Use of DNA injection for identification of slow nerve-dependent regions of the MLC2sgene

Abstract

It has been well established that expression of slow contractile protein genes in skeletal muscle is regulated, in part, by activity from slow motoneurons. However, very little is understood about the mechanism by which neural activity regulates transcription of slow isoform genes. The purpose of this investigation was first to more fully define the in vivo DNA injection technique for use in both fast-twitch and slow-twitch muscles and second to use the injection technique for the identification of slow nerve-dependent regions of the myosin light chain 2 slow ( MLC2s) gene. Initial experiments determined that the same amount of plasmid DNA was taken up by both the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles and that injection of from 0.5 to 10 μg DNA/muscle is ideal for analysis of promoter activity during regeneration. This technique was subsequently used to identify that the region from −800 to +12 base pairs of MLC2s gene directed ∼100 times higher activity in the innervated soleus than in innervated EDL, denervated soleus, or denervated EDL muscles. Placing the introns upstream of either the MLC2s or SV40 promoter increased expression 5- and 2.7-fold, respectively, in innervated soleus but not in innervated EDL, denervated soleus, or denervated EDL muscles. These results demonstrate that 1) in vivo DNA injection is a sensitive assay for promoter analysis in both fast-twitch and slow-twitch skeletal muscles and 2) both 5′ flanking and intronic regions of the MLC2s gene can independently and synergistically direct slow nerve-dependent transcription in vivo.

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

“…MLC2slow was sufficient to elicit slow nerve-dependent expression in regenerating skeletal muscle (9). Approximately 100-fold higher expression was detected in the SOLϩ compared with SOLϪ or innervated EDL muscles.…”

“…In Vivo DNA Injection-For promoter and overexpression assays, in vivo DNA injection during muscle regeneration was utilized following the protocol described previously (9). Briefly, 5 g of the promoter construct (Ϫ270MLC2s, Ϫ270MEF2, Ϫ270Ebox, or Ϫ270CACC) and 3 g of ␤-galactosidase control plasmid DNA (vector: SV40 promoter upstream of the ␤-galactosidase cDNA; CLONTECH Laboratories, Inc.) were mixed in a 10% sucrose-phosphate-buffered saline solution, pH 7.4.…”

“…Recently, it was determined that the 800-bp promoter of the rat MLC2slow gene was capable of directing slow nerve-dependent expression in regenerating muscles in vivo (9). As in the previous work, this study combined skeletal muscle regeneration with in vivo injection of plasmid DNA constructs into muscle.…”

“…These transitions in phenotype have been determined at the mRNA level, which suggests that alterations in slow neural activity transcriptionally regulate slow isoform gene expression (3,5,6). Regions of the MLC1slow, TnIslow, and MLC2slow genes have been identified that are responsive to slow innervation; however, the specific elements involved have not been isolated (7)(8)(9)(10). Potential cis-acting elements include those that are known to regulate qualitative and quantitative skeletal muscle gene expression.…”

Characterization of General Transcription Factor 3, a Transcription Factor Involved in Slow Muscle-specific Gene Expression

“…Fiber type specificity of muscle genes can be recapitulated in transgenic reporter mouse models or in vivo transfection assays by using respective transcription control regions (see, for example, Refs. [1][2][3][4][5][6][7][8]. Over the past years, signaling proteins such as calcineurin and Ras, and transcription factors GTF3 1 /MusTRD1, MEF-3, MEF-2, NFAT, and PGC-1␣ were implicated in the regulation of fiber type-specific expression in adult muscle (9 -17).…”