DIFFERENTIAL INTERACTION OF NUCLEAR RECEPTORS WITH THE PUTATIVE HUMANTRANSCRIPTIONAL COACTIVATOR HTIF1

Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. Intermediary factors interacting with their activation functions are required to mediate t ranscriptional stimulation. In search of such receptor interacting pro teins, we have screened a human cDNA expression library and isolated a human protein that interacts in vitro with transcriptionally active e strogen receptors (ER). Sequence analysis reveals that this protein is the human homolog of mouse TIF1 (transcription intermediary factor 1) shown to enhance nuclear receptor ligand-dependent activation functio n 2 (AF2) in yeast. We have characterized the nuclear receptor binding site on hTIF1 and shown that a region of 26 residues is sufficient fo r hormone-dependent binding to the estrogen receptor. As shown by poin t mutagenesis, the AF2 activation domain of ER is required for the bin ding of hTIF1 but not sufficient, since a short region encompassing th e conserved amphipathic alpha-helix corresponding to this domain fails to precipitate hTIF1. We also demonstrate that hTLF1 association with DNA-bound ER requires the presence of estradiol. Finally, we show tha t the interaction of hTLF1 with receptors is selective since strong in vitro hormone-dependent binding is only observed with some members of the nuclear receptor superfamily.