Fig. 7.

Brm and Brg1 bind to the Ric-8B promoter during osteoblast differentiation and downregulate its activity. (A and B) Proliferating MC3T3 cells were cotransfected with the construct p0.56Ric8B-Luc (100 ng) and increasing amounts of the plasmids coding for the SWI/SNF catalytic subunits Brm (A) or Brg1 (B) (100 to 800 ng). At 24 h after transfection, luciferase activity was determined. The graphs show the percentage of basal activity, calculated as described in for Fig. 3. Overexpression of Brm and Brg1 proteins was confirmed by Western blotting using specific antibodies (data not shown). The bars represent the averages ± standard errors of the means of three independent experiments, each performed in triplicate. (C to F) MC3T3 cells were cultured for up to 13 days in the presence of 50 μg/ml AA, starting at day 3 (DIV, days of differentiation in vitro). At the indicated times, cells were cross-linked with 1% formaldehyde, and the fragmented chromatin was immunoprecipitated using specific antibodies against Brm (C and D) and Brg1 (E and F). The enrichment levels of Ric-8B promoter sequences in the precipitated chromatin were determined by QPCR using specific primers. The DNA sequences analyzed are indicated at the top of each graph. The bars represent the means ± standard errors of the means of two independent experiments performed in duplicate. Statistically significant differences were determined by the analysis of variance test. ***, P < 0.001; **, P < 0.01.