Long-range <i>Pitx2c</i> enhancer–promoter interactions prevent predisposition to atrial fibrillation

Abstract

Genome-wide association studies found that increased risk for atrial fibrillation (AF), the most common human heart arrhythmia, is associated with noncoding sequence variants located in proximity to <i>PITX2</i> Cardiomyocyte-specific epigenomic and comparative genomics uncovered 2 AF-associated enhancers neighboring <i>PITX2</i> with varying conservation in mice. Chromosome conformation capture experiments in mice revealed that the <i>Pitx2c</i> promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9-mediated deletion of a 20-kb topologically engaged enhancer led to reduced <i>Pitx2c</i> transcription and AF predisposition. Allele-specific chromatin immunoprecipitation sequencing on hybrid heterozygous enhancer knockout mice revealed that long-range interaction of an AF-associated region with the <i>Pitx2c</i> promoter was required for maintenance of the <i>Pitx2</i>c promoter chromatin state. Long-range looping was mediated by CCCTC-binding factor (CTCF), since genetic disruption of the intronic CTCF-binding site caused reduced <i>Pitx2c</i> expression, AF predisposition, and diminished active chromatin marks on <i>Pitx2</i> AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at <i>PITX2</i>.