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Michael A Rudnicki - One of the best experts on this subject based on the ideXlab platform.
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MyoD induces myogenic differentiation through cooperation of its nh2 and cooh terminal regions
Journal of Cell Biology, 2005Co-Authors: Jeff Ishibashi, Michael A Rudnicki, Atsushi Asakura, Robert L. S. PerryAbstract:MyoD and Myf5 are basic helix-loop-helix transcription factors that play key but redundant roles in specifying myogenic progenitors during embryogenesis. However, there are functional differences between the two transcription factors that impact myoblast proliferation and differentiation. Target gene activation could be one such difference. We have used microarray and polymerase chain reaction approaches to measure the induction of muscle gene expression by MyoD and Myf5 in an in vitro model. In proliferating cells, MyoD and Myf5 function very similarly to activate the expression of likely growth phase target genes such as L-myc, m-cadherin, Mcpt8, Runx1, Spp1, Six1, IGFBP5, and Chrnβ1. MyoD, however, is strikingly more effective than Myf5 at inducing differentiation-phase target genes. This distinction between MyoD and Myf5 results from a novel and unanticipated cooperation between the MyoD NH2- and COOH-terminal regions. Together, these results support the notion that Myf5 functions toward myoblast proliferation, whereas MyoD prepares myoblasts for efficient differentiation.
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promoter specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression
Molecular Cell, 2002Co-Authors: Michael A Rudnicki, Stephen J Tapscott, Robert L. S. Perry, Bennett H Penn, Donald A Bergstrom, Andrew D StrandAbstract:We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.
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Activated MEK1 Binds the Nuclear MyoD Transcriptional Complex to Repress Transactivation
Molecular cell, 2001Co-Authors: Robert L. S. Perry, Maura H Parker, Michael A RudnickiAbstract:To elucidate the mechanism through which MAPK signaling regulates the MyoD family of transcription factors, we investigated the role of the signaling intermediate MEK1 in myogenesis. Transfection of activated MEK1 strongly repressed gene activation and myogenic conversion by the MyoD family. This repression was not mediated by direct phosphorylation of MyoD or by changes in MyoD stability or subcellular distribution. Deletion mapping revealed that MEK1-mediated repression required the MyoD amino-terminal transactivation domain. Moreover, activated MEK1 was nuclearly localized and bound a complex containing MyoD in a manner that is dependent on the presence of the MyoD amino terminus. Together, these data demonstrate that MEK1 signaling has a strong negative effect on MyoD activity via a novel mechanism involving binding of MEK1 to the nuclear MyoD transcriptional complex.
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the transition from proliferation to differentiation is delayed in satellite cells from mice lacking MyoD
Developmental Biology, 1999Co-Authors: Zipora Yablonkareuveni, Michael A Rudnicki, Anthony J Rivera, Michael Primig, Judy E Anderson, Priscilla NatansonAbstract:Satellite cells from adult rat muscle coexpress proliferating cell nuclear antigen and MyoD upon entry into the cell cycle, suggesting that MyoD plays a role during the recruitment of satellite cells. Moreover, the finding that muscle regeneration is compromised in MyoD−/− mice, has provided evidence for the role of MyoD during myogenesis in adult muscle. In order to gain further insight into the role of MyoD during myogenesis in the adult, we compared satellite cells from MyoD−/− and wildtype mice as they progress through myogenesis in single-myofiber cultures and in tissue-dissociated cell cultures (primary cultures). Satellite cells undergoing proliferation and differentiation were traced immunohistochemically using antibodies against various regulatory proteins. In addition, an antibody against the mitogen-activated protein kinases ERK1 and ERK2 was used to localize the cytoplasm of the fiber-associated satellite cells regardless of their ability to express specific myogenic regulatory factor proteins. We show that during the initial days in culture the myofibers isolated from both the MyoD−/− and the wildtype mice contain the same number of proliferating, ERK+ satellite cells. However, the MyoD−/− satellite cells continue to proliferate and only a very small number of cells transit into the myogenin+ state, whereas the wildtype cells exit the proliferative compartment and enter the myogenin+ stage. Analyzing tissue-dissociated cultures of MyoD−/− satellite cells, we identified numerous cells whose nuclei were positive for the Myf5 protein. In contrast, quantification of Myf5+ cells in the wildtype cultures was difficult due to the low level of Myf5 protein present. The Myf5+ cells in the MyoD−/− cultures were often positive for desmin, similar to the MyoD+ cells in the wildtype cultures. Myogenin+ cells were identified in the MyoD−/− primary cultures, but their appearance was delayed compared to the wildtype cells. These “delayed” myogenin+ cells can express other differentiation markers such as MEF2A and cyclin D3 and fuse into myotubes. Taken together, our studies suggest that the presence of MyoD is critical for the normal progression of satellite cells into the myogenin+, differentiative state. It is further proposed that the Myf5+/MyoD− phenotype may represent the myogenic stem cell compartment which is capable of maintaining the myogenic precursor pool in the adult muscle.
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reduced differentiation potential of primary MyoD myogenic cells derived from adult skeletal muscle
Journal of Cell Biology, 1999Co-Authors: Luc A Sabourin, Atsushi Asakura, Adele Girgisgabardo, Patrick Seale, Michael A RudnickiAbstract:To gain insight into the regeneration deficit of MyoD−/− muscle, we investigated the growth and differentiation of cultured MyoD−/− myogenic cells. Primary MyoD−/− myogenic cells exhibited a stellate morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD−/− myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo. Northern analysis indicated that proliferating MyoD−/− myogenic cells expressed fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain transcription factor expressed in newly activated satellite cells. Under conditions that normally induce differentiation, MyoD−/− cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD−/− myogenic cells. Mixing of lacZ-labeled MyoD−/− cells and wild-type myoblasts revealed a strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD−/− myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell.
Stephen J Tapscott - One of the best experts on this subject based on the ideXlab platform.
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Conversion of MyoD to a neurogenic factor: binding site specificity determines lineage
Cell reports, 2015Co-Authors: Abraham P. Fong, Lisa Maves, Zizhen Yao, Jun Wen Zhong, Nathan M. Johnson, Gist H. Farr, Stephen J TapscottAbstract:MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a "shared" E-box sequence (CAGCTG) and a "private" sequence (CAGGTG or CAGATG, respectively). To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.
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MyoD Is a Tumor Suppressor Gene in Medulloblastoma
Cancer research, 2013Co-Authors: Joyoti Dey, Stephen J Tapscott, Adrian M. Dubuc, Kyle Pedro, Derek Thirstrup, Brigham H Mecham, Paul A. Northcott, David Shih, Michael LeblancAbstract:While medulloblastoma, a pediatric tumor of the cerebellum, is characterized by aberrations in developmental pathways, the majority of genetic determinants remain unknown. An unbiased Sleeping Beauty transposon screen revealed MyoD as a putative medulloblastoma tumor suppressor. This was unexpected, as MyoD is a muscle differentiation factor and not previously known to be expressed in cerebellum or medulloblastoma. In response to deletion of one allele of MyoD, two other Sonic hedgehog-driven mouse medulloblastoma models showed accelerated tumor formation and death, confirming MyoD as a tumor suppressor in these models. In normal cerebellum, MyoD was expressed in the proliferating granule neuron progenitors that are thought to be precursors to medulloblastoma. Similar to some other tumor suppressors that are induced in cancer, MyoD was expressed in proliferating medulloblastoma cells in three mouse models and in human medulloblastoma cases. This suggests that although expression of MyoD in a proliferating tumor is insufficient to prevent tumor progression, its expression in the cerebellum hinders medulloblastoma genesis.
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pbx homeodomain proteins direct MyoD activity to promote fast muscle differentiation
Development, 2007Co-Authors: Lisa Maves, Ashlee Tyler, Andrew Jan Waskiewicz, Cecilia B. Moens, Biswajit Paul, Yi Cao, Stephen J TapscottAbstract:The basic helix-loop-helix (bHLH) transcription factor MyoD directly regulates gene expression throughout the program of skeletal muscle differentiation. It is not known how a MyoD-driven myogenic program is modulated to achieve muscle fiber-type-specific gene expression. Pbx homeodomain proteins mark promoters of a subset of MyoD target genes, including myogenin ( Myog ); thus, Pbx proteins might modulate the program of myogenesis driven by MyoD. By inhibiting Pbx function in zebrafish embryos, we show that Pbx proteins are required in order for MyoD to induce the expression of a subset of muscle genes in the somites. In the absence of Pbx function, expression of myog and of fast-muscle genes is inhibited, whereas slow-muscle gene expression appears normal. By knocking down Pbx or MyoD function in combination with another bHLH myogenic factor, Myf5, we show that Pbx is required for MyoD to regulate fast-muscle, but not slow-muscle, development. Furthermore, we show that Sonic hedgehog requires MyoD in order to induce both fast- and slow-muscle markers but requires Pbx only to induce fast-muscle markers. Our results reveal that Pbx proteins modulate MyoD activity to drive fast-muscle gene expression, thus showing that homeodomain proteins can direct bHLH proteins to establish a specific cell-type identity.
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global and gene specific analyses show distinct roles for MyoD and myog at a common set of promoters
The EMBO Journal, 2006Co-Authors: Yi Cao, Roshan M Kumar, Bennett H Penn, Charlotte A Berkes, Charles Kooperberg, Laurie A Boyer, Richard A Young, Stephen J TapscottAbstract:We used a combination of genome-wide and promoter-specific DNA binding and expression analyses to assess the functional roles of MyoD and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that MyoD and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, MyoD can bind and recruit histone acetyltransferases. At early targets, MyoD is sufficient for near full expression, whereas, at late expressed genes, MyoD initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without MyoD; however, transcriptional activation requires the combined activity of MyoD and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by MyoD.
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promoter specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression
Molecular Cell, 2002Co-Authors: Michael A Rudnicki, Stephen J Tapscott, Robert L. S. Perry, Bennett H Penn, Donald A Bergstrom, Andrew D StrandAbstract:We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.
S A Leibovitch - One of the best experts on this subject based on the ideXlab platform.
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Mutant MyoD Lacking Cdc2 Phosphorylation Sites Delays M-Phase Entry
Molecular and cellular biology, 2004Co-Authors: Lionel A. Tintignac, Marie Pierre Leibovitch, Valentina Sirri, Yann Lécluse, Maria Castedo, Didier Métivier, Guido Kroemer, S A LeibovitchAbstract:The transcription factors MyoD and Myf-5 control myoblast identity and differentiation. MyoD and Myf-5 manifest opposite cell cycle-specific expression patterns. Here, we provide evidence that MyoD plays a pivotal role at the G2/M transition by controlling the expression of p21Waf1/Cip1 (p21), which is believed to regulate cyclin B-Cdc2 kinase activity in G2. In growing myoblasts, MyoD reaccumulates during G2 concomitantly with p21 before entry into mitosis; MyoD is phosphorylated on Ser5 and Ser200 by cyclin B-Cdc2, resulting in a decrease of its stability and down-regulation of both MyoD and p21. Inducible expression of a nonphosphorylable MyoD A5/A200 enhances the MyoD interaction with the coactivator P/CAF, thereby stimulating the transcriptional activation of a luciferase reporter gene placed under the control of the p21 promoter. MyoD A5/A200 causes sustained p21 expression, which inhibits cyclin B-Cdc2 kinase activity in G2 and delays M-phase entry. This G2 arrest is not observed in p21−/− cells. These results show that in cycling cells MyoD functions as a transcriptional activator of p21 and that MyoD phosphorylation is required for G2/M transition.
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Critical Role for Lysine 133 in the Nuclear Ubiquitin-mediated Degradation of MyoD
The Journal of biological chemistry, 2003Co-Authors: Sabrina Batonnet, Marie Pierre Leibovitch, Lionel A. J. Tintignac, S A LeibovitchAbstract:Abstract The ubiquitin-proteasome system is responsible for the regulation and turnover of the nuclear transcription factor MyoD. The degradation of MyoD can occur via an NH2 terminus-dependent pathway or a lysine-dependent pathway, suggesting that MyoD ubiquitination may be driven by different mechanisms. To understand this process, deletion analysis was used to identify the region of MyoD that is required for rapid proteolysis in the lysine-dependent pathway. Here we report that the basic helix-loop-helix domain is required for ubiquitination and lysine-dependent degradation of MyoD in the nucleus. Site-directed mutagenesis in MyoD revealed that lysine 133 is the major internal lysine of ubiquitination. The half-life of the MyoD K133R mutant protein was longer than that of wild type MyoD, substantiating the implication of lysine 133 in the turnover of MyoD in myoblasts. In addition, the MyoD K133R mutant displayed activity 2–3-fold higher than the wild type in transactivation muscle-specific gene and myogenic conversion of 10T1/2 cells. Taken together, our data demonstrate that lysine 133 is targeted for ubiquitination and rapid degradation of MyoD in the lysine-dependent pathway and plays an integral role in compromising MyoD activity in the nucleus.
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Overexpression of Mos(rat) proto-oncogene product enhances the positive autoregulatory loop of MyoD.
FEBS letters, 1998Co-Authors: B Benayoun, K Pelpel, B Solhonne, M Guillier, S A LeibovitchAbstract:The myogenic b-HLH transcription factor MyoD activates expression of muscle-specific genes and autoregulates positively its own expression. Various factors such as growth factors and oncogene products repress transcriptional activity of MyoD. The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate myogenic differentiation by specific phosphorylation of MyoD which favors heterodimerization of MyoD and E12 proteins. Here we show that overexpression of Mos enhances the expression level of MyoD protein in myoblasts although phosphorylation of MyoD by Mos does not modify its stability but promotes transcriptional transactivation of the MyoD promoter linked to the luciferase reporter gene. Moreover, co-expression of MyoD with Mos(wt) but not with the kinase-inactive Mos(KM) greatly enhances expression of endogenous MyoD protein and the DNA binding activity of MyoD/E12 heterodimers in 10T1/2 cells. Our data suggest that Mos increases the ability of MyoD to transactivate both muscle-specific genes and its own promoter and could therefore participate in the positive autoregulation loop of MyoD and muscle differentiation.
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Overexpression of Mosrat proto‐oncogene product enhances the positive autoregulatory loop of MyoD
FEBS Letters, 1998Co-Authors: B Benayoun, K Pelpel, B Solhonne, M Guillier, S A LeibovitchAbstract:The myogenic b-HLH transcription factor MyoD activates expression of muscle-specific genes and autoregulates positively its own expression. Various factors such as growth factors and oncogene products repress transcriptional activity of MyoD. The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate myogenic differentiation by specific phosphorylation of MyoD which favors heterodimerization of MyoD and E12 proteins. Here we show that overexpression of Mos enhances the expression level of MyoD protein in myoblasts although phosphorylation of MyoD by Mos does not modify its stability but promotes transcriptional transactivation of the MyoD promoter linked to the luciferase reporter gene. Moreover, co-expression of MyoD with Mos(wt) but not with the kinase-inactive Mos(KM) greatly enhances expression of endogenous MyoD protein and the DNA binding activity of MyoD/E12 heterodimers in 10T1/2 cells. Our data suggest that Mos increases the ability of MyoD to transactivate both muscle-specific genes and its own promoter and could therefore participate in the positive autoregulation loop of MyoD and muscle differentiation.
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MOS ACTIVATES MYOGENIC DIFFERENTIATION BY PROMOTING HETERODIMERIZATION OF MyoD AND E12 PROTEINS
Molecular and cellular biology, 1997Co-Authors: B Benayoun, M Guillier, Marie Vandromme, Marie Pierre Leibovitch, S A LeibovitchAbstract:The activities of myogenic basic helix-loop-helix (bHLH) factors are regulated by a number of different positive and negative signals. Extensive information has been published about the molecular mechanisms that interfere with the process of myogenic differentiation, but little is known about the positive signals. We previously showed that overexpression of rat Mos in C2C12 myoblasts increased the expression of myogenic markers whereas repression of Mos products by antisense RNAs inhibited myogenic differentiation. In the present work, our results show that the rat mos proto-oncogene activates transcriptional activity of MyoD protein. In transient transfection assays, Mos promotes transcriptional transactivation by MyoD of the muscle creatine kinase enhancer and/or a reporter gene linked to MyoD-DNA binding sites. Physical interaction between Mos and MyoD, but not with E12, is demonstrated in vivo by using the two-hybrid approach with C3H10T1/2 cells and in vitro by using the glutathione S-transferase (GST) pull-down assays. Unphosphorylated MyoD from myogenic cell lysates and/or bacterially expressed MyoD physically interacts with Mos. This interaction occurs via the helix 2 region of MyoD and a highly conserved region in Mos proteins with 40% similarity to the helix 2 domain of the E-protein class of bHLH factors. Phosphorylation of MyoD by activated GST-Mos protein inhibits the DNA-binding activity of MyoD homodimers and promotes MyoD-E12 heterodimer formation. These data support a novel function for Mos as a mediator (coregulator) of muscle-specific gene(s) expression.
Anne Fernandez - One of the best experts on this subject based on the ideXlab platform.
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MyoD distal regulatory region contains an SRF binding CArG element required for MyoD expression in skeletal myoblasts and during muscle regeneration.
Molecular biology of the cell, 2003Co-Authors: Aurore L'honoré, Marie Vandromme, Gilles Carnac, Ned J. C. Lamb, Patric Turowski, Anne FernandezAbstract:We show here that the distal regulatory region (DRR) of the mouse and human MyoD gene contains a conserved SRF binding CArG-like element. In electrophoretic mobility shift assays with myoblast nuclear extracts, this CArG sequence, although slightly divergent, bound two complexes containing, respectively, the transcription factor YY1 and SRF associated with the acetyltransferase CBP and members of C/EBP family. A single nucleotide mutation in the MyoD-CArG element suppressed binding of both SRF and YY1 complexes and abolished DRR enhancer activity in stably transfected myoblasts. This MyoD-CArG sequence is active in modulating endogeneous MyoD gene expression because microinjection of oligonucleotides corresponding to the MyoD-CArG sequence specifically and rapidly suppressed MyoD expression in myoblasts. In vivo, the expression of a transgenic construct comprising a minimal MyoD promoter fused to the DRR and β-galactosidase was induced with the same kinetics as MyoD during mouse muscle regeneration. In contrast induction of this reporter was no longer seen in regenerating muscle from transgenic mice carrying a mutated DRR-CArG. These results show that an SRF binding CArG element present in MyoD gene DRR is involved in the control of MyoD gene expression in skeletal myoblasts and in mature muscle satellite cell activation during muscle regeneration.
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Mutation of MyoD-Ser237 abolishes its up-regulation by c-Mos.
FEBS letters, 2000Co-Authors: K Pelpel, Anne Fernandez, M Leibovitch, S A LeibovitchAbstract:Recently we have shown that Mos could activate myogenic differentiation by promoting heterodimerisation of MyoD and E12 proteins. Here, we demonstrate that MyoD can be efficiently phosphorylated by in vitro kinase assay with purified Mos immunoprecipitated from transfected cells. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that Mos phosphorylates MyoD on serine 237. Mutation of serine 237 to a non-phosphorylable alanine (MyoD-Ala237) abolished the positive regulation of MyoD by Mos following overexpression in proliferating 10T1/2 cells. Taken together, our data show that direct phosphorylation of MyoD-Ser237 by Mos plays a positive role in increasing MyoD activity during myoblast proliferation.
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cdk1- and cdk2-mediated phosphorylation of MyoD Ser200 in growing C2 myoblasts: role in modulating MyoD half-life and myogenic activity.
Molecular and cellular biology, 1999Co-Authors: Magali Kitzmann, Marie Vandromme, Valerie Schaeffer, Gilles Carnac, Jean-claude Labbé, Ned J. C. Lamb, Anne FernandezAbstract:We have examined the role of protein phosphorylation in the modulation of the key muscle-specific transcription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoes substantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by either purified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparative two-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1 and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatable alanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either in vitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of an E-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2 fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-type MyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts. Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integral role in compromising MyoD activity during myoblast proliferation.
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the muscle regulatory factors MyoD and myf 5 undergo distinct cell cycle specific expression in muscle cells
Journal of Cell Biology, 1998Co-Authors: Magali Kitzmann, Marie Vandromme, Gilles Carnac, Ned J. C. Lamb, Michael Primig, Anne FernandezAbstract:The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle–dependent regulation, thus establishing a correlation between the cell cycle–specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.
Atsushi Asakura - One of the best experts on this subject based on the ideXlab platform.
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increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Atsushi Asakura, Hiroyuki Hirai, Boris Kablar, Shigeru Morita, Jeff Ishibashi, Bryan A Piras, Amanda J Christ, Mayank VermaAbstract:MyoD is a myogenic master transcription factor that plays an essential role in muscle satellite cell (muscle stem cell) differentiation. To further investigate the function of MyoD in satellite cells, we examined the transplantation of satellite cell-derived myoblasts lacking the MyoD gene into regenerating skeletal muscle. After injection into injured muscle, MyoD(-/-) myoblasts engrafted with significantly higher efficiency compared with wild-type myoblasts. In addition, MyoD(-/-) myoblast-derived satellite cells were detected underneath the basal lamina of muscle fibers, indicating the self-renewal property of MyoD(-/-) myoblasts. To gain insights into MyoD gene deficiency in muscle stem cells, we investigated the pathways regulated by MyoD by GeneChip microarray analysis of gene expression in wild-type and MyoD(-/-) myoblasts. MyoD deficiency led to down-regulation of many muscle-specific genes and up-regulation of some stem cell markers. Importantly, in MyoD(-/-) myoblasts, many antiapoptotic genes were up-regulated, whereas genes known to execute apoptosis were down-regulated. Consistent with these gene expression profiles, MyoD(-/-) myoblasts were revealed to possess remarkable resistance to apoptosis and increased survival compared with wild-type myoblasts. Forced expression of MyoD or the proapoptotic protein Puma increased cell death in MyoD(-/-) myoblasts. Therefore, MyoD(-/-) myoblasts may preserve stem cell characteristics, including their resistance to apoptosis, expression of stem cell markers, and efficient engraftment and contribution to satellite cells after transplantation. Furthermore, our data offer evidence for improved therapeutic stem cell transplantation for muscular dystrophy, in which suppression of MyoD in myogenic progenitors would be beneficial to therapy by providing a selective advantage for the expansion of stem cells.
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MyoD induces myogenic differentiation through cooperation of its nh2 and cooh terminal regions
Journal of Cell Biology, 2005Co-Authors: Jeff Ishibashi, Michael A Rudnicki, Atsushi Asakura, Robert L. S. PerryAbstract:MyoD and Myf5 are basic helix-loop-helix transcription factors that play key but redundant roles in specifying myogenic progenitors during embryogenesis. However, there are functional differences between the two transcription factors that impact myoblast proliferation and differentiation. Target gene activation could be one such difference. We have used microarray and polymerase chain reaction approaches to measure the induction of muscle gene expression by MyoD and Myf5 in an in vitro model. In proliferating cells, MyoD and Myf5 function very similarly to activate the expression of likely growth phase target genes such as L-myc, m-cadherin, Mcpt8, Runx1, Spp1, Six1, IGFBP5, and Chrnβ1. MyoD, however, is strikingly more effective than Myf5 at inducing differentiation-phase target genes. This distinction between MyoD and Myf5 results from a novel and unanticipated cooperation between the MyoD NH2- and COOH-terminal regions. Together, these results support the notion that Myf5 functions toward myoblast proliferation, whereas MyoD prepares myoblasts for efficient differentiation.
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reduced differentiation potential of primary MyoD myogenic cells derived from adult skeletal muscle
Journal of Cell Biology, 1999Co-Authors: Luc A Sabourin, Atsushi Asakura, Adele Girgisgabardo, Patrick Seale, Michael A RudnickiAbstract:To gain insight into the regeneration deficit of MyoD−/− muscle, we investigated the growth and differentiation of cultured MyoD−/− myogenic cells. Primary MyoD−/− myogenic cells exhibited a stellate morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD−/− myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo. Northern analysis indicated that proliferating MyoD−/− myogenic cells expressed fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain transcription factor expressed in newly activated satellite cells. Under conditions that normally induce differentiation, MyoD−/− cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD−/− myogenic cells. Mixing of lacZ-labeled MyoD−/− cells and wild-type myoblasts revealed a strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD−/− myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell.
