Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge

Abstract

Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide experimental support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.

Figure 1.

Identification of M33 as a functional homolog of PSC. (A) Coomasie-stained gel of PcG proteins purified from overexpression in Sf9 cells. (B) Schematic representation of G5E4 nucleosomal array used in assays. (5S) 5S nucleosomal positioning sequence; (HhaI) unique HhaI restriction sequence that is inaccessible when packaged around a histone octamer. (C) Agarose gel of REA assay. After reactions were completed, uncut and cut Cy5 end-labeled G5E4 DNA was separated on an agarose gel, scanned using a Typhoon PhosphorImager and quantified using ImageQuant software (GE Healthcare). The slower-migrating band represents the DNA that was not cleaved by HhaI (uncut), while the faster-migrating band represents cut DNA (arrowhead indicates cut DNA). (D) Graph of the data obtained by quantification of DNA bands in C. Graphs were created in Kaleidagraph software (Synergy) using a nonlinear sigmoidal curve fit. Error bars represent the standard deviation of three technical replicates. Apparent inhibition of remodeling was calculated by the following equation:

Figure 2.

Structure/function analysis of M33-mediated repression. (A) Diagram of M33 truncation mutants tested for biochemical activity. (B) Graph of inhibition activity of selected M33 C-terminal truncation mutants. Data were analyzed as in Figure 1. (C) Graph of inhibition activity of selected M33 N-terminal truncation mutants.

Figure 3.

The role of charge in M33-mediated repression activity. (A) Graph of predicted protein charge at pH 7.0 versus IC₅₀ as determined using Kaleidagraph software and performing a linear fit. Red circles represent data from M33 truncation mutants, and blue circles represent data from M33 charge mutants. (B) Schematic representation of the charge mutant proteins that were tested. (C) Coomasie-stained gel of M33 charge mutants expressed and purified from E. coli. (D) Plot of the quantification from the REA done with M33 charge mutants. (E) Coomasie-stained gel of M33 charge mutants in the context of the core PRC1 complex. Proteins were expressed and purified from Sf9 cells. (F) Plot of the quantification from the REA done with the charge mutant complexes. (G) Charge characteristics of the non-PcG basic proteins cloned. The accession numbers for the proteins are MrpL2, NP_079578.1; and CTF8, AAH23107.1. (H) Coomasie-stained gel of the non-PcG basic proteins expressed and purified from E. coli. (I) Plot of the quantification from the REA done with the non-PcG basic proteins.

Figure 4.

Analysis of evolutionary conservation of PcG function. (A) Phylogenetic tree of species containing RING domain or CHD proteins from UniProtKb protein database. The tree is based on alignments of 18S rRNA from each of the species. Number of predicted PcG proteins represents the number of each class of proteins that was found in the UniProtKb database. Number predicted to inhibit remodeling is the number of proteins from each class that is expected to have inhibition activity based on overall protein charge and regional charge. The bar represents 0.02 substitutions per site. (B) Charge properties of PcG proteins selected for in vitro activity analysis. The accession numbers for the proteins are Pcgf2, NP_001084738.1; Cbx7, NP_001017853.1; Mig-32, NP_502293.2; Cbx6, NP_001088074.1; Cbx8, AAI54356.1; and Pc1, NP_001081900.1. (C) Coomasie-stained gel of PcG proteins expressed and purified from E. coli. (D) Agarose gel of REA assay reaction products. (E) Plot of the quantification of results obtained in D.

Figure 5.

Compaction of nucleosomal arrays by mouse PcG proteins. (A) Representative EM images of nucleosomal arrays incubated with the indicated PcG protein. (B) Box plot representation of the measured maximal diameter of nucleosomal array particles. Particle length is the diameter of the smallest circle that can entirely surround one nucleosomal array. The box represents the upper and lower quartile, and the line splitting the box represents the mode. The open circles represent outliers, and the asterisks indicate a P-value of <0.0001 using Student's t-test. No protein, n = 72; Bmi1, n = 50; M33, n = 30.

Figure 6.

Compaction of nucleosomal arrays by diverse PcG proteins. (A) Representative images of nucleosomal particles incubated with various PcG proteins from different species. (B) Box plots of images as described above. No protein, n = 79; GST-Pcgf2, n = 86; Cbx7, n = 113; GST-Mig-32, n = 90; GST-Cbx6, n = 88; GST-Cbx8, n = 87; Pc1, n = 89.

Figure 7.

A model for chromatin compaction by the mPCC. (A) The mPCC is recruited to target loci, potentially through a variety of mechanisms. (DBP) DNA-binding protein. (B) The charged region of M33 (indicated by plus signs) interacts with nucleosomes to compact chromatin. (C) Further protein–protein interactions from other proteins in the core PRC1 complex drive spreading of compacted chromatin.