Profiling of PRC1 and PRC2 occupancy reveals the preponderant role of PRC1 in regulating the limb development program [RNA-seq]

The Polycomb Repressive Complexes (PRC) are key players in the regulation of tissue-specific gene expression through their ability to epigenetically silence developmental genes. They are subdivided into two multicomponent complexes, PRC1 and PRC2, functioning through post-translational modifications of histone tails. A large body of work has revealed functional interactions between PRC1 and PRC2, whereby trimethylation of lysine 27 on histone H3 (H3K27me3) by PRC2 contributes to the recruitment of canonical PRC1 (cPRC1). In parallel, a PRC2-independent binding of PRC1 has been uncovered and referred to as non-canonical PRC1 or variant PRC1 (vPRC1). Moreover, PRC1-dependent ubiquitination of lysine 119 on histone H2A is involved in recruiting PRC2/propagating PRC2-dependent H3K27 trimethylation. While it was initially assumed that cPRC1 and vPRC1 bind distinct targets, subsequent evidences pointed to cPRC1 and vPRC1 sharing a significant subset of their targets. In turn, this raises the question of PRC2/cPRC1 contribution to gene regulation. Here, we show that, in the developing limb, PRC2 inactivation barely affects PRC1 occupancy, consistent with the fact that the majority of PRC2-bound loci can also be bound by vPRC1 (RYBP-PRC1), both in wild type and PRC2 mutant limbs. Consistent with this, we found that loci bound by CBX2, a PRC1 subunit involved in the recognition of H3K27me3 and thereby recruitment of cPRC1, are, for the vast majority, also bound by vPRC1. Intriguingly, analysis of PRC2 mutant limbs revealed that while a large part of CBX2 occupancy is lost in absence of PRC2 function, as expected from the absence of H3K27me3, there is a significant number of loci retaining CBX2 binding and even few loci gaining CBX2 binding. Importantly, these loci corresponds to developmental genes and include most genes known as playing a key role in limb morphogenesis. Based on the importance of vPRC1 in gene silencing, our findings explain why PRC2 inactivation affects rather moderately the limb genetic program and questions the specific functional role of cPRC1/PRC2 in gene regulation. RNA-seq analysis in proximal and distal limb tissue at E12.5多梳抑制复合物（Polycomb Repressive Complexes，PRC）是通过表观遗传沉默发育基因的能力，调控组织特异性基因表达的核心调控因子。该复合物可分为两种多组分复合物：PRC1与PRC2，二者通过对组蛋白尾端施加翻译后修饰发挥功能。大量研究已揭示PRC1与PRC2之间存在功能互作：PRC2介导组蛋白H3第27位赖氨酸三甲基化（H3K27me3），可招募经典型PRC1（canonical PRC1，cPRC1）。与此同时，研究人员还发现了PRC2非依赖型的PRC1结合模式，即非经典型PRC1或称变异型PRC1（variant PRC1，vPRC1）。此外，PRC1介导的组蛋白H2A第119位赖氨酸泛素化，可参与招募PRC2并维持PRC2依赖的H3K27三甲基化过程。尽管最初认为cPRC1与vPRC1结合的靶位点存在差异，但后续研究证据表明，二者共享大量共有靶位点。这一发现随即引出了PRC2/cPRC1对基因调控的贡献这一科学问题。本研究显示，在发育中的肢体组织中，PRC2失活几乎不会影响PRC1的占据情况，这与以下事实一致：无论是野生型还是PRC2突变型肢体中，绝大多数结合PRC2的基因座同样可被vPRC1（RYBP-PRC1）结合。与此一致的是，我们发现作为PRC1亚基的CBX2，其功能为识别H3K27me3并招募cPRC1，该蛋白结合的基因座绝大多数同样可被vPRC1结合。有趣的是，对PRC2突变型肢体的分析显示：尽管如预期般，在PRC2功能缺失时，大部分CBX2的占据位点因H3K27me3的缺失而丢失，但仍有大量基因座保留了CBX2的结合，甚至有少数基因座出现了CBX2结合的增强。重要的是，这些基因座对应发育基因，且包含绝大多数已知在肢体形态发生中发挥关键作用的基因。鉴于vPRC1在基因沉默中的重要作用，我们的研究结果解释了为何PRC2失活对肢体基因程序的影响较为温和，并对cPRC1/PRC2在基因调控中的特定功能角色提出了质疑。本研究对胚胎第12.5天（E12.5）的肢体近、远端组织开展了RNA测序（RNA-seq）分析。