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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 526-536 |
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doi:10.1111/j.1745-7270.2008.00428.x |
First intron of nestin gene
regulates its expression during C2C12 myoblast differentiation
Hua Zhong1,2, Zhigang Jin2, Yongfeng Chen2, Ting Zhang2, Wei Bian2, Xing Cui1*, and Naihe Jing2*
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
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, [email protected]
Xing Cui: Tel, 86-536-88382459; E-mail, [email protected]
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 291661 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 differentiation 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 that the second intron of the nestin gene can drive reporter gene expression in developing neural precursor cells, and the first intron can induce reporter gene expression in both neural and muscle precursor cells [8]. Further studies revealed that the second intron of the nestin gene contained a central nervous system tissue-specific enhancer [8], and the cis-elements of POU and Sox binding sites in the 3' half of the second intron were important for this neural enhancer activity [11,12]. However, the molecular mechanism that regulates 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 myoblast differentiation.
Materials and Methods
Cell culture
C2C12 mouse cells were obtained from American Type Culture Collection (No. CRL 1772; Rockville, USA). The cells were cultured in Dulbecco抯 modified Eagle抯 medium (Gibco, New York, USA) supplemented with 20% fetal bovine serum (HyClone, Logan, USA) at 37 篊 with 5% CO2. The medium was changed into Dulbecco抯 modified Eagle抯 medium supplemented with 2% horse serum (Invitrogen, Carlsbad, USA) as C2C12 cells were induced to form myotubes. The cells were routinely passaged every 2 d, with passage density 60%, and induce density 80%.
Generation of reporter gene
constructs
The first intron was cloned into pGL3-px and pGL3-TK vectors to generate pNH200 and pNH201, respectively. A series of luciferase reporter gene constructs was prepared by amplifying the first intron of mouse nestin gene with different sets of primers and inserting these polymerase chain reaction (PCR) fragments into pGL3-px vectors. The promoter of mouse nestin gene (pNesP-3809/+183) was cloned as previously described [8]. The generation of the site-mutated construct was carried out by PCR using two sets of primers, as described previously [13]. In these constructs (p476/492-MT and p539/555-MT), the sequence of one E-box, 5'-CAGATG-3', was mutated to 5'-CTCGAG-3', and the other, 5'-ACCTGC-3', was mutated to 5'-GAATTC-3'. The expression constructs of MyoD, myf5, and myogenin were obtained from Prof. Lin Li (Institute of Biochemistry and Biology, Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai, China).
Reverse transcription (RT)-PCR
Total RNA was extracted at different time points using Trizol reagent (Invitrogen). The RT-PCR was carried out in a 20 ml final volume containing 5 mg total RNA, 1 RT buffer, 1 ml random primer, 1 ml dNTP (10 mM), 2 ml dithiothreitol (0.1 M), and 1 ml SSRT at 42 篊 for 1 h. All RT reagents were purchased from Invitrogen. Aliquots of cDNA synthesized above were used as a template for semiquantitative PCR analysis. Nestin primers [5'-gaat�cagat�cgctcagatcc-3' (forward) and 5'-gcacgacac�cagt�ag�a�actgg-3' (reverse)] were used, and a cDNA fragment of nestin was ampilified (Tm, 56 篊, 30 cycles, PCR products 487 bp). As a loading control, the housekeeping gene b-actin was amplified using a primer set [5'-tcgtcgacaa�cg�gctccggcatgt-3' (forward) and 5'-cca�gccaggtc�cagacg�caggat-3' (reverse); Tm, 56 篊, 21 cycles, PCR products 520 bp].
Luciferase activity
For transfection of plasmids, 3104 C2C12 cells mixed with 1 ml growth medium were seeded into each well of 12-well plates and cultured at 37 篊. When cells reached 70%-80% confluence, 0.4 mg luciferase reporter plasmid with different DNA fragments of the first intron and 0.2 mg control pRL-TK plasmid (Promega, Madison, USA) were co-transfected into cells using FuGENE HD reagent (Roche, Basel, Switzerland) according to the manufacturer抯 protocol. After 7 h, the culture medium was changed into differentiated medium containing 2% horse serum to induce C2C12 cell differentiation. After 3 d of culture, the cells were harvested as the differentiation 3 d (D3) group. The control cells were continually cultured in growth medium containing 20% fetal bovine serum for 3 d, and harvested as the undifferentiated group (D0). Cells were washed with phosphate-buffered saline and lysed with passive lysis buffer (Promega), and the supernatant was analyzed for luciferase reporter activity on a 20/20n luminometer (Turner BioSystems, Sunnyvale, USA) by the dual luciferase reporter system (Promega). The transfection efficiency was normalized by Renilla luciferase. In MRF co-expressing experiments, 0.4 mg MRF-expressing plasmids plus 0.4 mg luciferase reporter construct and 0.2 mg pRL-TK plasmid were co-transfected into C2C12 cells. After 2 d, C2C12 cells were harvested and the supernatant was analyzed with luciferase assay.
Statistical analysis
The data were expressed in the form mean盨D. Student抯 t-test was applied to study the relationship between the different variables. All experiments were repeated at least three times and similar results were obtained.
Results
First intron of mouse nestin
gene regulates its expression during C2C12 differentiation
C2C12 myoblasts were derived from adult mouse muscle, and could be induced by 2% horse serum to differentiate into multinucleated myotubes in vitro [14]. RT-PCR was used to detect nestin gene expression during C2C12 cell differentiation. The results showed that nestin mRNA could be detected in un-induced C2C12 cells (D0) at a low level, and its expression gradually increased after induction with a peak at D3 [Fig. 1(A)].
To identify the regulator sequence of the nestin gene during C2C12 cell differentiation, luciferase reporter constructs with the nestin gene promoter (pNesP-3809/+183), the second intron (pNes2In32/1628) and the first intron of mouse nestin gene (pNes1In1/1003) were transfected into C2C12 cells. After inducing C2C12 cell differentiation for 3 d (D3), the luciferase activities were detected and compared with that of the uninduced C2C12 cells (D0). The first intron-containing construct pNes1In1/1003 in the D3 group showed much higher luciferase activity than that of the D0 group, whereas neither of the constructs containing the promoter (pNesP-3809/+183) nor the second intron (pNes2In32/1628) displayed any activity difference between the D3 and D0 groups [Fig. 1(B)].
To eliminate promoter interference with reporter gene expression, we inserted the first intron of mouse nestin gene behind the nestin promoter (pNH200), TK promoter (pNH201), or SV40 promoter [Fig. 1(C), upper panel] and introduced these constructs into C2C12 cells. All constructs showed a significant luciferase activity increase between the D3 and D0 groups [Fig. 1(C), lower panel]. To test the cell-type specificity of this enhancer, we introduced the pNes1In1/1003 construct into different cell lines, such as P19EC, F9EC, NIH3T3, CHO, and SH-SY5Y cells, and found that the first intron did not display any enhancer activity in these non-muscle cells (data not shown). Together, these results suggest that the first intron of mouse nestin gene possesses enhancer activity during C2C12 myoblast differentiation.
Core enhancer sequence in
first intron of nestin gene
To identify the core enhancer sequence in the first intron of the nestin gene, we generated a series of deletion and truncation reporter constructs and transfected them into C2C12 cells (Fig. 2). After 3 d of induced differentiation, luciferase assays were carried out. We found that the full-length first intron (pNes1In1/1003) and its 5' deletion constructs, pNes1In179/1003 and pNes1In291/1003, displayed higher luciferase activity in the D3 group than in the D0 group. For further 5' deletion, including constructs pNes1In454/1003 and pNes1In662/1003, much reduced enhancer activity was found when compared with the full-length first intron. The 3' truncation experiments showed that both constructs pNes1In1/661 and pNes1In185/661 possessed high luciferase activity, whereas 3' truncation to 476 (pNes1In1/477) abolished the enhancer activity [Fig. 2(A)]. These results suggest that the core enhancer sequence is within the 185-661 region of the first intron of the nestin gene.
To narrow down the enhancer sequence even further, we carried out 5' deletion to positions 290 and 476 and found that the pNes1In291/661 construct still displayed strong enhancer activity, whereas deletion to 476 (pNes1In477/661) totally eliminated enhancer activity [Fig. 2(B)]. Consistently, further deletion and truncation experiments within the 185-661 region showed that only construct pNes1In291/661 had full enhancer activity, and other constructs displayed reduced activities [Fig. 2(C)].
In summary, these data suggest that the minimal sequence for the muscle-specific enhancer of the nestin gene is located in the region 291661 in the first intron of mouse nestin gene.
Two E-boxes involved in
enhancer activity of first intron of nestin gene
To search for the cis-elements responsible for the enhancer activity if the first intron of the nestin gene, we analyzed the 291-661 region with Genomatix software (http://www.genomatix.de) and found several putative E-boxes (CANNTG) in the promoters or enhancers of muscle-specific genes. Among these putative E-boxes, two at the positions 476-492 and 539-555 attracted our attention because deletion [Fig. 2(A), pNes1In1/477] or truncation [Fig. 2(B), pNes1In477/661] in the 476-492 region always caused abolishment of enhancer activity of the nestin gene, and the E-box at position 539-555 was very similar to the MyoD binding site. To validate the importance of these two sites, we constructed plasmids p476/492-MT and p539/555-MT with mutations at the 476-492 and 539-555 binding sites, respectively. After transfecting them into C2C12 cells, we found that both mutations had reduced enhancer activity (Fig. 3), suggesting that these two E-boxes are involved in muscle-specific enhancer activity of mouse nestin gene.
MyoD protein involved in
regulating nestin gene expression in C2C12 cells
The MRFs, MyoD, myf5 and myogenin, play very important roles during determination and terminal differentiation of skeletal muscle [15], and they also have important functions in the differentiation of C2C12 myoblasts [16]. To determine which MRF is involved in regulating muscle-specific enhancer of the nestin gene, we co-transfected the full-length first intron with expression vectors enco�ding MyoD, myf5, and myogenin into C2C12 cells. Luciferase assays showed that overexpression of MyoD (pCMV-MyoD) was able to significantly increase enhancer activity, modest effects could be detected with myf5 (pCMV-myf5), and myogenin (pCMV-myogenin) produced very little increase [Fig. 4(A)]. Consistently, co-transfection of shorter fragments of the first intron, pNes1In185/661 and pNes1In291/661, with MyoD expression plasmid could also increase luciferase activity significantly [Fig. 4(B)], suggesting that MyoD protein was involved in regulating the muscle-specific enhancer activity of the nestin gene.
Discussion
Intermediate filament protein, nestin, is expressed specifically in neuroepithelial stem cells and muscle progenitor cells in the myotome of rat and mouse embryos [3]. It is reported that the second intron of the nestin gene contains the neural-specific enhancer [8]. However, the detailed relationship between the first intron of the nestin gene and its expression in developing muscles has not been explained. In this study, we identified a muscle cell-specific enhancer in the first intron of mouse nestin gene, and characterized the core enhancer activity in the region 291661 of the first intron. We also showed that two E-boxes were important for the expression of the nestin gene during induced C2C12 cell differentiation. Finally, we found that MyoD protein was involved in nestin gene expression in C2C12 cells.
It has been reported that MyoD binds to its consensus sequence CANNTG (E-box) in the regulatory region of muscle-specific genes and regulates their expression [17,18]. In this study, we showed that the first intron of mouse nestin gene possessed the muscle cell-specific enhancer activity (Fig. 1), and there were two E-boxes within this region (Fig. 3). Further studies showed that the two E-boxes and MyoD protein were involved in nestin gene expression in C2C12 cells (Figs. 3 and 4). Interestingly, we found that there was no enhancer activity when we co-transfected MyoD expression plasmid with reporter gene construct pNes1In477/661, in which one E-box (539555) was intact but the other (476-492) was disrupted (data not shown). This result suggests that both E-boxes are important for the expression of the nestin gene in C2C12 cells. It is also possible that MyoD protein needs to bind cooperatively to two sites at a distance, as is the case with muscle-specific enhancer of creatine kinase [19].
Acknowledgements
We thank Prof. Lin Li (Institute of
Biochemistry and Biology, Shanghai Institutes for Biological Science, Chinese
Academy of Sciences, Shanghai, China) for kindly providing the MRF expression
constructs.
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