Highlights d PRC1 catalysis drives PRC2 occupancy and H3K27me3 deposition at target sites d cPRC1 binding and PRC1-mediated chromatin interactions require PRC1 catalysis d DNA-binding vPRC1 complexes occupy target sites independently of PRC1 catalysis d PRC1 catalytic activity is essential for Polycomb-mediated gene repression

The Polycomb repressive system is an essential chromatin-based regulator of gene expression. Despite being extensively studied, how its target genes are selected and whether its histone modifying activities are required for transcriptional repression remains controversial. Here, we directly test the requirement for PRC1 catalytic activity in Polycomb system function. We demonstrate that a mutation widely used to disrupt PRC1 catalysis is hypomorphic, complicating the interpretation of previous studies. To overcome this, we develop a new inducible mutation system in embryonic stem cells that completely ablates PRC1 catalytic activity, revealing that catalysis by PRC1 drives Polycomb chromatin domain formation and higher-order chromatin interactions. In the absence of catalysis, we uncover the primary DNA-based targeting determinants that direct Polycomb target site selection. Finally, we discover that Polycomb-mediated gene repression requires PRC1 catalytic activity. Together these discoveries provide compelling new evidence supporting a PRC1-initiated pathway for Polycomb system function in gene regulation.

The precise regulation of gene transcription is required to establish and maintain cell type-specific gene expression programs during multicellular development. In addition to transcription factors, chromatin, and its chemical modification, play a central role in regulating gene expression. In vertebrates, DNA is pervasively methylated at CG dinucleotides, a modification that is repressive to transcription. However, approximately 70% of vertebrate gene promoters are associated with DNA elements called CpG islands (CGIs) that are refractory to DNA methylation. CGIs integrate the activity of a range of chromatin-regulating factors that can post-translationally modify histones and modulate gene expression. This is exemplified by the trimethylation of histone H3 at lysine 4 (H3K4me3), which is enriched at CGI-associated gene promoters and correlates with transcriptional activity. Through studying H3K4me3 at CGIs it has become clear that CGIs shape the distribution of H3K4me3 and, in turn, H3K4me3 influences the chromatin landscape at CGIs. Here we will discuss our understanding of the emerging relationship between CGIs, H3K4me3, and gene expression.

Transcription must be tightly controlled to regulate gene expression and development. However, our understanding of the molecular mechanisms that influence transcription and how these are coordinated in cells to ensure normal gene expression remains rudimentary. Here, by dissecting the function of the SET1 chromatin-modifying complexes that bind to CpG island-associated gene promoters, we discover that they play a specific and essential role in enabling the expression of low to moderately transcribed genes. Counterintuitively, this effect can occur independently of SET1 complex histone-modifying activity and instead relies on an interaction with the RNA Polymerase II-binding protein WDR82. Unexpectedly, we discover that SET1 complexes enable gene expression by antagonising premature transcription termination by the ZC3H4/WDR82 complex at CpG island-associated genes. In contrast, at extragenic sites of transcription, which typically lack CpG islands and SET1 complex occupancy, we show that the activity of ZC3H4/WDR82 is unopposed. Therefore, we reveal a gene regulatory mechanism whereby CpG islands are bound by a protein complex that specifically protects genic transcripts from premature termination, effectively distinguishing genic from extragenic transcription and enabling normal gene expression.

The Polycomb system plays fundamental roles in regulating gene expression during mammalian development. However, how it controls transcription to enable gene repression has remained enigmatic. Here we employ rapid degron-based depletion coupled with live-cell transcription imaging and single-particle tracking to uncover 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 new 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.