The Meta-analysis Of TBX5 SNP Rs3825214 And Atrial Fibrillation,and The Functional Study Of Tbx5 For Col10a1 Gene Expression And Chondrocyte Development

The T-box gene family refers to a group of transcription factors that share a highly conserved,sequence-specific DNA-binding domain(T-box)containing around 180-amino acids.According to HUGO gene nomenclature committee(HGNC),there are 18 T-box family members.These T-box genes have been implicated essential roles during embryogenesis and cardiac development,given their specific expression pattern in developing mammalian heart for several T-box genes,including TBX5.TBX5 is consisted of three transcriptional variants which cover 9 exons and encode two distinct isoforms that differ in N-terminus.TBX5 isprobably the most frequently studied T-box gene over the past decade due to the typical cardiac defects observed in Holt-Oram syndrome(HOS),which is caused by TBX5 mutation.Most of the mutations are within exons 3-7 where locate sequence coding for the T-box domain.Notably,a variety of cardiac defects,as well as abnormalities in limb and other organs have been seen in HOS syndrome with different kinds of TBX5 mutations,suggesting a heterogeneous disease mechanism.We have performed a meta-analysis of TBX5 and found a significant correlation between its single nucleotide polymorphism(SNP)rs3825214(A to G),and risk of atrial fibrillation and its subtypes,supporting TBX5 as a master transcription factor for cardiac development.In addition,bioinformatics analysis of this SNP identified several TFs that may be affected for their binding affinity with TBX5.Identification and characterization of more TBX5 mutations and SNPs hold promise for therapeutic strategy targeting TBX5 associated developmental abnormalities and diseases.Background: The type X collagen gene(COL10A1)is specifically and highly expressed in hypertrophic chondrocytes.Mutation or abnormal expression of human COL10A1 gene which is often accompanied with aberrant chondrocyte maturation or hypertrophy has been observed in multiple skeletal diseases.Previous studies from our lab have shown that Runx2 is an essential transcription factor(TF)controlling cell-specific mouse Col10a1 gene expression via direct interaction with its cis-enhancer.Notably,multiple transcription factors,including Tbx5,showed similar binding sites within Col10a1 cis-enhancer that are adjacent or overlap with that of Runx2.Based on the expression profiling of these candidate transcription factors in different mouse chondrogenic cell models,we hypothesized that Tbx5 may interact with Col10a1 cis-enhancer and negatively correlate with its expression and thereby,a potential inhibitor of Col10a1 gene regulation.Aims: We aim to investigate the correlation of Tbx5 and Col10a1 gene expression and in vitro function of Tbx5 on chondrocyte hypertrophy(or maturation)by using two different mouse chondrogenic cell models.Methods: 1),A web-based promoter analysis software,the MATCH program was used to identify candidate TFs that may bind to a known 150-bp mouse Col10a1 gene cis-enhancer.Expression of candidate transcription factors in mouse MCT and ATDC5 cells were examined using q RT-PCR.These mouse chondrocytes are classified as proliferative and hypertrophic chondrocytes because of their different culturing conditions and exprerssion of marker genes.2),The vectors of Pcmv-Tbx5 and Col10a1-Tbx5 were constructed.3),Using gene transfection si RNA and cell staining approaches to over-express or inhibit Tbx5 in MCT and ATDC5 cells,and then to evaluate the regulation of Tbx5 on Col10a1 gene expression and chondrocyte hypertrophic differentiation.Results: 1),Based on MATCH program prediction,there are 6 potential transcription factor binding sites(TFBS)within the 150-bp Col10a1 cis-enhancer.Only Tbx5 showed significantly decreased expression in hypertrophic MCT and ATDC5 cells which highlyexpress Col10a1 gene.2),It leads to a significant decrease of Col10a1 expression when Tbx5 is overexpressed in MCT and ATDC5 cells,while interference of Tbx5 results in a significant increase of Col10a1.This indicates that Tbx5 negatively regulates the expression of Col10a1,and is a potential inhibitor for chondrocyte hypertrophy.Conclusion: Based on MATCH program prediction,there are 6 potential transcription factor binding sites(TFBS)within the150-bp Col10a1 cis-enhancer.Tbx5 is negatively correlated with Col10a1 gene expression in MCT and ATDC5 chondrogenic cell models and potentially inhibits chondrocyte hypertrophic differentiation.Tbx5 may interact with Runx2 and other TFs to regulate Col10a1 expression,and thus,affect the process of chondrocyte maturation in relevant skeletal development and diseases.