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 hTIF1α (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) co...