Transcriptional control for genes which exhibit tissue-specific expression is recognized to be important, and the contribution by positive and negative regulatory elements is of particular interest. Keratocan and mimecan are proteins that are synthesized as major proteoglycans in the corneal stroma of the vertebrate eye, but are either not synthesized in other tissues or are synthesized as partially glycosylated glycoproteins. Recent cloning of bovine and human keratocan genes and bovine mimecan gene, as well as the availability of an in vitro system for maintenance and suppression of expression of these genes, provide a unique model for studying their transcriptional regulation. The overall objective of this project is to analyze the cis-acting transcriptional regulatory elements of keratocan and mimecan genes, to identify corneal proteins that interact with these elements, and to test for specific genetic mutations in both genes. The following questions will be addressed: How are the transcription rates of mimecan and keratocan controlled in ocular vs. non-ocular tissues? Which motifs of the keratocan and mimecan transcriptional regulatory regions account for the serum and FGF-2 effects on their expression? What transcription factors regulate the tissue-specific expression of these genes? What are the functions of multiple mimecan mRNA transcripts synthesized in the same tissue? What is the sequence of cis-acting regulatory/localization elements within the multiple mimecan mRNA transcripts and what proteins bind to these elements? Experiments performed in vitro include: DNase I hypersensitive site mapping, luciferase reporter gene assays, site-directed mutagenesis/deletion of individual elements from the regulatory region to determine their contributions to function, and the cloning and sequencing of new transcription factors that bind to the regulatory motif to characterize their roles in driving transcription. Given the importance of keratan sulfate proteoglycans in providing corneal transparency, the research proposed here may provide novel information about cornea-specific transcriptional regulation and thereby suggest alternative targets for therapeutic up-regulation of corneal KSPGs in pathological situations. In addition, knowledge about tissue-specific transcriptional regulation will be required for successful in vitro generation of tissues for transplantation.
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