Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 6 | Page 526 First intron of nestin gene regulates its expression during C2C12 myoblast differentiation Acta Biochim Biophys Sin (2008): 526-532 | © 2008 Institute of Biochemistry and Cell Biology, SIBS, CAS | All Rights Reserved 1672-9145 http://www.abbs.info; www.blackwellpublishing.com/abbs | DOI: 10.1111/j.1745-7270.2008.00428.x First intron of nestin gene regulates its expression during C2C12 myoblast differentiation Hua Zhong 1,2 , Zhigang Jin 2 , Yongfeng Chen 2 , Ting Zhang 2 , Wei Bian 2 , Xing Cui 1 *, and Naihe Jing 2 * 1 Shan Dong University Medical School, Jinan 250012, China 2 Laboratory of Molecular Cell Biology, Key Laboratory of Stem Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Nestin is an intermediate filament protein expressed in neural progenitor cells and in developing skeletal muscle. Nestin has been widely used as a neural progenitor cell marker. It is well established that the specific expression of the nestin gene in neural progenitor cells is conferred by the neural- specific enhancer located in the second intron of the nestin gene. However, the transcriptional mechanism of nestin expression in developing muscle is still unclear. In this study, we identified a muscle cell-specific enhancer in the first intron of mouse nestin gene in mouse myoblast C2C12 cells. We localized the core enhancer activity to the 291–661 region of the first intron, and showed that the two E-boxes in the core enhancer region were important for enhancer activity in differentiating C2C12 cells. We also showed that MyoD protein was involved in the regulation of nestin expression in the myogenic differentiation of C2C12 cells. Keywords nestin; C2C12 myoblast; muscle-specific enhancer; MyoD Skeletal 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, and the differen- tiation and maturation of these progenitor cells. Under normal growth conditions, newly formed somite rapidly partition into the ventral scelerotome compartment and the dorsal dermomyotome from which muscle cells and dermis are generated. 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, 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 class VI intermediate filament protein, is expressed specifically in neuroepithelial stem cells and neural progenitor cells. It has been widely used as a neural progenitor cell marker for the developing central nervous system [3,4]. Nestin expression is also found in myogenic progenitor cells in the dermomyotome of dorsal-lateral somites, and its expression persists in developing thigh muscle until postnatal day 4 of rats [3]. Nestin mRNA is found in developing thigh muscle of rat, from embryonic day 15.5 (E15.5) to postnatal day 21 [5], but not in adult skeletal muscles [3,5,6]. In situ hybridization also showed that nestin expression was up-regulated in the developing mouse limb bud during myogenesis, and down-regulated during chondrogenesis [7]. The 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 [6,8−10]. Studies in transgenic mice showed Received: April 2, 2008 Accepted: April 30, 2008 This work was supported by the grants from the National Nature Science of China (Nos. 30623003 and 30721065), the National Key Basic Research and Development Program of China (Nos. 2005CB522704, 2006CB943902, and 2007CB947101 to N.J.), and the National High Technology Research and Development Program of China (No. 2006AA02Z186 to N.J.) *Corresponding authors: Naihe Jing: Tel, 86-21-54921381; Fax, 86-21-54921011; E-mail, njing@sibs.ac.cn Xing Cui: Tel, 86-536-88382459; E-mail, cuixing77@sdu.edu.cn