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

Abstract

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 hTlF1α (human transcriptional intermediary factor 1α). 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 hTlF1α is sufficient to interact with ERE-bound ERα in a ligand-dependent manner and showed that binding of ERα onto DNA does not significantly affect its hormone-dependent association with TlF1α. Finally, we show that, mainly in the absence of hormone, hTlF1α interacts better with ERβ than with ERα independently of the presence of ERE.