FIGURE 6.

MTA2 participates in the down-regulation of FSHR expression upon FSH treatment by directly recruiting HDAC1 into FSHR promoter. A, effects of MTA1 siRNA or MTA2 siRNA treatment on HDAC activity in TM4 cells in response to oFSH stimulation. B, expression level of FSHR mRNA in MTA1 siRNA- or MTA2 siRNA-treated TM4 cells upon oFSH stimulation was determined using QRT-PCR. Values are the mean ± S.D. (error bars) of at least three determinations. C, SCs were transfected with FSHR(−100/+123) Luc and expression vectors for His-MTA2 (0.5 μg) and empty vector as indicated. Two days after transfection, the cells were stimulated with vehicle or oFSH (25 ng/ml) for 6 h, after which the cells were harvested, and luciferase activities were normalized for total protein as determined by a Bradford assay. Results represent the mean ± S.D. luciferase activity for four independent experiments using two replicates. D, direct association of MTA2 and HDAC1 in SCs was illustrated by a co-IP assay followed by Western blotting analysis (IB) at different time points after oFSH treatment. E, ChIP analysis showing recruitment of MTA1 and HDAC1 onto the mouse FSHR promoter in SCs upon FSH stimulation. F, double ChIP analysis of MTA2-HDAC1 complex onto the FSHR promoter in SCs at different time points after oFSH treatment. G, after SCs had been deprived of endogenous MTA2 by siRNA treatment, cells were incubated with oFSH for different durations. QRT-PCR analysis was then used to evaluate the MTA2 and ABP mRNA levels at different time points after oFSH treatment. Values are the mean ± S.D. of at least three determinations. *, p < 0.05 when compared with control group. The efficiency of MTA2 deletion was also monitored by immunoblotting analysis (inset).