Extended Data Fig. 8: Purification of catalytically active PRC2 complex and Swi6.
From: Intrinsic mesoscale properties of a Polycomb protein underpin heterochromatin fidelity
(a) Schematic representation of the PRC2 coexpression construct. Each gene expression cassette contains a polyhedrin promoter (PPH), a cDNA of the PRC2 component, and an SV40 terminator (term). The cDNAs are tagged as follows: 3XStrep-TagII-HRV3C-EZH2, 9XHis-HRV3C-EED1, FLAG-HRV3C-BND1, and 6XHis-TEV-MSL1. (b) Agarose gel of SwaI-digested construct in (a). (c) Coomassie stained SDS-polyacrylamide gel of the purified recombinant PRC2. (d) (Left) SAH standard curve (Right) Histone Methyltransferase assay of the recombinant PRC2 on the nucleosome assembled with Xenopus histone H3 (28SAPAT32 replaced for 28QTTTSAAA34 of C. neoformans histone H3). (Mean ± SEM, n = 3 independent replicates) (e) Purified 6XHis-Swi6. (f) Mass photometry analysis of Swi6 in 20 mM HEPES, pH 7.5, and 150 mM NaCl. (g) DIC images of concentration-dependent Swi6 condensate formation in 20 mM HEPES, pH 7.5, and 125 mM NaCl 30 min after plating. Scale bars, 10 µm. (h) Condensate fusion of 189 µM Swi6 at indicated time points. Scale bars, 10 µm. Data in b, c, e, g, and h are representative of three independent experiments.
