PCGF5 is required for neural differentiation of embryonic stem cells [ChIP-seq]. PCGF5 is required for neural differentiation of embryonic stem cells [ChIP-seq]

Polycomb-group proteins are key regulators of the transcriptional programs that maintain stem cell identity and dictate lineage specification. Polycomb repressor complex 1 (PRC1) contains the E3 ligases RING1A/B, which monoubiquitinate lysine 119 at histone H2A (H2AK119ub1) to regulate gene expression. PRC1 has been sub-classified into six major complexes based on the presence of a PCGF subunit. Here, we find that Pcgf5, one of six PCGF paralogs, is an important requirement in the differentiation of mouse embryonic stem cells (mESCs) towards a neural cell fate. Although PCGF5 is not required for mESC self-renewal, its loss blocks mESC neural differentiation by activating the SMAD2/TGF-β signaling pathway. Inhibition of SMAD2/TGF-β signaling or rescue by overexpression of Pcgf5 can restore the capability of mESCs to differentiate towards a neural cell fate. PCGF5 works by stimulating RING1B-dependent H2AK119ub1 both in vitro and in vivo, leading to the suppression of TGF-β signaling genes. PCGF5 loss-of-function prevents the reduction of H2AK119ub1 and H3K27me3 around neural specific genes and keeps them repressed. Our results showed that PCGF5 might function as both a repressor for SMAD2/TGF-β signaling pathway and a facilitator for neural differentiation. Together, our findings reveal a critical context-specific function for PCGF5 in directing PRC1 to control cell fate. Overall design: H2AK119ub1 and H3K27me3 ChIP-seq with a drosophila chromatin spike-in were performed in wild type, PCGF5-depleted mESCs and the differentiated NPCs at day 6 after neural differentiation of mESCs, respectively. PCGF5 ChIP-seq was performed in NPCs at day 6 after neural differentiation of mESCs.

多梳家族蛋白（Polycomb-group proteins）是维持干细胞身份并调控谱系特化的转录程序关键调控因子。多梳抑制复合体1（Polycomb repressor complex 1, PRC1）包含E3泛素连接酶（E3 ligases）RING1A/B，后者可单泛素化组蛋白H2A第119位赖氨酸，形成H2AK119ub1修饰以调控基因表达。PRC1可根据其所包含的PCGF亚基（PCGF subunit）的差异，被划分为6种主要亚型复合体。本研究发现，6种PCGF旁系同源蛋白之一的Pcgf5，在小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）向神经细胞命运（neural cell fate）分化的过程中发挥关键调控作用。尽管PCGF5并非mESC自我更新（self-renewal）所必需，但其缺失会通过激活SMAD2/TGF-β信号通路（SMAD2/TGF-β signaling pathway）阻断mESC的神经分化。抑制SMAD2/TGF-β信号通路，或通过过表达（overexpression）Pcgf5挽救该表型，均可恢复mESC向神经细胞分化的能力。PCGF5可通过在体外（in vitro）与体内（in vivo）促进RING1B介导的H2AK119ub1修饰，进而抑制TGF-β信号通路相关基因的表达。PCGF5功能缺失（loss-of-function）会阻碍神经特异性基因（neural specific genes）区域的H2AK119ub1与H3K27me3修饰水平下降，并维持这些基因处于转录沉默状态。本研究结果显示，PCGF5既可作为SMAD2/TGF-β信号通路的抑制因子，又可作为神经分化的促进因子。综上，本研究揭示了PCGF5通过指导PRC1调控细胞命运的关键情境特异性功能。整体实验设计：分别在野生型（wild type）小鼠胚胎干细胞、PCGF5敲除的mESCs，以及小鼠胚胎干细胞神经分化第6天的神经前体细胞（neural progenitor cells, NPCs）中，辅以果蝇染色质spike-in内参（drosophila chromatin spike-in）开展H2AK119ub1与H3K27me3的染色质免疫共沉淀测序（ChIP-seq）；同时在上述神经分化第6天的NPCs中进行PCGF5的ChIP-seq实验。