application): The renin-angiotensin system is a major regulator of blood pressure and electrolyte balance in animals and humans. Renin is a rate-limiting enzyme of the RAS and its synthesis is highly regulated both at the transcriptional and secretory levels. Despite many years of study, the molecular mechanism defining renin gene transcription is still poorly understood. Major advances in the understanding of the mechanisms regulating this important gene have been made during the past several years and include: 1. Identification of an enhancer of transcription regulating the mouse renin gene. 2. Identification of a functional homologous enahancer sequence upstream of the human renin gene. 3. Identification of a second renin gene enhancer present upstream of human renin gene which is functional in primary chorioducidal cells. 4. Development new transgenic models containing p1 artificial chromosome (PAC) transgenes which not only accurately emulate these tissue and cell specific pattern of human renin expression but also the exquisite regulation of the gene in response to physiological cues. 5. Significant technical progress allowing to generate the germ line knockout of the renin enhancer in mouse genome generate mutations of the human renin enhancers in large pack clones and generate transgenic mice containing mutated packs. The PI will test the following hypothesis: The enhancer sequences found upstream of the mouse and human renin genes along with their bound cognate transcription factors play a major role in controlling the transcriptional regulation of renin gene expression in vivo including cell and tissue specificity, developmental expression, hormonal responsiveness and transcriptional responses to physiological cues. To accomplish this, the PI proposes two specific aims. 1. Elucidate the molecular mechanisms regulating activity at the mouse renin enhancer in As4.1 cells, which are derived by JG tumors. 2. Elucidate the physiological significance of the mouse renin enhancer and the human renin kidney and chorionic enhancers in vivo. The PI anticipates that the experiments proposed in these aims should provide, for the first time, important information on the factors regulating renin expression and their relevance in in vivo setting.
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