Variants in a cis-regulatory element of TBX1 in conotruncal heart defect patients impair GATA6-mediated transactivation

• Published in: Orphanet journal of rare diseases Vol. 16; no. 1; p. 334
• Main Authors: Jiang, Xuechao, Li, Tingting, Liu, Sijie, Fu, Qihua, Li, Fen, Chen, Sun, Sun, Kun, Xu, Rang, Xu, Yuejuan
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 31.07.2021; BioMed Central; BMC
• Subjects: Animals; Binding sites; Chromosomes; Cis-regulatory element; Congenital defects; Congenital heart disease; Conotruncal defect; Development and progression; DiGeorge Syndrome - genetics; Electrophoretic mobility; Embryos; Etiology; GATA6; GATA6 Transcription Factor; Gene expression; Gene regulation; Genetic aspects; Genetic transcription; Genetic variation; Genomes; Haploinsufficiency; Health aspects; Heart; Heart Defects, Congenital; Humans; Immunofluorescence; Immunohistochemistry; Medical research; Medicine, Experimental; Mice; Mutation; Pharyngeal arches; Pharynx; Proteins; Rare diseases; T-Box Domain Proteins - genetics; T-Box Domain Proteins - metabolism; TBX1; Tbx1 protein; Thymic hypoplasia; Transcription; Transcription factors; Transcription Factors - genetics; Transcriptional Activation - genetics; Transfection; China; TBX1; Variant; Pharyngeal arches; Transcription; Conotruncal defect; GATA6; Cis-regulatory element
• ISSN: 1750-1172; 1750-1172
• DOI: 10.1186/s13023-021-01981-4

TBX1 (T-box transcription factor 1) is a major candidate gene that likely contributes to the etiology of velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS). Although the haploinsufficiency of TBX1 in both mice and humans results in congenital cardiac malformations, little has been elucidated about its upstream regulation. We aimed to explore the transcriptional regulation and dysregulation of TBX1. Different TBX1 promoter reporters were constructed. Luciferase assays and electrophoretic mobility shift assays (EMSAs) were used to identify a cis-regulatory element within the TBX1 promoter region and its trans-acting factor. The expression of proteins was identified by immunohistochemistry and immunofluorescence. Variants in the cis-regulatory element were screened in conotruncal defect (CTD) patients. In vitro functional assays were performed to show the effects of the variants found in CTD patients on the transactivation of TBX1. We identified a cis-regulatory element within intron 1 of TBX1 that was found to be responsive to GATA6 (GATA binding protein 6), a transcription factor crucial for cardiogenesis. The expression patterns of GATA6 and TBX1 overlapped in the pharyngeal arches of human embryos. Transfection experiments and EMSA indicated that GATA6 could activate the transcription of TBX1 by directly binding with its GATA cis-regulatory element in vitro. Furthermore, sequencing analyses of 195 sporadic CTD patients without the 22q11.2 deletion or duplication identified 3 variants (NC_000022.11:g.19756832C > G, NC_000022.11:g.19756845C > T, and NC_000022.11:g. 19756902G > T) in the non-coding cis-regulatory element of TBX1. Luciferase assays showed that all 3 variants led to reduced transcription of TBX1 when incubated with GATA6. Our findings showed that TBX1 might be a direct transcriptional target of GATA6, and variants in the non-coding cis-regulatory element of TBX1 disrupted GATA6-mediated transactivation.