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David S ParkDavid S Park

New York University Grossman School of Medicine

Circulation, Sep 29 2023, | https://doi.org/10.1161/CIRCULATIONAHA.123.065700 An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract

Naoko Yamaguchi ^{# 1}, Ernest W Chang ^{# 1}, Ziyan Lin ², Akshay Shekhar ³, Lei Bu ¹, Alireza Khodadadi-Jamayran ², Aristotelis Tsirigos², Yiyun Cen ¹, Colin K L Phoon ⁴, Ivan P Moskowitz ⁵, David S Park¹

¹The Leon H. Charney Division of Cardiology, New York University Grossman School of Medicine. (N.Y., E.W.C., L.B., Y.C., D.S.P.).

²NYU Applied Bioinformatics Labs, New York University Grossman School of Medicine. (Z.L., A.K.J., A.T.).

³Regeneron Pharmaceuticals, Inc. Biotechnology, Tarrytown, NY (A.S.).

⁴Division of Pediatric Cardiology, Hassenfeld Children's Hospital at NYU Langone, New York University Grossman School of Medicine, Fink Children's Center (C.K.L.P.).

⁵Department of Pediatrics, Pathology, and Human Genetics, The University of Chicago, IL (I.P.M.).

#Contributed equally.

Correspondence to: David S. Park, MD, PhD

Abstract

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, GATA6, and TBX1 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. 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 transcriptional start site of Nkx2-5 ( Nkx2-5 ^∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5 ^{∆enh/∆enh} mice were structurally characterized using histology and scanning electron micrography, 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 NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. Nkx2-5 ^{∆enh/∆enh} mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5 ^{∆enh/∆enh} mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 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 Tbx1, Pitx2, and Sema3c. 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 GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. 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.

논문정보

형식Research article
게재일2023년 09월 (BRIC 등록일 2023-10-31)
연구진국외 연구진
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