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

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.

Myocyte Enhancer Factor 2 Activates Promoter Sequences of the Human AβH-J-J Locus, Encoding Aspartyl-β-Hydroxylase, Junctin, and Junctate

“…In addition, there was diminished DNA binding activity of the muscle-specific transcription factor MEF-2. These data and other experimental evidence indicate that MEF-2 is involved in the activation of muscle-specific genes, including rat myosin light chain 2 slow, human myoglobin, rat muscle creatine kinase, mouse troponin C slow, and human beta enolase (14,15,20,26,50). In addition, MEF-2 has been proposed to serve as a nodal point in the mechanisms by which motor nerves control the program of gene expression in skeletal muscle fibers and in the pathway leading to cardiac hypertrophy.…”

Molecular Dissection of DNA Sequences and Factors Involved in Slow Muscle-Specific Transcription

“…We have previously shown the TnI SURE and FIRE E boxes to be functionally equivalent and not to confer muscle type specificity (12); the involvement of MEF-2 and CACC motifs in fiber type-specific transcription was unknown. Interestingly, one or more of these motifs are important for enhancer function of other slow and fast muscle genes, such as the MLC2v, MLC1/ 3f, and aldolase genes (20,37,51,64). In almost all cases, the mutation of any one of these sites results in the reduction or abolishment of overall enhancer activity but not in the loss of fiber type specificity.…”

High resolution structure of the HDGF PWWP domain: A potential DNA binding domain

“…SAAB results indicate some preference for a 5¢-CACC-3¢ sequence, found in 61% of the unique sequenced clones. CACC-binding elements have been previously identified in regulatory regions of musclespecific genes (Biesiada et al 1999;Esser et al 1999). In particular, zinc finger domains such as Sp1 and RIN ZF have been shown to regulate gene expression using this element.…”