Figure 5.
Effect of RIP140 methylation on its corepressor activities. (A) Trans-repressive activities of RIP140 full-length fused to Gal4BD in the presence of MTI in COS-1 cells. (B) Hypermethylation induced by PRMT1 abrogated the repressive activities of RIP140. (C) Hypermethylation induced by PRMT1 abolished RIP140 mediated repression on retinoic acid (RA) induced RAR-β2 reporter gene transcription, while the triple-Arg/Ala mutant was not affected by PRMT1. (D) In P19 cells, PRMT1 abolished the repressive activity of the wild-type RIP140, but not the triple-Arg/Ala mutant, on the endogenous RAR-β2 expression at mRNA level as determined by RT–PCR followed by southern blot analyses. (E) ChIP analyses of the endogenous RAR-β2 promoter in P19 cells. Quantitation of ChIP by densitometric analyses of amplified genomic DNA normalized to the input DNA was shown on the right panel. Both RIP140 (subpanel a) and HDAC3 (subpanel b) were effectively recruited to the endogenous RAR-β2 gene promoter even in the presence of CARM1 (lanes 1 and 2), but their recruitment was substantially reduced in the presence of PRMT1 (lane 3, subpanel d). As a result, the acetylation of histone H4 on the chromatin target (lane 3, subpanel c) was increased and RAR-β2 gene expression was enhanced (Figure 5D). While a significant amount of PRMT1 was recruited to the genomic target (lane 3, subpanel d) less RIP140 was associated with this region (lane 3, subpanel a), suggesting that RIP140 methylation might occur on the chromatin target by PRMT1, and that the methylated RIP140 was released from the chromatin target, which could also contribute to the reduction of its co-repressive activity.