Les complexes histones méthyltransférases SET1, hautement conservés de la levure aux mammifères, sont ciblés aux régions promotrices par la protéine CFP1/CXXC, résultant en l’implémentation de la méthylation de la lysine 4 de l’histone H3 (H3K4me), modification post-traductionnelle influençant l’expression des gènes selon le contexte chromatinien. La présence de plusieurs complexes SET1 distincts dans différents systèmes modèles eucaryotes a compliqué l’étude de leurs fonctions dans un contexte développemental. Caenorhabditis elegans contient une seule protéine homologue de SET1, SET-2, et d’uniques homologues des autres sous-unités du complexe, RBBP5, ASH2, WDR5, DPY30 et CFP1. Cependant, la composition biochimique du complexe n’a pas été décrite. En couplant des expériences de co-immunoprécipitation avec des analyses de spectrométrie de masse, j’ai identifié le complexe SET-2/SET1 dans les embryons de C. elegans. D’autre part, j’ai montré que le complexe SET-2/SET1 co-immunoprécipite aussi un autre complexe conservé modifiant la chromatine et j’ai mis en évidence les interactions mises en jeu entre ces deux complexes. Mon analyse génétique a démontré que les mutants de perte de fonction des sous-unités des deux complexes partagent des phénotypes communs, en cohérence avec des fonctions développementales communes. Le laboratoire a également entrepris des expériences de transcriptomique et d’immunoprécipitation de la chromatine montrant un nouveau rôle de CFP-1 dans le recrutement de ce complexe au niveau de sites spécifiques de la chromatine. / The highly conserved SET1 family complexes are targeted by CFP1/CXXC protein to promoter regions through multivalent interactions to implement methylation of histone H3 Ly4 (H3K4me), a modification that correlates with gene expression depending on the chromatin context. The presence of distinct SET1 complexes in multiple eukaryotic model systems has hampered studies aimed at identifying the complete array of functions of SET1/MLL regulatory networks in a developmental context. Caenorhabditis elegans contains one SET1 protein, SET-2, one MLL-like protein, SET-16, and single homologs of RBBP5, ASH2, WDR5, DPY30 and CFP1. The biochemical composition of the complex however, has not been described. Through the use of co-immunoprecipitation coupled to mass spectrometry-based proteomics, I identified the SET-2/SET1 complex in C. elegans embryos. Most importantly, I showed that the SET-2/SET1 complex also co-immunoprecipitates another conserved chromatin-modifying complex and I highlighted the interactions involved between these two complexes. My genetic analysis revealed that loss of function mutants of the two complex subunits share common phenotypes, consistent with common developmental functions. The laboratory has also undertaken transcriptomic and chromatin immunoprecipitation experiments showing that CFP-1 has a role in the binding of this complex at specific chromatin regions.

In higher eukaryotes, the DNA base Cytosine can exist in a variety of modified forms when in the dinucleotide CpG. Although a methylated form tends to dominate within the genome, approximately 1% of all CpG dinucleotides are found unmodified at high densities spanning around 1Kb and tend to co-localise to the 5’ ends of around 60% of annotated gene promoters. These unique DNA sequences are known as CpG islands (CGIs) and their role within the genome to date is largely unknown. Methylation of CGIs in cancers however has been linked to silencing of associated genes implying a role in gene regulation. Furthermore these sites are also interesting as they remain specifically nonmodified within a genome rich in methylated CpG. We set out to better understand the roles for CGIs through the characterisation of any specific CGI binding proteins. Digestion of nuclei with methyl sensitive restriction enzymes facilitates the purification of CGI fragments. Subsequent immunohistochemistry on the CGI chromatin fragments along with ChIP-PCR over several CGIs revealed an enrichment of the “active” histone modifications including H3K4me3, a depletion of the “silencing” marks such as H3K27me3, as well as a group of CGI specific binding factors. These latter proteins contained a domain previously shown to bind to non-methylated CpG dinucleotides (the CXXC domain) and as such were ideal candidates for CGI specific factors, in particular a protein called Cfp1. Genome wide sequencing revealed a striking correlation between Cfp1 and H3K4me3 which were both seen at around 80% of islands. Furthermore, the presence of Cfp1/H3K4me3 at islands tended to have a negative correlation with the presence of chromatin rich in the silencing histone modification H3K27me3. Closer investigation of the Cfp1 protein reveals it to be a true non-methyl CGI binding factor in vivo and shRNA reduction of Cfp1 levels to around 10% of wild type resulted in a precipitous drop in H3K4me3 levels over CGIs without a dramatic reduction in global H3K4me3 levels. As Cfp1 has been shown to be part of the Set1 histone H3K4 methyltransferase complex responsible for this modification, this CXXC protein may be attracting this histone modifying complex and as such represents a method whereby the underlying DNA sequence (CpG) can drive the overlying epigenetic state. This study may go some way to understanding the functional significance of CGIs within the genome.

Indiana University-Purdue University Indianapolis (IUPUI) / CXXC finger protein 1 (CFP1) is an epigenetic regulator of H3K4 and cytosine methylation. Due to its role in establishing and maintaining methylation patterns, CFP1 determines whether DNA is found in its euchromatin or heterochromatin state and as such whether genes are transcriptionally active or inactive. In stem cells, deficiency of CFP1 results in inability to differentiate and in murine embryos it results in periimplantation death. Despite the demonstrated importance in developing tissue, the role of CFP1 in mature tissues, such as the liver, has yet to be elucidated. This study examined the role of CFP1 in maintaining liver homeostasis under conditions involving hepatocellular stress by examining liver regeneration, pregnancy-induced hepatomegaly, and non-alcoholic steatohepatitis (NASH) disease progression. The liver’s ability to recover was analyzed through liver:body mass ratios, blood serum analysis, liver histology, and qualitative observations. Deficiency of CFP1 in the livers of animals subjected to partial hepatectomies (PH) resulted in decreased liver regeneration capacity with liver mass restoration becoming significantly different starting at 48H post-PH and remaining so until 10D post-PH. This decreased regeneration appeared to be the result of reduced hepatocyte mitosis. Mouse dams lacking hepatic CFP1 mated with males expressing CFP1 displayed a proclivity for dystocia. Mice subjected to a fast food diet resulting in NASH while lacking hepatic CFP1 experienced decreased weight gain and hepatic lipid accumulation compared to their CFP1 expressing counterparts. Through these three studies, the critical role of CFP1 for the maintenance of liver homeostasis was demonstrated.