Estrogen receptors (ER) mediate growth and differentiation in normal and neoplastic tissues. The presence of ER in breast cancers predicts for efficaceous therapy by estrogens or by antiestrogens, such as tamoxifen. The goal of this project is to elucidate mechanistic aspects whereby activated ER regulate gene expression. A short DNA segment found in estrogen regulated genes was able to confer estrogen responsiveness to another gene in transfection assays. We have synthesized a DNA segment with this sequence, 38 nucleotides long, and inserted it into a plasmid. The sequence bound partially purified calf ER with 400-fold greater efficiency than an equivalent length of plasmid DNA. Optimum binding occurred at 100-150 mM ionic strength, at pH 7.5-8.0, and plasmids with multiple sequence copies bound correspondingly more ER. We now plan to evaluate factors that regulate this binding and to correlate binding efficiency to capacity for induction of gene expression in vivo. We will explore the effects of ligand binding and transformation from the 4S to the 5S form on binding efficiency. We will examine the significance of ligand structure on binding efficiency by study of selected estrogens and an 4-OH tamoxifen, each known to evoke a different response in vivo. We intend to examine ER binding to plasmids containing 1, 2, 3, 4, etc., reiterated sequences, and from these results, infer the DNA length occupied by bound ER. The potential of ER for unwinding or helicase activity will be assessed; positive results will suggest a mechanism for ER action. The sequence contains an inverted repeat symmetry, asymmetrical regions and an A/T rich region. The importance of each region will be evaluated through use of synthetic sequence variants in the binding assay. Simultaneously, each sequence variant will be evaluated for its ability to induce increased gene expression in transfection assays (using ptk-CAT in MCF-7 cells). The correlation of these results will allow us to relate binding efficiency with transcriptional enhancement. Finally, we will ascertain whether communication between the ER-ERE complex and nearby promoters occurs by direct interactions (DNA looping model) or by conducting a signal down the DNA. Our approach will be to measure induction from two promoters placed downstream of an ERE. Also, the orientation of the ER site and promoter on the DNA helix will be altered by systematically changing the distance between them. This research should provide new insight into the characteristics of ER binding to DNA and its functional sequences.
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