MOLECULAR-CLONING OF UP-REGULATED CYTOSKELETAL GENES FROM REGENERATING SKELETAL-MUSCLE - POTENTIAL ROLE OF MYOCYTE ENHANCER FACTOR-2 PROTEINS IN THE ACTIVATION OF MUSCLE-REGENERATION-ASSOCIATED GENES

A subtractive hybridization and cloning strategy was used to identify genes that are up-regulated in regenerating compared with normal skele tal muscle. The gastrocnemius muscle of CD1 mice was injected with a m yotoxic agent (BaCl2). A cDNA library was constructed from the regener ating muscle, and was screened with subtracted probes enriched in gene s up-regulated during regeneration. Cofilin and vimentin cDNA clones w ere isolated. Both cofilin and vimentin were demonstrated to be overex pressed in regenerating compared with non-regenerating muscle (17-fold and 19-fold induction respectively). Cofilin and vimentin mRNAs also exhibited an increased expression in C2C12 myoblasts and a decreased e xpression in differentiated myotubes. Analysis of the regeneration-ind uced vimentin enhancer/promoter region revealed a consensus binding si te for the myocyte enhancer factor2 (MEF2) transcription factors. Elec trophoretic mobility-shift assays and in vivo reporter assays revealed that MEF2 DNA-binding activity and transcriptional activation are inc reased in regenerating skeletal muscle, indicating that they may play a role in the activation of muscle genes during regeneration. These da ta suggest that both cofilin (an actin-regulatory protein) and vimenti n (an intermediate filament) may be key components of the cytoskeletal reorganization that mediates muscle cell development and adult skelet al-muscle repair.