The N-terminal tails of histones are subject to many chemical modifications that are involved in a variety of biological functions. Histone methylation is a major epigenetic modification found in both single and multicellular organisms and is directly involved in the regulation of gene expression. Methylation of lysine 9 of histone 3 (H3K9) has been shown to have diverse functions depending on the number of methyl groups added; H3K9me1 marks the active promoters, while H3K9me2 and H3K9me3 are present within inactive gene promoters and pericentric heterochromatin. G9a, also known as euchromatic histone-lysine N-methyltransferase 2 (Ehmt2), is a histone methylase that catalyses addition of mono- and dimethyl groups to H3K9 in euchromatic regions of the genome to silence genes. Therefore, it is a vital component of the gene expression regulation machinery. In mouse embryonic stem (ES) cells, G9a forms a stable heterodimer with the G9a-like protein (GLP or Ehmt1), which is further stabilised by the C2H2-type zinc finger protein, widely interspaced zinc finger protein (WIZ). These three proteins form the core G9a complex, which is essential for mouse development. Lack of any G9a complex member leads to embryonic lethality at E9.5 with severe growth defects. The ankyrin repeat domain of G9a/GLP can bind to H3K9me1/2 with high affinity in vitro (Collins et al. 2008). This enables the self-recruitment of the G9a complex to sites with H3K9me1/2 and maintenance of the mark. However, the initial recruitment of the G9a complex to sites lacking H3K9me1/2 mark during differentiation is poorly understood. Neither G9a nor GLP has a DNA/RNA binding domain, so recruitment of the G9a complex to specific sites must be mediated by other binding partners of the G9a complex. Using mass spectrometry, I was able to identify a number of zinc finger proteins as binding partners of G9a. Among these, WIZ was identified in stoichiometric amounts to G9a and GLP, and is a potential DNA binding protein similar to other C2H2-type zinc fingers. The aim of this study was to determine the role of WIZ in the recruitment of the G9a complex to specific sites. I showed that knockdown of WIZ had no significant effect on the chromatin binding of G9a in undifferentiated mouse ES cells, which indicates WIZ is dispensable in the maintenance of H3K9me2. However, I observed a 30% decrease in the G9a levels upon WIZ knockdown, which shows that WIZ might have a role in stabilising G9a. Using recombinant WIZ zinc finger pairs, I was able to show that the 3rd and 4th zinc finger of WIZ bind DNA in vitro. Furthermore, using the systematic evolution of ligands exponential enrichment (SELEX) approach I demonstrated that the zinc fingers of WIZ preferentially bind to G-rich double-stranded DNA sequences. Binding site analysis with synthetic DNA indicated that WIZ ZF3-4 require two binding sites that are a certain distance apart from each other for efficient binding. In addition, ZF3-4 binds ssDNA with higher affinity than dsDNA, and binding to ssDNA is sequence-independent. This study shows for the first time that mouse WIZ zinc finger pairs can bind DNA and RNA in vitro. Therefore, sequence-specific recruitment of G9a might be mediated by WIZ during differentiation. Furthermore, DNA binding preference of WIZ might suggest that WIZ-mediated recruitment of G9a to establish H3K9me2 could occur at the R-loops where G-rich DNA forms a hybrid with newly transcribed RNA or at the G-rich repetitive sequences.

The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. While the active mark H3K4me3 is present around the transcription start site on the gene, the function of the H3K36me3 mark is unknown. A number of H3K36-specific histone methyltransferases (HMTs) have been identified, however the focus of this study is the HMT Hypb. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at a subset of genes. Upon differentiation, we observe that H3K36me3 becomes enriched at the 3’ end of several muscle-specific genes. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of the H3K36 HMT Hypb was performed in muscle myoblasts using shRNA. Upon Hypb knockdown, we were surprised to observe enhanced myogenesis. N-ChIP was also performed on differentiated Hypb knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. N-ChIP data show a drop in H3K36me3 enrichment levels on myogenin and Ckm genes. The possible occupancy of Hypb on the coding regions of muscle-specific genes was experimentally observed by cross-linked chromatin immunoprecipitation (X-ChIP) on differentiated C2C12 cells and subsequently confirmed by X-ChIP on knockdown lines where the occupancy was lost. A model is proposed that links the observed phenotype with H3K36me3.

The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. While the active mark H3K4me3 is present around the transcription start site on the gene, the function of the H3K36me3 mark is unknown. A number of H3K36-specific histone methyltransferases (HMTs) have been identified, however the focus of this study is the HMT Hypb. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at a subset of genes. Upon differentiation, we observe that H3K36me3 becomes enriched at the 3’ end of several muscle-specific genes. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of the H3K36 HMT Hypb was performed in muscle myoblasts using shRNA. Upon Hypb knockdown, we were surprised to observe enhanced myogenesis. N-ChIP was also performed on differentiated Hypb knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. N-ChIP data show a drop in H3K36me3 enrichment levels on myogenin and Ckm genes. The possible occupancy of Hypb on the coding regions of muscle-specific genes was experimentally observed by cross-linked chromatin immunoprecipitation (X-ChIP) on differentiated C2C12 cells and subsequently confirmed by X-ChIP on knockdown lines where the occupancy was lost. A model is proposed that links the observed phenotype with H3K36me3.

The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. While the active mark H3K4me3 is present around the transcription start site on the gene, the function of the H3K36me3 mark is unknown. A number of H3K36-specific histone methyltransferases (HMTs) have been identified, however the focus of this study is the HMT Hypb. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at a subset of genes. Upon differentiation, we observe that H3K36me3 becomes enriched at the 3’ end of several muscle-specific genes. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of the H3K36 HMT Hypb was performed in muscle myoblasts using shRNA. Upon Hypb knockdown, we were surprised to observe enhanced myogenesis. N-ChIP was also performed on differentiated Hypb knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. N-ChIP data show a drop in H3K36me3 enrichment levels on myogenin and Ckm genes. The possible occupancy of Hypb on the coding regions of muscle-specific genes was experimentally observed by cross-linked chromatin immunoprecipitation (X-ChIP) on differentiated C2C12 cells and subsequently confirmed by X-ChIP on knockdown lines where the occupancy was lost. A model is proposed that links the observed phenotype with H3K36me3.

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.

Epigenetic mechanisms are essential for normal cell development. Alteration in those normal processes leads to malignant cell transformation and with this to cancer development. Use of inhibitors that alter the epigenetics of DNA methylation and histone post translational modifications has lead to the exploration of the epigenetic mechanism involved in silencing of tumor suppressor genes in cancer, including acute myeloid leukemia (AML). Moreover, combinations of inhibitors that target various epigenetic enzymes have being recognized to be more effective in the re-activation of tumor suppressor genes than individual drug treatments. Here, we reported that p15, p21 and E-cadherin genes are more effectively re-expressed using a combination of DNA methyltransferase and histone methyltransferase inhibitors in AML cell lines. Re-expression of hypermethylated p15 and E-cadherin genes required reduced levels of promoter histone 3 lysine 9 (H3K9) methylation rather than inhibition of DNA methylation itself. Moreover, induction of p21 expression was associated with changes in promoter histone 3 lysine 9 methylation (H3K9Me) by achieving inhibition of the histone methyltransferase, SUV39H1, activity. Altogether, our results highlight the potential of combining epigenetic drugs in the re-activation of epigenetically silenced tumor suppressor genes and the need for evaluating histone methyltransferases as therapeutic targets for treatment of acute myeloid malignancies.

Adult skeletal muscles can regenerate after injury due to the presence of satellite cells, a quiescent population of myogenic progenitor cells characterized by expressing the transcription factor Pax7. Once activated, satellite cells repair the muscle damage and replenish the stem cell niche due to the coordinated function of several transcription factors including Pax7 and the myogenic regulatory factors (MRFs). MRFs are skeletal muscle-specific transcription factors that can convert non-muscle cells into the myogenic lineage. MRFs are known to cooperate with other transcription factors in regulating the complex transcriptional network driving myogenic differentiation of muscle progenitors. The Six4 transcription factor emerges as a strong candidate for cooperating with MRFs. Six4 is expressed in skeletal muscles; the lack of a muscle development phenotype in Six4-null mice has been attributed to compensation by other Six family members. However, this did not exclude a critical role for Six4 during muscle development as Six1;Six4 double mutant mice show a more severe muscle phenotype than Six1 mutant mice. Nevertheless, the role of Six4 during adult muscle regeneration has never been addressed. I combined a partial loss-of-function of Six4 with high-throughput approaches to address the role of Six4 during adult skeletal muscle regeneration. I observed an important function of Six4 during muscle regeneration in vivo and in in vitro cell models. Using RNA interference assays against Six4 in tibialis anterior muscle regeneration after cardiotoxin-induced muscle damage, I observed for the first time that Six4 plays a role in proper muscle regeneration. The ability of the MRF MyoD, a central regulator of skeletal myogenesis, to convert a non-muscle cell model into the myogenic lineage was impaired with attenuated Six4 expression. I employed genome-wide approaches by combining ChIP-sequencing with gene expression profiling and identified a set of muscle genes coordinately regulated by both Six4 and MyoD. Throughout the genome, the cooperation between Six4 and MyoD was associated with binding of the H3K27me3 demethylase Utx and depletion of the H3K27me3 repressive chromatin mark. Together, these results reveal an important role for Six4 during adult muscle regeneration, and suggest a widespread mechanism of cooperation between Six4 and MyoD that correlates with modifying the epigenetic landscape of the regulatory regions of a large set of genes needed for efficient myogenesis.

No description available.

L'empreinte génomique est un mécanisme épigénétique qui conduit à l'expression d'un seul des deux allèles parentaux pour une centaine de gènes autosomaux chez les mammifères. La majorité des gènes soumis à l'empreinte est regroupée en clusters et tous ces gènes sont sous le contrôle de séquences discrètes appelées ICR (Imprinting Control Region). Les ICRs sont marquées épigénétiquement par une méthylation d'ADN et des modifications des histones alléliques. La méthylation d'ADN au niveau de ces ICRs est un facteur clé de l'empreinte et va être établie dans les lignées germinales suivant le sexe de l'embryon. Après fécondation, le nouvel embryon portera les empreintes paternelles et maternelles, ces empreintes devront alors être maintenues pendant tout le développement et interprétés dans le but de conduire à l'expression allélique des gènes soumis à l'empreinte. Cependant, la méthylation d'ADN ne peut expliquer à elle seule tous les aspects de l'empreinte génomique. Ainsi, d'autres marques épigénétiques doivent agir dans le maintien et la lecture de ces empreintes. Nous avons mis en évidence dans un premier temps que le contrôle de l'expression allélique dans le cerveau de Grb10 repose sur la résolution d'un domaine bivalent allélique spécifiquement dans le cerveau. Ces résultats mettent en avant pour la première fois un domaine bivalent dans le contrôle de l'expression des gènes soumis à l'empreinte et propose un nouveau mécanisme dans l'expression tissu spécifique de ces gènes. D'autre part, bien que des études en cellules ES aient démontré un rôle de G9a dans le maintien des empreintes au cours du développement embryonnaire, nos données suggèrent que G9a ne serait pas essentielle a ce maintien dans un contexte in vivo. / Genomic imprinting is a developmental mechanism which leads to parent-of-origin-specific expression for about one hundred genes in mammals. Most of imprinted genes are clustered and all are under control of sequence of few kilobases called Imprinting Control Region or ICR. ICRs are epigenetically marked by allelic DNA methylation and histone modifications. DNA methylation on ICRs is a key factor which is established in germ cells according to the sex of the embryo. After fecundation, the new embryo will harbored both paternal and maternal imprints which have to be maintained during the development and read to lead to allelic expression of imprinted genes. However, allelic DNA methylation alone cannot explain every aspect of genomic imprinting. Thus, there should be other epigenetic marks which act in the maintaining and reading of the imprints.Our data first indicate that bivalent chromatin, in combination with neuronal factors, controls the paternal expression of Grb10 in brain, the bivalent domain being resolved upon neural commitment, during the developmental window in which paternal expression is activated. This finding highlights a novel mechanism to control tissue-specific imprinting. On an other hand, although previous studies in ES cells show a role for G9a in the maintaining of imprints during embryonic development, our data suggest that G9a would not be essential in an in vivo model.

Chez le cilié Paramecium tetraurelia, la différentiation du génome somatique à partir du génome germinal est caractérisée par la délétion massive et reproductible d'éléments transposables et de 45 000 courtes séquences en copie unique dispersées sur l'ensemble du génome. Des petits ARN non codants produits par la lignée germinale, les scanARN, sont impliqués dans la régulation épigénétique des délétions d'ADN mais les mécanismes sous-jacents sont peu compris. Nous avons montré que la triméthylation de H3 (H3K27me3 et H3K9me3) présente une localisation dynamique pendant le développement du noyau somatique qui est altérée si l'endonucléase requise pour les événements d'élimination d'ADN est déplétée. Nous avons identifié une histone méthyltransférase, Ezl1p, nécessaire à la méthylation de H3 et requise pour les réarrangements du génome. Des analyses à l'échelle du génome entier ont montré que Ezl1p et les scanARN sont nécessaires à l'élimination des longues séquences germinales répétées tandis que les courtes séquences uniques présentent des sensibilités différentes à la déplétion de ces facteurs. Des déterminants cis tels que la longueur de l'ADN à éliminer peuvent contribuer à définir les séquences délétées. Dans une seconde étude, nous avons montré que chez Paramecium, la fonction centromérique est restreinte aux chromosomes germinaux. Un processus d'inactivation des centromères se produit pendant le développement du noyau somatique. L'endonucléase requise pour la délétion des séquences germinales est nécessaire pour l'inactivation des centromères suggérant fortement que l'inactivation des centromères germinaux repose sur l'élimination physique de l'ADN centromérique. / In the ciliate Paramecium tetraurelia, differentiation of the somatic genome from the germline genome is characterized by massive and reproducible deletion of transposable elements and of 45,000 short, dispersed, single-copy sequences. A specific class of small RNAs produced by the germline during meiosis, the scnRNAs, are involved in the epigenetic regulation of DNA deletion but the underlying mechanisms are poorly understood. We showed that trimethylation of histone H3 (H3K27me3 and H3K9me3) displays a dynamic nuclear localization that is altered when the endonuclease required for DNA elimination is depleted. We identified the histone methyltransferase Ezl1p responsible for H3 methylations establishment and showed that it is required for correct genome rearrangements. Genome-wide analyses showed that scnRNA-mediated H3 methylation is necessary for the elimination of long, repeated germline DNA, while single copy sequences display differential sensitivity to depletion of the scnRNA pathway or Ezl1p. Our study reveals cis acting determinants such as DNA length that may contribute to define the deleted germline sequences. In a second study, we showed that in Paramecium cells, the centromeric function is restricted to the germline chromosomes. A process of centromere inactivation occurs during the development of the somatic lineage, concomitantly with the events of DNA elimination. Our genetic analyses show that the endonuclease required for DNA elimination and Ezl1p but not the scnRNA are necessary for centromere inactivation. Our data strongly suggest that centromere inactivation relies on the physical elimination of the centromeric sequences from the somatic genome.