The CACC box and myocyte enhancer factor-2 sites within the myosin light chain 2 slow promoter cooperate in regulating nerve-specific transcription in skeletal muscle

Previous experiments showed that activity of the -800-base pair MLC2slow pr omoter was 75-fold higher in the innervated soleus (SOL) compared with the noninnervated SOL muscles, Using in vivo DNA injection of MLC2slow promoter -luciferase constructs, the aim of this project was to identify regulatory sites and potential transcription factors important for slow nerve-dependen t gene expression. Three sites within the proximal promoter (myocyte enhanc er factor-2 (MEF2), E-box, and CACC box) were individually mutated, and the effect on luciferase expression was determined. There was no change in luc iferase expression in the SOL and extensor digitorum longus (EDL) muscles w hen the E-box was mutated. In contrast, the MEF2 mutation resulted in a 30- fold decrease in expression in the innervated SOL muscles (10.3 versus 0.36 normalized relative light units (RLUs)). Transactivation of the MLC2slow p romoter by overexpressing MEF2 was only seen in the innervated SOL (676,340 versus 2,225,957 RLUs; p < 0.01) with no effect in noninnervated SOL or ED L muscles. These findings suggest that the active MLC2slow promoter is sens itive to MEF2 levels, but MEF2 levels alone do not determine nerve-dependen t expression. Mutation of the CACC box resulted in a significant up-regulat ion in the EDL muscles (0.23 versus 4.08 normalized RLUs), With the CACC bo x mutated, overexpression of MEF2 was sufficient to transactivate the MLC2s low promoter in noninnervated SOL muscles (27,536 versus 1,605,797 RLUs), R esults from electrophoretic mobility shift and supershift assays confirm ME F2 protein binding to the MEF2 site and demonstrate specific binding to the CACC sequence. These results suggest a model for nerve-dependent regulatio n of the MLC2slow promoter in which derepression occurs through the CACC bo x followed by quantitative expression through enhanced MEF2 activation.