We propose detailed studies towards elucidation of the regulation of elastin gene expression, focusing on the role of cis-elements in the promoter region. The experiments are designed to identify elements which confer tissue-specificity and cytokine responsiveness to elastin promoter expression. This proposal is based on our extensive progress in this project, including (a) cloning of the full-length human elastin cDNA, and elucidation of alternative splicing; (b) characterization of the complete human elastin gene structure, and delineation of the 5'-flanking region; (c) mapping of the human elastin gene to the long arm of chromosome 7; (d) development of human elastin promoter-reporter gene constructs, and their expression in transient cell transfections; (e) development of transgenic mice expressing the human elastin promoter in a tissue-specific and developmentally regulated manner; (f) demonstration of cytokine and hormonal modulation of human elastin promoter activity in cell cultures and in transgenic mice. We now plan to precisely define the cisregulatory elements by developing additional promoter-CAT constructs which, by utilization of transient cell transfections and transgenic mice, will allow identification of discrete nucleotide sequences conferring tissue specificity and cytokine responsiveness. These cis-elements will be characterized by introducing nucleotide substitutions into the putative responsive sequences and by examining trans-acting factors in gel mobility shift assays. The focus in these studies is on TGF-beta, a modulator of extracellular matrix gene expression, and glucocorticosteroids, which may act through receptor-ligand binding to GREs identified in the human elastin promoter region. These experiments are expected to provide critical information on TGF-beta and glucocorticoid-responsive genes in general, not limited to elastin. In addition, we are testing the hypothesis that structural alterations in the genes encoding elastin or associated microfibrils are responsible for clinical phenotype of PXE. In particular, our preliminary linkage data suggest that a subset of PXE families is linked to the FBN2 locus on chromosome 5q23-q3l. After having identified candidate genes by genetic linkage analysis, we plan to establish strategies to scan for mutations by PCR amplification of gene segments, followed by heteroduplex analysis and nucleotide sequencing. We expect that these approaches will allow us to disclose specific defects in the genes involved in elastin fibrillogenesis. In summary, we expect that the proposed studies will have major implications toward illuminating the factors regulating the expression of the elastin gene in normal cells and tissues, and in diseases with alterations in elastic fibers.
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