CHD-associated enhancers direct human cardiomyocyte lineage commitment.
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ABSTRACT:
PROVIDER: PRJNA787259 | ENA |
REPOSITORIES: ENA
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Project description:Enhancers orchestrate gene expression programs that drive multicellular development and lineage commitment. Thus, genetic variants at enhancers are thought to contribute to developmental diseases by altering cell fate commitment. However, while many variant-containing enhancers have been identified, systematic studies to endogenously test the impact of these enhancers on lineage commitment have been lacking. We performed a single-cell CRISPRi screen to directly assess the roles of 25 enhancers and putative cardiac target genes implicated in genetic studies of congenital heart defects (CHD). We identified 16 enhancers whose repression leads to delayed differentiation of human cardiomyocytes. A focused analysis on 6 TBX5 enhancers showed that TBX5 enhancer perturbation delays the transcriptional switch from mid-CM to late-CM states. Knockout of two TBX5 enhancers resulted in X and Y, which phenocopy expected CHD phenotypes. Together, these results identify critical enhancers of cardiac development and suggest that misregulation of these enhancers could contribute to cardiac defects in human patients.
2022-05-15 | GSE190475 | GEO
Project description:The lineage-determining transcription factor ETV2 is necessary and sufficient for hematoendothelial fate commitment. We investigated how ETV2-driven gene regulatory networks promote hematoendothelial fate commitment. We resolved the sequential determination steps of hematoendothelial versus cardiac differentiation from mouse embryonic stem cells. Etv2 was strongly induced and bound to the enhancers of hematoendothelial genes in a common cardiomyocyte-hematoendothelial mesoderm progenitor. However, only Etv2 itself and Tal1, not other ETV2-bound genes, were induced. Despite ETV2 genomic binding and Etv2 and Tal1 expression, cardiomyogenic fate potential was maintained. A second wave of ETV2-bound target genes was up-regulated during the transition from the common cardiomyocyte-hematoendothelial progenitor to the committed hematoendothelial population. A third wave of ETV-bound genes were subsequently expressed in the committed hematoendothelial population for sub-lineage differentiation. The shift from ETV2 binding to productive transcription, not ETV2 binding to target gene enhancers, drove hematoendothelial fate commitment. This work identifies mechanistic phases of ETV2-dependent gene expression that distinguish hematoendothelial specification, commitment, and differentiation.
2023-06-16 | GSE136692 | GEO
Project description:About 45% of congenital heart disease (CHD) is caused by rare gene mutations. Non-coding mutations that perturb cis-regulatory elements (CREs) likely contribute to CHD among the remaining cases without clear etiology. However, identifying CHD-causing non-coding variants has been problematic. We combined human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) differentiation and a lentivirus-mediated massively parallel reporter assay (lentiMPRA) to create a high-throughput platform to measure human cardiac enhancer activity. We tested 2451 candidate human cardiac enhancers, identified 1185 with measurable activity, and functionally dissected 123 of these by systematic tiling mutagenesis. We functionally evaluated 6761 non-coding de novo variants (ncDNVs) prioritized from the whole genome sequencing (WGS) of 749 CHD trios. 397 ncDNVs significantly affected cardiac CRE activity. Remarkably, 53% of these ncDNVs increased enhancer activity, often at regions with undetectable enhancer activity in the reference sequence. We introduced 10 of these DNVs associated with CHD genes into iPSCs and found that 4 altered expression of neighboring genes. Moreover, these 4 DNVs also altered cardiomyocyte differentiation, as assessed by single nucleus RNA sequencing. Using the MPRA data, we developed a regression model to prioritize future DNVs for functional testing and demonstrate that this model finds enrichment of DNVs in a second, independent WGS cohort. Taken together, we developed a scalable system to measure the impact of non-coding DNVs on CRE activity and deployed this platform to systematically assess the contribution of non-coding DNVs to CHD.
2022-08-05 | GSE210376 | GEO
Project description:Direct reprogramming of fibroblasts into cardiomyocyte-like cells (iCM) holds great potential for heart regeneration and disease modeling and may lead to future therapeutic applications in human patients with heart disease. Currently, the application of this technology is limited by our lack of understanding of the molecular mechanisms which drive direct iCM reprogramming. Using a quantitative mass spectrometry-based proteomic approach we have identified the temporal global changes in protein abundance that occur during the initial phases of iCM reprogramming. Collectively, our results show systematic and temporally distinct alterations in the levels of specific functional classes of proteins during the initiating steps of reprogramming including extracellular matrix proteins, translation factors, and chromatin-binding proteins.
2018-02-19 | PXD006152 | Pride
Project description:Evaluate the change in transcription factors that have a role in human mesenchymal stem cell (hMSC) commitment to a cardiomyocyte lineage when co-cultured for 4 days with rat neonatal cardiomyocytes and before acquiring a recognizable cardiac phenotype. A myocardial microenvironment was generated by dissociating neonatal rat hearts and establishing cardiomyocyte primary cultures. HumanMSCs constitutively labeled with dsRed localized to the cell's mitochondria were either grown separately (control) or added to the cardiomyocyte primary cultures and grown for 4 days. dsRed fluorescent hMSCs were harvested from co-cultures at 4 days using a FACscan flow cytometer. The RNA for the microarray analysis was prepared from three biologically separate samples of hMSCs co-cultured for 4 days and from hMSCs grown separately for 4 days (control).
2011-09-15 | E-GEOD-32171 | biostudies-arrayexpress
Project description:Maintenance of circadian alignment between an organism and its environment is essential to ensure metabolic homeostasis. Synchrony is achieved by cell autonomous circadian clocks. Despite a growing appreciation of the integral relation between clocks and metabolism, little is known regarding the direct influence of a peripheral clock on cellular responses to fatty acids. To address this important issue, we utilized a genetic model of disrupted clock function specifically in cardiomyocytes in vivo (termed cardiomyocyte clock mutant (CCM)). CCM mice exhibited altered myocardial response to chronic high fat feeding at the levels of the transcriptome and lipidome as well as metabolic fluxes, providing evidence that the cardiomyocyte clock regulates myocardial triglyceride metabolism. Time-of-day-dependent oscillations in myocardial triglyceride levels, net triglyceride synthesis, and lipolysis were markedly attenuated in CCM hearts. Analysis of key proteins influencing triglyceride turnover suggest that the cardiomyocyte clock inactivates hormone-sensitive lipase during the active/awake phase both at transcriptional and post-translational (via AMP-activated protein kinase) levels. Consistent with increased net triglyceride synthesis during the end of the active/awake phase, high fat feeding at this time resulted in marked cardiac steatosis. These data provide evidence for direct regulation of triglyceride turnover by a peripheral clock and reveal a potential mechanistic explanation for accelerated metabolic pathologies after prevalent circadian misalignment in Western society.
2010-03-24 | GSE21028 | GEO
Project description:Background: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. Methods: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine–based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. Results: We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. Conclusions: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.
2023-06-24 | PXD042806 | Pride
Project description:6761 CHD non-coding DNVs were prioritized from 749 unexplained CHD trios and functionally assessed in iPSC-CMs using lentiMPRA.
2023-11-16 | GSE208285 | GEO
Project description:The present study aims to explore the expression profiles and biological functions of long-chain noncoding RNA (lncRNA) in coronary heart disease (CHD)
2021-03-21 | GSE169256 | GEO
Project description:Exposure to per- and polyfluoroalkyl substances (PFASs) such as perfluorooctanesulfonic acid (PFOS) has been associated with congenital heart disease (CHD) and decreased birth weight. PFAS exposure can disrupt signaling pathways relevant for cardiac development in stem cell derived cardiomyocyte assays, including PFOS-induced disruption to in vitro cardiac differentiation into contracting spheroids of cardiomyocytes; the PluriBeat assay. Notably, cell line origin can affect how the assay respond to PFAS exposure. Herein, we examine the effect of PFOS on cardiomyocyte differentiation by transcriptomics profiling of two different human induced pluripotent stem cell (hiPSC) lines, to see if they exhibit a common pattern of disruption. Two stages of differentiation, the cardiac progenitor stage (early) and the cardiomyocyte stage (late), were investigated.
2022-05-03 | GSE202077 | GEO