Development and application of a fluorescence anisotropy microplate assay for analyzing the interaction of steroid hormone receptors with DNA and coactivators

A key event in estrogen receptor (ER)-mediated transcription is the binding of the ER to its DNA recognition sequence, the estrogen response element (ERE). To analyze this interaction, we used ultra low-volume microplates to develop the simple and rapid fluorescence anisotropy microplate assay (FAMA). The FAMA was applied to the binding of estrogen and progesterone receptors to their respective DNA response elements. The FAMA is also the first system suitable for screening large compound libraries to identify novel compounds that modulate binding of steroid receptors to their DNA response elements. Additionally, the FAMA can monitor the time course of dissociation of pre-formed steroid-receptor-DNA complexes. This feature of the FAMA has confirmed the DNA intersegment transfer model for how steroid receptors search for their target sites. Following ER-ERE binding, a second key event in ER-mediated transcription is the recruitment of P160 coactivators by ER. The FAMA represents the first reliable in vitro assay for the binding of full-length P160 coactivators to the ERE-ER complex. Binding of full-length SRC1a to the ERE-ERalpha and ERE-ERbeta complex occurred only in the presence of 17beta-estradiol (E2), as previously reported. Differential inhibition of ERE-ER interaction with SRC1a was observed with different NR-Box peptide competitors. Furthermore, the profile of peptide-mediated inhibition differed between ERE-ERalpha and ERE-ERbeta. The ability of NR-Box peptide competitors to dissociate pre-formed ERE-ER-SRC1 a complexes provided evidence for the sequential model of coregulator utilization in transcriptional activation by steroid receptors. Finally, we used a modified Glutathione S-transferase (GST) pull down assay to investigate the role of the ERE in P160 coactivator recruitment. In the absence of the cERE, addition of whole cell extract caused a 3-4 fold reduction in ER-coactivator interaction, compared to binding observed without extract. Interestingly, a 2-3 fold recovery from extract-mediated repression was observed for ER(E2) in complex with the cERE. This effect is not due to degradation of ER and is specific for ERE DNA. Such results suggest a novel role for the cERE in protecting established ER-P160 coactivator interactions in the face of the complex cellular milieu.