Conjugated activation of myocardial-specific transcription of Gja5 by a pair of Nkx2-5-Shox2 co-responsive elements

2020 
The sinoatrial node (SAN) is the primary pacemaker in the heart. During cardiogenesis, Shox2 and Nkx2-5 are co-expressed in the junction domain of the SAN and regulate pacemaker cell fate through a Shox2-Nkx2-5 antagonism. Cx40 is a marker of working myocardium and an Nkx2-5 transcriptional output antagonized by Shox2, but the underlying regulatory mechanisms remain elusive. Here we characterized a bona fide myocardial-specific Gja5 (coding gene of Cx40) distal enhancer, formed by a pair of Nkx2-5 and Shox2 co-bound elements in the regulatory region of Gja5. Transgenic reporter assays revealed that neither each element alone, but the conjugation of both elements together, drives myocardial-specific transcription. Genetic analyses confirmed that this conjugated enhancer activation is dependent on Nkx2-5 but inhibited by Shox2 in vivo, and is essential for Gja5 expression in the myocardial but not the endothelial cells of the heart. Furthermore, chromatin conformation analysis showed an Nkx2-5-dependent loop formation between these two elements and the Gja5 promoter in vivo, indicating that Nkx2-5 bridges the conjugated activation of this enhancer by pairing the two elements to the Gja5 promoter.
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