A previously-unrecognized motif of transcription factor RYBP, hotspot of cancer-related mutations, is essential for the integrity of Polycomb repressive complex 1

Polycomb repressive complex 1 (PRC1) catalyzes monoubiquitination of histone H2A on Lys119, promoting gene silencing. Cells at different developmental stages and in different tissues express different PRC1 isoforms. All isoforms share the same catalytic core (subunits RING1B and PCGF) and vary in the composition of regulatory subunits, clustering in two major classes. Canonical isoforms (cPRC1) are regulated by CBX-like subunits, while variant isoforms (vPRC1) are regulated by RYBP-like subunits. The molecular bases for how regulatory subunits affect the structural assembly of the complex and its catalytic activity are still largely unknown. To fill this knowledge gap, here we have specifically studied how RYBP regulates vPRC1 structure and function. Integrating the machine-learning algorithm AlphaFold2 and NMR, we have identified novel vPRC1 structural motifs in RING1B and RYBP. While the new RING1B motif is dispensable for vPRC1 assembly, the RYBP motif is essential for mediating inter-subunit interactions between RYBP and the catalytic RING1B-PCGF4 heterodimer. Importantly, the RYBP motif harbors cancer-related mutations systematically positioned on the same face of a putative transiently-forming -helix. Biochemical, biophysical and enzymatic characterization of purified cancer-related mutants confirm that this region is crucial for the structural stability of the complex. Overall, our data offer novel insights into the molecular architecture of vPRC1 and the effects of its regulatory subunit on the biochemical, structural, enzymatic, and physio-pathological properties of the complex.