Dissecting Non-canonical PRC1 Functions In Embryonic Stem Cells

Polycomb group(PcG)proteins act primarily as epigenetic transcriptional repressors,regulating a series of embryonic developmental processes and playing a key role in the maintenance of embryonic stem(ES)cell characteristics.PcG proteins mediate developmental gene silencing via forming at least two distinct protein complexes known as Polycomb repressive complex 1(PRC1)and PRC2.PRC2 consists of the core subunits Eed,Suz12 and Ezhl/2.Ezhl/2 has methyl transferase activity that can catalyze di-and tri-methylation of histone H3 at lysine 27(H3K27me2/3).PRC1 contains the E3 ligases Ringl A/B,which monoubiquitinate lysine 119 at histone H2A(H2AK119ubl).PRC1 can be distinguished as canonical and non-canonical PRC1 based on their subcomponent composition.According to this classification,canonical PRC1 contains Cbx proteins(Cbx2/4/6/7/8),Phc 1/2/3,Pcgf2/4 and RinglA/B.By contrast,non-canonical PRC1 contains Rybp or Yaf2 instead of Cbxs.Further diversification of PRC 1 emerges from the mutually exclusive association of Ringl A/B with Pcgfl-6.There are at least six different groups of PRC1 complexes,PRC1.1-1.6,each comprising one of six Pcgf.However,there is no clear insight into how many functional PRC1 complexes exist and what is the biological relevance to such diversification.Here,we generated Yaf2 knockout and Pcgf3/5 single or double knockout ES cells via CRISPR/Cas9 genome edit tool.We found that Yaf2 null ES cells reveal growth retardation and display severe defects in ectoderm differentiation in both in vlvo and in vitro differentiation experiments.RNA-seq analysis revealed that Yaf2 is predominantly a transcriptional repressor of some genes that are involved in ectoderm development.Proteomics has shown that Yaf2 can be a core member of all types of PRC1 complexes.In addition,the activation of Yaf2 function requires phosphorylation of serine 166 by Gsk3,which contributes to the pronotion of the enzymatic activity of Ring1B.When mutation of phosphorylation site the monoubiquitination of H2AK119 was decreased.Therefore,our results shown that Yaf2 regulates H2AK119ubl via phosphorylation of s166,thereby inhibiting the expression of ectodermal-related genes in ES cells.In addition,in Pcgf 3/5 knockout ES cells,we found that although these mutant cells maintain their self-renewal and colony-forming ability,they show severe mesoderm differentiation defects during in vitro and in vivo differentiation.Using RNA-Seq to analyze transcriptional profiles of Pcgf3/5 single or double mutant ES cells,we reported that in contrast to the canonical role of the related PRC1 in gene repression,Pcgf3/5 mainly act as transcriptional activators involved in the expression of mesodermal differentiation-related genes.Proteomic approaches and ChIP-qPCR analyses helped establish an extended Pcgf3/5 interactome and identified several novel Pcgf3/5 interactors.These included Tex10,which can activate transcription by binding to transcriptional coactivator P300.Furthermore,Pcgf3/5 deletion in ES cells substantially reduced the occupancy of Tex10 and P300 at target genes.Finally,we demonstrate that Pcgf3/5 regulates the overall level of histone H2AK119ub1 in ES cells.In conclusion,our results show that Pcgf3/5 interacts with Tex10 and P300 to perform transcriptional activation in ES cells and functionally compensates in ES cell pluripotency maintenance.