Fig. 4.

Synergy among p300, CARM1 and GRIP1 is dependent on the AD1 and AD2 domains of GRIP1. HeLa cells were transiently transfected with the MMP-9-Luc reporter construct and 0.2 µg of various combinations of pSG5-CARM1, pCMVβ-p300, pSG5-GRIP1, pSG.5-GRIP1ΔAD1, pSG.5-GRIP1ΔAD2 and pSG.5-GRIP1ΔAD1+ΔAD2, and then MMP-9 promoter activity determined as described in Materials and Methods. The results are the mean ± S.E. of at least three independent experiments. * p ≤0.05 compared to empty vector only PMA treated samples (bar 2); † p ≤0.05 compared to CARM1 or p300 construct alone PMA treated samples (bars 4 & 6); # p ≤0.05 wild type GRIP1 transfected PMA treated samples compared to mutant GRIP1 transfected PMA treated samples. NS1, not significant compared to empty vector only PMA treated samples (bar 2); NS2, not significant between the two linked samples; NS3, not significant compared to corresponding single coactivator expressed PMA-treated samples (bars 4 & 6); NS4, not significant compared to CARM1 and p300 coexpressed PMA-treated samples (bar 16). Insert is a schematic diagram of GRIP1. bHLH/PAS: bHLH/Per/Ah receptor nuclear translocator (ARNT)/Sim domain involved in DNA binding and heterodimerization between proteins containing these motifs;²⁴ S/T: serine/threonine-rich regions; L1L2L3: three LXXLL (L, leucine; X, any amino acid) motifs for interaction with ligand-bound nuclear receptors;²⁴ AD1 and AD2: two intrinsic transcriptional activation domains. The AD1 domain is responsible for interaction with CBP and p300; The AD2 domain is responsible for interaction with CARM1 and PRMT1.¹⁷,²⁴–²⁸