CD-RAP is a small chondrocyte-characteristic gene that is co-expressed in cartilage with type II collagen and aggrecan. CD-RAP is co-expressed with type IIB collagen in chondrogenesis, making it an excellent model for chondrocyte-specific gene expression. This grant renewal will focus on continued analysis of CD-RAP gene expression and explore the trans-acting factors and co-regulators necessary for specific expression in chondrocytes and repressed expression in other cells. We have analyzed 12 kb of the CD-RAP gene locus, determined multiple regulatory domains and defined a 183-bp regulatory module that confers tissue specificity of CD-RAP gene expression. Within the 183 bp module, a negative regulatory element was discovered that represses gene expression in non-cartilaginous tissues. Our overall strategy is to analyze in detail the mechanism of transcriptional regulation in the CD-RAP gene, then to test these results in other chondrocyte genes, primarily type II procollagen.
The specific aims of this proposal are to: (1) Determine the mechanism of 183-bp-coordinated tissue specificity: activation in chondrocytes and repression in other tissues, and define the minimum """""""" chondrocyte regulatory module"""""""" (CRM). (2) Determine the role of additional negative/ positive regulatory elements (for example, the deltaEF-1 and USFs and the new domains A, B, C and D), in the control of gene transcription. (3) Screen and analyze transcriptional co-regulators (co-repressors and co-activators) that function with the CRM. (4) Extend this analysis of a single gene to the analysis of the chondrocyte phenotype by (a) indentification of novel cartilage genes regulated by the CRM and (2) Identification of novel motifs for chondrocyte-characteristic genes. The experimental approach will include in vivo studies in transgenic mice and in vitro studies using cell culture of primary chondrocytes and cell lines transfected with recombinant DNA constructs.
In Specific Aim 4, the experimental approach will be complimented by a computational analysis using the technique of phylogenetic mapping to determine conserved regulatory domains within a set of genes. These studies will help to elucidate the mechanism for directing gene transcription in chondrocytes and will provide the basic science data that will be necessary to develop strategies to control chondrogenesis during development, in tissue engineering and repair, and in diseases such as osteoarthritis.
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