Structural basis for PRC2 decoding of active histone methylation marks H3K36me2/3

Abstract

Repression of genes by Polycomb requires that PRC2 modifies their chromatin by trimethylating lysine 27 on histone H3 (H3K27me3). At transcriptionally active genes, di- and trimethylated H3K36 inhibit PRC2. Here, the cryo-EM structure of PRC2 on dinucleosomes reveals how binding of its catalytic subunit EZH2 to nucleosomal DNA orients the H3 N-terminus via an extended network of interactions to place H3K27 into the active site. Unmodified H3K36 occupies a critical position in the EZH2-DNA interface. Mutation of H3K36 to arginine or alanine inhibits H3K27 methylation by PRC2 on nucleosomes in vitro. Accordingly, Drosophila H3K36A and H3K36R mutants show reduced levels of H3K27me3 and defective Polycomb repression of HOX genes. The relay of interactions between EZH2, the nucleosomal DNA and the H3 N-terminus therefore creates the geometry that permits allosteric inhibition of PRC2 by methylated H3K36 in transcriptionally active chromatin.

The role of H3K36 methylation and associated methyltransferases in chromosome-specific gene regulation

“…The interpretation of histone replacement experiments may, however, be less straightforward if it is the un-methylated H3K36 that is required for the process, which Set2 may aim to prevent. Thus, recent structural studies suggest that unmodified H3K36 is required for unimpeded methylation of lysine 27 of histone H3 (H3K27) by the Polycomb Repressive Complex 2 (PRC2) (Jani et al 2019; Finogenova et al 2020). Consistently, flies in which the zygotic histone H3.2 is replaced with a variant that contains arginine instead of K36, show mild defects in Polycomb repression of homeotic selector genes (Finogenova et al 2020).…”

“…Thus, recent structural studies suggest that unmodified H3K36 is required for unimpeded methylation of lysine 27 of histone H3 (H3K27) by the Polycomb Repressive Complex 2 (PRC2) (Jani et al 2019; Finogenova et al 2020). Consistently, flies in which the zygotic histone H3.2 is replaced with a variant that contains arginine instead of K36, show mild defects in Polycomb repression of homeotic selector genes (Finogenova et al 2020). We, therefore, considered a possibility that reduced activity of PRC2 in the ΔHisC; 12x H3K36R mutant may lead to an increased transcription of the X-chromosome genes, which in turn, may mask the effect of the Set2 knock-out.…”

Allosteric regulation of histone lysine methyltransferases: from context-specific regulation to selective drugs

“…A mechanism for an allosteric inhibition has been proposed, based on the observation that the H3K4me3 modification directly affects the catalytic efficiency but not the affinity to the substrate [ 57 ]. More recently, a cryo-EM structure of PRC2 with a di-nucleosomal construct, complemented by functional assays, led to propose a molecular mechanism for the inhibition of PRC2 by the H3K36 methyl mark [ 58 ]: in the substrate nucleosome, the unmodified H3K36 site is sandwiched at the interface between EZH2 and the nucleosomal DNA [ 58 ]. According to the proposed mechanism, the correct positioning of the nucleosome substrate is allowed only when H3K36 is unmethylated, and this enables the correct presentation of the H3 tail to the catalytic centre in EZH2 [ 58 ].…”

“…More recently, a cryo-EM structure of PRC2 with a di-nucleosomal construct, complemented by functional assays, led to propose a molecular mechanism for the inhibition of PRC2 by the H3K36 methyl mark [ 58 ]: in the substrate nucleosome, the unmodified H3K36 site is sandwiched at the interface between EZH2 and the nucleosomal DNA [ 58 ]. According to the proposed mechanism, the correct positioning of the nucleosome substrate is allowed only when H3K36 is unmethylated, and this enables the correct presentation of the H3 tail to the catalytic centre in EZH2 [ 58 ]. While this mechanism does not fit with the definition of allosteric regulation followed in this review (see above), it does fit with a broader definition sometimes used, referred to the interaction of an effector with a site other than the active site of the enzyme.…”

“…The presence of such two conformations of the H3 tail, where only one of them allows catalysis, could explain why H3K4me3-modified nucleosome is a sub-optimal substrate comparing an unmodified nucleosome [ 59 ], despite both having a similar affinity to PRC2 [ 57 ]. Collectively, these recent structures imply that the H3K4me3 [ 59 ] and H3K36me3 [ 58 ] marks reduce the activity of PRC2 by inducing a suboptimal presentation of the H3 tail to the active site. Future kinetic assays with mutagenesis, guided by these recent structures, will likely allow deciphering these molecular mechanisms and to directly support them.…”