Thesis: The Study On The Biological Function Of PRC2 Complex In Mouse Embryonic Stem Cells

Embryonic stem cells(ESCs)characterized by self-renewal and pluripotency are derived from the inner cell mass(ICM)of mammalian blastocyst.Polycomb group(Pc G)proteins,as a paradigmatic model for epigenetic regulation of gene silencing,is evolutionarily conserved that playing a critical role in stem cell differentiation and cell fate commitment.Based on biochemical experiments,Pc G proteins can assemble into two major complexes,PRC1 and PRC2,which catalyze histone H2AK119 mono-ubiquitination and H3K27 methylation through their catalytic subunits RING1 and EZH1/2 respectively.In addition,PRC2 contains core subunits(EZH1/2,SUZ12,EED)and several auxiliary subunits.The PRC2.1 is comprised of the core complex with addition of a polycomb-like(PCL)homolog.The PRC2.2 contains core proteins as well as JARID2 and AEBP2.Extensive research over the past years has revealed that PRC2functions as a chromatin regulator according to the ability of chromatin binding and histone modification.However,the molecular mechanisms how PRC2 is recruited to chromatin and to maintain the level of H3K27 methylation are still poorly understood.Early studies reported that the loss of any of the core subunits of PRC2 results in lethality in early mouse embryos indicating the importance of the PRC2 complex in early embryonic development,however there is a lack of systematic studies on the function of PRC2 in mouse embryonic stem cells.In this study,we generated single and combined knockout cell lines of PRC2 core subunits and some auxiliary subunits using CRISPR-Cas9 technology in mouse embryonic stem cells(m ESCs).To explore the effect of the deletion of these genes on the ESC self-renewal and pluripotency,we performed colony formation assay,alkaline phosphatase staining,teratoma formation assay and flowcytometry analysis.We found only quadruple PRC2 knockout(Ezh1^?/?;Ezh2^?/?;Eed^?/?;Suz12^?/?)ESCs resulted in severe defects in self-renewal and pluripotency.Contrastively,colonies of each subunit of PRC2 m ESCs were normal in shape and size,and still had high alkaline phosphatase(AP)activity like those of WT;Then,we found Eed konckout or quadruple PRC2knockout teratomas lacked ectodermal structures;Moreover,cell-cycle and apoptosis analysis revealed cells lacking all the core subunits of PRC2 had a markedly extended G1 phase and a shortened S phase and displayed the same apoptotic rates as the WT ESCs.In addition,the PRC2.1/2.2 auxiliary subunits PCL1-3 and AEBP2/JARID2 had no significant differences compared with WT cells in these experiments.To further explore the molecular basis of impairment of pluripotency and self-renewal observed in quadruple PRC2 knockout ESCs,we performed RNA-seq of quadruple PRC2knockout ESCs.The expression of germ layer and germline genes was significantly increased in quadruple PRC2 knockout ESCs.Subsequently,the results were confirmed by quantitative real-time PCR and western blot.Notably,the analysis of published Ch IP-Seq data suggested PRC2 core subunits can directly bind to the promoters of germ layer genes.In summary,we can conclude that core proteins of PRC2 repress germ layer genes in the maintenance of ES cell self-renewal and pluripotency via a synergistic mechanism.