Long-range Pitx2c 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 PITX2. Cardiomyocyte-specific epigenomic and comparative genomics uncovered 2 AF-associated enhancers neighboring PITX2 with varying conservation in mice. Chromosome conformation capture experiments in mice revealed that the Pitx2c promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9-mediated deletion of a 20-kb topologically engaged enhancer led to reduced Pitx2c 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 Pitx2c promoter was required for maintenance of the Pitx2c promoter chromatin state. Long-range looping was mediated by CCCTC-binding factor (CTCF), since genetic disruption of the intronic CTCF-binding site caused reduced Pitx2c expression, AF predisposition, and diminished active chromatin marks on Pitx2. AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at PITX2.

Reduced left atrial cardiomyocyte PITX2 and elevated circulating BMP10 predict atrial fibrillation after ablation

“…These findings are consistent with other murine models of Pitx2 deficiency ( 10 – 12 ). In addition, recent findings in a mouse model that deactivated the enhancer region of Pitx2c found Bmp10 to be one of the most upregulated genes ( 37 ). These results suggest that a common repressor/enhancer transcriptional network may exist between Bmp10 and Pitx2 whereby the loss of one of these genes results in the reciprocal upregulation of the other ( 18 , 38 ).…”

Genetics and Epigenetics of Atrial Fibrillation

“…In most cases, those AF associated variants are located in intergenic regions, limiting our understanding of the molecular mechanisms behind those associations. The exception to the rule is represented by 4q25 variants, to which several studies, including ours, have demonstrated a pivotal role regulating Pitx2 expression [ 228 , 229 , 230 ] as well as demonstrating a functional role for PITX2 in AF pathophysiology [ 130 , 133 , 134 , 154 ]. Thus, one of the future challenges in the genetics of AF is to discover the molecular mechanism behind GWAS data and AF pathophysiology [ 231 ], reemphasizing the responsibility of epigenetics.…”

Genome-wide association studies of cardiac electrical phenotypes

“… 87 , 89 , 90 By demonstrating a functional connection between the upstream AF-associated variants, PITX2 expression, and predisposition to AF, a recent study by Zhang et al . 91 has now provided conclusive evidence for PITX2 being the causal gene at this locus. Specifically, they showed that AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory function directed at PITX2 , and that the deletion of these genomic regions leads to reduced Pitx2c transcription and AF predisposition.…”

“…Specifically, they showed that AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory function directed at PITX2 , and that the deletion of these genomic regions leads to reduced Pitx2c transcription and AF predisposition. 91 …”