The objective of this project is to perform a focused and detailed analysis of transcriptional regulation in one chondrocyte-specific gene, Col11a2, in order to identify protein factors that play a role in the activation of this and perhaps other chondrocyte-specific genes. This work will provide information that can be applied to the development of chondrocyte-targeted gene therapy vectors for cartilage disorders such as osteoarthritis. A thorough mutational analysis of the Col11a2 gene has already identified three chondrocyte-specific enhancer elements that are essential for the tissue-specific expression of the gene, and the critical protein-binding sites within each enhancer have been pinpointed. The goal now is to identify the proteins that bind at those critical sites. The work needed to accomplish this goal has been divided into three Specific Aims, as follows: The first Specific Aim is to use the yeast one-hybrid system to identify candidate DNA-binding proteins that may regulate the Col11a2 gene. The second Specific Aim is to use affinity chromatography and mass spectrometry to identify additional candidate DNA-binding proteins. Both approaches will be used because each offers advantages and strengths that complement the limitations of the other approach. The third Specific Aim is to use multiple complementary experiments to test the hypothesis that each one of the identified candidates directly binds the Col11a2 gene and activates expression in vivo. The multiple approaches will include electrophoretic mobility shift assays, transfection-based experiments, in vivo DNA-protein crosslinking, and targeted gene inactivation in mice. This project will result in the identification of multiple proteins that cooperate to achieve chondrocytes-specific expression of the CoI11a2 gene, thus filling a significant gap in our understanding of the mechanisms by which chondrocyte-specific gene expression is regulated. These results will be useful in the development of tissue-targeted gene therapy vectors for the treatment of cartilage disorders. The proteins identified will also become the subjects of future studies on the regulation of other cartilage genes. Finally, the genes for the proteins identified can be examined in future linkage analysis studies to see if they participate in a genetic predisposition to osteoarthritis.
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