Mtf2-PRC2 control of canonical Wnt signaling is required for definitive erythropoiesis. [ChIP-seq]. Mus musculus

genomic loci or increase enzymatic activity, while PRC2 core proteins are required for complex stability and global levels of trimethylation of histone 3 at lysine 27 (H3K27me3). Here, we demonstrate a role for the classical PRC2 accessory protein Mtf2/Pcl2 in the hematopoietic system that is more akin to that of a core PRC2 protein. Mtf2-/- erythroid progenitors demonstrate markedly decreased core PRC2 protein levels and a global loss of H3K27me3 at promoter-proximal regions. The resulting de-repression of transcriptional and signaling networks blocks definitive erythroid development, culminating in Mtf2-/- embryos dying by e15.5 due to severe anemia. Gene regulatory network (GRN) analysis demonstrated Mtf2 directly regulates Wnt signaling in erythroblasts, leading to activated canonical Wnt signaling in Mtf2-deficient erythroblasts, while chemical inhibition of canonical Wnt signaling rescued Mtf2-deficient erythroblast differentiation in vitro. Using a combination of in vitro, in vivo and systems analyses, we demonstrate that Mtf2 is a critical epigenetic regulator of Wnt signaling during erythropoiesis and recast the role of polycomb accessory proteins in a tissue-specific context. Overall design: ChIP-Seq based binding measurements of H3K4me3, PCL2 and IgG controls for mouse embryonic fetal liver cells; for PCL2 wt and knockout sorted for Ter119 presence/absence CD71+Ter119-/lo and CD71+Ter119high cell fractions from fetal livers of e14.5 WT and Pcl2-/- embryos were FACS-sorted and crosslinked by formaldehyde. ChIP-Seq was performed for H3K27me3 (and IgG control) in both cell fractions from both genotypes. Pcl2 ChIP-seq and its IgG control was performed on both cell fractions from wild-type embryos.

基因组位点或提升酶活性，而多梳抑制复合体2（PRC2）核心蛋白是维持复合体稳定性以及组蛋白3赖氨酸27三甲基化（H3K27me3）全局水平所必需的。本研究阐明了经典PRC2辅助蛋白Mtf2/Pcl2在造血系统中的功能，其功能更近似于PRC2核心蛋白。Mtf2基因敲除（Mtf2-/-）的红系祖细胞中，PRC2核心蛋白水平显著降低，且启动子近端区域的H3K27me3全局水平缺失。由此引发的转录网络与信号网络去抑制作用阻断了定型红系发育，最终导致Mtf2-/-胚胎于胚胎期15.5天（e15.5）因重度贫血死亡。基因调控网络（GRN）分析显示，Mtf2可在成红细胞中直接调控Wnt信号通路，使Mtf2缺陷型成红细胞中的经典Wnt信号通路激活；而通过化学手段抑制经典Wnt信号通路，可在体外挽救Mtf2缺陷型成红细胞的分化缺陷。本研究结合体外实验、体内实验与系统生物学分析，证实Mtf2是红细胞生成过程中Wnt信号通路的关键表观遗传调控因子，并重塑了多梳辅助蛋白在组织特异性语境中的功能。实验整体设计：针对小鼠胚胎胎肝细胞，基于染色质免疫共沉淀测序（ChIP-Seq）检测H3K4me3、PCL2的结合情况，并设置IgG对照；本研究选取胚胎期14.5天（e14.5）野生型（WT）与Pcl2基因敲除（Pcl2-/-）小鼠的胎肝细胞，通过荧光激活细胞分选（FACS）分选出Ter119阳性/阴性的CD71+Ter119-/lo与CD71+Ter119high细胞亚群，随后进行甲醛交联。对两种基因型小鼠的上述两种细胞亚群，均开展H3K27me3（及IgG对照）的ChIP-Seq检测；同时对野生型小鼠的上述两种细胞亚群，开展Pcl2的ChIP-Seq检测并设置IgG对照。