MULTIHORMONAL REGULATION OF GENE EXPRESSION

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Most eucaryotic genes are subject to multifactorial regulation by developmental, hormonal, tissue-specific, and/or environmental cues. The net amount of protein made from a gene must therefore reflect how these signals integrate. Our long term goal is to understand how these complex regulatory events are coordinated to elicit appropriate levels of gene expression. The chicken ovalbumin (Ov) gene serves as an excellent model for studying multifactorial regulation of gene expression because it is regulated at the transcriptional level by four classes of steroid hormones, by insulin, and by other positive and negative modulators in a strictly tissue-specific manner. The major focus of this proposal is to understand how steroid hormones activate the Ov gene. Intrinsic to the comprehension of this problem is an analysis of how steroids function synergistically to activate late response genes. As most genes induced by steroids are members of this class, it is essential that an understanding be achieved of the intermediary events between activation of the early genes by steroid receptors and the subsequent activation of the late genes. The Specific Aims of this grant are to I. Identify and characterize the steroid-responsive early response proteins that regulate the Ov gene, II. Investigate the synergistic effects of steroid hormones on the transcription of the Ov gene, and III. Examine the role of the negative regulatory element in modulating expression of the Ov gene. To this end, the gene regulatory proteins will be cloned and characterized that bind to the major regulatory elements in the Ov gene, the steroid-dependent regulatory element (SDRE) and the negative regulatory element (NRE). Transfection experiments with linker scanning mutations through the SDRE and NRE will be employed to define specific roles for each protein binding site. Genomic footprinting will be done to assist in determining how synergism is achieved by two steroids and how the SDRE and NRE act as a single functional entity. As steroid hormones have been implicated in the etiology of some cancers, a better understanding of the molecular events triggered 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 disorders.