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. 2020 Aug 7;432(17):4856-4871.
doi: 10.1016/j.jmb.2020.07.002. Epub 2020 Jul 3.

DNA Binding Reorganizes the Intrinsically Disordered C-Terminal Region of PSC in Drosophila PRC1

Jin Joo Kang  1 , Denis Faubert  2 , Jonathan Boulais  3 , Nicole J Francis  4
Affiliations

DNA Binding Reorganizes the Intrinsically Disordered C-Terminal Region of PSC in Drosophila PRC1

Jin Joo Kang et al. J Mol Biol. .

Abstract

Polycomb Group proteins regulate gene expression by modifying chromatin. 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 of PSC assembles into PRC1, including partnering with dRING to form the ubiquitin ligase. The intrinsically disordered C-terminal region of PSC compacts chromatin and inhibits chromatin remodeling and transcription in vitro. Both regions of PSC are essential in vivo. To understand how these two activities may be coordinated in PRC1, we used crosslinking mass spectrometry to analyze the conformations of the C-terminal region of PSC in PRC1 and how they change on binding DNA. Crosslinking identifies interactions between the C-terminal region of PSC and the core of PRC1, including between N and C-terminal regions of PSC. New contacts and overall more compacted PSC C-terminal region conformations are induced by DNA binding. Protein footprinting of accessible lysine residues reveals an extended, bipartite candidate DNA/chromatin binding surface in the C-terminal region of PSC. Our data suggest a model in which DNA (or chromatin) follows a long path on the flexible disordered region of PSC. Intramolecular interactions of PSC detected by crosslinking 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 intrinsically disordered regions that bind nucleic acids.

Keywords: Polycomb; chromatin; crosslinking mass spectrometry; intrinsically disordered region; protein footprinting.

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Figures

Figure 1.
Figure 1.. Assembly into PRC1 affects PSC activity.
A. PRC1ΔPh (left) and PSC alone interact differently with chromatin. PRC1ΔPh has ubiquitin ligase activity and can compact chromatin at a ratio of 1 PRC1:3-4 nucleosomes. In contrast, PSC alone self-interacts and compacts chromatin at a ratio of 1:1 with nucleosomes. See Introduction. B. Schematic of PSC. Ubiquitin ligase activity and assembly into PRC1 requires the Homology Region (HR), while the disordered PSC-CTR binds DNA and compacts chromatin. C. Distribution of lysine residues in PRC1ΔPh. Black lines indicate the positions of lysines; gray bars indicate predicted disordered regions in each subunit, and known domains are shaded and labelled.
Figure 2.
Figure 2.. Identification of crosslinked peptides in PRC1ΔPh.
A. Venn Diagram of overlap of crosslinking sites in PRC1ΔPh identified with high confidence by pLink, MeroX, and SIM-XL. B. Graph of inter- and intra-protein crosslinking sites of PRC1ΔPh identified in the absence or presence of DNA. Note that the scores are calculated differently by each algorithm so that different thresholds are used in each case, and they cannot be directly compared. C-E. Maps of intra-protein crosslinks identified in PSC by each program. F. Representative spectrum of crosslinked peptides identified by MeroX. See sFig. 2 for spectra generated by pLink and SIM-XL.
Figure 3.
Figure 3.. XL-MS analysis of PSC in the context of PRC1ΔPh with and without bound DNA.
A, B. Intra-protein crosslinks identified in PSC in the absence (A) or presence (B) of DNA. C. Intra-protein crosslinks identified in both the absence and presence of DNA. D. Intraprotein crosslinks identified only in the absence (red) or presence (blue) of DNA.
Figure 4.
Figure 4.. Protein footprinting by chemical acetylation of accessible lysines to identify putative DNA/chromatin binding regions in the PSC-CTR.
A. Schematic of protein footprinting assay. B. Heat map of accessibility of lysines in PSC in PRC1ΔPh alone or with DNA or chromatin bound. Asterisks indicate lysines with significantly decreased acetylation in samples with DNA (black) or chromatin (red) relative to PRC1ΔPh alone. Green asterisk indicates a site that has increased accessibility when PRC1ΔPh is bound to chromatin. Blue lines indicate DNA Patch (DP) 1 and 2, and gray lines control regions used in part C. C. Average accessibility of lysines in DP1 and DP2 compared with the same number of lysines from adjacent control regions (con.P1, con.P2). Bars show average + SEM. p-values are for unpaired, two-tailed t-tests.
Figure 5.
Figure 5.. Mutation of a subset of lysines identified by protein footprinting decreases the DNA binding affinity of PRC1ΔPh.
A. Schematic of PSC-KA indicating sites of K-->A mutations in DP1. B. SYPRO Ruby stained SDS-PAGE of PRC1ΔPh-PSC-KA indicating that the mutations do not affect complex formation. C. Representative membranes from filter binding assay. D. Summary of filter binding experiments using two independent preparations of PRC1ΔPh and PRC1ΔPh-PSC-KA (n=3 binding assays). Curve fits were done using the mean of the replicates using a least squares non-linear regression (Y=ABmax*X/(X+Kd)+b).
Figure 6.
Figure 6.. Model for organization of the PSC-CTR.
A. Intra-protein interaction regions (PP) and candidate DNA/chromatin binding surfaces (DP) are largely distinct. B. Conceptual model of PSC organization. Diagram is not precisely scaled, and is meant to indicate possible folding of PSC that is consistent with crosslinking and footprinting data. Long-range crosslinks are shown by dashed lines because it was too difficult to draw these regions apposed. The model is conceptual, and not meant to imply that all contacts detected by XL-MS occur simultaneously. C. Model of the Ring domains of PSC/dRING bound to a nucleosome (based on PDB 4r8p) indicating the position of lysines in PP1A that crosslink to the PSC-CTR. Although speculative, the positions of these residues suggest the PSC-CTR (and possibly DNA or chromatin bound to it) can be brought close to the core of PRC1 without disrupting the interaction of the E3 ligase motif with the nucleosome. H3=blue, H4=orange, H2A=yellow, H2B=magenta, PSC=Cyan, dRING=green.
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