FIG. 3.

MeCP2 and MBD4 preferentially bind to methylated nucleosomes. Nucleosomes were reconstituted using [γ³²P]ATP-end-labeled unmethylated (control) and methylated versions of a 5S rRNA gene fragment (see Materials and Methods) and were titrated with increasing amounts of either MeCP2 or MBD4. The electrophoretic shifts resulting from the complexes formed between these proteins and the nucleosomes were analyzed by electrophoresis in electrophoretic mobility shift assays. Approximately 30 to 40 nM concentrations of nucleosomes incubated for 20 min at room temperature with increasing amounts (indicated by triangles) of MeCP2 (A) or MBD4 (B) were analyzed by native 4% PAGE. Lanes: 2 and 8, 7.5 nM protein; 3 and 9, 15 nM protein; 4 and 10, 30 nM protein; 5 and 11, 60 nM protein; and 6 and 12, 120 nM protein. D, free DNA; N, nucleosomes; −, no protein.