DNA binding reorganizes the intrinsically disordered C-terminal region of PSC in Drosophila PRC1

Abstract

Polycomb Group (PcG) proteins regulate gene expression by modifying chromatin. A key PcG complex, Polycomb Repressive Complex 1 (PRC1), has two activities: a ubiquitin ligase activity for histone H2A, and a chromatin compacting activity. In Drosophila, the Posterior Sex Combs (PSC) subunit of PRC1 is central to both activities. The N-terminal homology region (HR) of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase for H2A. The intrinsically disordered C-terminal region of PSC (PSC-CTR) compacts chromatin, and inhibits chromatin remodeling and transcription in vitro. Both the PSC-HR and the PSC-CTR are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used cross-linking mass spectrometry (XL-MS) to analyze the conformations of the PSC-CTR in PRC1 and how they change on binding DNA. XL-MS identifies interactions between the PSC-CTR and the core of PRC1, including between the PSC-CTR and PSC-HR. New contacts and overall more compacted PSC-CTR conformations are induced by DNA binding. Protein footprinting of accessible lysine residues in the PSC-CTR reveals an extended, bipartite candidate DNA/chromatin binding surface. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible PSC-CTR. Intramolecular interactions of the PSC-CTR detected by XL-MS can bring the high affinity DNA/chromatin binding region close to the core of PRC1 without disrupting the interface between the ubiquitin ligase and the nucleosome. Our approach may be applicable to understanding the global organization of other large IDRs that bind nucleic acids. HighlightsO_LIAn intrinsically disordered region (IDR) in Polycomb protein PSC compacts chromatin C_LIO_LICross-linking mass spectrometry (XL-MS) was used to analyze topology of the PSC IDR C_LIO_LIProtein footprinting suggests a bipartite DNA binding surface in the PSC IDR C_LIO_LIA model for the DNA-driven organization of the PSC IDR C_LIO_LICombining XL-MS and protein footprinting is a strategy to understand nucleic acid binding IDRs C_LI O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/130492v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@346c5dorg.highwire.dtl.DTLVardef@1b672b3org.highwire.dtl.DTLVardef@1247bfdorg.highwire.dtl.DTLVardef@48448e_HPS_FORMAT_FIGEXP M_FIG C_FIG