The estrogen receptor (ER) binds to cis-acting elements termed estrogen response elements (ERE) within target genes with high affinity. Occupation of the ER protein with an estrogen results in the transcriptional regulation of these target genes. The estrogenic ligand is viewed as the inducer of ER function and the ERE has generally been relegated the structural role of a docking site. However, the speed, strength, and duration of a transcriptional response to 17beta-estradiol vary widely between different target genes such as the Xenopus Vitellogenin A2 and the rat prolactin genes. A major difference between the ER complexes formed on these target genes is the primary sequence, tertiary structure, and contextual location of the EREs. How do these differences affect ER function? Instead of a docking site, it is perhaps more productive to view the different EREs as DNA ligands for ER that allosterically affect protein function. The ability of the DNA binding site to induce conformational change in its protein has been demonstrated for a number of transcription factors including the recombinant DNA-binding domain of the glucocorticoid receptor. My colleagues and I have demonstrated changes in two properties of ER upon DNA binding: estrogen dissociation rate and protein surface chemistry. Little information is available on the dependence of the allosteric effect on the structure of the DNA ligand. The experiments detailed in this proposal will address the hypothesis that the DNA ligand allosterically regulates ER function and that this is dependent on the structure of the DNA ligand. Thus the location and structure of an ERE in a gene play a mechanistic role in transcriptional regulation. What are the binding characteristics for ER to a panel of DNA ligands? Sets of plasmids will be constructed that contain the ERE alone or in the context of sequence elements that can form non-B DNA (left-handed helical) structure upon supercoiling of the plasmid. A combination of equilibrium DNA-binding assays and surface plasmon resonance technology in the form of the BIAcore(TM) instrument marketed by Pharmacia will be used to conduct binding experiments. How does DNA ligand structure impact on ER function? Estrogen dissociation and aqueous two-phase partitioning assays will be used to access changes in ER upon binding the panel of DNA ligands. What are the transcriptional consequences of locating an ERE in a region of DNA with the potential for unusual topology? Transfection assays with reporter genes containing the panel of DNA ligands will be used to answer this question. The results of these experiments will define the role of DNA as an allosteric effector of the estrogen receptor.
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