Rationale : The evolutionary conserved Tbx3 / Tbx5 gene cluster encodes T-box transcription factors that play crucial roles in the development and homeostasis of the cardiac conduction system in human and mouse. Both genes are expressed in overlapping patterns and function in strictly tissue-specific and dose-dependent manners, yet, their regulation is poorly understood. Objective : To analyze the mechanism underlying the complex regulation of the Tbx3 / Tbx5 cluster. Methods and Results : By probing the 3-dimensional architecture of the Tbx3/Tbx5 cluster using high-resolution circular chromosome conformation capture sequencing in vivo, we found that its regulatory landscape is in a preformed conformation similar in embryonic heart, limbs, and brain. Tbx3 and its flanking gene desert form a 1 Mbp loop between CCCTC-binding factor (CTCF)-binding sites that is separated from the neighboring Tbx5 loop. However, Ctcf inactivation did not result in transcriptional regulatory interaction between Tbx3 and Tbx5 . Multiple sites within the Tbx3 locus contact the promoter, including sites corresponding to regions known to contain variations in the human genome influencing conduction. We identified an atrioventricular-specific enhancer and a pan-cardiac enhancer that contact the promoter and each other and synergize to activate transcription in the atrioventricular conduction system. Conclusions : We provide a high-resolution model of the 3-dimensional structure and function of the Tbx3 / Tbx5 locus and show that the locus is organized in a preformed, permissive structure. The Tbx3 locus forms a CTCF-independent autonomous regulatory domain with multiple combinatorial regulatory elements that control the precise pattern of Tbx3 in the cardiac conduction system.

BackgroundRecent 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.ResultsTo 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0138-0) contains supplementary material, which is available to authorized users.