Abstract
Previous experiments showed that activity of the -800-base pair MLC2slow promoter 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-dependent gene expression. Three sites within the proximal promoter (myocyte enhancer factor-2 (MEF2), E-box, and CACC box) were individually mutated, and the effect on luciferase expression was determined. There was no change in luciferase expression in the SOL and extensor digitorum longus (EDL) muscles when 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 promoter 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 EDL muscles. These findings suggest that the active MLC2slow promoter is sensitive to MEF2 levels, but MEF2 levels alone do not determine nerve-dependent expression. Mutation of the CACC box resulted in a significant up-regulation in the EDL muscles (0.23 versus 4.08 normalized RLUs). With the CACC box mutated, overexpression of MEF2 was sufficient to transactivate the MLC2slow promoter in noninnervated SOL muscles (27,536 versus 1, 605,797 RLUs). Results from electrophoretic mobility shift and supershift assays confirm MEF2 protein binding to the MEF2 site and demonstrate specific binding to the CACC sequence. These results suggest a model for nerve-dependent regulation of the MLC2slow promoter in which derepression occurs through the CACC box followed by quantitative expression through enhanced MEF2 activation.
Highlights
The acquisition and maintenance of an adult skeletal muscle phenotype is regulated, in part, by the activity of specific motor neurons [1,2,3]
Work on other slow isoform contractile protein genes, such as the TnIslow and TnCslow genes, has demonstrated that MEF2, E-box, and CACC box sites are important in directing slow skeletal muscle specific expression [15, 16, 33, 34]
From the results of this study, we describe a two-step model for slow nerve-dependent regulation of the MLC2slow promoter
Summary
The acquisition and maintenance of an adult skeletal muscle phenotype is regulated, in part, by the activity of specific motor neurons [1,2,3]. It has been well established that chronic low frequency electrical stimulation of fast muscle fibers, by stimulating either through the nerve or by direct stimulation of the muscle, induces the slow skeletal muscle phenotype [2,3,4] This phenotype is characterized by an increased expression of the slow contractile protein isoforms. Induction of MLC2slow mRNA occurs at a time that is coincident with the establishment of functional neuromuscular junctions [21] Because of this relatively clear on/off pattern of control, the MLC2slow promoter serves as a good molecular tool for the identification of promoter elements responsible for slow nerve-dependent regulation of transcription. The main objectives were 1) to find a smaller region of
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