January 1, 2021;
Structures of monomeric and dimeric PRC2:EZH1 reveal flexible modules involved in chromatin compaction.
Polycomb repressive complex 2 (PRC2) is a histone methyltransferase critical for maintaining gene silencing during eukaryotic development. In mammals, PRC2 activity is regulated in part by the selective incorporation of one of two paralogs of the catalytic subunit, EZH1 or EZH2. Each of these enzymes has specialized biological functions that may be partially explained by differences in the multivalent interactions they mediate with chromatin. Here, we present two cryo-EM structures of PRC2:EZH1, one as a monomer and a second one as a dimer bound to a nucleosome. When bound to nucleosome substrate, the PRC2:EZH1 dimer undergoes a dramatic conformational change. We demonstrate that mutation of a divergent EZH1/2 loop abrogates the nucleosome-binding and methyltransferase activities of PRC2:EZH1. Finally, we show that PRC2:EZH1 dimers are more effective than monomers at promoting chromatin compaction, and the divergent EZH1/2 loop is essential for this function, thereby tying together the methyltransferase, nucleosome-binding, and chromatin-compaction activities of PRC2:EZH1. We speculate that the conformational flexibility and the ability to dimerize enable PRC2 to act on the varied chromatin substrates it encounters in the cell.
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Fig. 1: Structures of monomeric and dimeric PRC2:EZH1. a Cartoon representation of the proteins used to generate PRC2:EZH1 complexes. Hinge points (HP) 1 (aa 535–561) and 2 (aa 146–155) are regions in SUZ12 that appear to allow the upper and lower lobes to rotate with respect to each other and the C2 domain to rotate with respect to RBAP48. JARID2 amino acids 96–367 were used in the monomeric PRC2 complex, while amino acids 1–367 were used in the PRC2–nucleosome complex. b Cryo-EM map of monomeric PRC2:EZH1 showing the upper and lower lobes, and the SUZ12”foot”/C2 domain. Colors are as in panel a. c Composite cryo-EM map of a PRC2:EZH1 dimer bound to a nucleosome containing the H3K27M and H2AUb modifications. Protein subunits are colored as in panel a. One unit of the PRC2 dimer “PRC2A” is outlined with a dashed line. d Model of the PRC2:EZH1 dimer bound to a nucleosome containing the H3K27M and H2AUb modifications with subunits labeled. The model was built using the composite map except for nucleosomal DNA, which was modeled based on the 7-Å resolution map. e Cryo-EM map of a single PRC2:EZH1 monomer from the structure of the nucleosome-bound PRC2:EZH1 dimer. Compare to map of monomeric PRC2:EZH1 in panel b. Note that the lower lobe rotates ~170° relative to the upper lobe, and the C2 domain rotates ~115° relative to RBAP48.
Fig. 2: The MCSS/SANT2L loop is important for PRC2 activity. a Composite map of one PRC2:EZH1 bound to nucleosome with regions that likely interact with DNA indicated by dashed circles. b Upper panel: Cartoon representation of EZH1 showing basic patches. Lower panel: Sequence alignment of EZH1 and EZH2 MCSS/SANT2L loops showing the basic patch (green box) and the acidic patch (red underline). c Nucleosome-binding and methyltransferase activities of PRC2:EZH1 containing five arginine-to-alanine substitutions (see green box in panel b). Experiments were done with core PRC2 containing SUZ12, EED, RBAP48, and wild-type or mutant versions of EZH1/2 as indicated. For graphs indicating nucleosome binding, each data point represents the average of three independent mobility-shift experiments and is shown as mean ± standard deviation. Methyltransferase assays measured the incorporation of 3H-labeled S-adenosylmethionine into histone H3 at lysine 27. Assays were done in triplicate using 300 nM of nucleosomes and titration of 5–60 nM PRC2. d Experiments done as in panel c, except that regions aligned in panel b were “swapped” between EZH1 and EZH2. e Methyltransferase assay using reciprocal point mutation swaps between EZH1 and EZH2 (EZH1-R363G vs EZH2-G356R) (asterisk in panel b). The experiment was done as in panel c. f Methyltransferase activity of PRC2 complexes with the acidic patch (see panel b) deleted from EZH2 or inserted into EZH1 (residues 387–401 from EZH2 deleted or inserted into EZH1 between residues 394 and 395). The experiment was done as in panel c.
Fig. 3: PRC2 dimers promote chromatin compaction. a Cryo-EM map showing the PRC2:EZH1 dimer. Subunits are colored as in Fig. 1. One PRC2 monomer is highlighted in color and by a red dashed line. b, left panel: Isolated view of the dimeric interface of PRC2:EZH1 showing that the C2 domain of SUZ12 (green) from one PRC2:EZH1 interacts with RBAP48 from the other PRC2:EZH1 (gray). Right panel: Zoomed-in view of the basic loop (blue) in the C2 domain interacting with the acidic surface of RBAP48. Electrostatics were calculated using APBS in PyMol. The sequence of the basic residues in the C2 domain is shown and colored blue. c Confocal micrographs of serial twofold dilutions of PRC2:EZH1 mixed with nucleosome arrays showing compaction of chromatin into droplets. Scale bar: 100 μm. The final concentration of PRC2 in the reactions is indicated. Molarity is based on the expected size of PRC2:EZH1 monomers. All samples contain 3 μM total nucleosomes, assembled into arrays with 12 nuclesomes spaced 40 bp apart. Experiments were repeated at least three times with independently purified complexes and were consistent with each other. d Chromatin compaction as promoted by PRC2:EZH15RtoA mutant complexes. Concentrations and scale are as in c. Experiments were repeated twice with independently purified complexes that were consistent with each other. e Model of chromatin compaction promoted by PRC2 dimers. Chromatin is compacted into a phase-separated state through multivalent interactions with basic patches of PRC2:EZH1 dimers, leading to the formation of liquid–liquid phase-separated droplets.
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