ANALYSIS OF NEURAL-RESPONSIVE MYOGENIN UPSTREAM SEQUENCES BY MYOBLASTIMPLANTATION

The expression of myogenin is suppressed during innervation and has be en implicated in determining properties of skeletal muscle which are r egulated by electrical activity. We previously reported that transcrip tion driven by 3700 bp of the mouse myogenin upstream sequence (MYG370 0) is activated by denervation in transgenic mice (Nucleic Acids Res. 21, 5684-5693, 1993). To extend our investigation of the activity depe ndence of the myogenin promoter, we have utilized myoblast implantatio n as a novel approach to in vivo reporter analysis. Myoblasts for hind limb injections were generated by stable transfection of chloramphenic ol acetyltransferase (CAT) reporters into a beta-galactosidase-express ing line of C2 cells. In vitro characterization of stable myoblast clo nes carrying myogenin-CAT deletion constructs revealed that while the proximal myogenin 5'-flanking sequence confers myotube specificity, hi gh-level expression requires a region upstream (-335 to -1102) which d epends on chromosomal integration for its function. for analysis by im plantation, incorporation of injected myoblasts into existing myofiber s was confirmed by histochemical staining. Using clonal myoblasts harb oring nicotinic receptor alpha-subunit (alpha 800) and myosin light ch ain reporters as positive and negative controls, respectively, for den ervation responsiveness, we determined that the nuclei of injected myo blasts 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 acti vities of MYG3700 and MYG1565 reporters in injected limbs increased up to fourfold within 4 days after denervation, whereas the activities o f MYG1102 and MYG335 were unchanged. By 10 days after denervation, all myogenin reporters displayed denervation responsiveness. These implan tation 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 rep orter myoblast Lines in vitro and in vivo by implantation provide an e fficient means of evaluating regulatory regions for high-level express ion and neural modulation of muscle gene transcription. (C) 1995 Acade mic Press, Inc.