How a disordered linker in the Polycomb protein Polyhomeotic tunes phase separation and oligomerization

Abstract

SummaryThe Polycomb Group (PcG) complex PRC1 represses transcription, forms condensates in cells, and modifies chromatin architecture. These processes are connected through the essential, polymerizing Sterile Alpha Motif (SAM) present in the PRC1 subunit Polyhomeotic (Ph).In vitro, Ph SAM drives formation of short oligomers and phase separation with DNA or chromatin in the context of a Ph truncation (“mini-Ph”). Oligomer length is controlled by the long disordered linker (L) that connects the SAM to the rest of Ph--replacingDrosophilaPhL with the evolutionarily diverged human PHC3L strongly increases oligomerization. How the linker controls SAM polymerization, and how polymerization and the linker affect condensate formation are not know. We analyzed PhL and PHC3L using biochemical assays and molecular dynamics (MD) simulations. PHC3L promotes mini-Ph phase separation and makes it relatively independent of DNA. In MD simulations, basic amino acids in PHC3L form contacts with acidic amino acids in the SAM. Engineering the SAM to make analogous charge-based contacts with PhL increased polymerization and phase separation, partially recapitulating the effects of the PHC3L. Ph to PHC3 linker swaps and SAM surface mutations alter Ph condensate formation in cells, and Ph function inDrosophilaimaginal discs. Thus, SAM-driven phase separation and polymerization are conserved between flies and mammals, but the underlying mechanisms have diverged through changes to the disordered linker.HighlightsDisordered linker connecting the SAM to the rest of Polyhomeotic has diverged over evolutionPHC3L promotes phase separation and changes the underlying mechanismPHC3L is predicted to contact the SAM through charge complementary interactionsEngineered charge complementarity between PhL and SAM promotes oligomerization and phase separationLinker interactions modify Ph condensates in cells and Ph function inDrosophilaimaginal discs