Conjugated activation of myocardial-specific transcription ofGja5by a pair of Nkx2-5-Shox2 co-responsive elements

Abstract

<jats:title>Abstract</jats:title><jats:p>The sinoatrial node (SAN) is the primary pacemaker in the heart. During cardiogenesis, <jats:italic>Shox2</jats:italic> and <jats:italic>Nkx2-5</jats:italic> 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 <jats:italic>Gja5</jats:italic> (coding gene of Cx40) distal enhancer consisting of a pair of Nkx2-5 and Shox2 co-bound elements in the regulatory region of <jats:italic>Gja5</jats:italic>. Transgenic reporter assays revealed that neither element alone, but the conjugation of both elements together, drives myocardial-specific transcription. Genetic analyses confirmed that the activation of this enhancer depends on Nkx2-5 but is inhibited by Shox2 <jats:italic>in vivo</jats:italic>, and its presence is essential for <jats:italic>Gja5</jats:italic> expression in the myocardium 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 <jats:italic>Gja5</jats:italic> promoter <jats:italic>in vivo</jats:italic>, indicating that Nkx2-5 bridges the conjugated activation of this enhancer by pairing the two elements to the <jats:italic>Gja5</jats:italic> promoter.</jats:p>