Genome wide gene-expression and mRNA splicing profile in human cardiomyocytes differentiated from Wt and QKI mutant embryonic stem cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE144008 | GEO | 2020/10/19
REPOSITORIES: GEO
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Project description:Alternative splicing is critical for development. However, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-specific RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes likely arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Collectively, our results thus uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.
2022-02-01 | MSV000088762 | MassIVE
Project description:We ablated RBM24 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques. Although RBM24-/- hESCs still differentiated into sarcomere-hosting cardiomyocytes, they exhibited disrupted sarcomeric structures with punctate Z-lines due to impaired myosin replacement during early myofibrillogenesis. Transcriptomics revealed >4000 genes regulated by RBM24. Among them, core myofibrillogenesis proteins (e.g. ACTN2, TTN, and MYH10) were misspliced. Consequently, MYH6 cannot replace non-muscle myosin MYH10, leading to myofibrillogenesis arrest at the early premyofibril stage and causing disrupted sarcomeres. Intriguingly, we found that the actin-binding domain (ABD; encoded by exon 6) of the Z-line anchor protein ACTN2 is predominantly excluded from early cardiac differentiation, whereas it is consistently included in human adult heart. CRISPR/Cas9-mediated deletion of exon 6 from ACTN2 in hESCs, as well as forced expression of full-length ACTN2 in RBM24-/- hESCs, further corroborated that inclusion of exon 6 is critical for sarcomere assembly. Overall, we have demonstrated that RBM24-facilitated inclusion of exon 6 in ACTN2 at distinct stages of cardiac differentiation is evolutionarily conserved and crucial to sarcomere assembly and integrity.
2021-11-22 | GSE186518 | GEO
Project description:To identify QKI targets, we performed QKI knockdown in BEAS2B cells and analyzed alternative splicing patterns by high-throughput RNA sequencing. The mRNA profiles of control- and QKI-knockdown BEAS2B cells were generated by deep sequencing using Illumina GAIIx sequencer.
2014-04-14 | E-GEOD-55215 | biostudies-arrayexpress
Project description:Background: Modulation of mRNA splicing acts as an important layer of gene regulation, in addition to transcriptional regulation and epigenetic modifications. RNA binding proteins (RBPs) play essential roles in mediating RNA splicing and are key regulators of heart development and function. Our previous studies demonstrated that RBPMS (RNA-binding protein with multiple splicing) regulates cardiac development through modulating mRNA splicing during embryogenesis. Here we explored the postnatal function of RBPMS in the heart. Methods: We ablated Rbpms in the heart by generating a cardiac-specific knockout mouse line (Myh6-Cre, Rbpmsfl/fl), and evaluated its cardiac functions by histology, echocardiography, and gene expression. Paired-end RNA sequencing and RT-PCR were performed to identify and validate splicing targets of RBPMS in adult mouse hearts. Proximity-dependent Biotin Identification (BioID) assay and mass spectrometry analysis were performed to identify RBPMS binding partners. We also measured contractility and calcium fluxes in isolated mouse cardiomyocytes, and contractile forces of cardiac papillary muscle. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) were also used as a model to explore the influence of RBPMS on contractility of human cardiomyocytes. Results: he absence of Rbpms in the heart led to dilated cardiomyopathy (DCM) and heart failure, causing early death in mice. Mice with cardiac-specific knockout of Rbpms showed myocardium noncompaction with reduced cardiomyocyte number at the neonatal stage and developed DCM with pervasive myocardial fibrosis in adulthood. We found that RBPMS mediates a largely distinct RNA splicing profile in adult mouse hearts compared to neonatal hearts, indicating a stage-specific modulation of alternative RNA splicing by RBPMS. In adult hearts, RBPMS mainly influenced alternative splicing of genes associated with sarcomere structures and cardiomyocyte contraction, such as Ttn, Pdlim5 and Nexn, to generate new protein isoforms. In neonatal hearts, RBPMS influenced the splicing of cytoskeletal genes. RBMPS was associated with spliceosome factors and other RNA binding proteins that play important roles in the heart, such as RBM20 and GATA4. Importantly, we found that the absence of Rbpms caused severe cardiomyocyte contractile defects and reduced calcium sensitivity in both mouse and hiPSC-CMs. Our results demonstrated that Rbpms is crucial for postnatal cardiac function and cardiomyocyte contractility by regulating RNA alternative splicing. Conclusions: Loss of Rbpms in the heart causes reduced cardiomyocyte number and impaired cardiomyocyte contraction, leading to DCM and heart failure.
2023-11-03 | GSE218672 | GEO
Project description:Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells. This is achieved by their strong suppression of the RNA binding protein Quaking (QKI), which is required to mediate the splicing changes regulated by these miRNAs. During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing events and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted both by QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing. The purpose of the CLIP experiment was to determine direct targets of QKI.
2018-03-15 | GSE111188 | GEO
Project description:To identify QKI targets, we performed QKI knockdown in BEAS2B cells and analyzed alternative splicing patterns by high-throughput RNA sequencing.
2014-04-14 | GSE55215 | GEO
Project description:The formation of new and functional cardiomyocytes requires a 3-step process: dedifferentiation, proliferation, and redifferentiation, but the critical genes required for efficient dedifferentiation, proliferation, and redifferentiation remain unknown. In our study, a circular trajectory using single-nucleus RNA sequencing of the pericentriolar material 1 positive (PCM1+) cardiomyocyte nuclei from hearts 1 and 3 days after surgery-induced myocardial infarction (MI) at postnatal day (P) 1 was reconstructed and demonstrated that actin remodeling contributed to the dedifferentiation, proliferation, and redifferentiation of cardiomyocytes after injury. We identified four top actin-remodeling regulators, namely Tmsb4x, Tmsb10, Dmd, and Ctnna3, which we collectively referred to as 2D2P. Transiently expressed changes of 2D2P, using a polycistronic non-integrating lentivirus driven by Tnnt2 (cardiac-specific troponin T) promoters (Tnnt2-2D2P-NIL), efficiently induced transiently proliferative activation and actin remodeling in P7 cardiomyocytes and adult hearts. Furthermore, the intramyocardial delivery of Tnnt2-2D2P-NIL resulted in a sustained improvement in cardiac function without ventricular dilatation, thickened septum, or fatal arrhythmia for at least 4 months. In conclusion, this study highlights the importance of actin remodeling in cardiac regeneration and provides a foundation for new gene-cocktail-therapy approaches to improve cardiac repair and treat heart failure using a novel transient and cardiomyocyte-specific viral construct.
2023-10-29 | GSE246250 | GEO
Project description:Human cardiomyocytes can be generated from human embryonic stem cells (hESCs) in vitro by a variety of methods, including co-culture with visceral endoderm-like cell lines and growth factor directed differentiation as monolayers or three-dimensional embryonic bodies. To enable the identification, purification and characterisation of human embryonic stem cell derived cardiomyocytes (CMs) and cardiac progenitor cells (CPCs), we introduced sequences encoding GFP into the NKX2-5 locus by homologous recombination. We found that NKX2-5GFP hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells and cardiomyocytes and the standardization of differentiation protocols.
2011-11-16 | E-MEXP-3371 | biostudies-arrayexpress
Project description:Genomewide analysis of QKI-dependent alternative splicing
| PRJNA238897 | ENA
Project description:Background: The histone demethylase KDM1A is a multi-faceted regulator of critical developmental processes, including mesodermal and cardiac tube formation during mice gastrulation. The fine-tuning of KDM1A splicing has been linked to regulating the transcriptional program of excitable cells such as neurons. However, it is unknown whether modulating the expression of KDM1A isoforms is crucial for the specification and maintenance of cell identity of other cell types sensitive to electrical cues such as the cardiomyocytes. Objective: We investigated the role of ubKDM1A and KDM1A+2a ubiquitous splice variants during cardiomyogenesis and evaluated their impact on the regulation of cardiac differentiation in vitro. Methods and Results: We discovered a temporal modulation of ubKDM1A and KDM1A+2a isoform levels during human and mouse fetal cardiac development. Therefore, we generated human embryonic stem cells (hESCs) exclusively devoid of one or both isoforms and assessed their potential to derive cardiomyocytes. KDM1A depletion severely impaired cardiac differentiation. Conversely, KDM1A+2a-/- hESCs give rise to functional cardiomyocytes, displaying increased beating amplitude and frequency compared to wild-type cells. Transcriptomic profiling revealed that KDM1A-/- cardiomyocytes fail to activate an effective cardiac transcriptional program, while the depletion of KDM1A+2a enhances the expression of key cardiogenic markers. Notably, the impaired cardiac differentiation of KDM1A-/- cells can be rescued by re-expressing ubKDM1A or catalytically deficient ubKDM1A-K661A, but not by KDM1A+2a or KDM1A+2a-K661A. These data demonstrate a divergent role of the two KDM1A isoforms that is independent of their enzymatic activity. Through an exhaustive biochemical and genome-wide binding characterization, we excluded that the opposite ubKDM1A- and KDM1A+2a-mediated regulation of cardiac differentiation resides into differential substrate specificity, H3K4 demethylation efficiency, core-partners binding affinity, or alternative genome binding profiles. Conclusions: Our findings suggest the existence of a divergent scaffolding role of KDM1A splice variants during hESC differentiation into cardiomyocytes.
2022-07-01 | GSE189944 | GEO