p300 mediates functional synergism between AF-1 and AF-2 of estrogen receptor α and β by interacting directly with the N-terminal A/B domains

Estrogen receptor (ER) alpha and beta mediate estrogen actions in target cells through transcriptional control of target gene expression. For 17 beta-estradiol-induced transactivation, the N-terminal A/B domain (AF-1) and the C-terminal E/F domain (AF-2) of ERs are required. Ligand binding is considered to induce functional synergism between AF-1 and AF-2, but the molecular mechanism remains unknown. To clarify this synergism, we studied the role of reported AF-2 coactivators, p300/ CREB binding protein, steroid receptor coactivator-1/ transcriptional intermediary factor-2 (SRC-1/TIF2) family proteins and thyroid hormone receptor-associated protein-220/(vitamin D3 receptor-interacting protein205-(TRAP220/DRIP205) on the AF-1 activity in terms of synergism with the AF-2 function. We found that neither any of the SRC-1/TIF2 family coactivators nor TRAP220/ DRIP205 is potent, whereas p300 potentiates the AF-1 function of both human ER alpha and human ERP. Direct interactions of p300 with the A/B domains of ER alpha and ER beta were observed in an in vitro glutathione S-transferase pull-down assay in accordance with the interactions in yeast and mammalian two-hybrid assays. Furthermore, mutations in the p300 binding sites (56-72 amino acids in ER alpha and 62-72 amino acids in ER beta) in the A/B domains caused a reduction in ligand-induced transactivation functions of both ER alpha and E beta. Thus, these findings indicate that ligand-induced functional synergism between AF-1 and AF-2 is mediated through p300 by its direct binding to the A/B regions of ER alpha and ER beta.