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. 2018 Jun 1;32(11-12):794–805. doi: 10.1101/gad.311936.118

Figure 5.

Figure 5.

The interaction between the PRC2 core and accessory proteins is required for efficient recruitment of PRC2 to chromatin. (A, left) Diagram of PRC2 subunits based on the negative stain electron microscopy (EM) structure (Ciferri et al. 2012). SUZ12, AEBP2, or PCL homolog proteins and EZH2 were tagged with 3xFlag, 3xHA, and 3xMyc, respectively. (Right) αHA, αMyc, and αFlag Western blots on input and elution samples from an αFlag immunoprecipitation. 3xFlag-SUZ12, 3xHA-AEBP2 or 3xHA-PHF1, and 3xMyc-EZH2 were expressed in HEK293T cells. The N terminus of SUZ12(2-560) is necessary to coimmunoprecipitate AEBP2 and PHF1. (B, top) Diagram of SUZ12 separation-of-function mutations. ABHmut (mutated AEBP2-binding helix) refers to SUZ12(95–106)2xNAAIRS, PHF1mut refers to SUZ12(338–353)GSGSGS, and VEFS refers to SUZ12(561–739). (Bottom) Western blots using the indicated primary antibody on input and elution samples from an αFlag immunoprecipitation of 3xFlag-SUZ12 mutants. PHF1 + ABHmut refers to SUZ12 containing both PHF1 and ABH mutations. ABHmut disrupts the interaction between SUZ12 and AEBP2, and PHF1mut disrupts the interaction between SUZ12 and PHF1, while neither mutation disrupts the assembly of SUZ12 with EZH2, EED, or RbAp46/48. (C) Quantification of the eluted Western signals. The HA signal was first normalized to the amount of 3xFlag-SUZ12 in each elution. The binding fraction is the fraction of HA signal in the SUZ12 mutant condition relative to the wild-type SUZ12 condition. Each gray bar represents the mean, and each dot represents the results from a biological replicate. (D) Dfree (left) and fraction bound (right) of 3xFlag-HaloTag-EZH2 in cells transfected with the indicated 3xFlag-SUZ12 variants, a siRNA pool targeting the SUZ12 3′ untranslated region (UTR), and a nuclear BFP as a transfection marker. NTC refers to cells transfected with a nontargeting pool of siRNA and nuclear BFP. 3xFlag-HaloTag-EZH2 cells were labeled with 5 nM JF646 and imaged at 97.5 Hz. SLIMfast and evalSPT were used to localize, track, and evaluate single-molecule trajectories, and Spot-On was used to calculate the Dfree and fraction bound of 3xFlag-HaloTag-EZH2. (E) Diffusion coefficients, chromatin-bound fractions, and molecular weights of the various PRC2 mutant complexes. D and E show the average and SD from n = 3 biological replicates using >12 cells per replicate. P-values were generated from a heteroscedastic t-test using a two-tail distribution.