Investigation On PRC2 Regulation Of Erythropoiesis And Its Mechanism

The transcriptome of a eukaryotic cell determines its cellular identity.During cell differentiation immature cell-specific genes need to be turned off and more mature cell-specific genes are turned on.Polycomb Repressive Complex 2（PRC2）plays critical regulatory roles in these processes.By methylation H3K27（H3K27me3）PRC2 marks genes for silencing.While PRC2 is known to be important in differentiation of stem cells,by suppressing both stem cell or differentiated cell-specific genes to maintain stemness or to ensure proper differentiation in different stem cells.It remains unclear,however,how PRC2 functions during somatic cell differentiation.In this thesis research we take erythroid differentiation as a model to investigate PRC2 regulation during somatic cell differentiation and hypothesize that:PRC2 suppresses the expressions of both immature cell-and mature cell-specific gene expression to ensure normal cell differentiation.PRC2 is consisted of 3 core subunits,Eed,Suz12 and Ezh1/Ezh2,which harbors the catalytic activity.In this study we generated a conditional allele of Ezh2,the main catalytic gene of PRC2,by crossing a conditional Ezh2 knockout mouse with a hematopoietic-specific Vav^Cre mouse.The conditional knockout mouse(Ezh2f^l/flVav^Cre,the so call KO mouse thereafter)exhibited anemic phenotype and was found to have significant accumulation of cells in R2 stage（Cd71⁺Ter119^-）and much few R3（Cd71⁺Ter119⁺）cells than in the WT mouse,suggesting defects in erythroid development.Ezh2 deletion unproportionally impacted R2 and R3 cells in their proliferation and apoptosis.Proportion of proliferating R3 cells was greatly reduced in KO mice,while only a mild reduction was observed in the R2 cells.Similarly,Ezh2 inactivation profoundly increased cell death in R3 cells,but it only mildly impacted the R2 cells.Thus,it appeared that R2 to R3 transition may be the most critical PRC2 regulation point during erythroid development.In consistence with this,Ezh2 knockout induced the most cell death at the transitional cells between R2 and R3,termed R2-3,and the impact of Ezh2 KO on their proliferation was comparable to the R3 cells.At the molecular level,Ezh2 knockout also induced the highest number of differential expression genes in R2-3 stage between KO and WT cells,among all the stages analyzed.Ezh2 knockout derepressed a group of genes in R2-3 and R3 cells,whose expression was normally down-regulated during R2 to R3 transition.These genes are marked by 3m H3K27 at R3 stage.Overexpression of some of these genes was known to cause defects in erythroid development.Failure to down-regulate these genes in the KO cells would lead to differentiation defects.Wild type R3 cells expressed high levels of autophagy genes,in consistence with elevated autophagy activity during terminal maturation of erythrocytes.Ezh2 deficiency,however,led to premature upregulation of autophagy genes and increased autophagy activity was observed in the KO R2-3 cells.Abnormally high autophagy activity may be detrimental to the cells.In this study we identify a critical transitional stage during erythroid development,R2-3,bridging the early and terminal maturations during erythroid development.PRC2 is crucial for these transitional cells as it plays a central role in governing their gene expression.On the one hand,PRC2 suppresses immature stage-specific genes,whose persistent expression blocks cell differentiation;on the other hand,PRC2 also suppresses more mature cell-specific genes,preventing them from premature expression,which can be detrimental to the cells.