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.
PY - 2019/8/30
Y1 - 2019/8/30
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- and 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) cisregulatory 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 LVspecific 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.
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- and 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) cisregulatory 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 LVspecific 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.
KW - haplotype
KW - introns
KW - myocardium
KW - phenotype
KW - transcription factors
UR - http://www.scopus.com/inward/record.url?scp=85071783633&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85071783633&partnerID=8YFLogxK
U2 - 10.1161/CIRCRESAHA.119.315313
DO - 10.1161/CIRCRESAHA.119.315313
M3 - Article
C2 - 31366290
AN - SCOPUS:85071783633
SN - 0009-7330
VL - 125
SP - 575
EP - 589
JO - Circulation research
JF - Circulation research
IS - 6
ER -