Research Article

Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2

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Science  16 Oct 2015:
Vol. 350, Issue 6258, aac4383
DOI: 10.1126/science.aac4383

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A tripartite gene silencing complex

The formation of specialized cell types during development involves the silencing of genes not required in those cell types. An important player in this silencing process is the polycomb repressive complex 2 (PRC2), which methylates histone H3 on lysine residue 27 (H3K27me). Jiao and Liu determined the x-ray crystal structure of a functional PRC2 complex from a thermophilic yeast species (see the Perspective by Schapira). The intimate association of the three subunits confers stability to PRC2. The structure also reveals how the reaction product, H3K27me, stimulates PRC2 allosterically and how a cancer-associated histone mutation blocks the PRC2 active site.

Science, this issue p. 10.1126/science.aac4383; see also p. 278

Structured Abstract


Polycomb-group (PcG) proteins are key epigenetic regulators of cell identity determination and maintenance. As one of the main PcG protein complexes, polycomb repressive complex 2 (PRC2) mediates trimethylation of histone H3 at lysine 27 (H3K27me3), a hallmark of gene silencing and facultative heterochromatin formation. Dysregulation of PRC2 function is broadly linked to human diseases, including hematological malignancies, Weaver syndrome, and childhood glioblastoma. PRC2 consists of four core subunits—Ezh2, Eed, Suz12, and Rbbp4—among which Ezh2 is the catalytic subunit, which minimally requires Eed and Suz12 for catalysis. Although the histone methyltransferase activity of PRC2 was discovered more than a decade ago, the catalytic mechanism of PRC2 remains poorly understood. In addition, the end product of PRC2 catalysis, H3K27me3, is known to interact with Eed to stimulate the enzymatic activity of PRC2 allosterically. The details of this positive feedback loop, which is believed to account for spreading of the repressive H3K27me3 histone mark on silent chromatin, are also not fully understood. Additionally, a histone H3K27M missense mutation found in some pediatric brain cancers leads to a global decrease in the amount of H3K27me3 by inhibiting PRC2 through a so far uncharacterized mechanism. To begin to address these outstanding questions regarding PRC2 function and regulation, we report the crystal structures of an active PRC2 complex of 170 kD from the fungus Chaetomium thermophilum in both basal and stimulated states at 2.7 and 2.3 Å resolution, respectively.


As an evolutionarily conserved complex, PRC2 proteins from different species share compositional and functional similarities. Indeed, some fundamental aspects of human PRC2 catalysis and regulation, in particular the H3K27me3-mediated enzyme stimulation and the H3K27M-mediated enzyme inhibition, were faithfully recapitulated in our assays with the reconstituted minimal fungal PRC2, containing Ezh2, Eed, and the VEFS domain of Suz12 [Suz12(VEFS)], which was also used for crystallization.


An S-adenosyl-l-homocysteine (SAH) cofactor and an inhibiting H3K27M cancer mutant peptide bound to the catalytic SET domain of Ezh2 were captured in the crystal structures of both basal and stimulated complexes. In addition, a stimulating H3K27me3 peptide bound to both Ezh2 and Eed was also resolved in the latter. Structural analysis and accompanying biochemical assays provided the following mechanistic insights into PRC2 catalysis and regulation. First, Ezh2, Eed, and Suz12(VEFS) associate intimately. The Eed subunit is engulfed by a belt-like structural feature of Ezh2, and Suz12(VEFS) contacts both of these two subunits to confer enzyme activity. Second, two separate regions of Ezh2, including the SET activation loop (SAL) and SET, are together required to form the active catalytic domain of PRC2. Eed and Suz12(VEFS) are structurally important in maintaining the positioning and local conformation of the SAL of Ezh2, which may explain, at least in part, the indispensable role of Eed and Suz12 in PRC2 catalysis. Third, the H3K27M cancer mutant inhibits PRC2 enzyme activity by a direct competition mechanism, with residue H3R26, in the context of the lysine-to-methionine mutation, occupying the lysine access channel of the active site and thus occluding substrate binding. Lastly, the flexible stimulation-responsive motif (SRM) of Ezh2 responds to H3K27me3-mediated enzyme stimulation by forming a sandwich-like assembly with the H3K27me3 peptide and Eed. The SRM exhibits a dramatic disorder-to-order conformational transition upon binding of this stimulating peptide. This initiates an allosterically regulated pathway that communicates with the active site.


This work has resolved some long-standing questions regarding PRC2 structure and function and provides a structural framework for future functional studies. PRC2 is a representative of a distinct family of lysine methyltransferases. The unique structural arrangement of PRC2 revealed here underlies PRC2-mediated H3K27 trimethylation. The enzymatic activity of PRC2 is subject to complex regulation by a plethora of protein factors and noncoding RNAs in cells. Regulatory signals transmitted from discrete, distant surfaces of PRC2, such as that transferred by the SRM of Ezh2, are interpreted and integrated at the enzyme active site to generate distinct cellular outputs.

Overall structure of an active PRC2 in the stimulated state.

The Ezh2, Eed, and Suz12(VEFS) subunits are shown as gray, light blue, and light brown surfaces, respectively, except for the SET activation loop (SAL), the stimulation-responsive motif (SRM), and the SET regions of Ezh2, which are highlighted as cartoons. The SAL is colored in green, SRM in pink, and SET in blue.


Polycomb repressive complex 2 (PRC2) catalyzes histone H3K27 trimethylation (H3K27me3), a hallmark of gene silencing. Here we report the crystal structures of an active PRC2 complex of 170 kilodaltons from the yeast Chaetomium thermophilum in both basal and stimulated states, which contain Ezh2, Eed, and the VEFS domain of Suz12 and are bound to a cancer-associated inhibiting H3K27M peptide and a S-adenosyl-l-homocysteine cofactor. The stimulated complex also contains an additional stimulating H3K27me3 peptide. Eed is engulfed by a belt-like structure of Ezh2, and Suz12(VEFS) contacts both of these two subunits to confer an unusual split active SET domain for catalysis. Comparison of PRC2 in the basal and stimulated states reveals a mobile Ezh2 motif that responds to stimulation to allosterically regulate the active site.

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