Transcription Factor Interactions in Inhibin Regulation

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The overall objective of this research proposal is to understand at a molecular level how interactions between transcription factors and their co-regulatory proteins mediate hormone-dependent gene expression in reproductive tissues. These studies will be performed using the gene encoding the alpha subunit of inhibin, a gonadal protein that modulates pituitary follicle-stimulating hormone (FSH) synthesis and secretion. Inhibin gene expression is regulated by FSH through a pathway involving the activation of the cAMP-dependent protein kinase (PKA) and the phosphorylation of the transcription factor CREB (cAMP-response element (CRE) binding protein), which binds to an atypical CRE in the inhibin alpha subunit promoter. FSH action is co-dependent on and augmented by the orphan nuclear receptor steroidogenic factor-1 (SF-1), which binds to an atypical target site adjacent to the CRE in the inhibin alpha subunit gene. It remains unknown what the nature of this interdependence is or how these functional interactions are regulated as a consequence of the hormonal and cellular changes that occur during a normal reproductive cycle. Experiments in aim 1 will address these issues by investigating the time course of promoter occupancy by CREB and SF-1 following hormone stimulation and by testing for direct interactions between the two proteins on the inhibin alpha subunit promoter. Experiments in aim 2 will investigate the hypothesis that the transcriptional coactivators CBP and SRC-1 may mediate in part the functional interdependence observed between CREB and SF-1 by facilitating formation of a higher-order protein complex including these co-regulatory proteins. Finally, experiments in aim 3 will investigate the structural basis for functionally important protein-DNA and protein-protein interactions using NMR-based approaches. These studies will provide mechanistic insights that will increase our knowledge of ovarian gene regulation during the reproductive cycle and will be applicable to understanding defects in gene expression and regulation that may occur in reproductive disease of importance to human health.