A core subunit of Polycomb repressive complex 1 is broadly conserved in function but not primary sequence

Abstract

Polycomb Group (PcG) proteins mediate heritable gene silencing by modifying chromatin structure. An essential PcG complex, PRC1, compacts chromatin and inhibits chromatin remodeling. In Drosophila melanogaster, the intrinsically disordered C-terminal region of PSC (PSC-CTR) mediates these noncovalent effects on chromatin, and is essential for viability. Because the PSC-CTR sequence is poorly conserved, the significance of its effects on chromatin outside of Drosophila was unclear. The absence of folded domains also made it difficult to understand how the sequence of PSC-CTR encodes its function. To determine the mechanistic basis and extent of conservation of PSC-CTR activity, we identified 17 metazoan PSC-CTRs spanning chordates to arthropods, and examined their sequence features and biochemical properties. PSC-CTR sequences are poorly conserved, but are all highly charged and structurally disordered. We show that active PSC-CTRs--which bind DNA tightly and inhibit chromatin remodeling efficiently--are distinguished from less active ones by the absence of extended negatively charged stretches. PSC-CTR activity can be increased by dispersing its contiguous negative charge, confirming the importance of this property. Using the sequence properties defined as important for PSC-CTR activity, we predicted the presence of active PSC-CTRs in additional diverse genomes. Our analysis reveals broad conservation of PSC-CTR activity across metazoans. This conclusion could not have been determined from sequence alignments. We further find that plants that lack active PSC-CTRs instead possess a functionally analogous PcG protein, EMF1. Thus, our study suggests that a disordered domain with dispersed negative charges underlies PRC1 activity, and is conserved across metazoans and plants.

Fig. 1.

Sequences of diverse metazoan PSC-CTRs are highly charged, intrinsically disordered and poorly conserved. (A) Schematic representation of the homology region (HR) (amino acids 264–463) and the large, disordered C-terminal region (CTR) (amino acids 456–1603) of PSC. Gray box (amino acids 263–302) represents the conserved RING-finger domain. (B) Far-UV circular dichroism spectrum of the intrinsically disordered D. melanogaster PSC-CTR. A minimum of ellipticity at 200 nm is observed, consistent with a disordered structure. The CD spectrum of the well-folded globular protein bovine serum albumin is shown for comparison. (C) PSC-CTR sequences are significantly less conserved than PSC-HR sequences (P = 1.6 × 10^-35, two sample t-test with unequal variance). (D) Charge density plots of experimentally tested PSC-CTRs (named PSC, Su(z)2, PSC1, and PSC2). The accompanying cladogram is based on published metazoan phylogenies (–47). (E) Coomassie-strained gel of PSC-CTRs purified from Baculovirus-infected Sf9 cells. Black circle denotes the band corresponding to B. malayi PSC-CTR. Asterisk on this and all gels indicates HSC70 contaminant. Errors in all figures and tables represent standard deviation. A similar contaminant was purified away from D. melanogaster PSC through gel filtration chromatography and shown not to be active (15). The region in each “parent” PSC protein corresponding to PSC-CTR is listed in Table S2B.

Fig. 2.

Most PSC-CTRs bind DNA tightly and inhibit chromatin remodeling. Summary of measured K_d from double-filter binding assays and 50% inhibition points from Restriction Enzyme Accessibility (REA) assays for all experimentally tested PSC-CTRs. K_d values represent the affinity of PSC-CTRs for free DNA, while 50% inhibition points represent the ability of PSC-CTRs to inhibit chromatin remodeling.

Fig. 3.

The extent of contiguous negative charge in PSC-CTR determines repressive activity. Repressive PSC-CTRs are depicted with box and whisker plots, while nonrepressive PSC-CTRs are represented by red crosses. The ends of the whiskers respectively represent the maximum and minimum data points; upper and lower bounds of the box represent the upper and lower quartiles; horizontal line through the box is the median. Nonrepressive PSC-CTRs are D. pulex PSC1 and PSC2, which were experimentally tested in this study, and M. musculus BMI1 and X. laevis PCGF2, which were previously tested (43). (A) Overall charge and local positive charge properties cannot distinguish the repressive activities of PSC-CTR. “Net charge” is calculated from each full-length PSC-CTR sequence. The average of the UniProt Knowledgebase is represented as a filled circle. “Max contiguous positive charge” represents the length of the longest positively charged stretch in the protein, normalized by total protein length. “Fractional positive charge” represents the fraction of 25-amino acid windows with charge greater than +3.5 in each PSC-CTR. “Max local positive charge” represents the maximum charge attained amongst all 25-amino acid windows in each PSC-CTR. (B) Maximum contiguous negative charge distinguishes repressive from nonrepressive PSC-CTRs. This metric represents the length of the longest negatively charged domain within PSC-CTR, normalized by the length of the protein. (C) Representative charge plots of repressive PSC-CTRs (D. melanogaster and C. intestinalis) and nonrepressive PSC-CTRs (D. pulex), with red horizontal lines denoting the longest stretch of contiguous negative charge. Charge plots were generated from consecutive 25-amino acid sliding windows. (D) Coomassie-strained gel of PSC-act1 and PSC-act2 purified from Baculovirus-infected Sf9 cells. Although PSC1 and PSC2 have identical molecular weights, they migrate slightly differently. (E) Box and whisker plot depicts maximum contiguous negative charge of PSC-CTRs, PSC-act1 and PSC-act2 are indicated by green circles and the nonrepressive ‘parent’ D. pulex PSC1-CTR by a red cross. (F) Charge plots of the ‘parent’ D. pulex PSC1-CTR and the ‘activated’ PSC-act1 and PSC-act2. (G) Measurements of K_d and inhibition of chromatin remodeling for PSC-act1, PSC-act2, and the nonrepressive ‘parent’ D. pulex PSC1-CTR. All error bars denote standard deviation.

Fig. 4.

Broad conservation of PSC-CTR in metazoans and identification of plant EMF1 as a PSC-CTR-like protein. (A) PSC-CTR activity is superimposed upon an established metazoan phylogeny (–36). Taxa with repressive PSC-CTRs are colored black, while taxa that lack repressive PSC-CTRs are colored red. PSC-CTR activity in bolded taxa was experimentally tested, while activity in nonbolded taxa was predicted from their maximum contiguous negative charge, intrinsic disorder, and amino acid composition. Yellow asterisks denote taxa that possess the PSC-like protein EMF. Data from M. musculus and D. rerio PSC-CTRs were obtained from (43). Note that the A. coerulea genome was queried for repressive PSC-CTRs, but subsequent affinity purification and biochemical analysis was performed on EMF1 from Aquilegia vulgaris (a very closely related species to A. coerulea). (B) Coomassie-strained gel of A. thaliana EMF1 and A. vulgaris EMF1 purified from Baculovirus-infected Sf9 cells. The asterisk denotes a an approximately 70 kDa band, likely to be HSC70. The closed bracket denotes approximately 55 kDa bands, likely to be β-tubulin. (C) Box and whisker plot depicting maximum contiguous negative charge of PSC-CTRs, relative to EMF1 proteins (yellow asterisks).

Fig. P1.

Conserved charge properties determine chromatin modifying activity of a disordered domain in Polycomb proteins. (A) The C-terminal region of Drosophila melanogaster Polycomb protein PSC (PSC-CTR) modifies chromatin structure to inhibit chromatin remodeling. (B) The PSC-CTR is highly charged and disordered; metazoan PSC homologues and a Polycomb protein in plants have similar properties (high overall charge and intrinsic disorder) but not sequence.