Structural basis for the inhibition of PRC2 by active transcription histone posttranslational modifications

Abstract

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator essential for embryonic development and maintenance of cell identity that trimethylates histone H3 at lysine 27 (H3K27me3) leading to gene silencing. PRC2 is regulated by association with protein cofactors and crosstalk with histone posttranslational modifications. Trimethylated histone H3 K4 (H3K4me3) and K36 (H3K36me3) localize to sites of active transcription where H3K27me3 is absent and inhibit PRC2 activity through unknown mechanisms. Using cryo-electron microscopy we reveal that histone H3 tails modified with H3K36me3 engage poorly with the PRC2 active site and preclude its effective interaction with chromatin, while the H3K4me3 modification binds to the allosteric site in the EED subunit, acting as an antagonist that competes with allosteric activators required for the spreading of the H3K27me3 repressive mark. Thus, the location along the H3 tail of the H3K4me3 and H3K36me3 modifications allow them to target two essential requirements for efficient trimethylation of histone H3K27. We further show that the JARID2 cofactor modulates PRC2 activity in the presence of these histone modifications.

Keywords: Polycomb; allostery; antagonist; chromatin; cryo-EM; epigenetics; histone modifications.

Figure 1.. Cryo-EM structures of PRC2_AJ1–450 bound to H3K36me3 modified nucleosomes

A) Schematic representation of protein domains in the PRC2-AEBP2-JARID2 complex used in this work, containing either JARID2_1–450 or JARID2₁₁₉. B) Cryo-EM structure of PRC2_AJ1–450 bound to a H3K36me3-modified nucleosome in which the H3 tail is engaged by EZH2 and PRC2 is in an allosterically stimulated state. C) Cryo-EM structure of PRC2_AJ1–450 bound to H3K36me3-modified nucleosomes in which the H3 tail is not engaged by EZH2. The structures shown in B) and C) co-exist in the sample. D) Comparison of the position of K36 with respect to the nucleosomal DNA in structures of PRC2 bound to unmodified H3K36 (shown in orange; PDB 6WKR) and H3K36me3-modified nucleosomes (shown in pink).

Figure 2.. Comparison of tail-engaged and tailless PRC2_AJ1–450 complexes bound to H3K36me3-modified nucleosomes.

A) Overlay of the cryo-EM density maps for the co-existing tail-engaged (blue) and tailless (green) PRC2_AJ1–450 / H3K36me3 structures identified by our analysis. Maps are aligned using the nucleosome to show the relative rotation of PRC2 on the nucleosome surface. B) Close up view of the EZH2 bridge helix showing its relative position with respect to the H3 tail (pink) and nucleosomal DNA (grey) for the tail-engaged (blue) and tailless (green) structures. C) Close up view of the EZH2 CXC domain showing its relative position with respect to the H3 tail and nucleosomal DNA for the tail-engaged (blue) and tailless (green) structures (in B and C the tail, as seen in the tail-engaged complex, is shown in pink).

Figure 3.. Cryo-EM structures of PRC2 bound to H3K4me3-modified nucleosomes

A) Cryo-EM structure of PRC2_AJ119–450 bound to H3K4me3-modified nucleosomes. B) Close up view of the cryo-EM density showing the N-terminus of histone H3 containing H3K4me3 bound to EED. Map is shown at threshold 0.487. For lower threshold refer to Figure S7A. C) Cryo-EM structure of PRC2 _AJ1–450 bound to H3K4me3-modified nucleosomes. D) Close up view of cryo-EM density showing JARID2_K116me3 bound to EED and the folding of the SRM helix. Map is shown at threshold 0.25.

Figure 4.. H3K4me3 acts as an allosteric antagonist by binding to EED.

A) Interactions between the region of histone H3 (pink) around the H3K4me3 modification and the aromatic cage of EED (light blue) B) Interactions between JARID2 (magenta), EED (light blue), and EZH2 (yellow for the SRM and SAL, and darker blue for the SET domain) in an allosterically activated PRC2. The coordinates used were from the PRC2_AJ1–450/H3K4me3 structure obtained in this study and shown in Fig. 3C. JARID2 R115 interacts with E137 and D140 of the EZH2 SRM. JARID2 R108 and R110 are positioned to interact with E162 and D160 of EZH2, while JARID2 L111 and EZH2 I150 are involved in hydrophobic contacts. C) Interactions between the peptide around H3K27me3 (pink) and EED (light blue) and EZH2 (yellow for the SRM and SAL, and darker blue for the SET domain). in the coordinates used are those for the X-ray crystal structure of an activated PRC2 catalytic lobe from C. thermophilum (PDB: 5KJH). Histone H3R26 (similarly to JARID2 R115) interacts with negatively charged residues in the EZH2 SRM, and histone H3R23 establishes additional contacts with the SRM. D) Sequence alignment with respect to the PRC2-methylated lysine for: the N-terminal region of the histone H3 tail around K4, JARID2 around K116, H3 around K27, and PALI1 around K1241. The PRC2-modified lysine is colored in red, residues that are involved in hydrophobic contacts are colored in tan, and residues involved in electrostatic interactions are colored in blue. E) Overlay of structures shown in panels A (colored) and B (grey/transparent) showing that histone H3R2 of H3K4me3 clashes with the SAL.

Figure 5.. A structural model for tuning the catalytic activity of PRC2 in vitro.

Trimethylation of histone H3K27 by PRC2 can be inhibited by H3K36me3 and H3K4me3 histone modifications and activated by H3K27me3 or JARID2 K116me3. The interplay between these regulators is a gradient of PRC2 activity (red to green arrow). From left to right: in the absence of JARID2, H3K36me3 prevents efficient tail engagement and results in the loss of a well-defined register between PRC2 and the nucleosome substrate, as depicted as the blurry model of PRC2.; histone H3K4me3 engages the allosteric site and competes with substrates that are already modified with H3K27me3; PRC2 on unmodified substrates in the absence of JARID2 has a basal level of activity, while its EED allosteric site remains open for possible engagement with nearby nucleosomes with H3K27me3; in the presence of JARID2, PRC2 is allosterically stimulated, resulting in increased H3K27me3 activity (right most); histone H3K36me3 and H3K4me3 reduce PRC2 activity through the same mechanisms described above, however, methylated JARID2 stabilizes the catalytic lobe of PRC2 and facilitates some tail-engagement in the presence of H3K36me3, and it can compete with H3K4me3 for the EED allosteric site.