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. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. 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. We systematically tested the role of each of these sequences in Ph condensates using live imaging of transfected Drosophila S2 cells. We identified three distinct low-complexity regions in Ph based on sequence composition. We therefore hypothesized that sequences outside of mini-Ph, which are predicted to be intrinsically disordered, are required for proper condensate formation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. 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. 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. The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies.
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