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P19 cells upregulated the expression of Nkx2-5, GATA4and MEF2C, enhanced cardiac muscle development, and activated a MEF2-responsive promoter. Moreover, inhibition of CaMK signaling downregulated GATA4 expression. Finally, P19 cells constitutively expressing a dominant-negative form of MEF2C, capable of binding class II HDACs, underwent cardiomyogenesis more efficiently than control cells, implying the relief of an inhibitor. Our results suggest that HDAC activity regulates the specification of mesoderm cells into cardiomyoblasts by inhibiting the expression of GATA4 and Nkx2-5 in a stem cell model system.

The homeobox transcription factor tinman is essential for heart vessel formation in Drosophila. In contrast, mice lacking the murine homologue Nkx2-5 are defective in cardiac looping but not in cardiac myocyte development. The lack of an essential role for Nkx2-5 in cardiomyogenesis in mammalian systems is most likely the result of genetic redundancy with family members. In this study, we used a dominant negative mutant of Nkx2

Two families of transcription factors, myogenic regulatory factors (MRFs) and myocyte enhancer factor 2 (MEF2), function synergistically to regulate myogenesis. In addition to activating structural muscle-specific genes, MRFs and MEF2 activate each other's expression. The MRF, myogenin, can activate MEF2 DNA binding activity when transfected into fibroblasts and, in turn, the myogenin promoter contains essential MEF2 DNA binding elements. To determine which MEF2 is involved in this regulation, P19 cells stably expressing MyoD and myogenin were compared for their ability to activate the expression of MEF2 family members. There was very little cross-activation of MyoD expression by myogenin and vice versa. Myogenin expression, and not MyoD, was found to up-regulate MEF2C expression. MEF2A, -B, and -D expression levels were not up-regulated by overexpression of either MyoD or myogenin. To examine whether MEF2C can differentially regulate MyoD or myogenin expression, P19 cell lines overexpressing MEF2C were analyzed. MEF2C induced myogenesis in P19 cells and up-regulated the expression of myogenin with 25-fold greater efficiency than that of MyoD. Therefore, myogenin and MEF2C participate in a regulatory loop in differentiating stem cells. This positive regulation does not extend to MyoD or the other MEF2 family members. Consequently, MEF2C appears to play a specific role in early events of myogenesis.Two families of transcription factors, the MEF2 1 family and the myogenic basic helix-loop-helix family (MRFs), interact to synergistically activate skeletal muscle-specific promoters (1-3). The four vertebrate MEF2 family members, MEF2A-D (4, 5), contain a conserved MCM1, agamous, deficiens, and serum response factor-box/MEF2 domain at their N termini. This domain mediates protein-protein interactions as well as DNA binding to an AT-rich MEF2 binding site. The four MRFs, MyoD, myogenin,, bind to E box sequences in the promoters of skeletal muscle-specific genes and can induce skeletal muscle development when expressed in fibroblasts (13). A positive feedback loop likely exists between MEF2 and myogenin because myogenin induces MEF2 DNA binding activity in various cell lines (14), and MEF2 DNA binding sites regulate myogenin expression (15, 16) but not MyoD expression (17) during embryogenesis.Insight into the roles of the MRFs has been gained from gene knockout studies in mice. Skeletal muscle development proceeded normally in homozygous null mice missing either MyoD (18) or myf-5 (19), but in double homozygous mice lacking both, myoblasts did not form (20). Mice lacking myogenin produced normal myoblast cells but displayed a marked reduction in secondary myofibers (21)(22)(23). Taken together these results indicate that MyoD and myf-5 play a role in the determination of skeletal muscle and that myogenin plays an essential in vivo role in the terminal differentiation of secondary muscle fibers.An essential role for D-MEF2 in the development of cardiac, skeletal, and smooth muscle has been demonstrated by the deficien...

DNA sequences have been obtained for embryonic chick feather and scale keratin genes. Strong homologies exist between the protein coding regions of the two gene types and between the deduced amino acid sequences of the keratin proteins. Scale keratins are larger than feather keratins and the size difference is mainly attributable to four 13‐amino acid repeats between residues 77 and 128 which compose a peptide sequence rich in glycine and tyrosine. The strong similarities between the two peptide structures for feather and scale in the homologous regions suggests a similar conformation within the protein filaments. A likely consequence is that the additional repeat region of the scale protein is located externally to the core filament. Tissue‐specific features of filament aggregation may be attributable to this one striking sequence difference between the constituent proteins. It is believed that the genes share a common ancestry and that feather‐like keratin genes may have evolved from a scale keratin gene by a single deletion event.

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The participation of host RNA polymerase II in the vaccinia life cycle was examined by comparing efficiency of multiplication after treating the Ama+ sensitive and Ama 102 drug resistant lines with a-amanitin. In the latter, resistance is due to a mutation in RNA polymerase II. The toxin profoundly reduces synthesis of virus-specified polypeptides and morphopoeisis in Ama+ but not in Ama 102 rat myoblasts without appreciably altering vaccinia The highly active toxin a-amanitin, derived from the toadstool Amanita phallodes, has been recognized as a specific inhibitor of DNA-dependent RNA polymerase II, hereafter referred to as polymerase II, of animal cells (1). By virtue of its inhibitory specificity, a-amanitin was used to demonstrate that certain DNA and RNA agents, such as papovaviruses, adenoviruses, and influenza viruses, having an obligatory developmental stage in the host nucleus, most probably require polymerase II activity for replication (2-4). By contrast, the poxviruses, which develop in the cytoplasm, were reported to be insensitive to this toxin (as cited in ref. 5) and to contain in the virion core an a-amanitin-insensitive DNA-dependent RNA polymerase (5). However, some requirement for the host nucleus is implied in the replication of vaccinia because virus development in cytoplasts is incomplete (6, 7). The availability of the rat myoblast L6 cell line, which our initial experiments showed can support the growth of vaccinia and from which a mutant was derived having a polymerase II resistant to a-amanitin (8), prompted us to examine the possible role of host transcriptional function(s) in the life cycle of poxviruses. MATERIALS AND METHODSCells and Viruses. Monolayers of L2mouse fibroblasts were used for virus propagation and assays of plaque-forming units (PFU) in nutrient medium and under culture conditions described (9). The viruses used were the hemagglutinin-inducing parental IHD-J or the syncytogenic IHD-W variant of vaccinia (10) and the Indiana strain of vesicular stomatitis virus (VSV). For inoculation, 10 PFU/cell usually were added as reported elsewhere (11).To investigate the role of host-derived functions in development of vaccinia we used (i) a clone L6H9, designated Ama+, and an a-amanitin-resistant mutant Ama 102 derived from this clone of a rat myoblast line (8), both kindly provided by M. E. Pearson (University of Toronto), and (ii) temperature-sensitive mutant 422E derived from hamster BHK21 fibroblasts, which is conditional-lethal for 28S ribosomal RNA formation and assembly of the 60S ribosomal subunit (12), provided by H. E. Meiss (New York University Medical School).Synthesis and Labeling. Cytoplasmic DNA synthesis in IHD-W vaccinia-infected Ama+ and Ama 102 cells was measured by continuous labeling, at 370C for 4 hr after inoculation, in the presence of 1 ,uCi of [methyl-3H]thymidine per ml (New England Nuclear) as described (13). Briefly, labeled cells were allowed to swell in hypotonic saline/buffer solution, then were disrupted in a Dounce homogenizer. The r...