The Polycomb system has fundamental roles in regulating gene expression during mammalian development. However, how it controls transcription to enable gene repression has remained enigmatic. Here, using rapid degron-based depletion coupled with live-cell transcription imaging and single-particle tracking, we show how the Polycomb system controls transcription in single cells. We discover that the Polycomb system is not a constitutive block to transcription but instead sustains a long-lived deep promoter OFF state, which limits the frequency with which the promoter can enter into a transcribing state. We demonstrate that Polycomb sustains this deep promoter OFF state by counteracting the binding of factors that enable early transcription pre-initiation complex formation and show that this is necessary for gene repression. Together, these important discoveries provide a rationale for how the Polycomb system controls transcription and suggests a universal mechanism that could enable the Polycomb system to constrain transcription across diverse cellular contexts.

Polycomb 系统在哺乳动物发育过程中调节基因表达中起着重要作用。然而，它如何控制转录以实现基因抑制仍然是个谜。在这里，使用基于 degron 的快速耗竭结合活细胞转录成像和单粒子跟踪，我们展示了 Polycomb 系统如何控制单细胞中的转录。我们发现 Polycomb 系统不是转录的组成型块，而是维持长寿命的深度启动子 OFF 状态，这限制了启动子进入转录状态的频率。我们证明 Polycomb 通过抵消使早期转录起始复合物形成的因子的结合来维持这种深度启动子 OFF 状态，并表明这是基因抑制所必需的。总之，这些重要发现为 Polycomb 系统如何控制转录提供了基本原理，并提出了一种通用机制，可以使 Polycomb 系统在不同细胞环境中限制转录。