FIG. 4.

Interaction between MBD1 and MCAF is required for transcriptional repression. (A) TRD mutants of MBD1 reduce the repressive activity. Wild-type (wt) and mutant TRDs of MBD1 in a GAL4 fusion were expressed in HeLa cells. The repression level in combination with wild-type GAL4-TRD of MBD1 was normalized to 100. The amino acid residues in Fig. 2A were subjected to mutagenesis (I576R, L579R, and I576R/L579R). (B) Loss of ability of TRD mutants to bind MCAF. Wild-type and mutant TRDs fused to GST were immobilized and incubated with His-tagged MCAF (Δ8). The input indicates 10% of the protein in the reaction mixture. (C and D) Localization of MBD1 and MBD1 (I576R) mutant in the nucleus. An immunofluorescence analysis of DsRed- or EGFP-fused MBD1 was performed in HeLa cells. MBD1 (MBD+NLS) expresses only the MBD and the nuclear localization signal. (E) Colocalization of MBD1 and native MCAF. (F) Dissociation of MCAF from MBD1 (I576R) mutant.