Role of the polycomb protein EED in the propagation of repressive histone marks

Abstract

Polycomb group proteins have an essential role in the epigenetic maintenance of repressive chromatin states. The gene-silencing activity of the Polycomb repressive complex 2 (PRC2) depends on its ability to trimethylate lysine 27 of histone H3 (H3K27) by the catalytic SET domain of the EZH2 subunit, and at least two other subunits of the complex: SUZ12 and EED. Here we show that the carboxy-terminal domain of EED specifically binds to histone tails carrying trimethyl-lysine residues associated with repressive chromatin marks, and that this leads to the allosteric activation of the methyltransferase activity of PRC2. Mutations in EED that prevent it from recognizing repressive trimethyl-lysine marks abolish the activation of PRC2 in vitro and, in Drosophila, reduce global methylation and disrupt development. These findings suggest a model for the propagation of the H3K27me3 mark that accounts for the maintenance of repressive chromatin domains and for the transmission of a histone modification from mother to daughter cells.

Figure 1. Trimethyl-lysine binding to an aromatic cage on Eed

Ribbons representation of the Eed/H3K27me3 complex where Eed is coloured grey and the histone peptide is coloured yellow with its methyl-lysine side chain shown in stick representation. The Cα positions of the aromatic cage are shown as blue circles, and the Cα position of tyrosine 358 by a red circle. The bottom panel shows the methyl-lysine binding site with 2fo-fc electron density for the four cage residues and the H3K27me3 peptide. Designed mutations to the cage are shown in red in parentheses. The side-chain of methionine 256 is also shown; this is equivalent to Met-236 in esc which has been identified from classical genetic screens in Drosophila as essential for the function of Eed.

Figure 2. Interactions between Eed and trimethylated histone peptides

(A) Schematic representation of the interaction between Eed and H3K27me3. For clarity, the aromatic methyl-lysine binding cage has been omitted and the methylated lysine side-chain shown as a yellow circle. Hydrogen bonds from the main-chain carbonyl of the methyl-lysine, and the residue immediately N-terminal to it, with Eed are shown as dashed lines. The green hatching indicates the hydrophobic pocket on EED which accommodates the alanine side chain two residues before the methyl-lysine. (B) Eed is shown in surface representation with a composite of two of its cognate peptides shown in sticks representation and coloured yellow (H3K9me3) and pink (H4K20me3). (C) shows the pocket on Eed that accommodates Ala (−2) from the H3K9me3 peptide while (D) shows the other pocket that contains and Leu (+2) from the H4K20me3 peptide. The Eed surface is coloured according to atom type.

Figure 3. Eed and PRC2 interaction with Chromatin

Pull-down experiment to analyze the interaction between Eed full-length, PRC2-Ezh2 wild type or reconstituted with Eed Tyr-365Ala and H3K27 modified chromatin (left) or between PRC2-Ezh2 wild type and H3K9 modified chromatin (right). Note that “beads only” control for interaction H3K9 modified chromatin is not shown but was identical to the control shown in the figure.

Figure 4. Peptide mimicking repressive marks stimulates PRC2 activity

(A) Left, coomassie blue staining of reconstituted PRC2-Ezh2 complex. Right, HMT assay with PRC2-Ezh2 alone or in the presence of 10 and 40 μM H3K27, unmodified, mono, di or tri methylated peptides. (B) Titration of the methyl donor (S-adenosyl-Methionine) in the presence of H3K27me0/1/2/3 peptides (C) Nucleosome titration in the presence of H3K27me0/3 peptides. (D) Left, Coomassie blue staining of reconstituted PRC2-Ezh2 Tyr365Ala complex and right, HMT assay with the corresponding complex in the same condition as (A) (E) Relative PRC2 histone methyltransferase activity in the presence of various peptides as indicated in the legend. (F) Table indicating the peptides used for the stimulation study as well as their Kd values (μM) for ΔEed binding (G) Relative PRC2 histone methyltransferase activity in the presence of various peptides as indicated in the legend.

Figure 5. The aromatic cage in Drosophila ESC is important for its in vivo function

(A) Left, Coomassie blue staining of reconstituted dPRC2-Ez complex. Right, HMT assay with dPRC2-Ez and H3K27, unmodified, mono, di or tri methylated peptides. (B) Top, amino acid residues Phe-77, Tyr-338 and Phe-345 that were mutated to alanine in Drosophila ESC and corresponding residues in Eed. Bottom, schematic representation of transposon constructs. The Myc-tagged ESC or its mutants are expressed under the control of the esc gene promoter. (C) Rescue experiment. Details of the crossing schemes are shown in Supplementary Figures S9 and S10. Several independent lines were examined for each transgenic construct and showed the same phenotype except for one line of Myc-ESC Tyr338Ala. In the case of Myc-ESC Phe345Ala, transgenes were inserted at •C31 att sites at 68E and 86Fb, respectively. For direct comparison, wild type Myc-ESC lines were also established at the same chromosomal location and showed the same results as other wild type Myc-ESC lines established by conventional P-element transformation (D) ESC aromatic cage mutation does not impair binding to E(Z). Both Myc-ESC and Myc-ESC Phe-77Ala co-immunoprecipitated E(Z) from ovarian extracts of heterozygous esc⁻escl⁻ flies. The double Myc-ESC bands are caused by the well known phosphorylation of ESC. (E) Scheme indicating the genomic location of primers used for ChIP. (F) ChIP analysis of E(Z) binding to the bxd PRE (FM4) in homozygous esc⁶, escl^d01514 carrying the same Myc-ESC transgenes. In the presence of the aromatic cage mutations, E(Z) binding is strongly reduced. yw indicates the wild type stock with endogenous wild type ESC and ESCL. (G) ChIP analysis of the H3K27me3 distribution at four sites in the Ubx gene. H3K27me3 is drastically reduced in the presence of the aromatic cage mutations. (H) Histone H3K27 methylation in esc⁶, escl^d01514 double mutant larvae expressing Myc-ESC transgenes. Total protein lysates from wild type and homozygous esc⁶, escl^d01514 3rd instar larvae expressing Myc-ESC transgenes were used for western blot analysis with anti-H3, anti-H3K27me2 and anti-H3K27me3 antibodies. The aromatic cage mutations Myc-ESC Phe77Ala and Myc-ESC Phe345Ala cause an almost complete loss of histone H3K27 di- and trimethylation while wild type Myc-ESC fully restores the loss of H3K27 di- and tri-methylation in esc⁶, escl^d01514 double mutant larvae.