Regulation of Polyhomeotic Condensates by Intrinsically Disordered Sequences That Affect Chromatin Binding.

The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph) forms phase-separated condensates with DNA or chromatin in vitro, suggesting that PcG bodies may form through SAM-driven phase separation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. We therefore hypothesized that sequences outside of mini-Ph, which are predicted to be intrinsically disordered, are required for proper condensate formation. We identified three distinct low-complexity regions in Ph based on sequence composition. We systematically tested the role of each of these sequences in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affected Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occurred when the central, glutamine-rich intrinsically disordered region (IDR) was removed, which resulted in large Ph condensates. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. Chromatin fractionation experiments indicated that the removal of the glutamine-rich IDR reduced chromatin binding and that the removal of either of the other IDRs increased chromatin binding. Our data suggest that all three IDRs, and functional interactions among them, regulate Ph condensate size and number. Our results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation. Our observations highlight the complexity of regulation of biological condensates housed in single proteins.

Phase separation by the polyhomeotic sterile alpha motif compartmentalizes Polycomb Group proteins and enhances their activity.

Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) has been shown to play an important role in chromatin compaction and large-scale chromatin organization. Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand the underlying mechanism, here we analyze the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. We show that overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, SAM-induced phase separation, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification.