Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we ...performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.
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•Epigenetic and transcriptional dynamics in hESCs and hESC-derived populations•Lineage-specific remodeling at regions bound by OCT4, SOX2, and NANOG in hESCs•Germ-layer-specific switch to H3K4me1 or H3K27me3 at sites of high DNA methylation•Epigenetic dynamics frequently precede transcriptional activation
The epigenetic and transcriptional landscapes of three cell types representing each embryonic lineage derived from human embryonic stem cells are profiled, revealing distinct histone modification and DNA methylation dynamics that accompany lineage specification.
Organogenesis involves integration of diverse cell types; dysregulation of cell-type-specific gene networks results in birth defects, which affect 5% of live births. Congenital heart defects are the ...most common malformations, and result from disruption of discrete subsets of cardiac progenitor cells
, but the transcriptional changes in individual progenitors that lead to organ-level defects remain unknown. Here we used single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular subpopulations has catastrophic consequences. A network-based computational method for single-cell RNA-sequencing analysis that predicts lineage-specifying transcription factors
identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite the failure of right ventricular formation in Hand2-null mice
. Temporal single-cell-transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium was specified but failed to properly differentiate and migrate. Loss of Hand2 also led to dysregulation of retinoic acid signalling and disruption of anterior-posterior patterning of cardiac progenitors. This work reveals transcriptional determinants that specify fate and differentiation in individual cardiac progenitor cells, and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework for investigating congenital heart defects.
DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases ...(DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.
In diseased organs, stress-activated signalling cascades alter chromatin, thereby triggering maladaptive cell state transitions. Fibroblast activation is a common stress response in tissues that ...worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains unclear
. Pharmacological inhibition of bromodomain and extra-terminal domain (BET) proteins alleviates cardiac dysfunction
, providing a tool to interrogate and modulate cardiac cell states as a potential therapeutic approach. Here we use single-cell epigenomic analyses of hearts dynamically exposed to BET inhibitors to reveal a reversible transcriptional switch that underlies the activation of fibroblasts. Resident cardiac fibroblasts demonstrated robust toggling between the quiescent and activated state in a manner directly correlating with BET inhibitor exposure and cardiac function. Single-cell chromatin accessibility revealed previously undescribed DNA elements, the accessibility of which dynamically correlated with cardiac performance. Among the most dynamic elements was an enhancer that regulated the transcription factor MEOX1, which was specifically expressed in activated fibroblasts, occupied putative regulatory elements of a broad fibrotic gene program and was required for TGFβ-induced fibroblast activation. Selective CRISPR inhibition of the single most dynamic cis-element within the enhancer blocked TGFβ-induced Meox1 activation. We identify MEOX1 as a central regulator of fibroblast activation associated with cardiac dysfunction and demonstrate its upregulation after activation of human lung, liver and kidney fibroblasts. The plasticity and specificity of BET-dependent regulation of MEOX1 in tissue fibroblasts provide previously unknown trans- and cis-targets for treating fibrotic disease.
Complex genetic mechanisms are thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human cardiac anomaly can be caused by a ...combination of rare, inherited heterozygous mutations. Whole-exome sequencing of a nuclear family revealed that three offspring with childhood-onset cardiomyopathy had inherited three missense single-nucleotide variants in the
,
, and
genes. The
and
variants were inherited from the affected, asymptomatic father and the rare
variant (minor allele frequency, 0.0012) from the unaffected mother. We used CRISPR-Cas9 to generate mice encoding the orthologous variants and found that compound heterozygosity for all three variants recapitulated the human disease phenotype. Analysis of murine hearts and human induced pluripotent stem cell-derived cardiomyocytes provided histologic and molecular evidence for the
variant's contribution as a genetic modifier.
Research on human pluripotent stem cells has been hampered by the lack of a standardized, quantitative, scalable assay of pluripotency. We previously described an assay called ScoreCard that used ...gene expression signatures to quantify differentiation efficiency. Here we report an improved version of the assay based on qPCR that enables faster, more quantitative assessment of functional pluripotency. We provide an in-depth characterization of the revised signature panel (commercially available as the TaqMan hPSC Scorecard Assay) through embryoid body and directed differentiation experiments as well as a detailed comparison to the teratoma assay. We further show that the improved ScoreCard enables a wider range of applications, such as screening of small molecules, genetic perturbations and assessment of culture conditions. Our approach can be extended beyond stem cell applications to characterize and assess the utility of other cell types and lineages.
Mapping the gene-regulatory networks dysregulated in human disease would allow the design of network-correcting therapies that treat the core disease mechanism. However, small molecules are ...traditionally screened for their effects on one to several outputs at most, biasing discovery and limiting the likelihood of true disease-modifying drug candidates. Here, we developed a machine-learning approach to identify small molecules that broadly correct gene networks dysregulated in a human induced pluripotent stem cell (iPSC) disease model of a common form of heart disease involving the aortic valve (AV). Gene network correction by the most efficacious therapeutic candidate, XCT790, generalized to patient-derived primary AV cells and was sufficient to prevent and treat AV disease in vivo in a mouse model. This strategy, made feasible by human iPSC technology, network analysis, and machine learning, may represent an effective path for drug discovery.
Ectopic expression of combinations of transcription factors (TFs) can drive direct lineage conversion, thereby reprogramming a somatic cell’s identity. To determine the molecular mechanisms by which ...Gata4, Mef2c, and Tbx5 (GMT) induce conversion from a cardiac fibroblast toward an induced cardiomyocyte, we performed comprehensive transcriptomic, DNA-occupancy, and epigenomic interrogation throughout the reprogramming process. Integration of these datasets identified new TFs involved in cardiac reprogramming and revealed context-specific roles for GMT, including the ability of Mef2c and Tbx5 to independently promote chromatin remodeling at previously inaccessible sites. We also find evidence for cooperative facilitation and refinement of each TF’s binding profile in a combinatorial setting. A reporter assay employing newly defined regulatory elements confirmed that binding of a single TF can be sufficient for gene activation, suggesting that co-binding events do not necessarily reflect synergy. These results shed light on fundamental mechanisms by which combinations of TFs direct lineage conversion.
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•Integrated scRNA-, ATAC-, and ChIP-seq analyses to interrogate cardiac reprogramming•Gata4, Mef2c, and Tbx5 both facilitate and limit one another's ability to bind to DNA•Mef2c and Tbx5 bind to inaccessible chromatin and promote its remodeling•Context-specific cooperative mechanisms guide cardiac reprogramming
Srivastava and colleagues integrate multiple (epi)genomic assays to dissect the mechanisms by which transcription factors function independently and combinatorially to initiate cell fate transitions. Heterogeneous binding relationships between Gata4, Mef2c, and Tbx5 highlight the context-specific mechanisms that dictate cardiac reprogramming.
Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein ...interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.
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•GATA4:TBX5 interactome in CPs is enriched in de novo variants associated with CHD•A method for scoring interactome variants identified GLYR1 as a candidate CHD gene•GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes•The GLYR1 CHD variant disrupted interaction with GATA4 and impaired cardiogenesis
The integration of human protein-protein interactome networks of endogenous transcription factors known to be involved in cardiac malformations with the largest whole-exome-sequencing dataset of trios with congenital heart disease identified an enrichment of rare de novo variants among interactome proteins, pointing to candidate disease genes.
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