Estradiol repression of tumor necrosis factor-α transcription requires estrogen receptor activation function-2 and is enhanced by coactivators

The tumor necrosis factor-alpha (TNF-alpha) promoter was used to explore the molecular mechanisms of estradiol (E-2)-dependent repression of gene transcription. E-2 inhibited basal activity and abolished TNF-alpha activation of the TNF-alpha promoter. The E-2-inhibitory element was mapped to the -125 to -82 region of the TNF-alpha promoter, known as the TNF-responsive element (TNF-RE), An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) beta is more potent than ER alpha at repressing the -1044 TNF-alpha promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ERP, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators, These studies demonstrate that E-2-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.