Fig. 3: MeCP2 binding to nucleosome is enhanced by the presence of H3K27me3.

a Interactions between MeCP2 and H3K27me3 are determined by in vitro pull-down assay. Pull-down MeCP2 is shown by both MeCP2 and GST bands. Comparable levels of mononucleosomes between land 2 and 3 are shown by Ponceau S staining and H3. The presence of H3K27me3 is validated in Lane 3. b Schematic representation of MeCP2 domain constructs. c Co-immunoprecipitation of histone H3 with Flag-tagged MeCP2 full length or truncated fragments. From 293T cell transfected with Flag-tagged constructs in b, total protein extract was immunoprecipitated with Flag antibody and the presence of histone H3 is shown by immunoblotting. d In vitro pull-down assays of bait (purified GST, GST-tagged full length MeCP2 or MBD of MeCP2) to prey protein (recombinant mononucleosomes, either containing unmodified H3 or H3K27me3). Pull-downed H3 or H3K27me3 were visualized by SDS-PAGE and immunoblotting with anti-Histone H3 or H3K27me3. The presence of GST-fusion protein in each construct was indicated by the asterisks. e Competition assays with unmodified H3K27 or H3K27me3 peptides to MBD/nucleosome interactions. GST-tagged MBD binding to nucleosomes (top blot) containing H3K27me3 (lane 4) were challenged with H3K27 (lane 5–7) and H3K27me3 (lane 8-10) peptide. Bindings of GST-tagged MBD to histone H3 (middle blot) and H3K27me3 (bottom blot, sharp) were evaluated by GST pull-down and immunoblotting. H3K27me3 peptides in the MBD/nucleosome complexes were also revealed (bottom blot, double sharp). f, g Quantification of normalized H3K27me3 (purple, sharp) and H3K27me3 peptide (green, double sharp) levels by densitometry analysis in e. The signal strength in each band is normalized to H3K27me3 signal strength lane 4 in e. Source data are provided as a Source data file (a, c, d and e).