Synergy between variant PRC1 complexes defines Polycomb-mediated gene repression (ChIP-Seq)

The Polycomb system modifies chromatin and plays an essential role in repressing gene expression to control normal mammalian development. However, the components and mechanisms that define how Polycomb protein complexes achieve this remain enigmatic. Here we use combinatorial genetic perturbation coupled with quantitative genomics to discover the central determinants of Polycomb-mediated gene repression in mouse embryonic stem cells. We demonstrate that canonical Polycomb repressive complex 1 (PRC1), which mediates higher order chromatin structures, contributes little to gene repression. Instead, we uncover an unexpectedly high degree of synergy between variant PRC1 complexes which is fundamental to gene repression. We further demonstrate that variant PRC1 complexes are responsible for distinct pools of H2A monoubiquitylation that are associated with repression of Polycomb target genes and silencing during X-chromosome inactivation. Together, these discoveries reveal a new variant PRC1-dependent logic for Polycomb-mediated gene repression. Mouse embryonic stem cells in which distinct PCGF-containing PRC1 complexes can be conditionally removed individually or in different combinations ( Pcgf4-/-; Pcgf2fl/fl, Pcgf1fl/fl, Pcgf3fl/fl, Pcgf5fl/fl, Pcgf6fl/fl, Pcgf3/5fl/fl, Pcgf1/3/5fl/fl, Pcgf1/3/5/2fl/fl, Pcgf1/3/5/6fl/fl and Ring1a-/-; Ring1bfl/fl) and Mus domesticus (129S1) x Mus castaneus F1 hybrid ESC line with inducible full-length Xist transgene were profiled for PRC1 (H2AK119ub1 and RING1B, as well as subunits specific for distinct PRC1 complexes - PCGF1, PCGF2, CBX7, and PHC1) and PRC2 (H3K27me3 and SUZ12) binding and activity using spike-in calibrated native (histone modifications) or cross-linked ChIP-seq (Polycomb factors).

多梳蛋白（Polycomb）系统可修饰染色质，并在抑制基因表达以调控正常哺乳动物发育过程中发挥核心作用。然而，界定多梳蛋白复合物如何实现该功能的组分与机制仍尚不明确。本研究通过组合遗传扰动结合定量基因组学技术，在小鼠胚胎干细胞中揭示了多梳蛋白介导基因沉默的核心决定因子。本研究证实，介导高阶染色质结构的经典多梳抑制复合物1（PRC1）对基因沉默的贡献极小。与之相反，本研究揭示了变异型PRC1复合物间存在出乎意料的高度协同效应，而这正是基因沉默的核心基础。本研究进一步证实，变异型PRC1复合物负责催化不同池的H2A单泛素化（H2A monoubiquitylation），该修饰与多梳蛋白靶基因的沉默以及X染色体失活（X-chromosome inactivation）过程中的基因沉默密切相关。综上，本研究的这些发现揭示了一条依赖于变异型PRC1的全新多梳蛋白介导基因沉默的调控逻辑。本研究针对两类样本开展了表征分析：一类是携带可被条件性单独或组合敲除的不同PCGF型PRC1复合物的小鼠胚胎干细胞（基因型包括：Pcgf4-/-；Pcgf2fl/fl、Pcgf1fl/fl、Pcgf3fl/fl、Pcgf5fl/fl、Pcgf6fl/fl、Pcgf3/5fl/fl、Pcgf1/3/5fl/fl、Pcgf1/3/5/2fl/fl、Pcgf1/3/5/6fl/fl以及Ring1a-/-; Ring1bfl/fl）；另一类是携带可诱导全长Xist转基因的家鼠（Mus domesticus，129S1品系）×卡斯蒂利亚小家鼠（Mus castaneus）F1杂交胚胎干细胞系。实验采用加入校准内参的天然染色质ChIP-seq（用于检测组蛋白修饰）或交联染色质ChIP-seq（用于检测多梳蛋白因子），对PRC1复合物（包含H2AK119ub1、RING1B，以及不同PRC1复合物的特异性亚基PCGF1、PCGF2、CBX7和PHC1）与PRC2复合物（包含H3K27me3和SUZ12）的结合模式与活性进行了表征。