Abstract 1558: Developmentally Regulated Alternative Splicing of TbX5 and its Implication for Phenotype-Genotype Correlation in Patients with Congenital Heart Disease
In humans, mutations of Tbx5, a cardiac-enriched member of the Tbox family of transcription factors, cause the Holt-Oram syndrome, an autosomal dominant disease characterized by variable congenital heart defects and upper limb abnormalities. Mutations that occur in the Tbox DNA binding domain resulted in null or hypomorphic alleles; however, the mechanism(s) underlying disease caused by mutations outside the T-box remain largely undefined. Here, we report that Tbx5 expression is developmentally regulated by alternative splicing that yields two protein isoforms with distinct transcriptional properties. In addition to the wild type mRNA that encodes a 516 amino acid (aa) protein, differential splicing of exon 7 results in a frameshift that produces a truncated protein of 256 aa, which lacks the entire C-terminal transactivation domain. RNA analysis revealed that the two transcripts are differentially expressed, with the long isoform predominantly found in embryonic tissues while the isoform encoding the truncated protein is highly expressed in adult tissues. This developmentally regulated splicing can be recapitulated in primary myocyte cultures by growth factors. Protein-DNA, and protein-protein analyses indicate that the short isoforms has markedly decreased DNA binding affinity, but maintains the ability to interact, like the long isoform, with cardiac-enriched cofactors including GATA-4 and Nkx2.5. However, this physical interaction produces distinct transcriptional outcomes on target genes. Finally gain-of-function studies reveal that the two isoforms have differential effects on cell growth and differentiation. These findings identify novel mechanisms regulating Tbx5 activity during development, and provide molecular insight that may help establish much needed genotype-phenotype correlations for congenital heart defects into Tbx5 function in normal and diseased hearts.