Abstract
Haploinsufficiency or mutation of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 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, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5′-AGGTGTGAAGGTGTGA-3′. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations (F148Y, H194Q and G310S) do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) to further elucidate the molecular pathogenesis of VCFS/DGS.
Highlights
T-box genes encode a large family of transcription factors that are required during embryonic development
Most can bind as monomers to a Brachyury consensus half site, or as dimers to a palindrome, while few can bind to a tandem repeat
We found two classes of binding sites; a perfect direct repeat (TR), consisting of two classic Brachyury halfsites and a second, imperfect direct repeat (KSPS), in which the 59 site is similar to the Brachyury half site, but the 39 half is different
Summary
T-box genes encode a large family of transcription factors that are required during embryonic development. Brachyury has an evolutionarily conserved DNA binding domain, termed the T-box, and can regulate transcription of a reporter gene in cell culture [4,5]. Since the original discovery of Brachyury, nineteen different T-box genes have been identified and are evolutionarily conserved from flies to humans [6,7,8]. Most T-box genes are dispersed on different chromosomes. They are classified based upon sequence homology to each other and are members of five different subfamilies [6,7,8]. As for Brachyury, most T-box transcription factors are required for embryonic development and many are sensitive to altered gene dosage for biological function. Mutations in TBX3 lead to Ulnar Mammary Syndrome, and mutations in TBX5 cause HoltOram Syndrome, both of these presenting disease specific limb and heart defects [9,10] among others [11]
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