MULTIHORMONAL REGULATION OF GENE EXPRESSION

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Most eucaryotic genes are subject to multifactorial regulation by signaling pathways that are triggered by developmental, hormonal, tissue- specific, and/or environmental cues. These pathways often activate complex transcriptional cascades that give rise to altered amounts of protein encoded by target genes hours and even days after the initial signal. Our long term goal is to understand how these complex regulatory events are coordinated to elicit appropriate levels of gene expression. The overall aim of this proposal is to investigate the transcriptional cascades that are evoked by the binding of steroid hormones to their cognate receptors. In particular, our goal is to investigate how estrogen and glucocorticoid induce "secondary" or late response genes. The ovalbumin gene is indirectly induced by estrogen, glucocorticoid, androgen, and progesterone. Thus, this gene is an excellent model system for investigating the molecular events linking the binding of steroid receptors to early response genes with the subsequent activation of late response genes. The Specific Aims of this grant are to: I. Ascertain how estrogen and corticosterone induce expression of the ovalbumin gene; and II. Define the role of the negative regulatory element in the positive and negative regulation of the ovalbumin gene. To address the first aim, the plan is to clone two steroid-responsive, cycloheximide-sensitive proteins by using the yeast one-hybrid system. In vivo footprinting techniques will provide the basis for investigating several important questions. These include determining how estrogen and corticosterone synergize to induce transcription of the ovalbumin gene and ascertaining whether single-stranded DNA binding proteins are involved in transcriptional regulation. Mutagenesis and transfection experiments will be employed to continue the functional analysis of the NRE. As steroid hormones have been implicated in the etiology of some cancers, a better. understanding of the molecular events provoked by steroids can only improve the treatment and prevention of these malignancies. Furthermore, such knowledge may provide insights into the development of better contraceptives and aid in the treatment of reproductive and metabolic disorders.