Genetic variation in T-box binding element functionally affects SCN5A/SCN10A enhancer
Malou van den Boogaard, … , Phil Barnett, Vincent M. Christoffels
Malou van den Boogaard, … , Phil Barnett, Vincent M. Christoffels
Published June 18, 2012
Citation Information: J Clin Invest. 2012;122(7):2519-2530. https://doi.org/10.1172/JCI62613.
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Research Article

Genetic variation in T-box binding element functionally affects SCN5A/SCN10A enhancer

Abstract

The contraction pattern of the heart relies on the activation and conduction of the electrical impulse. Perturbations of cardiac conduction have been associated with congenital and acquired arrhythmias as well as cardiac arrest. The pattern of conduction depends on the regulation of heterogeneous gene expression by key transcription factors and transcriptional enhancers. Here, we assessed the genome-wide occupation of conduction system–regulating transcription factors TBX3, NKX2-5, and GATA4 and of enhancer-associated coactivator p300 in the mouse heart, uncovering cardiac enhancers throughout the genome. Many of the enhancers colocalized with ion channel genes repressed by TBX3, including the clustered sodium channel genes Scn5a, essential for cardiac function, and Scn10a. We identified 2 enhancers in the Scn5a/Scn10a locus, which were regulated by TBX3 and its family member and activator, TBX5, and are functionally conserved in humans. We also provided evidence that a SNP in the SCN10A enhancer associated with alterations in cardiac conduction patterns in humans disrupts TBX3/TBX5 binding and reduces the cardiac activity of the enhancer in vivo. Thus, the identification of key regulatory elements for cardiac conduction helps to explain how genetic variants in noncoding regulatory DNA sequences influence the regulation of cardiac conduction and the predisposition for cardiac arrhythmias.

Authors

Malou van den Boogaard, L.Y. Elaine Wong, Federico Tessadori, Martijn L. Bakker, Lisa K. Dreizehnter, Vincent Wakker, Connie R. Bezzina, Peter A.C. ‘t Hoen, Jeroen Bakkers, Phil Barnett, Vincent M. Christoffels

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Figure 6

In vitro and in vivo analysis of genomic region F1–F2 and SNPs associated with conduction.

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In vitro and in vivo analysis of genomic region F1–F2 and SNPs associate...
(A) Luciferase assays performed in Cos7 showed that rs6801957 was involved in T-box factor response. Representative human major (G) alleles Maj1 and Maj2 responded to stimulation by TBX5 and repression by TBX3, whereas this effect on the minor (A) allele Min1 was significantly less. Mutating rs6801957 in these alleles showed a similar effect. *P < 0.01. Error bars represent SD. See Supplemental Table 10 for information on variants per allele. (B) EMSA showing relative oligonucleotide binding of MBP-TBX3 (+) compared with MBP only (–). MBP-TBX3 fusion associated well with both Nppa probe and human Maj1, but less with Min1 and Mut (containing a 3-bp mutation in the proposed T-box binding element). See Supplemental Figure 9 for quantification and statistical analysis. (C) Representative image of the zebrafish heart (dotted outline) containing ZED-Maj1, showing expression in the ventricle (V). A, atrium. Scale bar: 100 μm. (D) Percent GFP expression in hearts of zebrafish containing the major or minor allele for rs6801957. ZED represents the vector without genomic region F1–F2. Representative human major alleles Maj1 and Maj3 showed GFP expression in 60%–70% of the hearts of zebrafish containing these constructs. When major alleles were mutated into minor alleles (Maj1Mut and Maj3Mut), a significant reduction in GFP expression was found. The presence of rs6795970 in Maj3, also linked to conduction disease, had no significant effect on GFP expression in the heart.