A Large Permissive Regulatory Domain Exclusively Controls Tbx3 Expression in the Cardiac Conduction System
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 (CCS) 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 analyse the mechanism underlying the complex regulation of the Tbx3/Tbx5 cluster.
Methods and Results: By probing the 3D architecture of the Tbx3/Tbx5 cluster using high-resolution 4C-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 CTCF binding sites that is separated from the neighbouring 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 3D 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 CCS.
- Cardiac conduction system
- Chromatin topology
- Heart development
- gene expression/regulation
- transcription factor genetics
- cardiac development
- Received January 30, 2014.
- Revision received June 13, 2014.
- Accepted June 23, 2014.