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S J Tapscott

Publications and source records attributed to S J Tapscott.

At least 55 records · Page 3Linked to original sources
PubMed1993

Deficiency in rhabdomyosarcomas of a factor required for MyoD activity and myogenesis.

Rhabdomyosarcoma cells express the myogenic helix-loop-helix proteins of the MyoD family but do not differentiate into skeletal muscle cells. Gel shift and transient transfection assays revealed that MyoD in the rhabdomyosarcoma cells was capable of binding DNA but was relatively nonfunctional as a transcriptional activator. Heterokaryon formation with fibroblasts resulted in the restoration of transcriptional activation by MyoD and the differentiation of the rhabdomyosarcoma cells into skeletal muscle cells. These results suggest that rhabdomyosarcomas are deficient in a factor required for MyoD activity.

Animals↗
PubMed1993

Selective accumulation of MyoD and myogenin mRNAs in fast and slow adult skeletal muscle is controlled by innervation and hormones.

Each of the myogenic helix-loop-helix transcription factors (MyoD, Myogenin, Myf-5, and MRF4) is capable of activating muscle-specific gene expression, yet distinct functions have not been ascribed to the individual proteins. We report here that MyoD and Myogenin mRNAs selectively accumulate in hindlimb muscles of the adult rat that differ in contractile properties: MyoD is prevalent in fast twitch and Myogenin in slow twitch muscles. The distribution of MyoD and Myogenin transcripts also differ within a single muscle and correlate with the proportions of fast glycolytic and slow oxidative muscle fibres, respectively. Furthermore, the expression of a transgene consisting of a muscle-specific cis-regulatory region from the myoD gene controlling lacZ was primarily associated with the fast glycolytic fibres. Alteration of the fast/slow fibre type distribution by thyroid hormone treatment or by cross-reinnervation resulted in a corresponding alteration in the MyoD/Myogenin mRNA expression pattern. These findings show that the expression of specific myogenic helix-loop-helix regulators is under the control of innervation and humoral factors and may mediate differential control of contractile protein gene expression in adult muscle.

Animals↗
PubMed1992

Monoclonal antibodies to the myogenic regulatory protein MyoD1: epitope mapping and diagnostic utility.

Monoclonal antibodies (MoAbs) were developed against recombinant wild-type murine MyoD1 protein. Each of 4 MoAbs was immunologically reactive with recombinant MyoD1 protein by enzyme-linked immunosorbent assay, and each specifically stained the nuclei of myogenic cells. Epitopes were mapped using fusion protein constructs with specific deletions of defined regions of the MyoD1 molecule. MoAb 5.2F recognized an epitope in the amino terminal region between amino acid residues (AAR) 3 and 56, whereas epitopes for MoAbs 1.1A, 5.4G, and 5.8A were in the carboxyl terminus (AAR 167-318) of the MyoD1 protein. The epitope for MoAb 5.8A was further delineated to AAR 170-209 by Western analysis and immunoprecipitation of in vivo transcribed and translated MyoD1 protein having specific deletions in the carboxyl terminus. The 5.8A epitope was ultimately localized to the region between AAR 180 and 189 of the protein by enzyme-linked immunosorbent assay using 10-amino acid residue synthetic peptides. This sequence is apparently unique to MyoD1 and has little homology to other myogenic regulatory proteins (myogenin, Myf5, Myf6, and MRF4). Transfection of cDNA for murine MyoD1 into a nonmuscle cell line conferred 5.8A reactivity, confirming the specificity of this reagent. MoAb 5.8A was then used to examine the expression of MyoD1 in normal and malignant human tissues. MyoD1 was not detected in any normal adult tissue but was detected in 25 of 25 histologically confirmed rhabdomyosarcomas. Staining was localized to the nucleus and showed marked heterogeneity between cells as well as differential staining within nuclei. Specific subcellular localization of 5.8A was further determined by immunoelectron microscopy, where antibody was found to localize to electron-dense areas, more frequently associated with the nuclear submembranous region. In addition to rhabdomyosarcomas, MoAb 5.8A stained 2 of 5 Wilms' tumors and one ectomesenchymoma, neoplasms known to contain myogenic elements. The 5.8A reagent was also of value in the accurate histopathological classification of 2 of 4 tumors previously diagnosed as extraosseous Ewing's sarcoma and 2 of 3 tumors diagnosed as undifferentiated sarcomas.

Amino Acid Sequence↗
PubMed1992

Inhibition of protein phosphatases blocks myogenesis by first altering MyoD binding activity.

To examine the role of protein phosphatases in skeletal muscle differentiation, C2C12 myoblasts were treated with okadaic acid, a potent in vitro inhibitor of protein phosphatases 1 and 2A which regulate various cellular events in intact cells. We now show that okadaic acid treatment of the mouse myoblast C2C12 cell line reversibly altered the morphology of the cells and blocked differentiation. At a molecular level, it extinguished expression of the myogenic determination genes, MyoD1 and myogenin, but induced the expression of an inhibitor of differentiation, Id. Analysis of the MyoD1 promoter showed that inhibition of MyoD1 expression by okadaic acid occurs at the transcriptional level. These changes occur 10-20 h after okadaic acid treatment. However, within 1 h of treatment the ability of muscle extracts to support a specific MyoD-dependent gel mobility shift using a MyoD DNA binding site is lost. These data suggest that protein phosphatases play an important role during myogenic differentiation.

Animals↗
PubMed1992

Functional antagonism between c-Jun and MyoD proteins: a direct physical association.

The product of the proto-oncogene Jun inhibits myogenesis. Constitutive expression of Jun in myoblasts interferes with the expression and the function of MyoD protein. In transient transfection assays Jun inhibits transactivation of the MyoD promoter, the muscle creatine kinase enhancer, and a reporter gene linked to MyoD DNA-binding sites. Conversely, MyoD suppresses the transactivation by Jun of genes linked to an AP-1 site. We demonstrate that both in vivo and in vitro MyoD and Jun proteins physically interact. Mutational analysis suggests that this interaction occurs via the leucine zipper domain of Jun and the helix-loop-helix region of MyoD.

Animals↗
PubMed1992

A novel myoblast enhancer element mediates MyoD transcription.

The MyoD gene can orchestrate the expression of the skeletal muscle differentiation program. We have identified the regions of the gene necessary to reproduce transcription specific to skeletal myoblasts and myotubes. A proximal regulatory region (PRR) contains a conserved TATA box, a CCAAT box, and a GC-rich region that includes a consensus SP1 binding site. The PRR is sufficient for high levels of skeletal muscle-specific activity in avian muscle cells. In murine cells the PRR alone has only low levels of activity and requires an additional distal regulatory region to achieve high levels of muscle-specific activity. The distal regulatory region differs from a conventional enhancer in that chromosomal integration appears necessary for productive interactions with the PRR. While the Moloney leukemia virus long terminal repeat can enhance transcription from the MyoD PRR in both transient and stable assays, the simian virus 40 enhancer cannot, suggesting that specific enhancer-promoter interactions are necessary for PRR function.

Animals↗
PubMed1992

Sodium butyrate inhibits myogenesis by interfering with the transcriptional activation function of MyoD and myogenin.

Sodium butyrate reversibly inhibits muscle differentiation and blocks the expression of many muscle-specific genes in both proliferating myoblasts and differentiated myotubes. We investigated the role of the basic helix-loop-helix (bHLH) myogenic determinator proteins MyoD and myogenin in this inhibition. Our data suggest that both MyoD and myogenin are not able to function as transcriptional activators in the presence of butyrate, although both apparently retain the ability to bind DNA. Transcription of MyoD itself is extinguished in butyrate-treated myoblasts and myotubes, an effect that may be due to the inability of MyoD to autoactivate its own transcription. We present evidence that the HLH region of MyoD is essential for butyrate inhibition of MyoD. In contrast to MyoD and myogenin, butyrate does not inhibit the ubiquitous basic HLH protein E2-5 from functioning as a transcriptional activator.

Base Sequence↗
PubMed1991

The MCK enhancer contains a p53 responsive element.

p53 is an antioncogene that is defective or absent in a large number of human tumors. Its function in normal cells is not known. We show that co-transfection of mouse p53 with muscle-specific creatine kinase-chloramphenicol acetyltransferase reporter gene, containing 3.3 kilobase of upstream control sequence for the muscle-specific creatine kinase gene, results in a 10- to 80-fold activation. The p53 responsive element maps to a region distinguished from the known MyoD binding region. Identification of a p53 responsive element should allow a more focused analysis of the effects of p53 in controlling gene activity.

Animals↗
PubMed1991

Muscle-specific transcriptional activation by MyoD.

We focus on the mechanism by which MyoD activates transcription. Previous experiments showed that when the 13-amino-acid basic region of E12 replaced the corresponding basic region of MyoD, the resulting MyoD-E12Basic chimeric protein could bind specifically to muscle-specific enhancers in vitro and form dimers with E12, but could not activate a cotransfected reporter gene or convert 10T1/2 cells to muscle. Here we show that back mutation of this chimeric protein (with the corresponding residues in MyoD) re-establishes activation, and we identify a specific alanine involved in increasing DNA binding and a specific threonine required for activation. Using a reporter gene containing MyoD-binding sites located downstream from the transcription start site, we show that MyoD-E12Basic can bind in vivo and thereby inhibit expression of the reporter. In vivo binding is also supported by the fact that the addition of the "constitutive" VP16 activation domain to MyoD-E12Basic restores full trans-activation potential. The normal MyoD-activation domain maps within the amino-terminal 53 residues and can be functionally replaced by the activation domain of VP16. The activity of the MyoD-activation domain is dramatically elevated when deletions are made almost anywhere in the rest of the MyoD molecule, suggesting that the activation domain in MyoD is usually masked. Surprisingly, MyoD-E12Basic can activate transcription in CV1 and B78 cells (but not in 10T1/2 or 3T3 cells), suggesting that the activation function of the basic domain requires a specific factor present in CV1 and B78 cells. We propose that to function, the masked MyoD-activation domain requires the participation of a second factor that recognizes the basic region. We refer to such a factor as a recognition factor.

Amino Acid Sequence↗
PubMed1990

De novo methylation of the MyoD1 CpG island during the establishment of immortal cell lines.

CpG dinucleotides are unevenly distributed in the vertebrate genome. Bulk DNA is depleted of CpGs and most of the cytosines in the dinucleotide in this fraction are methylated. On the other hand, CpG islands, which are often associated with genes, are unmethylated at testable sites in all normal tissues with the exception of genes on the inactive X chromosome. We used Hpa II/Msp I analysis and ligation-mediated polymerase chain reaction to examine the methylation of the MyoD1 CpG island in adult mouse tissues, early cultures of mouse embryo cells, and immortal fibroblastic cell lines. The island was almost devoid of methylation at CCGG sites in adult mouse tissues and in low-passage mouse embryo fibroblasts. In marked contrast, the island was methylated in 10T 1/2 cells and in six other immortal cell lines showing that methylation of this CpG island had occurred during escape from senescence. The island became even more methylated in chemically transformed derivatives of 10T 1/2 cells. Thus, CpG islands not methylated in normal tissues may become modified to an abnormally high degree during immortalization and transformation.

Animals↗
PubMed1989

5-bromo-2'-deoxyuridine blocks myogenesis by extinguishing expression of MyoD1.

The pyrimidine analog 5-bromodeoxyuridine (BUdR) competes with thymidine for incorporation into DNA. Substitution of BUdR for thymidine does not significantly affect cell viability but does block cell differentiation in many different lineages. BUdR substitution in a mouse myoblast line blocked myogenic differentiation and extinguished the expression of the myogenic determination gene MyoD1. Forced expression of MyoD1 from a transfected expression vector in a BUdR-substituted myoblast overcame the block to differentiation imposed by BUdR. Activation of BUdR-substituted muscle structural genes and apparently normal differentiation were observed in transfected myoblasts. This shows that BUdR blocks myogenesis at the level of a myogenic regulatory gene, possibly MyoD1, not by directly inhibiting the activation of muscle structural genes. It is consistent with the idea that BUdR selectively blocks a class of regulatory genes, each member of which is important for the development of a different cell lineage.

Animals↗
PubMed1989

Positive autoregulation of the myogenic determination gene MyoD1.

Transfection of cDNA expression vectors encoding either MyoD1 or myogenin into 10T1/2 cells converts them to myogenic cells. We show that transfection of 10T1/2 cells with the MyoD1 cDNA activates expression of endogenous MyoD1 mRNA, indicating that MyoD1 is subject to positive autoregulation. This activation of endogenous MyoD1 mRNA was also observed in Swiss 3T6 cells, but not in several other fibroblast or adipoblast cell lines transfected with the MyoD1 cDNA. In addition, transfection of the MyoD1 cDNA leads to activation of myogenin expression, and transfection of the myogenin cDNA leads to activation of MyoD1 expression. Thus, MyoD1 and myogenin appear to function in a positive autoregulatory loop that could either: account for or contribute to the stability of myogenic commitment; or amplify the level of expression of both MyoD1 and myogenin above a critical threshold that is required for activation of the myogenic program.

Animals↗
PubMed1989

Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD.

MyoD is a master regulatory gene for myogenesis. Under the control of a retroviral long terminal repeat, MyoD was expressed in a variety of differentiated cell types by using either a DNA transfection vector or a retrovirus. Expression of muscle-specific proteins was observed in chicken, human, and rat primary fibroblasts and in differentiated melanoma, neuroblastoma, liver, and adipocyte lines. The ability of MyoD to activate muscle genes in a variety of differentiated cell lines suggests that no additional tissue-specific factors other than MyoD are needed to activate the downstream program for terminal muscle differentiation or that, if such factors exist, they are themselves activated by MyoD expression.

Adipose Tissue↗
PubMed1988

MyoD1: a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts.

Expression of a complementary DNA (cDNA) encoding the mouse MyoD1 protein in a variety of fibroblast and adipoblast cell lines converts them to myogenic cells. Polyclonal antisera to fusion proteins containing the MyoD1 sequence show that MyoD1 is a phosphoprotein present in the nuclei of proliferating myoblasts and differentiated myotubes but not expressed in 10T1/2 fibroblasts or other nonmuscle cell types. Functional domains of the MyoD1 protein were analyzed by site-directed deletional mutagenesis of the MyoD1 cDNA. Deletion of a highly basic region (residues 102 to 135) interferes with both nuclear localization and induction of myogenesis. Deletion of a short region (residues 143 to 162) that is similar to a conserved region in the c-Myc family of proteins eliminates the ability of the MyoD1 protein to initiate myogenesis but does not alter nuclear localization. Deletions of regions spanning the remainder of MyoD1 did not affect nuclear localization and did not inhibit myogenesis. Furthermore, expression of only 68 amino acids of MyoD1, containing the basic and the Myc similarity domains, is sufficient to activate myogenesis in stably transfected 10T1/2 cells. Genetic analysis maps the MyoD1 gene to mouse chromosome 7 and human chromosome 11.

Animals↗
PubMed1984

Differential binding of antibodies against the neurofilament triplet proteins in different avian neurons.

Using monospecific antisera against each of the three chicken neurofilament (NF) proteins, NF70, NF160 and NF180, the distribution of each of these proteins in several types of neurons was examined by immunohistochemistry. Striking differences were observed in the relative staining by the three antibodies when the soma of different types of neurons were compared, and also when the soma of some neurons were compared with their axons. Both the soma and axons of dorsal root sensory neurons were brightly stained by each of the antisera. The soma of spinal cord ventral horn neurons, however, were stained only by A-NF70 and A-NF180, not by A-NF160. The axons of these neurons were uniformly stained by A-NF70 and A-NF180, while only gradually becoming NF160-positive over the first several hundred microns. The lack of staining by A-NF160 was also observed in many neuronal soma in cultures of dissociated spinal cord cells. The soma and dendrites of adult cerebellar Purkinje cells were weakly stained by A-NF70 and A-NF180 and not at all by A-NF160, but both A-NF70 and A-NF180 yielded prominent staining of immature Purkinje cells and dendrites. These results suggest that the three NF proteins may be unequally distributed within the soma and processes of different types of neurons and/or may be subject to regionally selective modification.

Animals↗
PubMed1983

Effects of phorbol 12-myristate 13-acetate on the differentiation program of embryonic chick skeletal myoblasts.

The effects of phorbol 12-myristate 13-acetate (PMA) on three aspects of myogenesis have been analyzed: (a) fusion of mononucleated myogenic cells to form myotubes; (b) synthesis and accumulation of two muscle-specific proteins; and (c) DNA synthesis. Using autoradiography combined with immunofluorescent localization of muscle-specific light meromyosin and the muscle-specific intermediate filament protein desmin, we have found that embryonic chick myogenic cells cultured in the presence of PMA (50 nM) initiate the synthesis of both desmin and muscle-specific light meromyosin and, by these criteria, partially differentiate. These cells differ from normal definitive postmitotic myoblasts, however, since they (a) do not fuse; (b) do not assemble normal myofibrils; and (c) incorporate [3H]thymidine. PMA does not appear to induce DNA synthesis in postmitotic myoblasts, but it apparently permits cells to initiate expression of muscle-specific proteins while preventing complete withdrawal from the cell cycle. Inhibition of fusion by PMA has been reported, but continued incorporation of [3H]thymidine in nuclei of cells expressing muscle-specific proteins is a previously undescribed effect of PMA. This effect is not achieved by 4-alpha-phorbol-12, 13-didecanoate, a nonpromoting phorbol ester, and may be relevant to the action of PMA as a tumor promoter.

Animals↗