Polycomb group of proteins (PcGs) are essential multiprotein complexes that regulate, through chromatin repression and compaction, cell identity and cell-fate transitions ensuring the correct establishment of lineage-specific transcriptional programs. Due to their key roles in cellular homeostasis and proliferation, it is not surprising at all that their deregulation, in terms of expression levels or activity, has been linked to the development and sustainment of several types of human cancers. Aberrations affecting subunits of both Polycomb Repressive Complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2), the two major PcGs complexes, have been reported. EZH2, the catalytic subunit of PRC2 responsible for its methylation activity on lysine 27 of histone H3 (H3K27), is often over-expressed in human cancers. This correlates with global increased H3K27 trimethylation (H3K27me3) levels and with tumor prognosis. Recently, mutations affecting critical residues within EZH2 catalytic SET domain and thus impairing its activity have been described. Interestingly, both hyper-activating and inactivating mutations have been shown to affect EZH2 histone methylation activity. A complex scenario in which EZH2 can act as oncogene or tumor-suppressor depending on the cell-context, is emerging. Up to now very little is known about the biological role of the mutated forms of EZH2 and subsequent alteration of methylation patterns and, therefore, the aim of this thesis is to try to unravel the molecular mechanisms underlying these tumorigenic mutations. I took advantage of mouse embryonic stem cells (mESCs), representing a simple model system where PcGs activity is well characterized, and of the new powerful CRISPR/Cas9 genome-editing tool. At the beginning of this project CRISPR/Cas9 technology had just emerged as a versatile and powerful tool to perform highly efficient genome-editing in a variety of cell-types. I applied this approach to mESC to obtain relevant genetic cellular models to study the role of mutations affecting EZH2 activity. I generated isogenic mESC lines harboring physiological EZH2 Y726D and R685C-inactivating aminoacidic substitution. Moreover, a cellular model for K27M mutation, that affects EZH2 substrate histone H3.3 thus inhibiting its enzymatic activity, was obtained. Ezh2 and Ezh1 knock-out cells combined with homozygous EZH2 Y641N expressing cells allowed me to clarify several aspects regarding EZH1 and EZH2 interplay and cooperation within PRC2 activity, suggesting a context-dependent EZH1-compensative role. My preliminary results demonstrate that the differentiation capabilities of mESCs relies on H3K27me3 deposition whereas PRC2 recruitment to target loci occurs in an H3K27me3-independent manner. I coupled differentiation assays with location analyses (ChIP-qPCR and ChIP-seq), aimed to map chromatin association of Polycomb components and specific deposition of histone modifications, to elucidate the molecular mechanisms by which distinct mutations affect the activity of PRC2
