EHMT2 represses totipotency- and differentiation-associated genes in pluripotent stem cells through distinct regulatory modes [Cut & Run]. EHMT2 represses totipotency- and differentiation-associated genes in pluripotent stem cells through distinct regulatory modes [Cut & Run]

Mouse embryonic stem cells (mESCs) and other naïve pluripotent stem cells, under specific conditions, can reverse typical developmental trajectories and de-differentiate into 2-cell-like cells (2CLCs) that resemble the mammalian embryo during zygotic genome activation (ZGA). We implemented a multipurpose allele for acute protein depletion and efficient immunoprecipitation to dissect the molecular functions of EHMT2 in mESCs. This approach allowed us to define different types of EHMT2 target genes and distinct modes of chromatin engagement and repression. Most notably, EHMT2 directly represses large clusters of co-regulated gene loci that comprise a significant fraction of the 2CLC-specific transcriptome by initiating H3K9me2 spreading from distal LINE-1 elements. Conversely, the loss of EHMT2 allows the recruitment of the activator DPPA2/4 to promoter-proximal ERVs in 2CLCs, thus facilitating the mESC-to-2CLC transition. Repression of germ layer-associated transcripts by EHMT2 occurs outside of gene clusters in collaboration with ZFP462 and entails binding to non-repeat enhancers. Our data suggests that EHMT2 functions as an attenuator of the bidirectional differentiation potential of mouse naïve pluripotent stem cells and define molecular models for the gene-specific repressive activity of this essential histone methyltransferase. Overall design: MERVL GFP- EHMT dTAG lines were treated with DMSO/dTAG for 72hrs , Sorted for gfp positive and negative population. Cut and Run performed using anti-DPPA4 antibody (AF3730)

小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）及其他幼稚态多能干细胞（naïve pluripotent stem cells）在特定培养条件下，可逆转常规发育轨迹，去分化为类似合子基因组激活（zygotic genome activation, ZGA）阶段哺乳动物胚胎的2细胞样细胞（2-cell-like cells, 2CLCs）。
本研究构建了可用于急性蛋白耗竭与高效免疫沉淀的多用途等位基因，以解析EHMT2在mESCs中的分子功能。
该策略帮助我们明确了EHMT2的不同靶基因类型，以及染色质结合与基因抑制的不同模式。
尤为关键的是，EHMT2可通过从远端长散在核元件-1（LINE-1）起始H3K9me2（组蛋白H3赖氨酸9二甲基化）的扩散，直接抑制包含2CLC特异性转录组中相当一部分的大规模协同调控基因位点簇。
反之，EHMT2的缺失可使激活因子DPPA2/4被招募至2CLCs中启动子近端的内源性逆转录病毒元件（endogenous retroviruses, ERVs），从而促进mESC向2CLC的转化。
EHMT2与ZFP462协同作用，在基因簇之外抑制胚层相关转录本，该过程依赖于其与非重复序列增强子的结合。
本研究数据表明，EHMT2可作为小鼠幼稚态多能干细胞双向分化潜能的衰减因子，并为这一关键组蛋白甲基转移酶的基因特异性抑制活性构建了分子模型。
实验整体设计：将携带MERVL GFP标记的EHMT dTAG细胞系用二甲基亚砜（DMSO）或dTAG处理72小时，随后分选出GFP阳性与阴性细胞群；采用抗DPPA4抗体（AF3730）进行Cut&Run（切割与释放）测序实验。