The long-term goals of this project are to examine the mechanism of ER regulation in reproductive tissue. Much of the earlier work has focused on the Xenopus laevis ER and this proposal continues some of these studies, but emphasizes studies of the structure and function of the human ER.
Aim 1 is to test the hypothesis that tighter binding to the ERE in ER mutants partially activates the ER, resulting in estrogen-independent transcription by the ER, and enabling antiestrogens to activate the ER. To accomplish this, ER mutants will be produced exhibiting enhanced binding to ERE using a bacteriophage P22 challenge phage system. The relative affinity for the ERE will be correlated with the estrogen independent activity and antiestrogen activation. Finally, the role of the affinity of the ER for the ERE in ER phosphorylation by a DNA- dependent kinase will be examined.
Aim 2 is to determine whether intrinsically bent DNA near the vitellogenin EREs potentiates the ability of the EREs to activate transcription.
Aim 3 is to determine if intrinsic DNA bending sequences within the ERE can influence the ability of ER to bind to the ERE and to activate transcription.
Aim 4 will identify the DNA sequences and mechanisms responsible for the developmental induction of XER mRNA and the long-term autoregulation of XER gene expression.
Aim 5 will examine the role of chromatin assembly in transcriptional silencing of the vitellogenin promoter, determine whether unliganded ER or ER complexed to antiestrogens can bind to ERE- containing chromatin, and the role of the two ER transactivation domains in the transcription of chromatin templates.
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