The reversible process of histone acetylation and deacetylation is an important mechanism of epigenetic regulation in the control of gene expression and chromatin structure. In general, histone acetylation is related to gene activation, whereas histone deacetylation is associated with transcriptional gene silencing and maintenance of heterochromatin. A large number of histone deacetylases (HDACs), the enzymes that catalyze the reaction of histone deacetylation, have been identified in plants and other eukaryotes, and they were found to play crucial roles in plant growth and development. In Arabidopsis thaliana, histone deacetylase 1 (AtHD1) is a homolog of Saccharomyces cerevisiae Rpd3 that is a global transcriptional regulator. Downregulation of AtHD1 in transgenic Arabidopsis results in histone hyperacetylation and induces a variety of phenotypic and developmental defects, suggesting that AtHD1 is also a global regulator of many physiological and developmental processes. To characterize the expression pattern and distribution of AtHD1 in cells, the subcellular location of AtHD1 was determined by monitoring the expression of an AtHD1-GFP fusion protein in a transient expression assay and in transgenic Arabidopsis.The results show that AtHD1 is localized in the nucleus and appears to be excluded from the nucleolus. The histone deacetylase activity of AtHD1 was studied in an in vitro assay using radiolabeled histone peptides as a substrate. Recombinant AtHD1 produced by bacteria demonstrated a moderate but significant HDAC activity, whereas that produced by the baculovirus expression system did not have activity. This suggests that AtHD1 may require other cofactors or association with other proteins, rather than post-translational modifications, in order to have full HDAC activity. To study the possible interactions of AtHD1 with other proteins, a recombinant AtHD1 protein with two units of c-myc epitope fused to its C-terminus was expressed in transgenic Arabidopsis. We attempted to isolate proteins interacting with AtHD1 by co-immunoprecipitation (Co-IP). However, in the first few trials of Co-IP, a lot of contaminating proteins were present in the eluent along with the recombinant AtHD1-cmyc protein. Improvements in the experimental conditions are required for further investigation.

Les déacétylases dhistones (HDACs) constituent une famille denzymes identifiées initialement comme régulatrices du niveau dacétylation des histones, un processus important contrôlant la transcription génique. Comme leur nom lindique, il a longtemps été considéré que les seuls substrats des HDACs étaient les histones. Néanmoins, il est à présent démontré quelles peuvent aussi avoir dautres substrats nucléaires ou cytoplasmiques dont plusieurs facteurs de transcription et suppresseurs de tumeurs. Il a été montré que linhibition de lactivité des HDACs par des inhibiteurs globaux tels la trichostatine A (TSA) ou le suberoylanilide hydroxamique acide (SAHA) inhibe la croissance tumorale dans plusieurs modèles animaux. Linhibition des HDACs pourrait influencer tant les cellules tumorales que la néovascularisation de la tumeur (angiogenèse). Au cours des précédentes années de notre doctorat, nous avons étudié lune des HDACs de classe I, HDAC8. Nous avons découvert que cette HDAC, principalement cytoplasmique, est spécifiquement exprimée par les cellules de phénotype musculaire lisse et sassocie avec lactine ? des muscles lisses (? SMA) (Expression of histone deacetylase 8, a class I histone deacetylase, is restricted to cells showing smooth muscle differentiation in normal human tissues. Waltregny D, De Leval L, Glenisson W, Ly Tran S, North BJ, Bellahcene A, Weidle U, Verdin E, Castronovo V. American Journal of Pathology, 2004 Aug;165(2):553-64. - Histone deacetylase HDAC8 associates with smooth muscle alpha-actin and is essential for smooth muscle cell contractility. Glenisson W, Waltregny D, Tran SL, North BJ, Verdin E, Colige A, Castronovo V. FASEB Journal, 2005 Jun;19(8):966-8. - Novel smooth muscle markers reveal abnormalities of the intestinal musculature in severe colorectal motility disorders. T. Weidel, G.J.J.M. van Eys, W. Glénisson, D. Waltregny, V. Castronovo, J.-M. Vanderwinden. Neurogastroenterology and motility, 2006, 18(7):526-38.). Nous avions suspecté que HDAC8 pourrait jouer un rôle important dans la biologie de la cellule musculaire, potentiellement via la déacétylation de protéines cytosquelettiques ou encore par régulation de la transcription de gènes impliqués dans la différenciation musculaire lisse. Ainsi afin détudier limpact potentiel de HDAC8 et dautres HDACs dans ce processus, nous avons utilisé une approche dARN interférence dans un modèle de différenciation myofibroblastique (un modèle de différenciation musculaire lisse senso stricto nétant pas disponible). Les myofibroblastes (MFs) sont des cellules qui présentent des caractéristiques morphologiques et biochimiques intermédiaires entre des fibroblastes et des cellules musculaires lisses. La différenciation myofibroblastique, processus par lequel les fibroblastes aquièrent ces caractéristiques, peut être induite in vitro par des cytokines tel le TGF?1 (Transforming Growth Factor beta 1) mais sa régulation est encore mal connue. Les myofibroblastes jouent un rôle dans de multiples processus physiologiques, comme dans la cicatrisation, ainsi que dans des conditions pathologiques, comme dans linflammation chronique et le cancer. Plus précisément, les MFs pourraient jouer un rôle important dans le remodelage du stroma autour dun cancer, ce qui favoriserait la progression de certaines tumeurs (dont celles du colon). De récentes études ont montré que la TSA empêche, dans des cultures de fibroblastes de rat, linduction par le TGF?1 de la fibrogenèse (synthèse de procollagène ?1(I) et ?1(III) et d?-SMA). Cette observation suggère limplication dHDACs dans la régulation de la différenciation myofibroblastique. Nous avons donc entrepris didentifier la/les HDAC(s) régulant(s) spécifiquement(s) la différenciation myofibroblastique dans une culture primaire de fibroblastes de peau humaine grace à lutilisation de petits ARN interférants (siRNA) permettant de diminuer spécifiquement labondance de huit HDACs (HDAC1 à 8) tant au niveau de leur transcript que de leur protéine. Nous avons trouvé de manière reproductible que linduction de l?-SMA par le TGF?1 est bloquée par la transfection de siRNA spécifiques dHDAC4. Afin détudier quels sont les autres aspects de la fibrogenèse qui sont régulés par HDAC4, ainsi que le rôle quHDAC4 pourrait jouer dans la régulation de la signalisation du TGF?1, nous avons étudié lexpression de trois inhibiteurs endogènes de la voie de signalisation du TGF?. De manière intéressante, la diminution dHDAC4 induite par les siRNA entraine une augmentation de lexpression de TGIF et de TGIF2 mais pas de Smad7. Dans létat actuel de nos connaissance, cette observation serait la première à indiquer que le niveau dexpression de TGIF et de TGIF2 puisse être régulé par une HDAC. (Histone deacetylase 4 is required for TGF?1-induced myofibroblastic differentiation, W. Glénisson, D. Waltregny, V. Castronovo, BBA Mol cell Research, 2007).

The reversible process of histone acetylation and deacetylation is an important mechanism of epigenetic regulation in the control of gene expression and chromatin structure. In general, histone acetylation is related to gene activation, whereas histone deacetylation is associated with transcriptional gene silencing and maintenance of heterochromatin. A large number of histone deacetylases (HDACs), the enzymes that catalyze the reaction of histone deacetylation, have been identified in plants and other eukaryotes, and they were found to play crucial roles in plant growth and development. In Arabidopsis thaliana, histone deacetylase 1 (AtHD1) is a homolog of Saccharomyces cerevisiae Rpd3 that is a global transcriptional regulator. Downregulation of AtHD1 in transgenic Arabidopsis results in histone hyperacetylation and induces a variety of phenotypic and developmental defects, suggesting that AtHD1 is also a global regulator of many physiological and developmental processes. To characterize the expression pattern and distribution of AtHD1 in cells, the subcellular location of AtHD1 was determined by monitoring the expression of an AtHD1-GFP fusion protein in a transient expression assay and in transgenic Arabidopsis.The results show that AtHD1 is localized in the nucleus and appears to be excluded from the nucleolus. The histone deacetylase activity of AtHD1 was studied in an in vitro assay using radiolabeled histone peptides as a substrate. Recombinant AtHD1 produced by bacteria demonstrated a moderate but significant HDAC activity, whereas that produced by the baculovirus expression system did not have activity. This suggests that AtHD1 may require other cofactors or association with other proteins, rather than post-translational modifications, in order to have full HDAC activity. To study the possible interactions of AtHD1 with other proteins, a recombinant AtHD1 protein with two units of c-myc epitope fused to its C-terminus was expressed in transgenic Arabidopsis. We attempted to isolate proteins interacting with AtHD1 by co-immunoprecipitation (Co-IP). However, in the first few trials of Co-IP, a lot of contaminating proteins were present in the eluent along with the recombinant AtHD1-cmyc protein. Improvements in the experimental conditions are required for further investigation.

Thèse de doctorat : Biologie cellulaire et moléculaire des plantes : Strasbourg 1 : 2008. / Titre provenant de l'écran-titre. Bibliogr. 16 p.. Notes bibliogr.. Index.

Epigenetic regulation is a mechanism by which heritable changes in gene expression are controlled by chromatin status rather than primary DNA sequence. Changes in chromatin structure affect accessibility of DNA elements to the transcriptional machinery and thus affect transcription activity of the gene. A key event in this process is reversible modification of core histones, which is catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDs, HDAs, or HDACs). In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation is associated with gene activation.To study the role of histone deacetylase in plant gene regulation and development, we generated constitutive antisense histone deacetylase 1 (CASH) transgenic plants. AtHD1 is a homolog of RPD3 protein, a global transcriptional regulator in yeast. Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones. Down-regulation of histone deacetylation caused a variety of growth and developmental abnormalities and ectopic expression of tissue-specific genes. However, changes in genomic DNA methylation were not detected in repetitive DNA sequences in the transgenic plants.We also identified a T-DNA insertion line in exon 2 of AtHD1 gene (athd1-t1), resulting in a null allele at the locus. The complete inhibition of the AtHD1 expression induced growth and developmental defects similar to those of CASH transgenic plants. The phenotypic abnormalities were heritable across the generations in the mutants. When the athd1-t1/athd1-t1 plants were crossed to wild-type plants, the mutant phenotype was corrected in the F1 hybrids, which correlated with the AtHD1 expression and reduction of histone H4 Lys12 acetylation. Microarray analysis was applied to determine genome-wide changes in transcriptional profiles in the athd1-t1 mutant. Approximately 6.7% (1,753) of the genes were differentially expressed in leaves between the wild-type (Ws) and the athd1-t1 mutant, whereas 4.8% (1,263) of the genes were up- or down-regulated in flower buds of the mutant. These affected genes were randomly distributed across five chromosomes of Arabidopsis and represented a wide range of biological functions. Chromatin immunoprecipitation assays indicated that the activation for a subset of genes was directly associated with changes in acetylation profiles.

Among mammalian histone deacetylases (HDACs) identified so far, HDAC6 is unique in that it possesses tandem deacetylase domains, as well as a carboxyl-terminal zinc finger motif implicated in ubiquitin binding. HDAC6 has also been demonstrated to deacetylate tubulin and thus potentially contribute to regulation of cell motility. Although HDAC6 has been reported to interact with a variety of nuclear factors, this enzyme is predominantly localized to the cytoplasm. / Here I show that the subcellular localization of human HDAC6 is governed by additional elements not present in orthologues of this protein from other species. Human HDAC6 contains a unique insertion of eight or ten repeats of a Ser/Glu-containing tetradecapeptide sequence, here termed the SE14-repeat domain, which is responsible for mediating the Leptomycin B-resistant cytoplasmic retention of human HDAC6. Human HDAC6 also contains a second functional leucine-rich nuclear export signal when compared to murine HDAC6, and a region near the amino terminus of human HDAC6 is capable of mediating nuclear import. / I have also identified a previously uncharacterized protein containing a ubiquitin-like domain, here termed Ubl90, as a novel binding partner for HDAC6. Interestingly, Ubl90 binds to the deacetylase domains of HDAC6, rather than to the carboxyl-terminal zinc finger motif previously implicated in ubiquitin association. Ubl90 also interacts with several class I HDACs (HDAC1, HDAC2 and HDAC8), although more weakly than with HDAC6. When Ubl90 is overexpressed, it leads to the redistribution of HDAC1 from a nuclear to a pancellular localization, although this does not affect HDAC1-dependent transcriptional repression in a transient transfection assay. Like HDAC6, Ubl90 is capable of repressing Runx2-mediated transcriptional activation, and, when overexpressed, it leads to cell cycle disturbances similar to those seen with HDAC6 overexpression. These results suggest that Ubl90 and HDAC6 interact functionally, as well as physically.

Skeletal muscle specification and differentiation programs are regulated by the myogenic regulatory factors which include Myf5, MyoD, myogenin and Mrf4. Upstream of the MRFs, the transcription co-activators and other intracellular and extracellular signals play crucial roles in regulating skeletal myogenesis. Histone acetyltransferase activity of p300 is required for Myf5 and MyoD expression. Furthermore, the MyoD core enhancer region is indispensable for MyoD expression. However, the mechanism by which p300 activates MyoD gene expression is to be determined. The histone acetyltransferase activity of p300 can be inhibited by small molecule inhibitors such as curcumin. Thus, using the inhibitor approach on stem cells is useful to investigate the role of p300 in activating MyoD expression during myogenesis. We here show that curcumin was able to inhibit stem cell determination and differentiation into skeletal myocytes. We also show that p300 is present, and histone acetylation is high at the core enhancer region. Therefore, we provide evidence that p300 is directly involved in MyoD gene expression during skeletal myogenesis.

Recent studies have focused their attention on the structure and function of histone chaperones involved in different aspects of somatic chromatin assembly and disassembly. However, one of the most dramatic chromatin remodeling processes takes place immediately after fertilization and is mediated by proteins in metazoan eggs that function as histone chaperones and histone storage proteins. These include members of the nucleoplasmin (NPM) family and the nuclear autoantigenic sperm protein (NASP) families. While it had been know for some time that these proteins function as “histone sinks”, new studies are shedding light on their role as histone chaperones. NASP was first identified in Xenopus laevis eggs where it accumulated in the nucleoplasm and was found to bind histones H3 and H4 at which time it believed to act simply as a histone storage protein. Interestingly, in addition to binding and providing storage to H3-H4 in the egg and in somatic cells, our studies have shown NASP to be the first characterized chaperone for histone H1. The members of the histone H1 family (linker histones) are essential to maintaining the structure of chromatin with respect to the folding of the chromatin fiber, nucleosome spacing, chromatin remodeling, gene transcription and progression through the cell cycle. Until recently there has been no histone H1 chaperone characterized and no evidence of a storage protein to which linker histones are bound, when not associated with DNA of NCPs. By using recombinant NASP, to incorporate linker histones onto the nucleosome arrays in a chromatin fiber, we studied the dynamics and conformation of chromatin in a more biologically relevant and precise method than presented in any previous chromatin research. Like NASP, nucleoplasmin was identified as a factor in X. laevis eggs that binds histones and loads them onto DNA. Subsequently, the nucleoplasmin protein family has been to be universally represented throughout metazoans where it plays a similar role in chromatin metabolism. Members of this family include nucleophosmin (NPM1), nucleoplasmin (NPM2, NPM or Np), the newly characterized NPM3 and nucleoplasmin-like proteins (NPM-like or NLP). We have been able to demonstrate that NPM2 that is highly phosphorylated in X. laevis eggs can unfold sperm and somatic chromatin by facilitating the removal of linker histones and other chromosomal proteins from linker DNA regions between nucleosomes in the absence of any stable interaction with the nucleosome core particle (NCP) itself. In addition, our studies reveal that NPM2 is a pentameric chaperone, as opposed to a decameric chaperone, that regulates the condensation state of sperm and somatic chromatin by removing linker histones and specific nuclear basic proteins and depositing histone H2A-H2B dimers on the distal face of the NPM2. / Graduate

Thesis (M. Med. Sc.)--University of Hong Kong, 2008.

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xix, 278 p.; also includes graphics (some col.) Includes bibliographical references (p. 256-278).