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

Abstract

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.

Tbx5 overexpression favors a first heart field lineage in murine embryonic stem cells and in Xenopus laevis embryos

“…The observed up-regulation of other cardiac factors such as Nkx2.5, Mef2c, Bmp2, and endogenous Tbx5 upon Tbx5 overexpression is agreement to results in other cell lines (Hiroi et al, 2001;Sun et al, 2004;Takeuchi et al, 2005). Taken together these data indicate that Tbx5 is involved in an initial regulatory network displaying mutual regulation with Nkx2.5 (Sun et al, 2004) and supports with Mef2c the induction of downstream factors of terminal differentiation .…”

“…Taken together these data indicate that Tbx5 is involved in an initial regulatory network displaying mutual regulation with Nkx2.5 (Sun et al, 2004) and supports with Mef2c the induction of downstream factors of terminal differentiation . In addition, MLC1a, an atrial marker gene, is up-regulated upon Tbx5 overexpression, while Hand1, a transcription factor that is expressed in the left ventricle and prevents terminal differentiation (Firulli et al, 1998), is down-regulated.…”

Fbxo25 controls Tbx5 and Nkx2–5 transcriptional activity to regulate cardiomyocyte development

“…6A). Intriguingly, this finding supports the reports that Tbx5 and Nkx2-5 regulate their transcriptional activities by binding to their own promoters to increase mRNA and proteins in an auto-regulation fashion [30,31]. Additionally, Fbxo25 reduction led to downregulation of the mRNA expression levels of Myh6 and Myh7 compared with shRNA Luc (Fig.…”

TBX3 Is Overexpressed in Breast Cancer and Represses p14ARF by Interacting with Histone Deacetylases

“…Transfection was performed using lipofectamine according to the manufacturer's protocol (24). Briefly, the day before transfection, 8 Â 10 4 Cos-1 cells were plated in each well.…”