Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 526-536
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? modified Eagle? medium (Gibco, New York, USA)
supplemented with 20% fetal bovine serum (HyClone, Logan, USA) at 37 ? with 5% CO2. The medium was changed into Dulbecco? modified Eagle? 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 ? 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‘-gaatcagatcgctcagatcc-3‘ (forward)
and 5‘-gcacgacaccagtagaactgg-3‘ (reverse)] were used, and a
cDNA fragment of nestin was ampilified (Tm, 56 ?, 30 cycles, PCR products
487 bp). As a loading control, the housekeeping gene b-actin was amplified using a primer set [5‘-tcgtcgacaacggctccggcatgt-3‘
(forward) and 5‘-ccagccaggtccagacgcaggat-3‘ (reverse); Tm, 56 ?, 21 cycles, PCR products 520 bp].
Luciferase activity
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 ?. 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? 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?D. Student? 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 encoding 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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