TBX1 Preferentially Binds and Regulates Transcription of Downstream Targets Via a Tandem T-site Repeat
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Haploinsufficiency of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial syndrome/DiGeorge syndrome/ 22q11 deletion syndrome. TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins bind to similar but not identical consensus DNA sequences, consisting of two half-sites in a palindromic orientation, indicating that there are specific binding preferences. Binding preferences are important to delineate to understand how transcription factors belonging to the same family distinguish their correct direct downstream targets.;We hypothesized that TBX1 has a preferential DNA binding. In addition, having this consensus sequence would help us identify novel direct downstream targets of TBX1, of which very few are known. TBX1 protein was found to bind strongly to a tandem repeat, 5'-AGGTGTGAAGGTGTGA-3', unlike most other family members that prefer binding to a palindrome. A second motif, 5'-AGGTGTGATCGCGTCAT-3', sharing the same orientation as the tandem repeat was also identified and consisted of a more degenerate second half site. In silico genome wide binding site analysis identified putative mouse TBX1 target genes. Three candidate loci, Fgf8, MyoD, Bmper, were bound and activated by TBX1 and mRNA expression was reduced in Tbx-/- embryos, suggesting direct transcriptional regulation.;Cases of patients with point mutations in TBX1 have been reported. I have tested three of these mutations (F148Y, H194Q and G310S) and found that they do not alter binding to the consensus sequence but lead to a decrease in the activation of transcription (F148Y, G310S) in cell culture reporter assays thereby explaining the basis of the defects. Collectively the present studies show a novel DNA binding mechanism of TBX1, provide new insights into disrupted function of mutated TBX1 proteins and identified 3 potential direct transcriptional target genes.
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