BACKGROUND

Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT.

METHODS

We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of () using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes.

RESULTS

mice recapitulated cyanotic conotruncal defects seen in patients with , and mutations. mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as , and . Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on gene dosage in human induced pluripotent stem cell-derived cardiomyocytes.

CONCLUSIONS

Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.