrons que cette marque est spécifiquement induite sur la région codante de gènes activés, nos résultats indiquent que cette phosphorylation serait impliquée dans la dynamique de la chromatine au cours de l’élongation de la transcription. / Chromatin, which basic unit is the nucleosome, is a very dynamic structure that requires remodeling during nuclear processes that need to access the DNA such as replication, transcription and DNA damage repair. A number of remodeling factors have been characterized and include histone chaperone, ATP-dependent remodelers, histone variants and post-translational histone modifiers. The NuA4 acetyltransferase complex, responsible for H4 and H2A acetylation, participates in chromatin and nucleosome dynamics associated to these nuclear processes. It was shown to promote chromatin relaxation during transcription activation and DNA repair. NuA4 is the only acetyltransferase complex essential for viability in the yeast Saccharomyces cerevisiae. The objective of my Ph.D. project was to understand the mechanisms that regulate NuA4’s activity. We brought to the forefront tree different ways to control NuA4’s activity, via its auto-acetylation, its specific targeting to the chromatin and via its histone substrate. While acetylation of the Yng2 subunit of NuA4 by NuA4 itself is important for maintenance of the integrity and activity of the complex, we discovered that NuA4 is capable of in vivo and in vitro auto-acetylation of several of its subunits. In addition, we showed that the acetylation of a single lysine residue located in the MYST domain of the catalytic subunit Esa1 is essential for the activity of the complex and has no effect on its integrity. On another hand, we have characterized a sub-complex of NuA4 composed by the Eaf5, Eaf7 and the chromodomain-containing Eaf3 proteins that are implicated in NuA4 targeting to the chromatin. This trimeric complex is also found in the cell independent of NuA4 and preferentially associated to gene coding region. Finally, published works from our laboratory showed that post-translational modifications of H4 N-ter tail can regulate NuA4 activity. Phosphorylation of H4 serine1 inhibits its acetylation on adjacent lysines by NuA4. We demonstrated that this mark is specifically induced on coding region of active genes and that this phosphorylation would be implicated in chromatin dynamics associated with the transcription elongation process.

L'ADN des cellules eucaryotes est enroulé autour de protéines appelées histones pour former la chromatine. Le niveau de compaction de la chromatine est dynamique. Ceci permet de réguler via l'accessibilité de l'ADN, les processus comme la transcription. Les histones peuvent subir des modifications post traductionnelles comme la méthylation, qui influencent le niveau de compaction de la chromatine. Par exemple, au niveau des promoteurs des gènes, la méthylation sur la lysine 9 de l'histone H3 (H3K9) est associée à la répression transcriptionnelle, tandis que la méthylation sur la lysine 4 (H3K4) est associée a l'activation transcriptionnelle. Ces marques sont mises en place par des histones méthyltransférases et enlevées par des histones déméthylases qui sont spécifiques des résidus méthylés. Ma thèse a porté sur l'étude d'histone déméthylases dans la régulation transcriptionnelle de gènes clé de la prolifération cellulaire, les gènes cibles de E2F et l'ADN ribosomique. Les facteurs E2Fs régulent des gènes comme CCNE et CDC6 impliques dans l'entrée et la progression en phase S. Ces gènes sont activés au début de la phase S. Le contrôle de la transcription de ces gènes est crucial pour un cycle cellulaire normal et leur dérégulation est associée à l'apparition de cancers. La répression et l'activation des gènes cibles de E2F au cours du cycle cellulaire fait intervenir le contrôle de la méthylation des résidus H3K4 et H3K9. Cependant, les histone déméthylases impliquées sont mal connues. Nous avons montré que les histones déméthylases JARID1A et JARID1B, spécifiques de H3K4, régulent la transcription de CCNE et CDC6 en phase S. JARID1A et JARID1B sont recrutées au promoteur de ces gènes. Elles sont importantes pour limiter leur activation lors de la progression en phase S. Cette étude montre pour la première fois l'implication de ces histones déméthylases dans la régulation fine des gènes cibles de E2F au cours du cycle cellulaire. La transcription des gènes ribosomiques ou ADNr par l'ARN Polymérase I (Pol-I) est la première étape de la biogénèse des ribosomes. Elle a lieu dans les nucléoles. Ce processus est étroitement lié à la croissance et la prolifération cellulaire. Une transcription Pol-I accrue et des nucléoles hypertrophiés sont des caractéristiques communes à un grand nombre de cellules cancéreuses. La transcription Pol-I est adaptée à la disponibilité en facteurs de croissance. Ainsi, elle est réprimée lorsque les cellules sont privées en facteurs de croissance et activée en leur présence. Cette réponse est sous le contrôle de cascades de signalisation cellulaire comme la voie Phosphatidyl-Inositol-3-Phosphate (PI3K). Il est connu que des événements dynamiques de méthylation d'histones participent à cette régulation. Cependant, on sait peu de choses sur comment les voies de signalisation régulent ces événements. En collaboration avec l'équipe du Dr. Konstantin Panov, nous avons observé que l'histone déméthylase JMJD2A, spécifique de H3K9, est présente dans les nucléoles de cellules humaines. JMJD2A, via sa capacité à déméthyler H3K9, est requise pour activer la transcription Pol-I en réponse aux facteurs de croissance. Nous montrons également que PI3K régule cette réponse chromatinienne en déclenchant l'accumulation de JMJD2A dans les nucléoles en réponse aux facteurs de croissance. Cette étude indique que la régulation de la localisation subnucléraire de JMJD2A en réponse à la voie PI3K est un des mécanismes par lesquels les cellules adaptent leur capacité de synthèse protéique à la disponibilité de facteurs de croissance. Mes travaux de thèse renforcent notre compréhension des mécanismes impliquant des histones déméthylases dans la régulation de la prolifération cellulaire. Comprendre ces mécanismes est crucial et permettra de cibler ces enzymes dans le traitement des pathologies de la prolifération cellulaire comme le cancer. / In eukaryote nuclei, DNA is wrapped around histone proteins. This structure is called the chromatin. The compaction level of chromatin is highly dynamic. This allows the regulation of gene transcription which requires free access to the DNA. Histone proteins undergo several post translational modifications including methylation that impact chromatin compaction. For example, at genes promoters, methylation on the lysine 9 of histone H3 (H3K9) is associated with chromatin compaction and thereby transcription repression, whereas methylation on histone H3 lysine 4 (H3K4) is associated with transcriptional activation. Histone methylation is set by enzymes called histone methyltransferases and removed by histone demethylases which are specific for methylated residues. During my PhD, I studied the role of histone demethylases in the transcriptional regulation of cell proliferation master genes, E2F-regulated genes and rDNA transcription. E2F transcription factors regulate genes like CCNE or CDC6 involved in entry and progression through S phase. Those genes must be activated at the onset of S phase. The transcriptional control of those genes is crucial for a normal cell cycle, and their deregulation is associated with cancer development. Histone methylation events are involved in the repression and activation of E2F target genes during cell cycle progression. However the histone demethylases involved are still unclear. We have shown that the H3K4-specific histone demethylases JARID1A and JARID1B are involved in the fine-tuning of CCNE and CDC6 transcription during S phase. JARID1A and JARID1B are recruited on the promoter of those genes and help limiting their activation at the beginning of S phase. This study shows for the first time the role of those histone demethylases in the fine tuned regulation of E2F targets genes during S phase. Ribosomal DNA (rDNA) transcription is the first step of ribosome biogenesis. rDNA is transcribed in the nucleolus by RNA polymerase I (Pol-I). Pol-I transcription is tightly linked to cell growth and proliferation. High levels of Pol-I transcription along with hypertrophied nucleoli is a hallmark of several cancers cells. Pol-I transcription must be regulated according to the availability of growth factors. It is repressed when the cells are deprived of growth factors and activated when growth factors are available. This regulation is under the control of cellular signaling pathways including the Phosphatidyl-Inositol-3-Kinase (PI3K) pathway. Histone methylation events are known to play a role in this regulation. However little is known about how the cell signaling pathways modulate the chromatin response in this process. In collaboration with the team of Dr. Konstantin Panov, we observed that the H3K9-specific histone demethylase JMJD2A is present in the nucleoli of human cells. We showed that JMJD2A, through its ability to demethylate H3K9, is required for the activation of Pol-I transcription in response to growth factors. We further show that PI3K regulate this chromatin response by triggering accumulation of JMJD2A in the nucleoli in response to growth factors. This study demonstrates a yet unknown role for JMJD2A in Pol-I transcription and suggests that the control of JMJD2A localization by the PI3K pathway is a crucial mechanism by which cells adapt protein synthesis to the availability of growth factors. My PhD work helps strengthening our understanding of the mechanisms that involve histone demethylases in the regulation of cell proliferation genes. Understanding those mechanisms is crucial as it might help targeting those enzymes for the treatment of cell proliferation-associated diseases like cancer.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 146 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 125-142).

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1988. / Extra t.p. with thesis statement inserted. Includes bibliographical references.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Histone lysine demethylases comprise an important family of epigenetic regulatory enzymes. They catalyse the demethylation of tri-, di- and monomethylated lysine residues on histone H3, thus contributing to either silencing or activation of chromatin. Their biological roles are widespread and have just begun to be elucidated. Among other functions, they contribute to establishment and maintenance of pluripotent states in embryonic stem cells, and also to cellular differentiation during development. Abnormal expression or mutation of some demethylases has been linked to diverse diseases, from prostate and oesophageal cancers to X-linked mental retardation. The development of small molecule inhibitors of histone demethylases is therefore of interest, both from the therapeutic perspective, and with the aim of developing chemical probes to understand the diverse functions of the demethylases in vivo. Most histone lysine demethylases belong to the 2-oxoglutarate and ferrous iron dependent dioxygenase superfamily. This family utilises molecular oxygen to catalyse hydroxylation of substrates, together with oxidation/decarboxylation of the 2-oxoglutarate cofactor. In the work outlined in this thesis, the JMJD2 family of histone demethylases was characterised biochemically, with attention given to mechanism, substrate selectivity and the role of eo factors. JMJD2E was identified herein as a novel histone demethylase in H. sapiens, and was shown to be selective for the demethylation of tri-, di- and monomethylated lysine 9 in histone H3. JMJD2E was also found to be particularly amenable to mechanistic and inhibition studies in vitro. A variety of mechanistic investigations established details of the catalytic cycle, its substrate selectivity and the role of iron and ascorbic acid as cofactors. Crystallographic analyses were also employed to compare its substrate selectivity to other JMJD2 family members. Assays suitable for the evaluation of inhibitors of the JMJD2 demethylases were then developed. These included a coupled enzyme assay suitable for kinetic measurements, and two mass spectrometric assays for observing inhibitor binding and catalytic activity. A critical review of the 20G oxygenase inhibitor literature carried out, and was then used as a basis for the identification of inhibitor scaffolds for the JMJD2 demethylases. These were characterised both in vitro (using kinetic assays, mass spectrometry and crystallography), and in cell culture. Some were further developed to achieve selective inhibition of the JMJD2 demethylases over the related prolyl hydroxylase PHD2; crystallography was again employed to understand the mode of inhibition of these potent inhibitors. The kinetic assays developed were optimised for use in a high-throughput screen, and a library of 240 000 compounds was screened against JMJD2E. This was the first instance of high-throughput screening against these promising therapeutic targets. Several hit compounds were identified and characterised further in vitro. Finally, alternative means of inhibiting the JMJD2 demethylases were investigated. Compounds were identified that inhibited JMJD2A by ejection of its unique structural zinc ion, thus demonstrating that selective inhibition of the JMJD2 demethylase family is possible. In summary, this work contains the first detailed investigation of a histone demethylase subfamily, and also the first steps towards identifying potent, selective inhibitors of these epigenetic regulatory enzymes.

Cheung, Wai Kuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 66-76). / Abstracts in English and Chinese. / List of Figures --- p.v / List of Tables --- p.vi / List of Abbreviations --- p.vii / Acknowledgements --- p.ix / Abstract --- p.xi / 摘要 --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Centromere and Kinetochore --- p.1 / Chapter 1.2 --- The Kinetochore Subunits: Centromeric Nucleosomes --- p.2 / Chapter 1.3 --- CenH3: The Centromere Specific Histone --- p.5 / Chapter 1.4 --- The Centromeric DNA: Tandem Repeats and Retrotransposons --- p.8 / Chapter 1.5 --- The Genetic and Epigenetic Nature of the Centromeres --- p.9 / Chapter 1.6 --- Point Centromeres and Regional Centromeres --- p.10 / Chapter 1.7 --- Holocentric Chromosomes --- p.11 / Chapter 1.8 --- Hypothesis --- p.13 / Chapter Chapter 2 --- Materials and methods --- p.15 / Chapter 2.1 --- Chemicals --- p.15 / Chapter 2.2 --- Bacterial strains in routine cloning --- p.15 / Chapter 2.3 --- Plant materials in cloning and transformation --- p.15 / Chapter 2.4 --- Construction of LnCENH3-GFP and CeHCP3-DsRED chimeric gene cassettes for rice transformation --- p.15 / Chapter 2.5 --- Cloning of CENH3 gene of Luzula spp --- p.22 / Chapter 2.6 --- Construction of full length OsCENH3 RNAi and 150bp OsCENH3 RNAi constructs for rice transformation --- p.22 / Chapter 2.7 --- Agrobacterium-mediated transformation of rice (Oryza sativa L.japonica. cv. Nipponbare) --- p.24 / Chapter 2.8 --- Gene gun transformation of rice (Oryza sativa L.japonica. cv. Nipponbare) by Biolistic PDS-1000/He´ёØ System (Bio-rad) --- p.26 / Chapter 2.9 --- Detection of transgenes expression --- p.28 / Chapter 2.10 --- Nuclear protein extraction --- p.29 / Chapter 2.11 --- Protein-DNA Binding Assay --- p.30 / Chapter 2.12 --- Protein precipitation by methanol-chloroform --- p.32 / Chapter 2.13 --- Western blot analysis of proteins from Protein-DNA binding assay --- p.33 / Chapter 2.14 --- Tubulin immunolocalization of root tips --- p.33 / Chapter 2.15 --- Bioinformatics analysis --- p.34 / Chapter Chapter 3 --- Results --- p.36 / Chapter 3.1 --- Plant transformation vectors construction --- p.36 / Chapter 3.2 --- Rice transformation --- p.38 / Chapter 3.3 --- Transgenic plants screening --- p.39 / Chapter 3.4 --- Analysis of the codon usages of CeHCP-3 gene in C. elegans and O. sativa --- p.42 / Chapter 3.5 --- In vitro Protein-DNA binding assays --- p.44 / Chapter 3.6 --- Subcellular localization study of LnCENH3 in rice --- p.46 / Chapter 3.7 --- Chromosome morphology of the transgenic rice expression LnCENH3 --- p.47 / Chapter 3.8 --- Tubulin immunolocalization of LnCENH3-GFP transgenic rice --- p.49 / Chapter 3.9 --- Cloning of CENH3s in Luzula genus --- p.51 / Chapter 3.10 --- Bioinformatics analysis of Luzula CENH3s --- p.53 / Chapter Chapter 4 --- Discussion --- p.57 / Chapter 4.1 --- Expression of fusion proteins in rice --- p.57 / Chapter 4.2 --- Incorporation of LnCENH3-GFP in nucleosomes --- p.57 / Chapter 4.3 --- Expression pattern of LnCENH3-GFP in rice --- p.58 / Chapter 4.4 --- Formation of additional kinetochores on transgenic rice chromosome --- p.60 / Chapter 4.5 --- Deletion of 8 amino acids in LeCENH3 --- p.62 / Chapter Chapter 5 --- Conclusion --- p.65 / References --- p.66 / Chapter 5.1 --- Appendix --- p.77

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2009 Aug 16

Chromatin remodelers such as CHD1 (chromodomain, helicase/ATPase, DNA-binding domain) regulates histone dynamics and allows for higher order of chromatin compaction.CHD1 has been found to be important in fertility, wing development, and it colocalizes with elongating RNA polymerase II in Drosophila melanogaster. CHD1 is also important in embryonic stem cell pluripotency in mice and chd1 is the second most deleted gene in prostate cancer in humans. Furthermore, CHD1 suppresses the level of H3 dimethylated on lysine 9 (H3K9me2) and heterochromatin protein 1a (HP1a) to antagonize repressive chromatin. To complement these studies, I am seeking to determine the possible role of CHD1 on H3K9me2 and H3K56ac. Influence of CHD1 on histone dynamics is examined by using native chromatin immunoprecipitation at CrebA. We observed that H3K9me2 levels do not significantly increase over a single active gene in Drosophila salivary glands with the loss of CHD1, which implies CHD1’s effect might be only limited to heterochromatic regions. Additionally, I have preliminary evidence that the loss of CHD1 leads to an increase in the level of H3 acetylated on lysine 56, a mark of newly deposited histones. This evidence together with yeast studies provides a model for how CHD1 regulates nucleosome turnover.