TOWARD STRUCTURAL INSIGHTS INTO THE RECOGNITION OF H3K27 TRIMETHYLATION BY CANONICAL PRC1

Polycomb group (PcG) proteins are chromatin modifiers that epigenetically regulate the transcription of key developmental genes involved in embryogenesis, pluripotency and tumorigenesis. Alterations in their activity are hallmarks in developmental disorders and cancer as chromatin modifiers are one of the most frequently mutated groups of genes in human tumors. Polycomb Repressive Complex 1 (PRC1) catalyzes the monoubiquitination of histone H2A at lysine 119 (H2AK119ub1) and are classified as canonical (PRC1.2 and PRC1.4) and variant (PRC1.1, PRC1.3 and PRC1.6), which differ in subunit composition, chromatin recruitment and catalytic efficiency. Canonical PRC1 complexes are recruited to PRC2 target sites through the recognition of H3K27me3 by the CBX chromodomain, while variant PRC1 complexes are defined for the presence of RYBP instead of CBX and their chromatin recruitment is independent of PRC2 localization and activity. Although the enzymatic function of PRC1 has been characterized, the structural determinants underlying the functional heterogeneity of canonical and variant assemblies remain poorly understood. In this study, all PRC1 cores were reconstituted in vitro and purified as homogeneous complexes, enabling a direct comparison of their biochemical and structural properties. Ubiquitination assays revealed intrinsic differences in catalytic efficiency, with variant complexes displaying higher activity than canonical counterparts. These findings demonstrate that PRC1 catalytic activity is not determined solely by the presence of CBX or RYBP but depends on the identity of the associated PCGF subunit. Since cPRC1.2 both reads the H3K27me3 mark through the CBX7 chromodomain and writes the H2AK119ub1 modification via its RING1B–PCGF catalytic module, cryo-electron microscopy (cryo-EM) was employed to investigate the structural organization of cPRC1.2 bound to nucleosome substrates either lacking or carrying the H3K27me3 modification. In parallel, biolayer interferometry (BLI) measurements revealed a markedly increased binding affinity of cPRC1.2 for H3K27me3-modified nucleosomes compared to unmodified substrates. Together, these approaches support a model in which recognition of H3K27me3 stabilizes cPRC1.2 on chromatin, promoting a more productive engagement of the catalytic module and facilitating H2AK119 ubiquitination.