FIG. 5.

Association of MBD1 and MCAF on endogenous gene promoter. (A) Methylation-specific PCR. The promoter region of the p16 tumor suppressor gene was studied in human lung cancer cell lines NCI-H1299 and SBC-5. W, U, and M indicate specifically amplified fragments corresponding to unmodified, unmethylated, and methylated sequences, respectively. (B) Link of MCAF by MBD1 to methylated promoter. The cells were treated with a protein-protein cross-linker (DTBP) and formaldehyde. Specific fragments of p16 promoter were detected by PCR amplification by using a set of W primers in the immunoprecipitates. HA-tagged MBD1 was expressed for a chromatin immunoprecipitation with anti-HA antibodies (upper). Wild-type or mutant (I576R) HA-MBD1 was coexpressed together with FLAG-MCAF, followed by the chromatin immunoprecipitation with anti-FLAG antibodies (lower). (C) Competition of MBD1 with Sp1 for binding to MCAF. His-MCAF Δ3 (2 μg) (Fig. 2E) on nickel-chelating resin was incubated with GST-TRD of MBD1 and GST-Sp1 Δ1 containing the residues 90 to 785 of Sp1 (0 to 2 μg each). Bound proteins on the resin were eluted by imidazole. The input indicates 10% of the protein in the reaction mixture.