Relatively little is known about the molecular mechanisms involved in human uterine decidualization. Over the past few years, our laboratory has developed an in vitro model system of human decidualization that indicates that prolactin is a model gene with which to delineate the molecular mechanisms involved in the regulation of decidualization. We have identified several DNA binding proteins in nuclear extracts of undecidualized endometrial stromal cells that are downregulated during decidualization and several binding proteins that are induced during decidualization. Two of these binding proteins appear to be decidua- specific. Our central hypothesis is that delineation of the cis- and transacting factors regulating the expression of decidual prolactin during decidualization will provide new insights into the molecular mechanisms involved in the decidualization process.
Our specific aims are to 1) determine the cis-acting DNA elements on the decidual prolactin promoter that are critical for induction of prolactin gene expression during decidualization, 2) identify and characterize the transcription factors that mediate induction of the prolactin gene during decidualization, and 3) test the hypothesis that transcription factors that mediate induction of prolactin gene expression during decidualization are also involved in the determination of decidualization and the associated changes in gene expression. Particular emphasis will be given to characterization of novel decidua- specific transcription factors.
Aim 1 will utilize EMSAs, DNase 1 footprinting and transfection studies with deletion and site-directed mutants of the decidual prolactin promoter in primary cultures of human endometrial cells.
Aim 2 will utilize supershift and competitive binding assays, and the sequence of a novel transcription factor(s) will be determined after expression cloning of a human decidual library.
In Aim 3, undecidualized endometrial stromal cells will be transfected with expression plasmids that overexpress the transcription factors identified in Aim 2 to determine whether these factors, alone and in combination, induce the cells to express prolactin and acquire other phenotypic changes characteristic of decidualized endometrial stromal cells. Taken together, these investigations should provide new insights into the molecular mechanisms involved in the cascade of transcription factors involved in prolactin gene expression and the regulation of human decidualization and. Such investigations are of practical importance since decidualization is essential for successful uterine implantation and abnormalities of decidualization are frequent causes of spontaneous human abortion.
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