Functional characterization of the Holt-Oram syndrome associated transcription factor Tbx5 during embryonic heart development

Holt-Oram Syndrome is a congenital disorder characterized by limb malformations and heart defects, which include incomplete atrial and ventricular septation and conduction system anomalies. Mutations in the human TBX5 gene, a member of the T-box family of transcription factors, are associated with Holt-Oram Syndrome. It is hypothesized that these mutations disrupt embryonic development by inhibiting transcriptional function of the Tbx5 protein. However, the genes regulated by Tbx5 in heart development and the transcriptional mechanisms by which this occurs are not fully characterized. To understand how Tbx5 regulates transcription, an in vitro approach was utilized. Biochemical assays demonstrated that Tbx5 is a transcriptional activator, harbors a potent activation domain, and synergistically activates transcription in cooperation with cardiac transcription factors. When comparing the transcriptional activity with Tbx20, a related T-box gene expressed in the heart, it was demonstrated that its transcriptional activity is distinct from that of Tbx5. Specific cis-elements of the nppa promoter that mediate the transcriptional activities of Tbx5 and Tbx20 were also identified. In vivo, the expression patterns and signaling pathways that regulate expression of Tbx5 and Tbx20 are distinct, which is consistent with their divergent transcriptional function. In order to identify novel downstream targets of Tbx5 transcriptional activation a genome wide microarray screen was performed on cardiac-derived cells overexpressing Tbx5. A number of Tbx5-induced genes were identified that also are expressed similarly to Tbx5 during heart development. In addition, it was demonstrated that the transcriptional activities of Tbx5 defined in these studies are significantly compromised by Holt-Oram Syndrome associated mutations. Collectively, the results described herein characterize transcriptional activities of Tbx5 and give insight into how mutations in Tbx5 can lead to cardiac defects.