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Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. Loss of function variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2 and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.
Background: Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure, a major cause of morbidity and mortality. This causality indicates that even two-fold changes in the amount of TTN can profoundly disturb cardiac physiology. Although a critical role of TTN in sarcomere formation and cardiomyocyte contractility is well established, the mechanisms regulating transcription of the TTN gene remain poorly understood. Methods: We performed bioinformatics analysis to identify a putative transcriptional enhancer of TTN . Next, we created biallelic deletion of the enhancer in human induced pluripotent stem cell derived cardiomyocytes and performed enhancer reporter assays both in vitro and in vivo to demonstrate necessity and sufficiency of the enhancer in TTN gene expression, respectively. Furthermore, we performed massive parallel reporter assay to define critical transcriptional factors of the TTN enhancer activity and analyzed whole genome sequencing (WGS) data of human patients with unexplained dilated cardiomyopathy (DCM). Results: We identified an intron mediated enhancer that promotes cardiac-specific TTN expression. Global deletion of this element downregulated TTN expression in cardiomyocytes and impaired sarcomere development, while transgenic expression promoted cardiac expression in mice. Using mutational scanning we defined key transcription factor binding sites, including NKX2-5 and MEF2 motifs that promote TTN expression in cardiomyocytes. Consistent with these functions, analyses of WGS data in 69 patients with unexplained DCM revealed one rare variant that disrupted the conserved MEF2 transcriptional factor binding motif. Conclusions: Discovery of a TTN enhancer advances our understanding of cardiomyocyte development, provides an opportunity to modulate TTN transcriptional activity, and ultimately develop therapeutic strategies to treat dilated cardiomyopathy caused by TTN haploinsufficiency.
Dilated cardiomyopathy (DCM), a disorder that occurs in 1:250 individuals, is associated with rates of mortality of 20% within 5 years of diagnosis and is a leading cause for heart failure and cardiac transplantation. Mutations in the massive sarcomere protein titin (encoded by TTN ) are the most common genetic cause of DCM, occurring in 10-20% of cases. As dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency, we predict that strategies to increase the expression of the wild type (WT) TTN allele might attenuate the damaging effects of TTNtv. We used bioinformatic analyses to identify a putative TTN enhancer within intron 1. To confirm its function, we deleted 658 bp from intron 1 that encompasses the putative TTN enhancer in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing. We used qPCR and RNA sequencing of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs) and demonstrated that a homozygous deletion in this region leads to decreased TTN gene expression compared to the WT control (0.344 fold change, p < 0.001), and also to decreased expression of other sarcomeric genes such as TNNT2 (0.074 fold change, p < 0.001), MYH6 (0.18 fold change, p < 0.001), MYH7 (0.008 fold change, p < 0.001), and ACTN2 (0.118 fold change, p < 0.001). The expression of transcription factors (TF) that have binding sites in this region is also affected, such as MEIS2 (0.4 fold change, p < 0.001) and KLF6 (0.33 fold change, p < 0.001), both of which are known to be involved in cardiogenesis. These TF may act as a link between the deletion in TTN Intron 1 and a decreased transcription of other cardiac-relevant genes. We also utilized Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) on WT hiPSC-CMs to identify open regions of chromatin that are accessible for TF binding. This provided additional evidence that this 658 bp region in TTN intron 1 has enhancer activity. Ongoing studies are aiming to refine the TTN Intron 1 enhancer by further studies using CRISPR/Cas9, CRISPR droplet sequencing (CROP-Seq), and luciferase-based enhancer activity assays. If confirmed, we expect that increasing the activity of this 658 bp region with small molecules may provide a novel therapeutic target for DCM caused by TTNtv.
The discovery of damaging gene mutations in congenital heart disease (CHD) patients enables identification of regulators of cardiac development. Exome sequencing identified de novo heterozygous loss-of-function (LoF) and missense variants in GATA6 among CHD probands, most with outflow tract malformations. Other subjects with GATA6 LoF mutations developed pancreatic agenesis. To elucidate the molecular basis for the predominance of this heart defect, we modeled GATA6 mutations in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). GATA6 variants were introduced into isogenic hiPSCs using CRISPR/Cas9 genome editing. Genome-wide molecular profiles including chromatin accessibility (ATAC-Seq) and gene expression (single cell and bulk RNA-Seq) were evaluated during hiPSC-CM differentiation. Analyses of GATA6 mutant hiPSC-CMs showed deficits in hiPSC-CM differentiation, chromatin accessibility and transcriptional profiles. Heterozygous GATA6 LoF hiPSCs made hiPSC-CMs but exhibited reduced expression of second heart field genes. Homozygous GATA6 LoF hiPSCs failed to differentiate and adopted fibroblast expression profiles. hiPSCs carrying a homozygous GATA6 missense variant, R456G, which altered a DNA-binding domain residue, showed enhanced capacity to differentiate into neuroepithelial-like cells. Chromatin-accessibility studies confirmed that GATA6 normally binds to genes in the promoter region and other genes at distal enhancers. Human GATA6 haploinsufficiency disrupts developmental transcriptional responses driving cardiac morphogenesis. The HAND2 -dependent genetic program, operant during outflow tract development, is particularly sensitive to GATA6 dosage. The mixed differentiation patterns observed in mutation-carrying hiPSCs likely contributes to vascular phenotypes observed in CHD patients. GATA6 haploinsufficiency preferentially alters binding of distal enhancers to promoters in genes where GATA6 normally binds the enhancer rather than the promoter. We speculate that pathogenicity of GATA6 haploinsufficiency is mediated by weaker binding of GATA6 to distal enhancers than to promoter elements, altering expression of these genes in GATA6 haploinsufficient patients.
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