A single exposure of male Xenopus laevis to estradiol-17-beta induces hepatic transcription of the genes coding for the egg yolk precursor protein, vitellogenin (VIT), and elicits the long-term induction of Xenopus estrogen receptor (XER) mRNA and serum retinol binding protein gene transcription. To study estrogen regulation of gene expression we developed stable Xenopus liver and fibroblast cell lines, cloned and expressed the Xenopus estrogen receptor (XER), and used mutagenesis and cotransfection to analyze the domain structure of the XER, and to identify regulatory sequences in the vitellogenin promoter. We have identified a far upstream palindrome related to the consensus estrogen response element (ERE) which may mediate autoregulation of XER gene expression. The putative XER ERE, and the native ERE, will be compared in transfections, and in binding and competition studies using our purified mutant XER. A specific transcription activation domain has not been identified in the XER or human ER. We shall test the ability of acidic amphipathic helices located in the hormone binding domain of the XER, and synthetic transcription activators, to increase the activity of wild type and mutant XER. We have overexpressed and purified a 111 amino acid XER mutant, which binds to the ERE and retains considerable ability to activate transcription. In a collaborative study, this mutant, and other XER mutants, will be bound to EREs separated by a few hundred nucleotides. We will examine the ability of the ERE-bound XER mutants to establish direct protein-protein contact by looping out the DNA. Electrophoresis, protein cross-linking using bifunctional reagents, and electron microscopy will be used to identify and characterize the complexes. Using purified XER mutants and our high efficiency HeLa cell transcription extract we will: (A) Correlate in vivo activity of mutant XERs with their ability to activate transcription in extracts. (B) Use our powerful synthetic ERE-TATA promoters as immobilized templates to begin to characterize the components of a simplified XER regulated transcription complex. (C) Use an immobilized native VIT promoter to determine whether the XER remains bound to the promoter during transcription, or dissociates after facilitating formation of an initiation complex involving an essential activator element we have identified. (D) The activator element linked to a TATA box is a powerful transcription activator, yet it is inactive in the VIT promoter unless XER is bound. We shall determine whether its activity is suppressed by unliganded XER, or by nucleosome phasing.
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