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. Overall design: 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).