FIG. 7.

SMAR1 recruits the repressor complex at the cyclin D1 promoter. Chromatin immunoprecipitation was performed as described in Materials and Methods. (A) Endogenous recruitment of SMAR1, HDAC1, Sin3A/Sin3B, p107, and p130 on the cyclin D1 promoter probe II and III region was studied in the 293 cell line (left panel). SMAR1 protein is undetectable in MCF-7 cells, thus ChIP for recruitment of SMAR1 on the probe II and III region was studied in untransfected MCF-7 (0 h) or cells transfected (24 h) with Flag-SMAR1 (right panel); two lanes for each time point indicate two different concentrations of templates (1 and 0.5 μl, respectively) used for amplification of immunoprecipitated DNA. (B) Recruitment of SMAR1 on the cyclin D1 promoter upon overexpression of Flag-SMAR1 in a time course experiment from the time of transfection was studied in 293 and MCF-7 cells (left and right panels, respectively). Chromatin was immunoprecipitated at various time points, and pulled DNA was amplified using primers for probe II. Densitometric analysis for SMAR1 recruitment in each cell line is represented in the bar graph. (C) 293 and MCF-7 cells were transfected with 1 μg Flag-SMAR1, and cells were cross-linked 24 h posttransfection. ChIP assay using various antibodies against the proteins associated with SMAR1 was performed in 293 and MCF-7 cells (left and right panels, respectively), and immunoprecipitated DNA was amplified using primers for probe II and probe III. For every PCR, 1 and 0.5 μl of the template was used for amplification. Ab, antibody.