Cut& Tag of quiescent muscle stem cells (MuSCs)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE266725 | GEO | 2024/06/11
REPOSITORIES: GEO
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Project description:The maintenance of cell lineage and cell fate are essential for the function of adult stem cells. Despite this, the epigenetic mechanisms that regulate muscle stem cell (MuSC) identity are not well understood. In this study, we performed Cut&Tag of the activating histone marks H3K4me3 and H3K27ac on freshly isolated quiescent MuSCs and found that a large number of genes without transcription still maintained the permissive mark H3K4me3 but lacked the marker of active enhancers, H3K27ac. These genes included those that are essential for non-myogenic lineage determination. We found that many of the genes that were not transcribed but enriched for H3K4me3, were also enriched for the RE-1 binding motif, the motif recognized by the repressive transcription factor REST. Using a genetic mouse model where REST is conditionally knocked out of MuSCs, we further investigated the role of REST in the maintenance of quiescent MuSC cell identity. Investigation of the transcriptome and chromatin accessibility of WT and REST deficient MuSCs determined that the loss of REST results in the gain of expression of key genes of several non-myogenic tissues, particularly neuronal genes. Additionally, the loss of REST led to the dramatic reduction of the MuSC pool and the induction of muscle atrophy. The unstable cell identity caused by the genetic deletion of REST results in the MuSCs undergoing apoptosis and is the main driver of the observed loss of the MuSC pool. Together, the data presented in this study establishes a novel function of the transcription factor REST, where it safeguards the identity and myogenic lineage of MuSCs, through the repression of alternative lineages.
2024-06-11 | GSE266720 | GEO
Project description:The maintenance of cell lineage and cell fate are essential for the function of adult stem cells. Despite this, the epigenetic mechanisms that regulate muscle stem cell (MuSC) identity are not well understood. In this study, we performed Cut&Tag of the activating histone marks H3K4me3 and H3K27ac on freshly isolated quiescent MuSCs and found that a large number of genes without transcription still maintained the permissive mark H3K4me3 but lacked the marker of active enhancers, H3K27ac. These genes included those that are essential for non-myogenic lineage determination. We found that many of the genes that were not transcribed but enriched for H3K4me3, were also enriched for the RE-1 binding motif, the motif recognized by the repressive transcription factor REST. Using a genetic mouse model where REST is conditionally knocked out of MuSCs, we further investigated the role of REST in the maintenance of quiescent MuSC cell identity. Investigation of the transcriptome and chromatin accessibility of WT and REST deficient MuSCs determined that the loss of REST results in the gain of expression of key genes of several non-myogenic tissues, particularly neuronal genes. Additionally, the loss of REST led to the dramatic reduction of the MuSC pool and the induction of muscle atrophy. The unstable cell identity caused by the genetic deletion of REST results in the MuSCs undergoing apoptosis and is the main driver of the observed loss of the MuSC pool. Together, the data presented in this study establishes a novel function of the transcription factor REST, where it safeguards the identity and myogenic lineage of MuSCs, through the repression of alternative lineages.
2024-06-11 | GSE266726 | GEO
Project description:In Duchenne Muscular Dystrophy (DMD), the absence of the subsarcolemmal dystrophin protein leads to repeated myofiber damages inducing cycles of muscle regeneration that is driven by muscle stem cells (MuSCs). With time, MuSC regenerative capacities are overwhelmed, leading to fibrosis and muscle atrophy. Whether MuSCs from DMD muscle have intrinsic alterations or are primed by their degenerative/regenerative environment is still debated. We investigated cell behavior and gene expression in human using MuSCs derived from DMD or healthy muscles. We found that proliferation, differentiation and fusion were not altered in DMD-MuSCs, but with time, they lost the expression of the myogenic marker CD56 twice as fast as healthy MuSCs. The rapid drift towards a fibroblast-like cell identity was observed at the clonal level, and resulted from the altered expression of epigenetic enzymes required to maintain the myogenic cell fate. Indeed, the reexpression of CBX3, SMC3, H2AFV and H3F3B prevented the MuSC identity drift. Amongst the epigenetic changes, a closing of chromatin at the gene encoding the transcription factor MEF2B caused a down-regulation of its expression and a loss of the myogenic fate. Thus, MEF2B is a key mediator of the myogenic identity in human MuSCs, that is altered in DMD pathology.
2024-06-30 | GSE232667 | GEO
Project description:Quiescent adult muscle stem cells (MuSCs) regenerate skeletal muscle upon injury throughout life. However, aged skeletal muscles fail to maintain stem cell quiescence, leading to declines in MuSC number and functionality. Although autophagy plays an important role in the maintenance of MuSC quiescence, how quiescent MuSCs and their autophagy levels are maintained throughout life is largely unknown. The current study reveals how GnRH, a hypothalamic hormone, maintains the quiescence of adult MuSCs by preventing the onset of senescence and how the decline of sex steroids in organismal ageing is implicated in MuSC ageing.
2019-10-05 | GSE121016 | GEO
Project description:Background: Skeletal muscle function crucially depends on motor innervation and after injury on the resident muscle stem cells (MuSCs). However, it is poorly understood how innervation affects MuSC properties. Methods: We investigated the alterations of MuSCs and their immediate niche, the myofiber, after denervation in a surgery-based mouse model of unilateral sciatic nerve transection. FACS-isolated MuSCs were subjected to transcriptomics and proteomics analyses to investigate which changes occur after denervation. We performed Cardiotoxin-induced muscle injury, MuSC transplantation and floating myofiber cultures to assess MuSC functionality after denervation in addition to bioinformatics and histological analyses. Results: We observed a significant increase in the number of MuSCs (Pax7 positive; p-value= 0.0441), proliferating MuSCs (Pax7/Ki67 positive; p-value= 0.0023), activated MuSCs (MyoD positive; p-value= 0.0016) and differentiating MuSCs (Myog positive; p-value= 0.0057) after denervation. This aberrant activation and premature commitment of MuSCs to the myogenic lineage was accompanied by profound alterations on the mRNA (2613 differentially expressed genes, adj. p-value <0.05) and protein (1096 differentially abundant proteins, q-value <0.05) level after denervation. MuSCs from denervated hosts still engrafted and fused to form new myofibers irrespective of the innervation status of the recipient, suggesting the MuSC niche is driving alterations in MuSCs after denervation. The myofiber transcriptome after denervation showed massive changes in the general expression profile (10492 DEGs, p-value <0.05) and in several predicted secreted factors. Incubation of myofiber-associated MuSCs with supernatant from denervated myofibers increased cluster formation, reinforcing myofibers as a source of secreted factors driving MuSC alterations after denervation. Opn and Tgfb1 showed an increased secretion by denervated myofibers (30-fold and 6000-fold, respectively), and incubation with Tgfb1 alone induced Junb expression in myogenic cells, one of the genes highly upregulated in MuSCs after denervation (p-value= 1.85e-18, log2fc= 3.27), demonstrating that myofiber-secreted ligands influence MuSC gene expression. A combination of skeletal muscle injury and denervation led to reduced numbers of proliferating MuSCs (Sham: 47 vs DEN: 19.75 cells per cross section 10 days post-injury) and sustained high levels of developmental myosin heavy chain (Sham: 1 % vs DEN: 40 % of all myofibers 21 days post-injury), indicating hampered MuSC functionality due to changes in the microenvironment. Conclusion: Denervation of skeletal muscle causes alterations in myofiber secretion, leading to activation and profound changes of MuSCs, ultimately resulting in a reduced regenerative capacity. As these alterations are partially reversible, MuSCs are a promising target for novel treatment options for neuromuscular disorders and peripheral nerve injuries.
2024-03-04 | GSE217929 | GEO
Project description:Background: Skeletal muscle function crucially depends on motor innervation and after injury on the resident muscle stem cells (MuSCs). However, it is poorly understood how innervation affects MuSC properties. Methods: We investigated the alterations of MuSCs and their immediate niche, the myofiber, after denervation in a surgery-based mouse model of unilateral sciatic nerve transection. FACS-isolated MuSCs were subjected to transcriptomics and proteomics analyses to investigate which changes occur after denervation. We performed Cardiotoxin-induced muscle injury, MuSC transplantation and floating myofiber cultures to assess MuSC functionality after denervation in addition to bioinformatics and histological analyses. Results: We observed a significant increase in the number of MuSCs (Pax7 positive; p-value= 0.0441), proliferating MuSCs (Pax7/Ki67 positive; p-value= 0.0023), activated MuSCs (MyoD positive; p-value= 0.0016) and differentiating MuSCs (Myog positive; p-value= 0.0057) after denervation. This aberrant activation and premature commitment of MuSCs to the myogenic lineage was accompanied by profound alterations on the mRNA (2613 differentially expressed genes, adj. p-value <0.05) and protein (1096 differentially abundant proteins, q-value <0.05) level after denervation. MuSCs from denervated hosts still engrafted and fused to form new myofibers irrespective of the innervation status of the recipient, suggesting the MuSC niche is driving alterations in MuSCs after denervation. The myofiber transcriptome after denervation showed massive changes in the general expression profile (10492 DEGs, p-value <0.05) and in several predicted secreted factors. Incubation of myofiber-associated MuSCs with supernatant from denervated myofibers increased cluster formation, reinforcing myofibers as a source of secreted factors driving MuSC alterations after denervation. Opn and Tgfb1 showed an increased secretion by denervated myofibers (30-fold and 6000-fold, respectively), and incubation with Tgfb1 alone induced Junb expression in myogenic cells, one of the genes highly upregulated in MuSCs after denervation (p-value= 1.85e-18, log2fc= 3.27), demonstrating that myofiber-secreted ligands influence MuSC gene expression. A combination of skeletal muscle injury and denervation led to reduced numbers of proliferating MuSCs (Sham: 47 vs DEN: 19.75 cells per cross section 10 days post-injury) and sustained high levels of developmental myosin heavy chain (Sham: 1 % vs DEN: 40 % of all myofibers 21 days post-injury), indicating hampered MuSC functionality due to changes in the microenvironment. Conclusion: Denervation of skeletal muscle causes alterations in myofiber secretion, leading to activation and profound changes of MuSCs, ultimately resulting in a reduced regenerative capacity. As these alterations are partially reversible, MuSCs are a promising target for novel treatment options for neuromuscular disorders and peripheral nerve injuries.
2024-03-04 | GSE217928 | GEO
Project description:Abstract: Transcription factors (TFs) play key roles in regulating differentiation and function of stem cells, including muscle satellite cells (MuSCs), a resident stem cell population responsible for postnatal regeneration of the skeletal muscle. Sox11 belongs to the Sry-related HMG-box (SOX) family of TFs that play diverse roles in stem cell behavior and tissue specification. Analysis of single-cell RNA-sequencing (scRNA-seq) datasets identify a specific enrichment of Sox11 mRNA in differentiating but not quiescent MuSCs. Consistent with the scRNA-seq data, Sox11 levels increase during differentiation of murine primary myoblasts in vitro. scRNA-seq data comparing muscle regeneration in young and old mice further demonstrate that Sox11 expression is reduced in aged MuSCs. Age-related decline of Sox11 expression is associated with reduced chromatin contacts within the topologically associated domains. Unexpectedly, Myod1Cre-driven deletion of Sox11 in embryonic myoblasts has no effects on muscle development and growth, resulting in apparently healthy muscles that regenerate normally. Pax7CreER or Rosa26CreER driven (MuSC-specific or global) deletion of Sox11 in adult mice similarly has no effects on MuSC differentiation or muscle regeneration. These results identify Sox11 as a novel myogenic differentiation marker with reduced expression in quiescent and aged MuSCs, but the specific function of Sox11 in myogenesis remain to be elucidated.
2023-09-21 | GSE226907 | GEO
Project description:Muscle stem cells (MuSC) exhibit distinct behaviors during successive phases of developmental myogenesis. However, how their transition to adulthood is regulated is poorly understood. Here we show that fetal MuSC resist progenitor specification and exhibit altered division dynamics, intrinsic features that are progressively lost postnatally. Following transplantation, fetal MuSC more efficiently expand and contribute to muscle repair. Conversely, the efficiency of niche colonization increases in adulthood, indicating a balance between muscle growth and stem cell pool repopulation. Gene expression profiling identified several extracellular matrix (ECM) molecules preferentially expressed in fetal MuSC, including tenascin-C, fibronectin and collagen VI. Loss-of-function experiments confirmed their essential and stage-specific role in regulating MuSC function. Finally, fetal-derived paracrine factors were able to enhance adult MuSC regenerative potential. Together, these findings demonstrate that MuSC change the way in which they remodel their microenvironment to direct stem cell behavior in support of the unique demands of muscle development or repair. MuSCs were isolated through fluorescent-activate cell sorting usinf alpha7-integirn and CD34 as markers to identify the cell population. Total mRNA was then isolated, and samples at different developmental times were compared.
2016-01-20 | E-GEOD-76989 | biostudies-arrayexpress
Project description:Background: Skeletal muscle crucially depends on motor innervation, and, when damaged, on the resident muscle stem cells (MuSCs). However, the role and function of MuSCs in the context of denervation remains poorly understood. Methods: Alterations of MuSCs and their myofiber niche after denervation were investigated in a surgery-based mouse model of unilateral sciatic nerve transection. FACS-isolated MuSCs were subjected to RNA-sequencing and mass spectrometry for the analysis of intrinsic changes after denervation and in vivo assays, such as Cardiotoxin-induced muscle injury or MuSC transplantation, were performed to assess MuSC functions after denervation. Bioinformatic and histological analyses were conducted to further examine MuSCs and their myofiber niche after denervation. Results: Muscle cross section analysis revealed a significant increase in Pax7 (p-value= 0.0441), Pax7/Ki67 (p-value= 0.0023), MyoD (p-value= 0.0016) and Myog (p-value= 0.0057) positive cells after denervation, illustrating a break of quiescence and commitment to the myogenic lineage. An Omics approach showed profound intrinsic alterations on the mRNA (2613 differentially expressed genes, p-value <0.05) and protein (1096 differentially abundant proteins, q-value <0.05) level of MuSCs 21 days after denervation. Skeletal muscle injury together with denervation surgery caused deregulated regeneration, indicated by the reduced number of proliferating MuSCs and sustained high levels of developmental myosin heavy chain (Sham: 1 % vs DEN: 40 % of all myofibers), at 21 days post-surgery. In a transplantation assay, MuSCs from a denervated host were still able to engraft and fuse to form new myofibers, irrespective of the innervation status of the recipient muscle. Analysis of myofibers revealed not only massive changes in the expression profile (10492 differentially expressed genes, p-value <0.05) after denervation, but it was also shown that secretion of Opn and Tgfb1 from denervated myofibers was increased 30-fold and 6000-fold, respectively. Bioinformatic analyses indicated strong upregulation of gene expression of the transcription factor Junb in MuSCs from denervated muscles (log2 fold change = 3.27). Of interest, Tgfb1 recombinant protein was able to induce Junb gene expression in vitro, demonstrating that myofiber-secreted ligands can induce gene expression changes in MuSCs, which might result in the phenotypes observed after denervation. Conclusion: Skeletal muscle denervation is altering myofiber secretion, causing MuSC activation and profound intrinsic changes, leading to reduced regenerative capacity. As MuSCs possess a remarkable regenerative potential, they might represent a promising target for novel treatment options for neuromuscular disorders and peripheral nerve injuries.
2024-02-27 | PXD036993 | Pride
Project description:Regulation of quiescence is essential for adult stem cell maintenance, longevity and sustained regeneration potential. Our studies have uncovered that physiological and transient changes in mitochondrial shape regulate the quiescent state of adult muscle stem cells (MuSCs). We show that mitochondria in MuSCs rapidly fragment in response to an activation stimulus, via a systemic HGF/mTOR mechanism, to drive the exit from deep quiescence. Deletion of the mitochondrial fusion protein OPA1 and forced mitochondrial fragmentation is sufficient to transition MuSCs into G-alert quiescence causing premature activation and depletion upon a stimulus. Loss of OPA1 activates a glutathione (GSH) redox signaling pathway that promotes cell-cycle progression, myogenic gene expression and commitment. MuSCs with chronic OPA1 loss and mitochondrial fragmentation, leading to mitochondrial dysfunction, continue to reside in a primed state but acquire severe cell cycle defects. Additionally, we provide evidence that OPA1 decline and impaired mitochondrial dynamics may contribute to age-related MuSC dysfunction. These findings reveal a fundamental role for OPA1 and mitochondrial dynamics in establishing the quiescent state and activation potential of adult stem cells.
2022-08-24 | GSE208326 | GEO