Functional Analysis of Craniofacial Genes TWIST1 and SNAI3

Objectives: We recently identified significant associations (p<0.0001) between SNPs rs9931509, 3’UTR of SNAI3 with severe antero-posterior maxillo-mandibular discrepancies and rs2189000, 5’ UTR of TWIST1 with mandibular body length in patients with moderate or severe malocclusion. Evidence suggests that PITX2 and SOX2 regulate TWIST1 during early development and that predicted transcription factor bindings sites (TFBS) for PITX2 and SOX2 exist upstream of TWIST1. Further, our in sílico analysis predicted that the TWIST1 SNP rs2189000 disrupts a TFBS for PITX2. We also found various microRNA binding sites within the 3’UTR of SNAI3. This study evaluates the associated regions for their potential to regulate SNAI3 and TWIST1 function.
Methods: The 4.7 kb TWIST1 5’ UTR region was cloned into a TK-Luciferase vector to test for changes in Luciferase expression in the presence of PITX2 or SOX2. Also an 800 bp region in the 3’ UTR of SNAI3 was cloned into a pMIR-REPORT vector to test effects of predicted microRNAs on the post-transcriptional regulation of SNAI3. Further, a Luciferase transfection reporter plasmid and assays were performed in three cell lines (LS8, CHO and 293T) to uncover regulatory mechanism(s) affecting TWIST1 and SNAI3.
Results: Preliminary results indicate that in the presence of PITX2 or SOX2, Luciferase expression is activated significantly (p<0.05) in LS8 and CHO cell lines. We are testing if the rare allele for rs2189000 alters Luciferase expression responsiveness to PITX2 or SOX2 compared to the wild type and also if predicted microRNAs can affect SNAI3 reporter construct and protein levels.
Conclusions: In silico results predicted that our associated regions in TWIST1 and SNAI3 have regulatory potential. Preliminary results on 2 cell lines suggest that PITX2 and SOX2 regulate TWIST1 expression. Ongoing efforts will test if rare alleles of rs2189000 and rs9931509 will alter TWIST1 expression and SNAI3 post-transcription levels respectively and therefore explain our association results.