TBX5, a gene mutated in Holt-Oram syndrome, is regulated through a GC box and T-box binding elements (TBEs).

TBX5 is a member of the T-box gene family and encodes a transcription factor that regulates the expression of other gene(s) in the developing heart and limbs. Mutations of TBX5 cause Holt-Oram syndrome (HOS), an autosomal dominant condition characterized by congenital heart defects and limb anomalies. How TBX5 gene expression is regulated is still largely unknown. In order to identify transcription factors regulating TBX5 expression, we examined the 5'-flanking region of the human TBX5 gene. We determined that up to 300 bp of the 5'-flanking region of the TBX5 gene was necessary for promoter activity in mouse cardiomyocyte ECL2 cells. One GC box, three potential T-box-like binding elements (TBE-A, -B, and -C), and one NKX2.5 binding site were identified. Site-directed mutagenesis of the potential binding sites revealed that the GC box, TBE-B, TBE-C, and NKX2.5 are functionally positive for the expression of TBX5. DNA footprint analysis showed that these binding regions are resistant to DNaseI digestion. Electrophoretic mobility shift assays (EMSAs) further demonstrated the protein-DNA interactions at the GC box and the potential TBE-B, TBE-C, and NKX2.5 sites in a sequence-specific manner. The ability of TBX5 to regulate its own promoter was demonstrated by the ability of ectopically expressed human TBX5 to increase reporter expression. We conclude that the GC box, T-box-like binding elements, and NKX2.5 binding site play important roles in the regulation of TBX5 expression, and that TBX5 is likely to be autoregulated as part of the mechanism of its transcription. Copyright 2004 Wiley-Liss, Inc.