Figure 7.

SR-0065 treatment leads to increased recruitment of the corepressor SMRT to RARγ in both Lanthascreen TR-FRET assays as well as ChIP/ReChIP analysis. A) Lanthascreen TR-FRET corepressor recruitment assay was used to determine ligand dependent recruitment of the corepressor SMRT to RARγ. 5nM GST RARγLBD, 5nM Tb-Anti GST antibody, and 800nM fluorescein labeled SMRT peptide was incubated with either 10μM SR-0065, 10μM SR-1758 or DMSO vehicle only for 4 hours at room temperature in the dark as described in Material and Methods. Following incubation, fluorescence measurement was taken with Ex=340nm, Em1=490nm and Em2= 520nm. FRET measurements were determined by dividing the readings from 520nm/490nm. FRET signals for compound treatments were normalized to fold change over vehicle only. Treatment with SR-0065 resulted in significant recruitment of the SMRT peptide compared to vehicle only. In contrast, the acid form, SR-1758, was not able to recruit peptide compared to vehicle only. B) ChIP/reChIP analysis of the RARγ target gene Cyp26A1 in F9 cells. F9 cells were plated at 30% confluence 24 hours prior to treatment with 10μM ATRA, 10μM SR0065 or vehicle only for 24 hours and then harvested for Re-ChIP assay. Re-ChIP assays were performed as described in Materials and Methods section using anti-RARγ for the first immunoprecipitation step followed by anti-SMRT in the second immunoprecipitation. Anti-rabbit IgG was used as a negative control and anti-acetyl-histone H3 was used as a positive control in this experiment. Treatment with the RAR agonist ATRA resulted in significant loss of SMRT occupancy on the Cyp26A1 promoter while treatment with SR-0065 demonstrated a significant increase in recruitment of SMRT compared to DMSO only treatment consistent with its function as an RARγ inverse agonist. Densitometry analysis was performed to calculate percent input for ChIP signal using anti-SMRT ab for each condition. (DMSO=109%, ATRA=41%, SR-0065=191%).