This study will identify the structure and test the functional significance of DNA sequences responsible for the strict temporal and spatial transcription of two natriuretic peptides genes, the type-B(BNP) and type-C (CNP) of the mouse. For many genes such DNA regulatory elements are located proximal to the protein encoding region. However, distal elements can also regulate transcription, particularly for genes that constitute a multigene family locus, such as the alpha nd beta- globin gene clusters. Studies of natriuretic peptide hormone gene transcription to date have focused on DNA sequences located proximal to the type-A gene (ANF) of the rat and human. Analysis of the transcriptional mechanisms regulating the other two members of this gene family have not been reported. The applicant recently isolated and characterized the DNA sequence of genomic clones harboring the mouse BNP gene. In addition, the GNP gene was mapped to mouse chromosome 4, the same location as the ANF gene. This novel observation could have significant mechanistic implications for natriuretic peptide gene expression. The present study will extend these observations to the third member of the mouse NP gene family, the CNP gene. Genomic and cDNA clones will be isolated, sequenced, and the CNP gene mapped. The tissue specificity of BNP and CNP gene expression will be analyzed in adult mice. The DNA elements that regulate expression of the mouse BNP will be studied in depth, using several complementary structural-functional methods. These assays include transfection studies of BNP-reporter genes in AT-1 cardiomyocytes. Interactions between BNP DNA elements and proteins present in cardiomyocyte nuclear extracts will be characterized by several in vitro methods including electrophoretic mobility shift assays, and DNA footprinting. In addition, the BNP regulatory elements will be examined in transgenic mice. The CNP gene also will be studied in transgenic mice, but instead of using a relatively benign reporter gene, the SV40T-antigen oncogene will be used in an attempt to derive a cell line that synthesizes CNP. These studies should increase our understanding of DNA elements that regulate expression of the members of the natriuretic peptide gene family, In addition, this study may provide information about unique tissue-specific transcriptional regulatory elements and proteins.
