title: Mammalian TBX1 preferentially binds and regulates downstream targets via a tandem T-site repeat.
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.
authors.affiliation: Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors.forename: Raquel
authors.initials: R
authors.lastname: Castellanos
authors.affiliation: Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America; Department of Ophthalmology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors.forename: Qing
authors.initials: Q
authors.lastname: Xie
authors.affiliation: Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America; Department of Neurology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors.forename: Deyou
authors.initials: D
authors.lastname: Zheng
authors.affiliation: Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America; Department of Ophthalmology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors.forename: Ales
authors.initials: A
authors.lastname: Cvekl
authors.affiliation: Department of Genetics, Albert Einstein College of Medicine, Bronx, New York, United States of America.
authors.forename: Bernice E
authors.initials: BE
authors.lastname: Morrow
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004062:DiGeorge Syndrome / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D055106:Genome-Wide Association Study; D006801:Humans; D051379:Mice; D008817:Mice, Mutant Strains; D020125:Mutation, Missense; D011485:Protein Binding; D020218:Response Elements; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism*; D020080:Tandem Repeat Sequences; D014158:Transcription, Genetic
chemical_list: D020825:T-Box Domain Proteins; C491573:Tbx1 protein, mouse