TY - JOUR
T1 - Variation in a Left Ventricle-Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function
AU - Vincentz, Joshua W.
AU - Firulli, Beth A.
AU - Toolan, Kevin P.
AU - Arking, Dan E.
AU - Sotoodehnia, Nona
AU - Wan, Juyi
AU - Chen, Peng Sheng
AU - De Gier-De Vries, Corrie
AU - Christoffels, Vincent M.
AU - Rubart-Von Der Lohe, Michael
AU - Firulli, Anthony B.
N1 - Publisher Copyright:
© 2019 American Heart Association, Inc.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020
Y1 - 2020
N2 - Rationale: The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5′ to the gene encoding the basic helix-loop-helix transcription factor HAND1 (heart- A nd neural crest derivatives-expressed protein 1). Objective: Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. Methods and Results: We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. Conclusions: Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans. Visual Overview: An online visual overview is available for this article.
AB - Rationale: The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5′ to the gene encoding the basic helix-loop-helix transcription factor HAND1 (heart- A nd neural crest derivatives-expressed protein 1). Objective: Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. Methods and Results: We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. Conclusions: Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans. Visual Overview: An online visual overview is available for this article.
KW - Haplotype
KW - introns
KW - myocardium
KW - phenotype
KW - transcription factors
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U2 - 10.1161/CIRCRESAHA.119.315313
DO - 10.1161/CIRCRESAHA.119.315313
M3 - Article
C2 - 31366290
AN - SCOPUS:85071783633
SP - 575
EP - 589
JO - Circulation Research
JF - Circulation Research
SN - 0009-7330
ER -