NOVEL FUNCTIONS OF POLYCOMB PROTEINS AND OGT GLYCOSYLTRANSFERASEIN CHROMATIN REGULATION AND TRANSCRIPTION CONTROL IN EMBRYONIC STEM CELLS

During my PhD course, I have been involved in studies aiming to identify novel properties of proteins having fundamental roles in the regulation of chromatin modifications and gene transcription. One of the two project was focused on the functional characterization of the products of the catalytic activity of Polycomb Repressive Complex 2 (PRC2). In spite of the wide knowledge about PRC2-dependent trimethylation of Lysine 27 of histone H3 (H3K27me3), the other forms of methylation on H3K27, namely mono (me1) and di-methylation (me2), are still poorly characterized. Using mouse embryonic stem cells (mESC) as model system, we were able to provide an extensive characterization of the functional properties of these methylation forms, highlighting their differential deposition along the genome, their fundamental role in the mechanisms of transcriptional regulation in mESC, and their potential implications during differentiation program. Our data demonstrated that while H3K27me1 was required for efficient transcription of genes and positively correlated with the deposition of H3K36me3, H3K27me2 was broadly deposited and protects the genome from aberrant firing of non specific cell type enhancers. My second project focused on the activity of the O-linked glycosyltransferase Ogt which is the only enzyme capable to catalyze O-linked GlcNAcylation within the cell. Sxc protein, which is the Drosophila orthologue of mammalian Ogt, is essential for Polycomb (PcG) function. Sxc null embryos showed lethal phenotypes at early developmental stages like those observed in PcG null embryos. Starting from this observation we found appealing to investigate whether Sxc function in Drosophila were conserved in mammals. In particular, we were interested in the discovery of possible role for Ogt in the context of PcG recruitment to chromatin and the mechanisms governing transcriptional regulation in mESC. With our study we have identified a novel partnership between Ogt and ten-eleven translocation (TET) proteins, which catalyze hydroxylation of methylated cytosine. We have shown that Tet1 protein recruits Ogt to target genes in proximity of transcription start sites (TSS) rich in cytosine-guanine dinucleotides (CpG). Tet1-Ogt co-localization at target genes correlated with low levels of cytosine modification, suggesting a role in the regulation of CpG island (CpGi) methylation.