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1.
Semin Cell Dev Biol ; 72: 10-18, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29127045

RESUMO

The Myogenic Regulatory Factors (MRFs) Myf5, MyoD, myogenin and MRF4 are members of the basic helix-loop-helix family of transcription factors that control the determination and differentiation of skeletal muscle cells during embryogenesis and postnatal myogenesis. The dynamics of their temporal and spatial expression as well as their biochemical properties have allowed the identification of a precise and hierarchical relationship between the four MRFs. This relationship establishes the myogenic lineage as well as the maintenance of the terminal myogenic phenotype. The application of genome-wide technologies has provided important new information as to how the MRFs function to activate muscle gene expression. Application of combined functional genomics technologies along with single cell lineage tracing strategies will allow a deeper understanding of the mechanisms mediating myogenic determination, cell differentiation and muscle regeneration.


Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Desenvolvimento Muscular/genética , Músculo Esquelético/metabolismo , Fatores de Regulação Miogênica/genética , Regeneração/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Fatores de Regulação Miogênica/classificação , Filogenia
2.
Nat Commun ; 13(1): 3961, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35803939

RESUMO

Satellite cells are required for the growth, maintenance, and regeneration of skeletal muscle. Quiescent satellite cells possess a primary cilium, a structure that regulates the processing of the GLI family of transcription factors. Here we find that GLI3 processing by the primary cilium plays a critical role for satellite cell function. GLI3 is required to maintain satellite cells in a G0 dormant state. Strikingly, satellite cells lacking GLI3 enter the GAlert state in the absence of injury. Furthermore, GLI3 depletion stimulates expansion of the stem cell pool. As a result, satellite cells lacking GLI3 display rapid cell-cycle entry, increased proliferation and augmented self-renewal, and markedly enhanced regenerative capacity. At the molecular level, we establish that the loss of GLI3 induces mTORC1 signaling activation. Therefore, our results provide a mechanism by which GLI3 controls mTORC1 signaling, consequently regulating muscle stem cell activation and fate.


Assuntos
Células Satélites de Músculo Esquelético , Diferenciação Celular/fisiologia , Proliferação de Células , Alvo Mecanístico do Complexo 1 de Rapamicina , Músculo Esquelético , Células-Tronco , Internalização do Vírus
3.
Nat Commun ; 12(1): 3253, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059674

RESUMO

Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue.


Assuntos
Autorrenovação Celular/genética , Músculo Esquelético/lesões , Fator de Transcrição PAX7/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/fisiologia , Acetilação , Animais , Células COS , Sistemas CRISPR-Cas , Cardiotoxinas/administração & dosagem , Cardiotoxinas/toxicidade , Diferenciação Celular/genética , Chlorocebus aethiops , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Mutagênese , Cultura Primária de Células , Regiões Promotoras Genéticas , Células Sf9 , Sirtuína 2/genética , Sirtuína 2/metabolismo , Spodoptera , Ativação Transcricional
4.
Biomolecules ; 10(9)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872229

RESUMO

The Wnt Inhibitory Factor 1 (Wif1), known to inhibit Wnt signaling pathways, is composed of a WIF domain and five EGF-like domains (EGF-LDs) involved in protein interactions. Despite the presence of a potential O-fucosylation site in its EGF-LDs III and V, the O-fucose sites occupancy has never been demonstrated for WIF1. In this study, a phylogenetic analysis on the distribution, conservation and evolution of Wif1 proteins was performed, as well as biochemical approaches focusing on O-fucosylation sites occupancy of recombinant mouse WIF1. In the monophyletic group of gnathostomes, we showed that the consensus sequence for O-fucose modification by Pofut1 is highly conserved in Wif1 EGF-LD III while it was more divergent in EGF-LD V. Using click chemistry and mass spectrometry, we demonstrated that mouse WIF1 was only modified with a non-extended O-fucose on its EGF-LD III. In addition, a decreased amount of mouse WIF1 in the secretome of CHO cells was observed when the O-fucosylation site in EGF-LD III was mutated. Based on sequence comparison and automated protein modeling, we suggest that the absence of O-fucose on EGF-LD V of WIF1 in mouse and probably in most gnathostomes, could be related to EGF-LD V inability to interact with POFUT1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência Consenso , Fator de Crescimento Epidérmico/química , Evolução Molecular , Fucose/metabolismo , Animais , Fator de Crescimento Epidérmico/metabolismo , Fucosiltransferases/metabolismo , Camundongos , Modelos Moleculares , Filogenia , Domínios Proteicos , Proteínas Recombinantes/metabolismo
5.
Cell Stem Cell ; 24(3): 419-432.e6, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30713094

RESUMO

Loss of dystrophin expression in Duchenne muscular dystrophy (DMD) causes progressive degeneration of skeletal muscle, which is exacerbated by reduced self-renewing asymmetric divisions of muscle satellite cells. This, in turn, affects the production of myogenic precursors and impairs regeneration and suggests that increasing such divisions may be beneficial. Here, through a small-molecule screen, we identified epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation.


Assuntos
Aurora Quinase A/metabolismo , Polaridade Celular , Distrofina/deficiência , Receptores ErbB/metabolismo , Distrofia Muscular Animal/metabolismo , Regeneração , Células-Tronco/metabolismo , Animais , Divisão Celular , Células Cultivadas , Distrofina/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Distrofia Muscular Animal/patologia , Transdução de Sinais , Células-Tronco/patologia
6.
Nat Commun ; 10(1): 4256, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31534153

RESUMO

PAX7 is a paired-homeobox transcription factor that specifies the myogenic identity of muscle stem cells and acts as a nodal factor by stimulating proliferation while inhibiting differentiation. We previously found that PAX7 recruits the H3K4 methyltransferases MLL1/2 to epigenetically activate target genes. Here we report that in the absence of Mll1, myoblasts exhibit reduced H3K4me3 at both Pax7 and Myf5 promoters and reduced Pax7 and Myf5 expression. Mll1-deficient myoblasts fail to proliferate but retain their differentiation potential, while deletion of Mll2 had no discernable effect. Re-expression of PAX7 in committed Mll1 cKO myoblasts restored H3K4me3 enrichment at the Myf5 promoter and Myf5 expression. Deletion of Mll1 in satellite cells reduced satellite cell proliferation and self-renewal, and significantly impaired skeletal muscle regeneration. Pax7 expression was unaffected in quiescent satellite cells but was markedly downregulated following satellite cell activation. Therefore, MLL1 is required for PAX7 expression and satellite cell function in vivo. Furthermore, PAX7, but not MLL1, is required for Myf5 transcriptional activation in committed myoblasts.


Assuntos
Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Mioblastos/metabolismo , Fator Regulador Miogênico 5/metabolismo , Fator de Transcrição PAX7/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Feminino , Masculino , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator Regulador Miogênico 5/genética , Fator de Transcrição PAX7/genética , Regiões Promotoras Genéticas/genética
8.
Methods Mol Biol ; 1686: 149-159, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29030819

RESUMO

Adult satellite cells are quiescent, but are poised for activation in response to exercise, injury, or disease allowing adult muscle growth or repair. Once activated, satellite cells proliferate extensively to produce enough myogenic progenitors in order to regenerate the muscles. In order to self-renew, a subset of activated satellite cells can resist the myogenic differentiation and return to quiescence to replenish the satellite cell pool. These cellular processes that normally occur during skeletal muscle regeneration can be recapitulated ex vivo using isolated and cultured myofibers. Here, we describe a protocol to isolate single myofibers from the extensor digitorum longus muscle. Moreover, we detail experimental conditions for analyzing satellite cells in quiescence and progression through the myogenic lineage.


Assuntos
Separação Celular/métodos , Fibras Musculares Esqueléticas/citologia , Células Satélites de Músculo Esquelético/citologia , Animais , Diferenciação Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/fisiologia , Regeneração , Células Satélites de Músculo Esquelético/fisiologia
9.
Cell Stem Cell ; 23(5): 653-664, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388423

RESUMO

Muscle stem cells, or satellite cells, are required for skeletal muscle maintenance, growth, and repair. Following satellite cell activation, several factors drive asymmetric cell division to generate a stem cell and a proliferative progenitor that forms new muscle. The balance between symmetric self-renewal and asymmetric division significantly impacts the efficiency of regeneration. In this Review, we discuss the relationship of satellite cell heterogeneity and the establishment of polarity to asymmetric division, as well as how these processes are impacted in homeostasis, aging, and disease. We also highlight therapeutic opportunities for targeting satellite cell polarity and self-renewal to stimulate muscle regeneration.


Assuntos
Envelhecimento , Doença , Homeostase , Músculo Esquelético/citologia , Células-Tronco/citologia , Animais , Humanos , Células Satélites de Músculo Esquelético/citologia
10.
Cell Stem Cell ; 22(5): 755-768.e6, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29681515

RESUMO

Asymmetrically dividing muscle stem cells in skeletal muscle give rise to committed cells, where the myogenic determination factor Myf5 is transcriptionally activated by Pax7. This activation is dependent on Carm1, which methylates Pax7 on multiple arginine residues, to recruit the ASH2L:MLL1/2:WDR5:RBBP5 histone methyltransferase complex to the proximal promoter of Myf5. Here, we found that Carm1 is a specific substrate of p38γ/MAPK12 and that phosphorylation of Carm1 prevents its nuclear translocation. Basal localization of the p38γ/p-Carm1 complex in muscle stem cells occurs via binding to the dystrophin-glycoprotein complex (DGC) through ß1-syntrophin. In dystrophin-deficient muscle stem cells undergoing asymmetric division, p38γ/ß1-syntrophin interactions are abrogated, resulting in enhanced Carm1 phosphorylation. The resulting progenitors exhibit reduced Carm1 binding to Pax7, reduced H3K4-methylation of chromatin, and reduced transcription of Myf5 and other Pax7 target genes. Therefore, our experiments suggest that dysregulation of p38γ/Carm1 results in altered epigenetic gene regulation in Duchenne muscular dystrophy.


Assuntos
Epigênese Genética , Músculo Esquelético/citologia , Fator Regulador Miogênico 5/metabolismo , Fator de Transcrição PAX7/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Células-Tronco/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Músculo Esquelético/metabolismo , Fator Regulador Miogênico 5/genética , Fator de Transcrição PAX7/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
12.
Stem Cells Transl Med ; 5(3): 282-90, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26798058

RESUMO

Skeletal muscle regeneration is initiated by satellite cells, a population of adult stem cells that reside in the muscle tissue. The ability of satellite cells to self-renew and to differentiate into the muscle lineage is under transcriptional and epigenetic control. Satellite cells are characterized by an open and permissive chromatin state. The transcription factor Pax7 is necessary for satellite cell function. Pax7 is a nodal factor regulating the expression of genes associated with satellite cell growth and proliferation, while preventing differentiation. Pax7 recruits chromatin modifiers to DNA to induce expression of specific target genes involved in myogenic commitment following asymmetric division of muscle stem cells. Emerging evidence suggests that replacement of canonical histones with histone variants is an important regulatory mechanism controlling the ability of satellite cells and myoblasts to differentiate. Differentiation into the muscle lineage is associated with a global gene repression characterized by a decrease in histone acetylation with an increase in repressive histone marks. However, genes important for differentiation are upregulated by the specific action of histone acetyltransferases and other chromatin modifiers, in combination with several transcription factors, including MyoD and Mef2. Treatment with histone deacetylase (HDAC) inhibitors enhances muscle regeneration and is considered as a therapeutic approach in the treatment of muscular dystrophy. This review describes the recent findings on epigenetic regulation in satellite stem cells and committed myoblasts. The potential of epigenetic drugs, such as HDAC inhibitors, as well as their molecular mechanism of action in muscle cells, will be addressed.


Assuntos
Diferenciação Celular/genética , Epigênese Genética , Desenvolvimento Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , Linhagem da Célula , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/genética , Músculo Esquelético/metabolismo , Especificidade de Órgãos , Fator de Transcrição PAX7/genética , Regeneração , Células Satélites de Músculo Esquelético/metabolismo
13.
Cell Metab ; 22(1): 54-6, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26003784

RESUMO

Identifying candidates that rejuvenate aged muscle stem cells is an important strategy toward developing therapies to treat age-related diseases. In this issue, Egerman et al. (2015) re-investigate the activity of GDF11 in myogenesis, recently suggested as an anti-aging agent, and instead find a potent inhibitory effect on skeletal muscle regeneration.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Músculo Esquelético/fisiologia , Regeneração , Animais , Humanos
14.
Nat Med ; 21(12): 1455-63, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26569381

RESUMO

Dystrophin is expressed in differentiated myofibers, in which it is required for sarcolemmal integrity, and loss-of-function mutations in the gene that encodes it result in Duchenne muscular dystrophy (DMD), a disease characterized by progressive and severe skeletal muscle degeneration. Here we found that dystrophin is also highly expressed in activated muscle stem cells (also known as satellite cells), in which it associates with the serine-threonine kinase Mark2 (also known as Par1b), an important regulator of cell polarity. In the absence of dystrophin, expression of Mark2 protein is downregulated, resulting in the inability to localize the cell polarity regulator Pard3 to the opposite side of the cell. Consequently, the number of asymmetric divisions is strikingly reduced in dystrophin-deficient satellite cells, which also display a loss of polarity, abnormal division patterns (including centrosome amplification), impaired mitotic spindle orientation and prolonged cell divisions. Altogether, these intrinsic defects strongly reduce the generation of myogenic progenitors that are needed for proper muscle regeneration. Therefore, we conclude that dystrophin has an essential role in the regulation of satellite cell polarity and asymmetric division. Our findings indicate that muscle wasting in DMD not only is caused by myofiber fragility, but also is exacerbated by impaired regeneration owing to intrinsic satellite cell dysfunction.


Assuntos
Divisão Celular Assimétrica , Polaridade Celular , Distrofina/metabolismo , Músculo Esquelético/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Separação Celular , Distrofina/deficiência , Citometria de Fluxo , Camundongos Endogâmicos mdx , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Fuso Acromático/metabolismo
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