Wnt signaling regulates the function of MyoD and myogenin

The myogenic regulatory factors (MRFs), MyoD and myogenin, can induce myogenesis in a variety of cell lines but not efficiently in monolayer cultures of P19 embryonal carcinoma stem cells. Aggregation of cells expressing MRFs, termed P19[MRF] cells, results in an approximately 30-fold enhancement of myogenesis. Here we examine molecular events occurring during P19 cell aggregation to identify potential mechanisms regulating MRF activity. Although myogenin protein was continually present in the nuclei of >90% of P19[myogenin] cells, only a fraction of these cells differentiated. Consequently, it appears that post-translational regulation controls myogenin activity in a cell lineage-specific manner. A correlation was obtained between the expression of factors involved in somite patterning, including Wnt3a, Wnt5b, BMP-2/4, and Pax3, and the induction of myogenesis. Co-culturing P19[Wnt3a] cells with P19[MRF] cells in monolayer resulted in a 5- to 8-fold increase in myogenesis. Neither BMP-4 nor Pax3 was efficient in enhancing MRF activity in unaggregated P19 cultures. Furthermore, BMP-4 abrogated the enhanced myogenesis induced by Wnt signaling. Consequently, signaling events resulting from Wnt3a expression but not BMP-4 signaling or Pax3 expression, regulate MRF function. Therefore, the P19 cell culture system can be used to study the link between somite patterning events and myogenesis.     

Characterization of the DNA-binding properties of the origin-binding domain of simian virus 40 large T antigen by fluorescence anisotropy

The affinity of the origin-binding domain (OBD) of simian virus 40 large T antigen for its cognate origin was measured at equilibrium using a DNA binding assay based on fluorescence anisotropy. At a near-physiological concentration of salt, the affinities of the OBD for site II and the core origin were 31 and 50 nM, respectively. Binding to any of the four 5'-GAGGC-3' binding sites in site II was only slightly weaker, between 57 and 150 nM. Although the OBD was shown previously to assemble as a dimer on two binding sites spaced by 7 bp, we found that increasing the distance between both binding sites by 1 to 3 bp had little effect on affinity. Similar results were obtained for full-length T antigen in absence of nucleotide. Addition of ADP-Mg, which promotes hexamerization of T antigen, greatly increased the affinity of full-length T antigen for the core origin and for nonspecific DNA. The implications of these findings for the assembly of T antigen at the origin and its transition to a non-specific DNA helicase are discussed.     

Growth factor responsiveness: role of MyoD and myogenin.

A differentiation-defective variant (DD-1) of the MM14 myoblasts acquired the ability to synthesize DNA in response to treatment with epidermal growth factor (EGF) (R. W. Lim and S. D. Hauschka, 1984, Dev. Biol. 105, 48) and no longer expressed myogenic determinant genes (i.e., MyoD and myogenin) (P.R. Mueller, and B. Wold, 1989, Science 246, 780). To determine the effect of expression of MyoD on EGF responsiveness, DD-1 cells were cotransfected with a MyoD expression vector and with pRSVneo. A clone, MyoDD-1 cells, which was G418 resistant, formed multinuclear syncitia, and also expressed MyoD and myogenin, was further characterized. EGF responsiveness, as assessed by DNA synthesis, was decreased 5- to 10-fold in the MyoDD-1 cells from that in G418-resistant control DD-1 cells, despite similar EGF receptor numbers and binding affinities of the receptors. Responsiveness of MyoDD-1 cells to fibroblast growth factor (FGF) was also diminished although to a lesser extent. To determine the effects of decreased myogenic determinant gene expression on mitogen responsiveness, MM14 myoblasts were grown in medium supplemented with 5 microM 5-bromo-2'-deoxyuridine (BUdR-MM14). BUdR-MM14 cells had decreased expression of MyoD and myogenin, did not fuse, and had an altered morphology, from round to flat. The BUdR effect on fusion and cell shape was reversed by growth in control medium. BUdR-MM14 cells were responsive to EGF and had enhanced responsiveness to FGF. The combined studies support the view that expression of MyoD and/or myogenin contributes to negative regulation of mitogen responsiveness.     

MyoD and myogenin protein expression in skeletal muscles of senile rats

We analyzed the level of protein expression of two myogenic regulatory factors (MRFs), MyoD and myogenin, in senile skeletal muscles and determined the cellular source of their production in young adult (4 months old), old (24, 26, and 28 months old), and senile (32 months old) male rats. Immunoblotting demonstrated levels of myogenin approximately 3.2, approximately 4.0, and approximately 5.5 times higher in gastrocnemius muscles of 24-, 26-, and 32-month-old animals, respectively, than in those of young adult rats. Anti-MyoD antibody recognized two major areas of immunoreactivity in Western blots: a single MyoD-specific band (approximately 43-45 kDa) and a double (or triple) MyoD-like band (approximately 55-65 kDa). Whereas the level of MyoD-specific protein in the 43- to 45-kDa band remained relatively unchanged during aging compared with that of young adult rats, the total level of MyoD-like immunoreactivity within the 55- to 65-kDa bands was approximately 3.4, approximately 4.7, approximately 9.1, and approximately 11.7 times higher in muscles of 24-, 26-, 28-, and 32-month-old rats, respectively. The pattern of MRF protein expression in intact senile muscles was similar to that recorded in young adult denervated muscles. Ultrastructural analysis of extensor digitorum longus muscle from senile rats showed that, occasionally, the area of the nerve-muscle junction was partially or completely devoid of axons, and satellite cells with the features of activated cells were found on the surface of living fibers. Immunohistochemistry detected accumulated MyoD and myogenin proteins in the nuclei of both fibers and satellite cells in 32-month-old muscles. We suggest that the up-regulated production of MyoD and myogenin proteins in the nuclei of both fibers and satellite cells could account for the high level of MRF expression in muscles of senile rats.     

Differential binding of quadruplex structures of muscle-specific genes regulatory sequences by MyoD, MRF4 and myogenin

Four myogenic regulatory factors (MRFs); MyoD, Myf-5, MRF4 and Myogenin direct muscle tissue differentiation. Heterodimers of MRFs with E-proteins activate muscle-specific gene expression by binding to E-box motifs d(CANNTG) in their promoters or enhancers. We showed previously that in contrast to the favored binding of E-box by MyoD-E47 heterodimers, homodimeric MyoD associated preferentially with quadruplex structures of regulatory sequences of muscle-specific genes. To inquire whether other MRFs shared the DNA binding preferences of MyoD, the DNA affinities of hetero- and homo-dimeric MyoD, MRF4 and Myogenin were compared. Similarly to MyoD, heterodimers with E47 of MRF4 or Myogenin bound E-box more tightly than quadruplex DNA. However, unlike homodimeric MyoD or MRF4, Myogenin homodimers associated weakly and nonpreferentially with quadruplex DNA. By reciprocally switching basic regions between MyoD and Myogenin we demonstrated dominance of MyoD in determining the quadruplex DNA-binding affinity. Thus, Myogenin with an implanted MyoD basic region bound quadruplex DNA nearly as tightly as MyoD. However, a grafted Myogenin basic region did not diminish the high affinity of homodimeric MyoD for quadruplex DNA. We speculate that the dissimilar interaction of MyoD and Myogenin with tetrahelical domains in muscle gene promoters may differently regulate their myogenic activities.     

Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity.

The levels of mRNAs coding for the myogenic factors MyoD and myogenin were measured during synapse formation in developing muscle and in adult muscle, after denervation and reinnervation and after muscle paralysis induced by blocking of neuromuscular transmission by neurotoxins known to alter the density and localization of synaptic proteins such as the acetylcholine receptor (AChR). The mRNA levels of both factors depend on usage of the neuromuscular synapses, but they change to different extents. Myogenin mRNA levels decrease drastically with innervation and increase strongly following blocking of transmission whereas the level of MyoD mRNA showed only a small decrease in response to innervation, denervation or muscle paralysis by neurotoxins. Neither mRNA showed a synapse-related cellular distribution. The results suggest that nerve-induced electrical muscle activity determines the cellular ratio of MyoD and myogenin mRNAs in adult muscle.     

Differential regulation of the promoter activity of the mouse UCP2 and UCP3 genes by MyoD and myogenin

UCP2 and UCP3 are members of the uncoupling protein family, which may play roles in energy homeostasis. In order to determine the regulation of the predominant expression of UCP3 in skeletal muscle, the effects of differentiation and myogenic regulatory factors on the promoter activities of the mouse UCP2 and UCP3 genes were studied. Reporter plasmids, containing approximately 3 kb of the 5'-upstream region of the mouse UCP2 and UCP3 genes, were transfected into C2C12 myoblasts, which were then induced to differentiate. Differentiation positively induced the reporter expression about 20-fold via the UCP3 promoter, but by only 2-fold via the UCP2 promoter. C2C12 myoblasts were cotransfected with expression vectors for myogenin and/or MyoD as well as reporter constructs. The simultaneous expression of myogenin and MyoD caused an additional 20-fold increase in the reporter expression via the UCP3 promoter, but only a weak effect via the UCP2 promoter. In L6 myoblasts, only MyoD activated the UCP3 promoter, but in 3T3-L1 cells neither factor activated the UCP3 promoter, indicating that additional cofactors are required, which are present only in C2C12 myoblasts. The expression of UCP2 and UCP3 is differentially regulated during muscle differentiation due to the different responsiveness of their promoter regions to myogenin and MyoD.