Les modifications post-traductionnelles (MPTs) d’histones sont apparues comme un acteur majeur de la régulation de l’expression des gènes. Cependant peu de choses sont connues sur le réel impact des MPTs sur la chromatine. Il a été suggéré que les MPTs d’histones (H2A, H2B, H3 et H4) ont le potentiel de moduler la fonction chromatinienne selon un « codehistone » en recrutant des protéines spécifiques de liaison. L’objectif de mon projet est d’approfondir la fonction de l’acétylation du domaine globulaire de l’histone H3 et de comparer cette modification avec celles des queues N-terminale in vivo sur une lignée ES cellulaire. Pour étudier l’impact de ces MPTs in vivo, toutes les copies endogènes du gène H3 sauvage (WT) doivent être remplacées par des copies mutées. Ainsi la première étape de mon projet est d’établir une lignée cellulaire exprimant seulement H3 mutée (e.g reproduisant une acétylation permanente) afin d’étudier les effets des modifications sur le domaine globulaire de H3 sur (a) l’expression génique, (b) l’architecture chromatinienne mais également pour étudier (c) les effets réciproques et synergiques entre les différentes modifications du domaine globulaire et (d) comparer ces effets avec les modifications sur la queue N-terminale dans un système in vivo. / Post-translational modifications (PTMs) of histones have emerged as key players in the regulation of gene expression. However, little is known to what extent PTMs can directly impact chromatin. It has been suggested that PTMs of core histones (H2A, H2B, H3 and H4) have the potential to govern chromatin function according to the so called ‘‘histone code’’ hypothesis by recruiting specific binding proteins. The goal of my project is to gain insight in the function acetylation within the globular domain of H3 and to compare these modifications with histone tail modifications, in vivo by using the CRISPR in mouse embryonic stem cells (ES). To study the impact of PTMs in vivo, all endogenous wild type (WT) H3 gene copies have to be replaced with mutant copies. Hence, the primary focus of my project is to establish cell lines that exclusively express mutated H3 (e.g. mimicking acetylation) in order to study effects of H3 globular domain modifications on (a) gene expression (b) chromatin architecture as well as to study (c) cross talks and synergisms between globular domain modifications and (d) compare the effects with tail modifications in an vivo system.

Apicomplexan parasites are leading causes of human and livestock diseases such as toxoplasmosis and malaria caused by Toxoplasma gondii and Plasmodium falciparum respectively. These organisms are varied in their morphologies and astoundingly complex on their life cycles that include infections of more than one host organism, differentiation through several morphologically distinct forms, and both sexual and asexual replication. What we and others have initially proposed was that the control of gene expression and cellular differentiation are particularly interesting in these organisms, as the apparent lack of large families of recognizable transcription factors typically found in other eukaryotic organisms suggests that they may be unusually reliant on epigenetic mechanisms. The initial hypothesis had to be re-assessed in light of the discovery in Apicomplexa of an expanded family of plant-like transcription factors (TFs) harbouring APETALA2 (AP2)-like domains. Yet, a growing body of evidence tends to favor epigenetic as one of the main contributor to parasite developmental programs and adjustments to fluctuant environment. One way to examine dynamic changes in post-translations modifications (PTMs) patterns is to alter the histone code writing. We therefore took advantage of HDAC inhibitors and showed that specific inhibition of TgHDAC3 by the cyclopeptide FR235222 disrupts the genome wide steady-state level of histone H4 acetylation inducing derepression of stage-specific genes. Yet, many questions about TgHDAC3 modus operandi remain unanswered. During my thesis, I uncovered the TgHDAC3-regulated proteome-wide acetylome typified by the presence of non-histone proteins including AP2 TFs and novel PTMs, e.g. the acetylation at Lys31 within the globular domain of histone H4. H4K31ac promotes a relaxed chromatin state at the promoter of active genes through nucleosome disassembly in both parasites. We identified TgGCN5B and TgHDAC3 as two antagonist enzymes regulating H4K31 acetylation in T. gondii. In contrast, H4K31monomethylation is enriched throughout the gene body of T. gondii active genes and contributes to transcription, whereas it is enriched at transcriptionally inactive pericentromeric heterochromatin regions in P. falciparum, a region that is lacking H3K9me3 and heterochromatin protein 1 in this parasite. We also showed that treating T. gondii cystogenic strains with a low dose of FR235222 induces the levels of proteins known to be expressed exclusively in cat (sporozoite and merozoite) or in murine chronic stage (bradyzoite). Lastly, we determined the specific interactome of TgHDAC3 and found as partners a MORC protein (CR230), several AP2 TFs, and ELM2 domain-containing scaffolding proteins. Collectively, these data established TgHDAC3 family as a central regulator of gene expression and stage conversion in T. gondii and, likely, other Apicomplexa. / Apicomplexan genome architecture is typified by a binary chromatin structure, with a major fraction of the bulk genome packaged as transcriptionally permissive euchromatin while few loci are embedded in silenced heterochromatin. There is evidence that histone modifications occurring at the lateral surface of the nucleosome play a substantial role in shaping chromatin structure, yet our understanding of the exact mechanism of action is poor. Here, we address how versatile modifications at Lys31 within the globular domain of histone H4 contribute to genome organization and expression in Apicomplexa. H4K31 acetylation was found at the promoter of active genes. The residue lies where the DNA wraps around the histone and its acetylation may enhance nucleosome disassembly, thereby favoring a more relaxed, open chromatin state. This residue tends also to be monomethylated and depending of the parasite examined different patterns were found. H4K31me1 was enriched in the core body of Toxoplasma active genes, yet its occupancy was inversely correlated with transcripts levels likely because the mark by reducing histone turnover impedes RNA polymerase progression across transcribed units. In contrast to the methylation of H3, it is the first time that a methylated residue of H4 has been clearly associated with transcriptional regulation. In Plasmodium, H4K31me1 was exclusively enriched at transcriptionally inactive genomic regions and peculiarly at pericentromeric heterochromatin, likely to replace the missing H3K9me3 that commonly decorated pericentric nucleosomes in other species.