First Intron Of Nestin Gene Regulates Its Expression During C2C12 Myoblast Differentiation

Nestin First Intron Regulates its Expression during C2C12 myoblast differentiationBackgroundSkeletal muscle in mammals is a mesodermal derivative and comes from precursor cells present in the somite of embryos[1].Myogenesis includes generation of the myogenic progenitor cells in the somite,differentiation and maturation of this progenitor cells.Under normal growth condition,newly formed somite rapidly partition into the ventral scelerotome compartment and the dorsal dermomyotome from which muscle cells and dermis generate.Peripheral muscles,such as those in the limb,are derived from cells that migrate from the lateral part of the somite[1]. The myogenic progenitor cells or myoblasts in the limb bud express the determination-class muscle regulatory factors(MRFs),then exit the cell cycle,and finally differentiate into myocytes.Most myocytes subsequently fuse with each other to form multinucleate myotubes,and then mature into myofibers[1].Myogenesis is regulated by morphogens and myogenic determination factors[2].Skeletal muscle development is accompanied by changes in the composition of intermediate filaments,where myogenic progenitor cells express nestin and vementin; myocytes express nestin,vementin and desmin;but mature myofibers only express desmin.Nestin,a classâ…¥intermediate filament protein,is expressed specifically in the neuroepthelial stem cells and neural progenitor cells.It has been widely used as a neural progenitor cell marker for the developing central nervous system(CNS)[3 4]. Nestin expression is also found in the myogenic progenitor cells in the dermomyotome of dorsal-lateral somites,and its expression persists in the developing thigh muscle until postnatal day 4 of rats[3].Nestin mRNA is found in the developing thigh muscle of rat,from embryonic day 15.5(E15.5)to postnatal day 21 rats[5],but not in the adult skeletal muscles[3 5 10].In situ hybridization also showed that nestin expression was upregulated in the developing mouse limb bud during myogenesis,and down-regulated during chondrogenesis[6].Nestin gene has been cloned from human,rat and mouse.It shares considerable similarity in gene structure between different species,and contains three introns and four extrons[7 8 9 10].Studies in transgenic mice showed that the second intron of nestin gene can drive the reporter gene expression in developing neural precursor cells,and the ftrst intron can induce reporter gene expression in both neural and muscle precursor cells[7].Further studies revealed that the second intron of nestin gene contained a CNS-tissue specific enhancer[7],and the cis-elements of POU and Sox binding sites in the 3' half of second intron were important for this neural enhancer activity[20 21].However,the molecular mechanism which regulates the nestin expression in developing muscles and in myogenesis is still unclear.In this study,we showed that a muscle-specific enhancer,localized in the first intron of mouse nestin gene,drove reporter gene expression during C2C12 myoblasts differentiation. Nestin First Intron Regulates its Expression during C2C12 myoblast differentiationNestin is an intermediate filament protein,and is expressed in the neural progenitor cells and in the developing skeletal muscle.Nestin has been widely used as a neural progenitor cell marker.It is well established that the specific expression of nestin gene in the neural progenitor cells is conferred by the neural specific enhancer located in the second intron of nestin gene.However,the transcriptional mechanism of nestin expression in the developing muscle is still unclear.In this study,we identified a muscle cell-specific enhancer in the 1st intron of mouse nestin gene in mouse myoblast C2C12 cells.Moreover,we localized the core enhancer activity to the region of 291 to 661 of the 1st intron.We further showed that the two E-boxes in the core enhancer region were important for the enhancer activity in differentiating C2C12 cells.Finally,we also showed that MyoD protein was involved in the regulation of nestin expression in the myogenic differentiation of C2C 12 cells.