The expression of Gli3, regulated by HOXDI3, may play a role in idiopathic congenital talipes equinovarus

Background: Idiopathic congenital talipes equinovarus (ICTEV) is a congenital limb deformity. Based on extended transmission disequilibrium testing, Gli-Kruppel family member 3 (Gli3) has been identified as a candidate gene for ICTEV. Here, we verify the role of Gli3 in ICTEV development. Methods: Using the rat ICTEV model, we analyzed the differences in Gli3 expression levels between model rats and normal control rats. We used luciferase reporter gene assays and ChIP/EMSA assays to analyze the regulatory elements of Gli3. Results: Gli3 showed higher expression levels in ICTEV model rats compared to controls (P < 0.05). We identified repressor and activator regions in the rat Gli3 promoter. The Gli3 promoter also contains two putative HOXDI3 binding sites. Using EMSA, the HOXDI3 binding site 2 was found to directly interact with HOXDI3 in vitro. ChIP assays of the HOXDI3-Gli3 promoter complex from a developing limb confirmed that endogenous HOXDI3 interacts with this region in vivo. Conclusion: Our findings suggest that HOXDI3 directly interacts with the promoter of Gli3. The increase of Gli3 expression in ICTEV model animal might result from the low expression of HOXDI3.