Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD). However, underlying CHD gene regulatory network (GRN) imbalances are unknown.Here, we define transcriptional consequences of reduced dosage of the CHD-linked transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5dependent pathways-including lineage decisions and genes associated with cardiomyocyte function and CHD genetics-in discrete subpopulations of cardiomyocytes. GRN analysis identified vulnerable nodes enriched for CHD genes, indicating that cardiac network stability is sensitive to TBX5 dosage. A GRN-predicted genetic interaction between Tbx5 and Mef2c was validated in mouse, manifesting as ventricular septation defects. These results demonstrate exquisite sensitivity to TBX5 dosage by diverse transcriptional responses in heterogeneous subsets of iPSC-derived cardiomyocytes. This predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.
Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. Accordingly, in undifferentiated and pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. How global H3K9 methylation levels are coupled with the loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of Suv39h1as, an antisense long non-coding RNA to Suv39h1. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, affecting all heterochromatic regions, particularly peri-centromeric major satellites and retrotransposons. This increase in heterochromatinization efficiency leads to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the genetic control of pluripotency with the global efficiency of H3K9 methylation associated with a major cell fate restriction, the irreversible loss of pluripotency.
30Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD) 1 . This observation predicts gene regulatory network (GRN) imbalances 2 , but the nature 32 of dosage-vulnerable GRNs and their contribution to human cardiogenesis and CHDs are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD 34 transcription factor TBX5 during human cardiac differentiation from induced pluripotent stem (iPS) cells. Single cell RNAseq revealed that transcriptional responses to reduced TBX5 levels 36 are not homogeneous, and instead, discrete sub-populations of cardiomyocytes exhibit dysregulation of distinct TBX5 dose-sensitive genes related to cellular phenotypes and CHD-38 associated genetics. Cellular trajectory inference revealed TBX5 dosage-dependent differentiation paths, with implications for cardiac developmental identity. GRN analysis of the 40 single cell RNAseq data identified vulnerable nodes enriched for CHD genes, implicating a critical sensitivity to TBX5 dosage in cardiac network stability. A novel GRN-predicted genetic 42 interaction between TBX5 and MEF2C was validated in mouse, revealing a highly dosagesensitive pathway for CHD. Our results reveal unforeseen complexity and exquisite sensitivity to 44 TBX5 dosage in discrete sub-populations of iPSC-derived cardiomyocytes, providing mechanistic insights into human CHDs and quantitative transcriptional regulation in disease. linked to CHD predict reduction in dosage of transcriptional regulators, including transcription factors (TFs) and chromatin-modifying genes 1 . Despite advances in tracing the roles of 52 individual factors, how altered dosage of transcriptional regulators translates to altered transcriptional activity is not known, nor is it known how these altered GRNs disrupt heart 54 development, resulting in CHD.Heterozygous mutations in the T-box TF gene TBX5 cause Holt-Oram syndrome (HOS) 56 5,6 , which presents with upper limb defects, CHDs, diastolic dysfunction, or arrhythmias. In humans, homozygous TBX5 loss of function is presumed to cause fetal demise. Altered 58 expression of select genes in mice in vivo demonstrate a stepwise sensitivity to reductions in Tbx5 dosage 7,8 . These findings suggest that a reduction in TBX5 dosage perturbs downstream 60 gene expression, but the overall disrupted networks and mechanisms are not understood.Mechanistic investigation of human CHD has been hampered by a lack of relevant and 62 tractable models. Human heart tissue is largely inaccessible for molecular analysis, and pathological or surgical specimens are limited. However, induced pluripotent stem (iPS) cells, 64 genome editing, directed differentiation, and single cell RNAseq together provide a human cellular platform for gene-centered cardiac disease modeling at single cell resolution. In 66 considering how transcriptional regulator haploinsufficiency might cause CHD, at least two scenarios are possible: 1. That reduced dosage affects genes only in specific anatomical 68 locations, such as the ...
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