Binding of different histone marks differentially regulates the activity and specificity of polycomb repressive complex 2 (PRC2)

Abstract

The polycomb repressive complex 2 (PRC2) is the major methyltransferase for H3K27 methylation, a modification critical for maintaining repressed gene expression programs throughout development. It has been previously shown that PRC2 maintains histone methylation patterns during DNA replication in part through its ability to bind to H3K27me3. However, the mechanism by which PRC2 recognizes H3K27me3 is unclear. Here we show that the WD40 domain of EED, a PRC2 component, is a methyllysine histone-binding domain. The crystal structures of apo-EED and EED in complex respectively with five different trimethyllysine histone peptides reveal that EED binds these peptides via the top face of its β-propeller architecture. The ammonium group of the trimethyllysine is accommodated by an aromatic cage formed by three aromatic residues, while its aliphatic chain is flanked by a fourth aromatic residue. Our structural data provide an explanation for the preferential recognition of the Ala-Arg-Lys-Ser motif-containing trimethylated H3K27, H3K9, and H1K26 marks by EED over lower methylation states and other histone methyllysine marks. More importantly, we found that binding of different histone marks by EED differentially regulates the activity and specificity of PRC2. Whereas the H3K27me3 mark stimulates the histone methyltransferase activity of PRC2, the H1K26me3 mark inhibits PRC2 methyltransferase activity on the nucleosome. Moreover, H1K26me3 binding switches the specificity of PRC2 from methylating H3K27 to EED. In addition to determining the molecular basis of EED-methyllysine recognition, our work provides the biochemical characterization of how the activity of a histone methyltransferase is oppositely regulated by two histone marks.

Fig. 1.

Preferential binding of EED to trimethylated lysine histone peptides. (A) The top surface of EED harbors an aromatic cage consisting of Phe97, Tyr148, Trp364, and Tyr365, distinct from the EZH2-binding site shown in the crystal structure of EED in complex with an EZH2 fragment (30). The aromatic cage residues on the top surface of EED are displayed in a stick model. EED and the EZH2 peptide are colored in green and blue, respectively. (B) Binding of different trimethylated histone peptides to EED measured by fluorescence polarization. (C) Tabulated binding affinities (K_d) of different histone peptides for EED, measured by fluorescence polarization binding assays. The target lysines are colored in red. NB, no detectable binding; ND, not determined.

Fig. 2.

The H3K27me3 peptide specifically stimulates, but the H1K26me3 peptide inhibits, the activity of PRC2. (A) Effects of different histone peptides on the activity of PRC2 on histone H3 methylation. Top is an autoradiograph of the Middle (Coomassie blue staining). Quantification of two independent experiments is shown in Bottom. Error bars are standard deviations of the two experiments. (B) Effects of different methylation states of the H1K26 peptide on the methylation activity of PRC2. (C) The H1K26me3 peptide containing an A24E mutation lost its ability to inhibit PRC2 HMT activity on nucleosome.

Fig. 3.

Structure of EED in complex with a trimethylated H3K27 peptide. (A) Top view of the EED WD40 domain in complex with a trimethylated H3K27 peptide. EED is shown in a cartoon representation and colored green, and the H3K27me3 peptide is shown in a stick model. (B) The complex structure of EED and H3K27me3. EED is shown in a surface representation with positive electrostatic potential denoted in blue and negative potential by red. The H3K27me3 peptide is depicted in a stick model. (C) Detailed view of intermolecular interactions between EED and H3K27me3. EED residues in contact with the H3K27me3 peptide are shown in sticks. Hydrogen bonds are marked with black dashed lines. (D) Superposition of the crystal structures of EED in complex with different trimethylated histone peptides. Methyllysines are shown in a stick model. (E) Top view of WDR5 in complex with an H3K4me2 peptide (23). The H3K4me2 peptide and the H3R2-binding residues in WDR5 are shown in stick models. Hydrogen bonds are shown in red dashed lines.