Characterization of the Role of SOX9 in Cartilage-Specific Gene Regulation
Although advances have been made toward understanding the complex mechanisms that regulate the process of DNA transcription, the specific mechanisms of activation for many individual genes remain unknown. In this study, we focus on the role the transcription factor SOX9 plays in activating cartilage-specific genes, specifically Col9a1 and Cartilage Link Protein (CRTL1). Previously, enhancers of these genes containing single SOX9 binding sites were shown to be activated through SOX9 binding. However, the hypothesis was made that in cartilage-specific genes dimeric SOX9, as opposed to monomeric SOX9, is necessary for activation. We identified a putative binding site adjacent to each of the known single SOX9 binding sites in the Col9a1 D and E enhancers and in the CRTL1 enhancer. Electrophoretic Mobility Shift Assays (EMSAs) were performed to determine whether SOX9 bound to these putative sites. Transient transfections were then performed using wild-type and mutant enhancer- reporter plasmids to determine whether these putative SOX9 binding sites were important for activation in vivo. Although dimeric SOX9 bound to each of the enhancers in vitro, several different effects were seen in vivo. In the presence of the wild-type Col9a1 D enhancer, no activation was seen. However, when the enhancer was extended to include an additional pair of newly found SOX9 binding sites, expression was increased 10-fold. When any of the four SOX9 binding sites within this enhancer were mutated, expression was completely eliminated, suggesting that interdependent dimers or a tetramer of SOX9 is necessary for the activation of transcription. The weaker Col9a1 enhancer E was found to increase gene expression minimally through binding of either dimeric or monomeric SOX9. However, dimeric SOX9 was required for the activation of gene expression by the CTRL1 enhancer. Through this study we validate the importance of not just monomeric but of dimeric and possibly tetremeric SOX9 as an activator of cartilage-specific gene expression.
SOX9, cartilage gene regulation, Microbiology
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