Analysis of neural-responsive myogenin upstream sequences by myoblast implantation.
The expression of myogenin is suppressed during innervation and has been implicated in determining properties of skeletal muscle which are regulated by electrical activity. We previously reported that transcription driven by 3700 bp of the mouse myogenin upstream sequence (MYG3700) is activated by denervation in transgenic mice (Nucleic Acids Res. 21, 5684-5693, 1993). To extend our investigation of the activity dependence of the myogenin promoter, we have utilized myoblast implantation as a novel approach to in vivo reporter analysis. Myoblasts for hindlimb injections were generated by stable transfection of chloramphenicol acetyltransferase (CAT) reporters into a beta-galactosidase-expressing line of C2 cells. In vitro characterization of stable myoblast clones carrying myogenin-CAT deletion constructs revealed that while the proximal myogenin 5'-flanking sequence confers myotube specificity, high-level expression requires a region upstream (-335 to -1102) which depends on chromosomal integration for its function. For analysis by implantation, incorporation of injected myoblasts into existing myofibers was confirmed by histochemical staining. Using clonal myoblasts harboring nicotinic receptor alpha-subunit (alpha 800) and myosin light chain receptors as positive and negative controls, respectively, for denervation responsiveness, we determined that the nuclei of injected myoblasts are susceptible to regulatory signals imposed by nerve-induced electrical activity of the myofiber into which they incorporate. In in vivo analysis of myogenin upstream sequence by implantation, CAT activities of MYG3700 and MYG1565 reporters in injected limbs increased up to fourfold within 4 days after denervation, whereas the activities of MYG1102 and MYG335 were unchanged. By 10 days after denervation, all myogenin reporters displayed denervation responsiveness. These implantation data suggest an early phase of denervation activation, one that is mediated by control elements residing within -1102 to -1565 of the myogenin upstream sequence. Thus, the combined analyses of stable reporter myoblast lines in vitro and in vivo by implantation provide an efficient means of evaluating regulatory regions for high-level expression and neural modulation of muscle gene transcription.
Gibney
G
G
Laboratory of Developmental Neurobiology, National Institutes of Health, Bethesda, Maryland 20892, USA.
Buonanno
A
A
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D003714:Denervation; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006614:Hindlimb / Q000196:embryology* / Q000294:innervation; D051379:Mice; D018482:Muscle, Skeletal / Q000196:embryology* / Q000294:innervation / Q000637:transplantation; D018008:Myogenin / Q000502:physiology*; D017421:Sequence Analysis; D015533:Transcriptional Activation
C503801:Myog protein, mouse; D018008:Myogenin