Regulation of Polyhomeotic condensates by intrinsically disordered sequences that affect chromatin binding

Abstract

The Polycomb group (PcG) complex PRC1 localizes in the nucleus in the form of 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 PcG bodies may form by SAM-driven phase separation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. We therefore hypothesize 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 affects Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occur when the central, glutamine rich IDR is removed, which results in large Ph condensates. Like mini-Ph condensates, these condensates exclude chromatin. Chromatin fractionation experiments indicate that removal of the glutamine rich IDR reduces chromatin binding, while removal of either of the other IDRs increases chromatin binding. Our data suggest 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 and highlight the complexity of regulation of biological condensates housed in single proteins.