FIGURE 4.

Chromatin binding module of ZMYND8 is essential for its histone interaction. A, HeLa cells were transiently transfected with FLAG-WT ZMYND8 or FLAG-ΔPBP ZMYND8, co-stained with α-FLAG and either α-H3K36Me2 or α-H4K16Ac antibodies. The Pearson's coefficient for FLAG-WT ZMYND8 and H3K36Me₂/H4K16Ac was >0.5, whereas that for FLAG-ΔPBP ZMYND8 and H3K36Me₂/H4K16Ac was <0.5. B, HEK293 cells were transiently transfected with FLAG-WT or FLAG-ΔPBP ZMYND8, and whole cell extracts were subjected to M2-agarose FLAG pull down and immunoblotted with α-FLAG, α-H3K36Me2, α-H4K16Ac, α-HDAC1, and α-CHD4 antibodies. C, interaction of GST-PBP or GST-MYND with biotinylated H4K16Ac and H3.1K36Me2 peptides. Western blot analysis was done with α-GST antibody. D, ChIP assay was performed after transiently transfecting FLAG-WT or FLAG-ΔPBP ZMYND8 in HEK293 cells with α-FLAG antibody. Relative fold enrichment at TP53 (11 kb) and RRAS2 (24 kb) gene loci was scored by quantitative PCR normalized to IgG. At least three separate experiments were performed. Error bars show standard deviation.