Fig. 5

The MPP8 chromodomain recognizes methylated ATF7IP. a A scheme for the identification of the proteins bound to ATF7IP WT or K16R mutant. The three cell lines expressing 3xFLAG-ATF7IP, K16R mutant or nothing were used for the immunoprecipitation with anti-FLAG M2 beads followed by LC–MS/MS analysis. A Venn diagram shows the number of bindings proteins specifically for ATF7IP WT. b Top three proteins identified as ATF7IP WT-only-bound proteins. c A schematic of mouse MPP8 protein. Known domains and an alignment of mouse and human MPP8 and mouse HP1a are shown. An asterisk indicates a key residue (W80) for the interaction with H3K9me3. d Co-IP assay using Atf7ip KO mESCs rescued by ATF7IP WT, K16R, or Δ(1–16) mutant. Bound endogenous MPP8 was detected by western blot analysis. e The interaction of MPP8 with ATF7IP via its chromodomain. Co-IP assay with anti-V5 antibody was performed using WT mESCs expressing V5-MPP8 WT or W80A mutant (CD, chromodomain dead). Bound endogenous ATF7IP was detected by western blot analysis. f The interaction of MPP8 with ATF7IP requires G9a and GLP. Co-IP assay with anti-V5 antibody was performed using WT or G9a/Glp DKO mESCs expressing V5-MPP8 WT. Bound endogenous ATF7IP was detected by western blot analysis. g In vitro methylation followed by in vitro binding assay with anti-FLAG. Recombinant proteins were produced in and purified from E. coli. The interaction of MPP8 with ATF7IP was detected by western blot analysis. KR, K16 R mutant. CD, chromodomain dead (W80A mutant). h Co-IP assay with anti-ATF7IP antibody using a chromatin fraction from WT or Atf7ip KO mESCs. The endogenous interaction of MPP8 with ATF7IP was detected by western blot analysis