MyoD1: A Nuclear Phosphoprotein Requiring a Myc Homology Region to Convert Fibroblasts to Myoblasts

Abstract

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.

Temporal expression of regulatory and structural muscle proteins during myogenesis of satellite cells on isolated adult rat fibers

“…Nuclei reacting with the anti-PCNA, or subsequently with the anti-myogenin, were almost completely eliminated (data not shown). We also demonstrated that exposure of fiber cultures to 5-bromo-2-deoxyuridine (50 μM), a drug which was shown to prevent differentiation of myoblasts in mass culture upon continuous incorporation into the DNA (Stockdale et al, 1964;Tapscott et al, 1988), drastically reduced the numbers of MyoD+ nuclei and extinguished myogenin+ nuclei; no effect was observed on the appearance and disappearance of PCNA+ cells on the fibers. Collectively, the above described experiments which have indicated that there is a link between DNA replication and expression of MyoD and myogenin provide further support that the two myogenic regulatory factor proteins are expressed in cells during or subsequent to their proliferation and not in myonuclei.…”

“…The similarity in the temporal appearance and disappearance of PCNA+ and MyoD+ cells on intact fibers (with or without bFGF) suggests that it is the proliferating satellite cells which express the myogenic regulatory factor protein MyoD. Analyzing the myogenic cell line C2, Tapscott et al (1988) concluded that MyoD protein was expressed by proliferating myoblasts, but not all proliferating myoblasts expressed MyoD. We also monitored the reactivity of mass cultures of rat satellite cells dissociated from isolated fibers and of C2 cells with the antibody against MyoD used in the present fiber study.…”

“…We further reasoned that upon activation, satellite cells may undergo a primordial program of myogenesis and thus may express specific muscle characteristics which are not expressed by the neighboring fiber; perhaps this expression would allow a distinction between the satellite cell and the fiber. With this in mind we analyzed the expression of the myogenic regulatory factor proteins MyoD (Davis et al, 1987;Tapscott et al, 1988) and myogenin (Wright et al, 1989;Edmondson and Olson, 1989)-members of the basic helix-loop-helix family of myogenic transcription factors. The four known members of this family are thought to be important in myogenic determination and in the progression from proliferation to differentiation during myogenesis (Rudnicki et al, 1993;reviewed in Olson, 1992reviewed in Olson, , 1993Sassoon, 1993;Weintraub, 1993;Emerson, 1993).…”

MyoD protein accumulates in satellite cells and is neurally regulated in regenerating myotubes and skeletal muscle fibers

“…The amount or conformation of MyoD needed to activate genes in muscle fibers and satellite cells may thus be different. MyoD can be phosphorylated (Tapscott et al, 1988) and form heterodimers with other regulatory factors (Farmer et al, 1992): this illustrates the plausibility of MyoD being able to activate different genes in different cellular environments. Second, we believe that the level of MyoD protein in fibers decreases with age and that non-growing innervated fibers contain insignificant levels of MyoD protein.…”

Helix‐loop‐helix transcription factors regulate Id2 gene promoter activity

“…Different HLH transcription factors potentially interact with each other by forming heterodimers [3]. The HLH transcription factors which form heterodimers are grouped into four classes: class A includes MEI (HEB, REB, HTF4), ME2 (ITF2), and E2A which are expressed ubiquitously [3 10]; class B includes cell type and tissue specific proteins such as NSCL, NEX, MASH-l, MASH-2, MyoD and others [11,14]; HES-family includes negatively acting transcriptional regulators HES-1, HES-2, HES-3, HES-5 [15 18]; and Id-like family includes transcriptional regulators lacking the basic, DNA binding domain such as Idl, Id2, Id3, and Id4 [19][20][21][22]. The activity of each individual HLH transcription factor depends on the complexes formed, and how these complexes bind to DNA and interact with the basic transcriptional complex.…”