ATAC-seq analysis in 5F cells and AGM cells with EZH1 depletion

Blood develops in distinct stages. Haematopoietic progenitors in the embryo manifest restricted differentiation potential relative to definitive haematopoietic stem cells in adult bone marrow, which support lifelong multilineage haematopoiesis. To identify regulators of embryonic haematopoiesis, we screened chromatin modifiers and identified the Polycomb group protein EZH1 as a barrier to multilineage potential from pluripotent stem cells (PSCs). EZH1 was directly bound to bivalently poised, yet restricted, HSC and lymphoid genes in primitive progenitors; knockdown enabled robust generation of multilineage progenitors. Moreover, EZH1 haploinsufficiency promoted the generation of HSCs with long-term, multilineage and self-renewal potential from sites of embryonic haematopoiesis in vivo. Together, this work identifies EZH1 as a key epigenetic barrier to definitive haematopoiesis during embryonic development, and highlights the utility of chromatin modifiers as cell engineering targets to enhance blood differentiation from PSCs. Overall design: ATAC-seq was performed to determine the chromatin accessibility in 5F cells transduced with shRNAs for luciferase (control) or EZH1, or in Ezh1 WT, HET or KO embryonic day (E)10.5 AGM cells.血液发育具有明确的阶段性特征。胚胎来源的造血祖细胞，相较于成体骨髓内的定型造血干细胞（definitive haematopoietic stem cells），其分化潜能更为受限——后者可支撑终生多谱系造血过程。为鉴定胚胎造血的调控因子，本研究对染色质修饰因子进行了筛选，发现多梳家族蛋白EZH1是阻碍多能干细胞（pluripotent stem cells, PSCs）获得多谱系分化潜能的表观遗传屏障。EZH1可直接结合原始造血祖细胞中处于二价 poised 状态且功能受限的造血干细胞（haematopoietic stem cells, HSC）与淋巴样基因；敲低EZH1可高效生成多谱系造血祖细胞。此外，EZH1单倍体剂量不足可在体内促进胚胎造血位点生成具备长期多谱系自我更新潜能的造血干细胞。综上，本研究证实EZH1是胚胎发育过程中阻碍定型造血的关键表观遗传屏障，并凸显了染色质修饰因子作为细胞工程靶点以增强多能干细胞向血液细胞分化的应用价值。实验设计：本研究通过ATAC测序（ATAC-seq）检测染色质开放状态，受试样本涵盖转染荧光素酶（luciferase）靶向短发夹RNA（short hairpin RNA, shRNA，对照组）或EZH1靶向shRNA的5F细胞，以及Ezh1野生型（wild type, WT）、杂合型（heterozygote, HET）或敲除型（knockout, KO）的胚胎第10.5天（embryonic day 10.5, E10.5）主动脉-性腺-中肾（aorta-gonad-mesonephros, AGM）区域细胞。