GATA‐6 gene enhancer contains nested regulatory modules for primary myocardium and the embedded nascent atrioventricular conduction system

Abstract

The cGATA-6 gene is flanked by an enhancer that selectively marks the atrioventricular conduction system (AVCS) in transgenic mice. This enhancer reads anterior/posterior and medial/lateral positional information very early in the cardiogenic program and remains active in progressively more restricted regions of primary myocardium leading up to the emergence of a histologically distinct AVCS. We undertook to parse this enhancer to resolve how the respective AVCSspecific transcription program is regulated at the molecular level. We determined that this AVCS enhancer includes a 102 bp module that is sufficient to restrict expression to primary nonchamber myocardium. This offers a novel tool to analyze the early molecular delineation of primary and chamber myocardium, which subsequently give rise to components of the central and peripheral conduction system, respectively. Furthermore, we show that this 102 bp module in turn contains a nested 47 bp core module that has the potential to direct expression specifically to the AVCS domain of primary myocardium, albeit with low efficiency. Accordingly, we show that a GATA site and a GC-rich site in the 102 bp region bolster the activity of the nested 47 bp AVCS core region even within the context of the parental 1,478 bp enhancer. These are the first functional elements to be reported for a cardiac conduction system-specific control region.

Development of the Pacemaker Tissues of the Heart

“…The Cx30.2-enhancer in addition requires binding sites for T-box genes. 93 32,95 Together, these data indicate Tbx5 and Gata4 are responsible for Cx30.2 activity in the AVC. Tbx5 and Gata4 are expressed in both the AVC and working myocardium, 24,95 suggesting the existence of additional cofactors for AVC-restricted activity of Cx30.2 and the chicken Gata6-enhancer.…”

“…17,24,75,[93][94][95] Cardiac troponin I and Gata6 are not specifically expressed in the AVC, indicating that these genes have multiple regulatory modules that define their complete pattern. In contrast to Tbx3 itself, the Tbx3 genomic fragment was not active in the AVB (Figure 5B), showing that gene expression in the AVC and the AVB depends on distinct regulatory sequences and pathways.…”

The formation and function of the cardiac conduction system

“…the AVC, AVB) from early embryonic stages onwards (Davis et al, 2001). Within the enhancer, a core unit of 47 bp is sufficient to restrict the expression of a reporter gene to the AVCS (Adamo et al, 2004). Interestingly, the core enhancer is active in the entire heart tube, but during chamber differentiation becomes confined to the non-chamber myocardium, including the AVC (Christoffels et al, 2004b), suggesting that it is suppressed in the differentiating chamber myocardium.…”

GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling

“…For example, expression of Nkx2.5 within the right ventricle, interventricular septum, and atrioventricular canal is controlled by enhancer modules that are distinct from those that direct expression in the atria (11). Likewise, enhancer sequences in the GATA6 locus that drive that gene's expression in conduction tissue differ from those responsible for expression within working myocardium (1,14). In these cases, it is important to realize that the expression profile of a reporter transgene under control of individual enhancer modules does not necessarily correspond to domains exhibiting higher levels of expression of the endogenous gene.…”

“…This genetic region contains consensus binding sites for several heart-associated factors, including Nkx2.5, MEF2C, SRF, GATA-4/5/6, HAND1, and Hand2 (15), whose combination may account for the differential activity of this enhancer (1,15). Previous studies (15) examining development of these mice demonstrated that lacZ expression driven by this GATA6 distal enhancer is associated with the developing myocardium.…”