Muscle progenitor specification and myogenic differentiation are associated with changes in chromatin topology.
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PROVIDER: PRJNA633082 | ENA |
REPOSITORIES: ENA
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Project description:Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterized at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. To further investigate transcriptional regulatory mechanisms governed by Pax7, we used immuno-affinity purification and mass spectrometric sequencing to identify the compendium of Pax7-associated proteins in muscle progenitors. Although Pax7-associated proteins have been identified in myoblasts, it is likely that such an approach would fail to uncover progenitor
specific interactions with this protein. We therefore isolated chromatin from iPax7 cells
engineered with a single, inducible copy of the Flag-tagged Pax7 transgene integrated next to the Hprt locus, which is expected to maintain expression levels comparable
to those of satellite cells in the presence of Dox. Through purification of Flag-Pax7, we
identified a cohort of factors and complexes involved in gene activation or repression and remodeling of chromatin and genome architecture that were substantially enriched compared to the uninduced control. We also identified a large cohort of sequence-specific TFs, including several that were shown to play an essential role in muscle stem cells (e.g., Foxk1, Six1, Runx1, Tead1, Nfix) and that are recruited to Pax7-bound enhancers in muscle cells. Importantly, we also confirmed multiple interactions from our proteomic screen through immunoprecipitation and western blotting.
2020-10-30 | MSV000086392 | MassIVE
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| S-EPMC7718254 | biostudies-literature
Project description:Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in inducible Pax7-expressing skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterized at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. We identified key promoter-enhancer interaction motifs, namely, cliques and networks, and interactions that were dependent on Pax7 binding. Remarkably, we found that the majority of super-enhancers were bound by Pax7 and a cadre of associated factors that maintained an epigenetic memory of active enhancers in the absence of Pax7. Lastly, we identified a previously uncharacterized Pax7-bound enhancer hub that simultaneously regulates the essential myosin heavy chain cluster during skeletal muscle cell differentiation. Our studies lay the groundwork for understanding the three-dimensional conformation of chromatin in muscle stem cells.
2020-10-31 | GSE150638 | GEO
Project description:Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and directly reprograms human pluripotent stem cells into skeletal myoblast progenitor cells. Compared to the exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin+ myofibers compared to exogenous PAX7 overexpression. RNA-seq analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for progenitor cell specification and their potential for regenerative medicine.
2020-04-10 | GSE145575 | GEO
Project description:Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-based Transcriptional Activators
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Project description:Understanding the mechanisms of myogenesis in human induced pluripotent stem cells (hiPSCs) is a prerequisite to achieving patient-specific therapy for diseases of skeletal muscle. hiPSCs of different origin show distinctive kinetics and ability to differentiate into myocytes. To address the unique cellular and temporal context of hiPSC differentiation, we perform a longitudinal comparison of the transcriptomic profiles of three hiPSC lines that display differential myogenic specification, one robust and two blunted. We detail temporal differences in mechanisms that lead to robust myogenic specification. We show gene expression signatures of putative cell subpopulations and extracellular matrix components that may support myogenesis. Further, we show that targeted knockdown of ZIC3 at the outset of differentiation leads to improved myogenic specification in blunted hiPSC lines. Our study suggests that β-catenin transcriptional cofactors mediate crosstalk between multiple cellular processes and exogenous cues to facilitate specification of hiPSCs to mesoderm lineage, leading to robust myogenesis.
2021-03-01 | GSE161025 | GEO
Project description: Not available
| S-EPMC5723218 | biostudies-other
Project description:Skeletal muscle is a highly organized and regenerative tissue that maintains its homeostasis primarily by activation and differentiation of muscle stem cells. Mimicking an in vitro skeletal muscle differentiation program that contains self-renewing adult muscle stem cells and aligned myotubes has been challenging. Here, we set out to engineer a biomimetic skeletal muscle construct that can self-regenerate and produce aligned myotubes using induced myogenic progenitor cells (iMPCs), a heterogeneous culture consisting of skeletal muscle stem, progenitor and differentiated cells. Utilizing electrospinning, we fabricated polycaprolactone (PCL) substrates that enabled iMPC-differentiation into aligned myotubes by controlling PCL fiber orientation. Newly-conceived constructs contained highly organized multinucleated myotubes in conjunction with self-renewing muscle stem cells, whose differentiation capacity was augmented by Matrigel supplementation. Additionally, we demonstrate using single cell RNA sequencing (scRNA-seq) that iMPC-derived constructs faithfully recapitulate a step-wise myogenic differentiation program. Notably, when the constructs were subjected to a damaging myonecrotic agent, self-renewing muscle stem cells rapidly differentiated into aligned myotubes, akin to skeletal muscle repair in vivo. Taken together, we report on a novel in vitro system that mirrors myogenic regeneration and muscle fiber alignment, and can serve as a platform to study myogenesis, model muscular dystrophies or perform drug screens.
2023-09-27 | GSE222162 | GEO
Project description:Direct lineage reprogramming provides a unique system to study cell fate transitions and unearth molecular mechanisms that safeguard cellular identity. We previously reported on direct conversion of mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by transient MyoD overexpression in concert with small molecules treatment. Here we employed integrative multi-omic approaches to delineate the molecular landscape of fibroblast reprogramming into iMPCs in comparison to transdifferentiation into myogenic cells solely by MyoD overexpression. Utilizing bulk RNA-sequencing and mass spectrometry, we uncovered molecular regulators and pathways that endow a myogenic stem cell identity on fibroblasts only in the presence of small molecule treatment. In addition, we demonstrate that Pax7+ cells in iMPCs share molecular attributes with myoblasts, however in addition express unique genes, proteins and pathways that are indicative of a more activated satellite cell-like state in vitro. Collectively, this study charts a molecular blueprint for reprogramming fibroblasts into muscle stem and progenitor cells and further establishes the fidelity of stable iMPC cultures in capturing skeletal muscle regeneration in vitro for disease modeling and basic research applications.
2022-05-19 | PXD029379 | Pride
Project description:Background Quiescent (non-dividing) muscle satellite cells (SCs) transition into proliferating progenitor cells that differentiate to repair skeletal muscle after injury. Recently, the AP-1 family member, FOS, was found to be rapidly and transiently induced in SCs in response to muscle damage, and its activity is required for effective SC activation and muscle repair. However, why FOS is rapidly down-regulated before SCs enter the cell cycle as progenitor cells remains unclear. In addition, whether boosting FOS expression in primary cycling muscle progenitor cells can enhance their myogenic properties needs to be evaluated. Methods In this study, we established a lentiviral, doxycycline-inducible system to explore the molecular and functional consequences of continued FOS expression in SC-derived, cycling muscle progenitor cells ex vivo. Specifically, we performed cell growth and EdU incorporation assays to measure cellular proliferation; myotube formation assays to evaluate terminal differentiation potential; and RNA-Sequencing (RNA-Seq) in conjunction with high-throughput chromosome conformation capture (Hi-C) to uncover changes in gene expression and long-range chromatin interactions. Results Persistent FOS activity in cycling muscle progenitor cells modestly enhances progenitor cell proliferation, but severely antagonizes their ability to differentiate and form myotubes. Genome-wide RNA-Seq analysis revealed that ectopic FOS activity in cycling muscle progenitor cells suppresses a global pro-myogenic gene expression program, while activating a stress-induced Mitogen-Activated Protein Kinase (MAPK) transcriptional signature. Importantly, we found various chromosomal re-organization events in A/B compartments, TADs, and long-range genomic loops near FOS-regulated differentially expressed genes in cycling muscle progenitor cells ectopically expressing FOS. Conclusions Altogether, our results suggest that elevated FOS activity in proliferating muscle progenitor cells perturbs cellular differentiation by altering the 3-dimensional (3D) chromosome organization near critical pro-myogenic genes. This work highlights the crucial importance of tightly controlling FOS expression in the muscle lineage, and raises the possibility that in states of chronic stress or disease, persistent FOS activity in muscle stem and/or progenitor cells may actually disrupt the muscle forming process.
2022-08-15 | GSE166241 | GEO