Fig. 5
From: Liquid–liquid phase separation of H3K27me3 reader BP1 regulates transcriptional repression
IDR2 of BP1 drives PRC2 interaction and H3K27me3 binding. A BP1 interaction with Suz12, a component of PRC2, requires IDR2 of BP1 in a yeast two-hybrid (Y2H) assay. Yeast cells harboring the indicated bait and prey constructs were assayed for growth on synthetic defined (SD) medium lacking leucine, tryptophan, histidine, and adenine SD (–Leu–Trp–His–Ade). The plasmid pair pGBKT7-53 and pGADT7 was used as the positive control. Another pair of plasmids, pGBKT7-Lam and pGADT7, was used as the negative control. Images were taken after 3 days of incubation at 30 °C. B SDS-PAGE analysis of recombinant MBP-BP1 and BP1 IDR deletion variants (MBP-BP1ΔIDR1 and MBP-BP1ΔIDR2) purified from E. coli. C Pull-down assay of the interaction between MBP-BP1ΔIDR1 or MBP-BP1ΔIDR2 and His-Suz12. Full-length MBP-BP1 was used as a positive control. D, E Interaction between BP1ΔIDR1-GFP (D), BP1ΔIDR2-GFP (E), and Suz12-Flag as examined by co-immunoprecipitation (Co-IP) in F. graminearum. GAPDH used as the loading control. F Peptide pull-down assays using the H3K27me3 peptide and either recombinant MBP-BP1 or its deletion variants (MBP-BP1ΔIDR1 and MBP-BP1ΔIDR2). MBP served as a negative control. Immunoprecipitation by biotinylated H3K27me3 was analyzed with an anti-MBP antibody. G–I Isothermal titration calorimetry (ITC) assays performed to measure the binding affinity of MBP-BP1 (G), MBP-BP1ΔIDR1 (H), or MBP-BP1ΔIDR2 (I) to the H3K27me3 peptide. NDB, no detectable binding