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 tissuespecific 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.

We focus on the mechanism by which MyoD activates transcription. Previous experiments showed that when the 13-amino-acid basic region of El2 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 El2, but could not activate a cotransfected reporter gene or convert 10T1A 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" VPI6 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 10T1A 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.

Myotonic dystrophy is caused by an expansion of a CTG triplet repeat sequence in the 3' noncoding region of a protein kinase gene, yet the mechanism by which the triplet repeat expansion causes disease remains unknown. This report demonstrates that a DNase I hypersensitive site is positioned 3' of the triplet repeat in the wild-type allele in both fibroblasts and skeletal muscle cells. In three unrelated individuals with myotonic dystrophy that have large expansions of the triplet repeat, the allele with the triplet repeat expansion exhibited both overall DNase I resistance and inaccessibility of nucleases to the adjacent hypersensitive site. These results indicate that the triplet repeat expansion alters the adjacent chromatin structure, establishing a region of condensed chromatin, and suggests a molecular mechanism for myotonic dystrophy.

ABSTRACT9L rat glioma cells have been used as a model for brain tumor therapies. It has been reported that in vivo infection of 9L cells with a replication-defective retrovirus expressing the herpes simplex thymidine kinase gene resulted in decreased tumor formation following treatment with the antiviral drug ganciclonr. the study reported here, rats were injected either intracerebrally or subcutaneously with 9L glioma cells expressing a chimeric hygromycin phosphotransferase-thymidine kinase fusion protein or with unmodified 9L cells. Tumor formation was decreased in the rats receiving modified cells, even in the absence of treatment with gacc vir. Suppression of tumor growth was also observed with cells modified to express the intracellular selectable marker neomycin phosphotransferase. These results indicate that an intracellular selectable marker, in the absence of pharmacologic selection, can inhibit tumor growth of 9L cells. The demonstration that intracellular marker genes can negatively influence the survival of transplanited cells has important implications for in vivo studies that use genetically modified cells.The median survival ofpatients with a malignant glioma is x1 year following diagnosis, even with aggressive surgery, radiation therapy, and chemotherapy (1). New treatment regimens for gliomas are frequently tested in animal models, often using the 9L rat glioma cell line. Several reports have used retroviral vectors to demonstrate in vivo transduction of C6 or 9L gliosarcoma tumors (2-5). Culver et al. (2) reported the effective treatment of an experimentally implanted 9L rat glioma by infection with a retrovirus conferring sensitivity to the drug ganciclovir. The investigators demonstrated that intracranially implanted 9L cells could be infected with a replication-defective retrovirus expressing the herpes simplex virus thymidine kinase (tk) gene following injection of the tumor with the viral packaging cell line. All animals were treated with ganciclovir, and 11 of 14 animals that received the virus-producing packaging cell line did not develop tumors in the 15-day observation period prior to sacrifice. Subsequent studies extended these findings and demonstrated that ganciclovir treatment was necessary for the prevention of tumor formation during the first 3 weeks after cell injection (3). These results and related studies (4-7) have provided the basis for human trials in which intratumor injection of a packaging cell line that produces an amphotropic retrovirus expressing the tk gene is followed by treatment with ganciclovir (8). We have attempted to reproduce the findings of Culver et al. (2)

Immunocytochemical analysis of dystrophin in genetically altered non-muscle cells is feasible and may be applicable to the prenatal and postnatal diagnosis of Duchenne's muscular dystrophy when conventional DNA analysis is not informative.

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.

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