Purpose: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in humans. Recent genome-wide association studies (GWAS) have revealed AF associated variants on chromosome 4q25, in the vicinity of PITX2, which encodes a crucial transcription factor for cardiovascular development. Our aim is to establish whether this region harbours cis-regulatory elements that could be acting on neighbouring genes, such as PITX2.Methods: We tested the regulatory activity of several genomic fragments from 4q25 by transfections assays in cultured cells and transgenic mouse embryos. We also analysed the chromatin architecture of the region by studying physical interactions by means of Chromosome Conformation Capture (3C).Results: We found a genomic fragment of ~7kb containing rs2200733, the most significant variant associated with AF, and sub-fragments of this region, drive reporter expression in HL-1 mouse cardiomyocytes, as well as in the mouse embryo in sites of expression of endogenous Pitx2 in a heterogenous way, such as facial mesenchyme, limb muscles, left gonad and the heart. These fragments are also active in two cell types unrelated to the cardiac lineage (mouse pluripotent P19 cells, and human HEK cells), showing they do not act as classical tissue-specific enhancers, but rather as accessory element that could potentiate the activity of tissue-specific enhancers located elsewhere in the locus. Furthermore, 3C analyses show a complex pattern of long range interactions in the locus, with different regions interacting specifically with the different promoters located there, including the neighbouring Enpep gene.Conclusions: We show that cis-regulatory elements are located in the region of 4q25 associated with an increased risk for AF. These elements establish specific long-distance interactions with various promoters, and therefore could regulate the transcription of these genes. Our results suggest that de-regulation of either one or both PITX2 and ENPEP might have a causal role in the development of AF.