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Histone deacetylases (HDAC) 1 and 2 are highly similar enzymes that help regulate chromatin structure as the core catalytic components of co-repressor complexes. Although tissue specific deletion of HDAC1 and HDAC2 has demonstrated functional redundancy, germline deletion of HDAC1 in the mouse causes early embryonic lethality, whereas HDAC2 does not. To address the unique requirement for HDAC1 in early embryogenesis I have generated conditional knock-out mouse embryonic stem (mES) cells in which HDAC1 or HDAC2 genes can be inactivated. Deletion of HDAC1, but not HDAC2, causes a significant reduction in the HDAC activity of Sin3A, NuRD and CoREST co-repressor complexes. This reduced co-repressor activity results in a specific 1.6-fold increase in histone H3 K56 acetylation, providing genetic evidence that H3K56Ac is a substrate of HDAC1. In culture, loss of HDAC1 but not HDAC2 leads to precocious mES cell differentiation. This genetic study of HDAC1 and HDAC2 in mES cells, which mimic the embryonic epiblast, has identified a unique requirement for HDAC1 in the optimal activity of HDAC1/2 co-repressor complexes and cell fate determination during differentiation. Given previous demonstrations of the roles of HDAC1/2 known co-repressor complexes in T cell development and the functional redundancy between HDAC1 and -2 in a number of tissues, a conditional knock-out approach was undertaken in murine T cells. I have demonstrated that HDAC1 and -2 exert pleiotropic effects on T-cell development. I have also shown, for the first time in a physiological system, that HDAC1/2 activity is critical for maintaining genome stability. The occurrence of tumours (and the developmental block) is dose dependent, occurring in cells with the least amount of deacetylase activity, indicating that regulation of the acetyl-proteome, a balance between HAT and HDAC enzymes is crucial for normal cell development and viability.

Natural products have been the source of numerous leads for several drugs. As these natural products are often isolated in small quantities, it is necessary to produce them synthetically to allow testing for biological activity. Furthermore, synthesis allows the preparation of unnatural analogues for SAR studies. Cyclic peptides represent an important family of biologically active natural products. The hepta- and octacyclopeptides sanguinamide A and sanguinamide B were recently isolated in submicromolar amounts by the Molinski group. The lack of material prevented biological evaluation of the natural products. For this reason and to confirm the structural elucidation we have targeted the total synthesis of sanguinamides. In addition to two proline residues, sanguinamides A and B include heterocycles and natural L-amino acid residues. We have completed the total syntheses of sanguinamides A and B; however the synthetic rotamers differed in both cases from the natural rotamers. We have investigated the influence of macrocyclisation on cis/trans conformational preference of the proline residues for the synthesis of sanguinamide A. We attempted several isomerisations and calculated the relative energies of the different sanguinamide conformers. [D-Ile]-Sanguinamide A, Cys(tBu) analogue of sanguinamide A and the synthetic sanguinamide B displayed antibacterial activity while the synthetic trans, trans-sanguinamide A displayed mild tyrosine kinase inhibitory activity. While extracted stylissamide A showed inhibition of translation during the elongation step, even though being structurally identical to the natural product, the synthetic compound prepared by macrocyclisation from a linear precursor was found to be totally inactive. Histones undergo different types of covalent modifications on the N-terminal tails such as acetylation, phosphorylation and methylation. Histone modification is a major mechanism of regulation in gene expression, replication and repair. Deregulation of histone modifications leads to cancer progression and therefore, inhibitors of enzymes which are able to catalyse the addition and removal of these epigenetic marks have therapeutic potential for treating cancer. An enzyme of particular interest is the family of zinc-dependent histone deacetylases (HDACs) that remove acetyl groups from acetylated lysine residues. Depsipeptides were prepared as HDAC inhibitors. We will ii present our total synthesis of largazole along with a range of analogues and discuss the SAR obtained from HDAC and cell proliferation assays. We elucidated the stereochemistry of burkholdac B by total synthesis of three diastereomers. The diastereomers made along with the natural product were tested as HDAC inhibitors. We are interested in inhibitors of lysine-specific demethylase 1 (LSD1) which is a different kind of epigenetic enzyme involved in demethylation of histone proteins in chromatin. Tranylcypromine is known to be an LSD1 inhibitor. Analogues of PCPA have been synthesised in order to explore the structure-activity relationships of this inhibitor. Analogues were also prepared and tested as LSD1 inhibitors.

En influençant le degré de compaction de la chromatine ainsi que ses interactions avec différents partenaires protéiques, les modifications post-traductionnelles des histones sont impliquées dans la régulation de l’expression des gènes. Avec les différents variants d’histones incorporés dans la chromatine, ces modifications dynamiques et sensibles à l’environnement sont constitutives du code histone. Ce travail présente une approche globale de criblage baptisée approche histonomique, visant à révéler une perturbation épigénétique à l’échelle des histones. Cette approche originale offre une comparaison rapide et fiable des abondances relatives des variants d’histones et de leurs modifications post-traductionnelles dans des cellules humaines en une seule analyse LC-MS. Comme preuve de concept, des cellules BeWo issues de choriocarcinome humain ont été exposées au butyrate de sodium, un inhibiteur non spécifique d’histones désacétylases. Les histones extraites des échantillons témoins ou traités au butyrate de sodium à 1 ou 2,5 mM ont été analysées par chromatographie liquide ultra performante couplée à un spectromètre de masse de type Q-TOF. Les analyses statistiques multivariées ont permis de discriminer les échantillons témoins des échantillons traités sur la base des différences de degrés d’acétylation observés sur plusieurs formes d’histones. La même approche a ensuite été appliquée à des cellules exposées au B[a]P à 1 μM et a révélé deux principaux marqueurs caractéristiques d’un remodelage de la chromatine induit par les effets génotoxiques duB[a]P. En résumé, cette approche histonomique globale pourrait se révéler être un outil complémentaire très utile pour explorer une potentielle perturbation du code histone lors d’exposition à des xénobiotiques environnementaux. / While acting upon chromatin compaction, histone post-translational modifications (PTMs) are involved in modulating gene expression through histone–DNA affinity and protein–protein interactions. These dynamic and environment-sensitive modifications are constitutive of the histone code that reflects the transient transcriptional state of the chromatin. Here we describe a global screening approach for revealing epigenetic disruption at the histone level. This original approach enables fast and reliable relative abundance comparison of histone PTMs and variants in human cells within a single LC-MS experiment. As a proof of concept, we exposed BeWo human choriocarcinoma cells to sodium butyrate (SB), a universal histone deacetylase (HDAC) inhibitor. Histone acide xtracts equally representing 3 distinct classes, Control, 1 mM and 2.5 mM SB, we reanalyzed using ultra-performance liquid chromatography coupled with a hybrid quadrupoletime-of-flight mass spectrometer (UPLC-QTOF-MS). Multivariate statistics allowed us to discriminate control from treated samples based on differences in the acetylation level of several histone forms. We then applied the same procedure to cells treated with 1 μMB[a]P and suceeded in revealing two markers of chromatin remodeling in relation withgenotoxic properties of B[a]P. Indeed, this untargeted histonomic approach could be auseful exploratory tool in many cases of environmental xenobiotic exposure when histone code disruption is suspected.