This research proposal describes experiments designed to reveal the mechanism by which the connexin43 gene is regulated in the myometrium. This gene codes for the gap junction protein that forms cell-cell channels. These channels are responsible for the propagation of electrical pulses through the muscle to allow synchronous contraction of the uterine muscle during labor. In contrast to heart muscle, where connexin43 is present at all times and were it is expressed constitutively, the myometrial cell-cell channels appear only at the onset of labor and disappear shortly after parturition. This induction of cell-cell channel formation is under the control of estrogen and progesterone. The proposed study of connexin43 gene regulation will be done with a rat connexin43 genomic clone that contains 6.7 kb of upstream regulatory sequence. It includes 6 half-palindromic estrogen and 3 progesterone response elements. The binding of transcriptional proteins to these and other regulatory elements will be studied by DNA footprinting analysis and mobility shift assays. Promoter studies will be performed with constructs that contain various segments of the promoter, including upstream regulatory sequences, linked to the reporter gene for luciferase. These constructs will be expressed in transgenic mice, and the effect of different hormones will be determined. The contribution of individual promoter elements will be assessed by mutagenic analysis. Finally, trans-acting transcription factors will be isolated from a cDNA expression library and characterized. The long-term goal of this research is to gain an understanding of the regulation of connexin43 gene expression in the myometrium during pregnancy and at parturition. This research is clinically relevant because it has been shown that premature labor is associated with the premature appearance of myometrial gap junctions. Once the interaction of different hormones with the regulatory elements of the connexin43 gene is better understood, better regimens for the treatment and prevention of premature labor may be designed.
