A Large Permissive Regulatory Domain Exclusively Controls Tbx3 Expression in the Cardiac Conduction System

Abstract

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.

Multi-ethnic genome-wide association study for atrial fibrillation

“…TBX3 and the adjacent gene TBX5 encode transcription factors, that have been shown to regulate the development of the cardiac conduction system. 19 Similarly, the NKX2–5 encodes a transcription factor, that is an early cue for cardiac development and has been associated with congenital heart disease 20 and heart rate 21 ( Supplementary Table 14 ). Further, reduced function of the transcription factor encoded by PITX2 has been associated with AF, shortening of the left atrial action potential, and with modulation of sodium channel blocker therapy in the adult left atrium.…”

Regulation of Vertebrate Conduction System Development

“…Interestingly, these data revealed that the regulatory landscape is in a preformed conformation that is similar in embryonic heart, brain and limb. Rather than the de novo formation of enhancer-promoter loops upon binding by cell-type relevant transcription factors to initiate transcription, the locus is in a fixed, permissive structure in which enhancer-promoter loops are pre-existing [40]. Such a permissive structure has previously been described for different loci, including the Hox and Shh gene loci.…”

“…Among the multiple sites contacting Tbx3 in the gene desert upstream of the gene, two evolutionary conserved enhancers have been identified that also contact each other. They are bound by cardiac-specific transcription factors Nkx2-5, Gata4, Tbx5 and Tbx3 and were shown to respond to a BMP-mediated signalling pathway to drive atrioventricular conduction system expression of Tbx3 [40].…”

“…Red and dark blue depict a high contact frequency, whereas light blue and grey depict a low contact frequency. The contact profiles of Tbx3 and Tbx5 hardly overlap, indicating that both genes do not share regulatory sequences and suggesting that common variants in humans upstream of TBX3 as identified by GWAS (star) can be exclusively assigned to TBX3 [40]. (b) Model of the 3D conformation of the Tbx3/Tbx5 locus.…”

“…Gja5 (Cx40). Despite the strict regulation on the chromatin level, Tbx5 also directly regulates Tbx3 by binding target sequences to activate transcription in, for example, the SAN Genomic regions within the Tbx3 loop solely contact Tbx3 but not Tbx5 and vice versa, indicating enhancer sharing between these evolutionary conserved clusters is unlikely to occur [40]. The strict separation of the regulatory landscapes of Tbx3 and Tbx5 is not only cell type-independent, but also evolutionary conserved between mouse and human.…”