Long-range regulatory interactions at the 4q25 atrial fibrillation risk locus involve PITX2c and ENPEP

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Recent genome-wide association studies have uncovered genomic loci that underlie an increased risk for atrial fibrillation, the major cardiac arrhythmia in humans. The most significant locus is located in a gene desert at 4q25, approximately 170 kilobases upstream of PITX2, which codes for a transcription factor involved in embryonic left-right asymmetry and cardiac development. However, how this genomic region functionally and structurally relates to PITX2 and atrial fibrillation is unknown. [Results]: To characterise its function, we tested genomic fragments from 4q25 for transcriptional activity in a mouse atrial cardiomyocyte cell line and in transgenic mouse embryos, identifying a non-tissue-specific potentiator regulatory element. Chromosome conformation capture revealed that this region physically interacts with the promoter of the cardiac specific isoform of Pitx2. Surprisingly, this regulatory region also interacts with the promoter of the next neighbouring gene, Enpep, which we show to be expressed in regions of the developing mouse heart essential for cardiac electrical activity. [Conclusions]: Our data suggest that de-regulation of both PITX2 and ENPEP could contribute to an increased risk of atrial fibrillation in carriers of disease-associated variants, and show the challenges that we face in the functional analysis of genome-wide disease associations.This study was funded by the CNIC Translational Grant Programme (CNIC-08-2009 to MM and DF), the Spanish Ministerio de Economia y Competitividad (grants BFU2011-23083 to MM, BFU2013-41322-P to JLGS, BFU2012-38111 to AA, and CSD2007-00008 to JLGS and MM), the Comunidad Autónoma de Madrid (grant CELLDD-CM to MM), and the Andalusian Government (grant BIO-396 to JLGS). The CNIC is supported by the Spanish Ministerio de Economia y Competitividad and the Pro-CNIC Foundation.Peer Reviewe

Long-range regulatory interactions at the 4q25 atrial fibrillation risk locus involve PITX2c and ENPEP

Background: Recent genome-wide association studies have uncovered genomic loci that underlie an increased risk for atrial fibrillation, the major cardiac arrhythmia in humans. The most significant locus is located in a gene desert at 4q25, approximately 170 kilobases upstream of PITX2, which codes for a transcription factor involved in embryonic left-right asymmetry and cardiac development. However, how this genomic region functionally and structurally relates to PITX2 and atrial fibrillation is unknown. Results: To characterise its function, we tested genomic fragments from 4q25 for transcriptional activity in a mouse atrial cardiomyocyte cell line and in transgenic mouse embryos, identifying a non-tissue-specific potentiator regulatory element. Chromosome conformation capture revealed that this region physically interacts with the promoter of the cardiac specific isoform of Pitx2. Surprisingly, this regulatory region also interacts with the promoter of the next neighbouring gene, Enpep, which we show to be expressed in regions of the developing mouse heart essential for cardiac electrical activity. Conclusions: Our data suggest that de-regulation of both PITX2 and ENPEP could contribute to an increased risk of atrial fibrillation in carriers of disease-associated variants, and show the challenges that we face in the functional analysis of genome-wide disease associations.We thank Miguel Torres and members of the Manzanares lab for support and comments; Christine Mummery, Jose Luis de la Pompa and Joaquin Rodriguez-Leon for reagents; the CNIC Transgenic Unit for generation of embryos; Stuart Pocock for statistical advice; and Simon Bartlett for English editing. This study was funded by the CNIC Translational Grant Programme (CNIC-08-2009 to MM and DF), the Spanish Ministerio de Economia y Competitividad (grants BFU2011-23083 to MM, BFU2013-41322-P to JLGS, BFU2012-38111 to AA, and CSD2007-00008 to JLGS and MM), the Comunidad Autonoma de Madrid (grant CELLDD-CM to MM), and the Andalusian Government (grant BIO-396 to JLGS). The CNIC is supported by the Spanish Ministerio de Economia y Competitividad and the Pro-CNIC Foundation.S