title: Long-range regulatory interactions at the 4q25 atrial fibrillation risk locus involve PITX2c and ENPEP.
abstract: 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.
authors.affiliation: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain. laguirre04@gmail.com.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Aguirre
authors.affiliation: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain. mevaalonso@gmx.net.
authors.forename: M Eva
authors.initials: ME
authors.lastname: Alonso
authors.affiliation: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain. cbadia@cnic.es.
authors.forename: Claudio
authors.initials: C
authors.lastname: Badía-Careaga
authors.affiliation: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain. isabelclara.rollan@cnic.es.
authors.forename: Isabel
authors.initials: I
authors.lastname: Rollán
authors.affiliation: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain. cris_amberes@yahoo.es.
authors.forename: Cristina
authors.initials: C
authors.lastname: Arias
authors.affiliation: Centro Andaluz de Biología del Desarrollo (CABD), CSIC-Universidad Pablo de Olavide-Junta de Andalucía, ctra. de Utrera km1, 41013, Seville, Spain. amfermin@upo.es.
authors.forename: Ana
authors.initials: A
authors.lastname: Fernández-Miñán
authors.affiliation: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain. elena.lopez@cnic.es.
authors.forename: Elena
authors.initials: E
authors.lastname: López-Jiménez
authors.affiliation: Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaen, Paraje de las Lagunillas s/n, 23071, Jaén, Spain. aaranega@ujaen.es.
authors.forename: Amelia
authors.initials: A
authors.lastname: Aránega
authors.affiliation: Centro Andaluz de Biología del Desarrollo (CABD), CSIC-Universidad Pablo de Olavide-Junta de Andalucía, ctra. de Utrera km1, 41013, Seville, Spain. jlgomska@upo.es.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Gómez-Skarmeta
authors.affiliation: Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaen, Paraje de las Lagunillas s/n, 23071, Jaén, Spain. dfranco@ujaen.es.
authors.forename: Diego
authors.initials: D
authors.lastname: Franco
authors.affiliation: Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029, Madrid, Spain. mmanzanares@cnic.es.
authors.forename: Miguel
authors.initials: M
authors.lastname: Manzanares
mesh_terms: D000818:Animals; D001281:Atrial Fibrillation / Q000235:genetics*; D002843:Chromatin / Q000378:metabolism; D002894:Chromosomes, Human, Pair 4 / Q000235:genetics*; D004622:Embryo, Mammalian / Q000378:metabolism; D005260:Female; D056426:Genetic Loci; D020022:Genetic Predisposition to Disease; D043384:Glutamyl Aminopeptidase / Q000235:genetics*; D057809:HEK293 Cells; D006325:Heart Atria / Q000196:embryology / Q000378:metabolism / Q000473:pathology; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D032383:Myocytes, Cardiac / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012307:Risk Factors; D014157:Transcription Factors / Q000235:genetics*; D000097577:Homeobox Protein PITX2
chemical_list: D002843:Chromatin; D043384:Glutamyl Aminopeptidase; D018398:Homeodomain Proteins; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2; C513548:Enpep protein, mouse