Figure 2.

MPP8 binds to methylated histone H3K9 through chromodomain. (A) Diagram of full-length MPP8 and chromodomain alignment among human MPP8, HP1α and PC2. Conserved amino acids are shaded. The conserved W80 is indicated with a star. (B, C) MPP8 specifically recognizes methyl-H3K9 in vitro. ³⁵S-labelled in vitro translated MPP8-wt or W80A mutant were tested for binding with immobilized biotin-H3 peptides with or without methylation at K9 (B) or K27 (C). Methylation states are indicated. ³⁵S-labelled in vitro translated HP1α serves as controls in the parallel. ‘In' represents 10% of total input, ‘B' and ‘Ft' represent bound and flow through fractions, respectively. (D) H3K9me3 marks recruit MPP8 onto heterochromatin in cells. Immortalized MEF cells derived from wild-type or Suv39h1/h2 knockout mice were transfected with vectors expressing Flag-MPP8 or W80A mutant. Cells were fixed and stained with mouse anti-Flag (red) and chicken anti-H3K9me3 (green) antibodies. Blue colour (DAPI) shows the nuclei. (E) MPP8 preferentially binds to chromatin containing H3K9me2 and me3 marks in cells. MPP8 stable knockdown 293T cells (KD2) were transfected with vectors expressing shRNA resistant Flag-MPP8 or W80A mutant. MPP8 and bound chromatins were IPed by anti-Flag antibody and analysed by western blot. Antibodies are indicated and a non-specific cross-react band is labelled with a star.