Effect of ligand and DNA binding on the interaction between human transcription intermediary factor 1α and estrogen receptors

Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. To achieve efficient regulation of gene expression, these receptors must interact with different type of molecules: 1) the steroid hormone, 2) the DNA response element, and 3) various proteins acting as transcriptional cofactors, In the present study, we have investigated how ligand and DNA binding influence the in vitro interaction between estrogen receptors (ERs) and the transcription intermediary factor hTIF1 alpha (human transcriptional intermediary factor 1 alpha). We first optimized conditions for the coactivator-dependent receptor ligand assay to lower ED50, and we then analyzed the ability of various natural and synthetic estrogens to allow the binding of the two types of proteins. Results were compared with the respective affinities of these ligands for the receptor. We then developed a protein-protein-DNA assay allowing the quantification of cofactor-ER-estrogen response element (ERE) complex formation in the presence of ligand and used measurements of fluorescence anisotropy to define the equilibrium binding parameters of the interaction. We demonstrated that the leucine-charged domain of hTIF1 alpha is sufficient to interact with ERE-bound ER alpha in a ligand-dependent manner and showed that binding of ER alpha onto DNA does not significantly effect its hormone-dependent association with TIF1 alpha. Finally, we show that, mainly in the absence of hormone, hTIF1 alpha interacts better with ERP than with ER alpha independently of the presence of ERE.