MULTIHORMONAL REGULATION OF GENE EXPRESSION

The long range goal of this proposal is to understand the complex regulatory events involved in the multihormonal control of gene expression and cellular differentiation with particular emphasis on understanding the mechanism of action of steroid hormones. The chicken ovalbumin gene represents and excellent model for these studies because two steroid hormones, estrogen and any second steroid, synergize with the peptide hormone insulin to increase expression of this gene over 200-fold in primary oviduct cell cultures. The hormonal regulation of another egg white gene lysozyme will be used to distinquish between gene-specific regulatory mechanisms and more generally applicable events. The specific aims of this proposal are to (1) identify and characterize the regulatory elements responsible for steroid-dependent transcription of the ovalbumin and lysozyme genes in primary oviduct cells, (ll) investigate the multihormonal interactions tht regulate the ovalbumin gene, (lll) define and characterize the regulatory elements in the ovalbumin and lysozyme genes responsible for tissue -specific expression, and (lV) determine how estrogen increases the stability of mRNAs from target genes. These objectives will rely initially on gene trasfer techniques to identify functionally relevent regions in the ovalbumin and lysozyme genes. Genes have already been created containing the 5'-flanking regions of the ovalbumin or lysozyme genes fused to the marker gene bacterial chloramphenicol acetyltransferase (CAT). Deletion and linker scanning mutants will be made in the fusion genes, and they will be transfected into primary oviduct cell. The cells will be cultured in the appropriate hormonal milieu prior to assay for expression of the transfected and endogenous egg white genes. Once regulatory elements have been identified, studies will focus on isolating and characterizing the proteins that bind to these regions. Information about the mechanism of action of steroid hormones and about the multihormonal regulation of gene expression should prove invaluable for understanding complex regulatory events in higher eucaryotes. As steroid hormones and growth factors have been implicated in the etiology of cancers of the reproductive tissues such as breast and prostate, a better understanding of these molecular events can only improve the treatment and prevention of these malignancies. In addition, such knowledge may provide insights into the development of better contraceptives and aid in the treatment of reproductive disorders.