TRANSFORMATION INDUCED CHANGES IN MATRIX GENE EXPRESSION

The research proposed in this application is designed to further elucidate the mechanisms controlling type I collagen gene expression in both normal and transformed cells. Type I collagen is the major interstitial collagen and is responsible for the structural integrity of most vertebrate tissues. Alterations in collagen production occur in a variety of pathologic and physiologic conditions, but the molecular mechanisms underlying these changes are not fully understood. Both in vitro and in vivo work is proposed in phase I of this study. Initial experiments will identify DNA/protein interactions in regulatory regions of the alpha 1(I) collagen gene. Sequence elements mediating these interactions will be identified and specific mutations created by site-directed mutagenesis; the resultant change in expression of the alpha 1(I) gene will be assessed by transfection or in vitro transcription. trans-acting factors which bind to regulatory sequences in the alpha 1(I) gene will be identified and their role in regulating transcription or stability of the alpha 1(I) gene characterized. The functions of these cis-elements and trans-acting factors will then be assayed under conditions in which the alpha 1(I) gene is known to be transcriptionally or posttranscriptionally downregulated. Concurrently, two specific questions will be examined in vivo using a transgenic mouse model: 1) what are the minimal sequence elements necessary for tissue-specific expression of the human alpha 1(I) collagen gene, and 2) what role do intronic sequences play in regulating alpha 1(I) transcription in vivo? In phase II, the role of various oncogenes in regulating alpha 1(I) gene expression will be explored in detail. DNA/protein interactions in regulatory regions of the alpha 1(I) gene will be studied before and after transformation by different oncogenes. It is hoped that these studies will not only broaden our understanding of collagen gene regulation, but also further elucidate the mechanisms by which a single transforming gene can effect the dramatic changes in cellular phenotype associated with malignant transformation.