PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency [RNA-seq]

The Polycomb repressive system plays a fundamental role in controlling gene expression during mammalian development. To achieve this, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) bind target genes and use histone modification-dependent feedback mechanisms to form Polycomb chromatin domains and repress transcription. The interrelatedness of PRC1 and PRC2 activity at these sites has made it difficult to discover the specific components of Polycomb chromatin domains that drive gene repression and to understand mechanistically how this is achieved. Here, by exploiting rapid degron-based approaches and time-resolved genomics we kinetically dissect Polycomb-mediated repression and discover that PRC1 functions independently of PRC2 to counteract RNA polymerase II binding and transcription initiation. Using single-cell gene expression analysis, we reveal that PRC1 acts uniformly within the cell population, and that repression is achieved by controlling transcriptional burst frequency. These important new discoveries provide a mechanistic and conceptual framework for Polycomb-dependent transcriptional control. Overall design: Mouse embryonic stem cells in which PRC1 can be depleted via the auxin-inducible degron (AID) system were profiled for gene expression using spike-in calibrated nuclear RNA-seq. Cells were treated with auxin for 2, 4, 8 or 24 hours in three independent biological replicates, and gene expression changes were analysed relative to the control untreated cells. Another mouse ESC line in which PRC2 can be depleted via the dTAG-inducible degron system was profiled in a similar manner to compare gene expression before and after 2 hours treatment with dTAG-13 compound.

多梳蛋白抑制系统在哺乳动物发育过程中对基因表达的调控发挥着基础性核心作用。为达成这一功能，多梳蛋白抑制复合体1和2（Polycomb repressive complexes 1 and 2，PRC1与PRC2）结合靶基因，并依托组蛋白修饰依赖的反馈机制组装形成多梳染色质结构域，进而抑制转录过程。PRC1与PRC2在上述位点的活性相互关联，这使得研究者难以明确多梳染色质结构域中驱动基因抑制的特定组分，也无法从机制层面阐明该抑制过程的实现路径。本研究借助快速降解子相关实验策略与时间分辨率基因组学技术，对多梳蛋白介导的基因抑制过程开展了动力学解析，并发现PRC1可独立于PRC2发挥功能，直接拮抗RNA聚合酶II的结合及转录起始过程。通过单细胞基因表达分析，我们进一步揭示出PRC1在整个细胞群体中均呈现均匀的作用模式，且基因抑制效应是通过调控转录爆发频率得以实现的。这些重要的全新研究发现为多梳蛋白依赖的转录调控提供了兼具机制阐释性与概念创新性的理论框架。实验整体设计：本研究针对可通过生长素诱导降解子（auxin-inducible degron，AID）系统敲低PRC1的小鼠胚胎干细胞，采用经spike-in校准的核RNA测序技术开展基因表达谱分析。实验设置三组独立生物学重复，分别使用生长素处理细胞2、4、8或24小时，并以未处理的细胞作为对照，分析基因表达的变化情况。另一株可通过dTAG诱导降解子系统敲低PRC2的小鼠胚胎干细胞系，采用类似的实验流程进行表达谱分析，用于对比经dTAG-13化合物处理2小时前后的基因表达差异。