CRABP2 Promotes Myoblast Differentiation and Is Modulated by the Transcription Factors MyoD and Sp1 in C2C12 Cells

Cellular retinoic acid binding protein 2 (CRABP2), a member of a family of specific carrier proteins for Vitamin A, belongs to a family of small cytosolic lipid binding proteins. Our previous study suggested that CRABP2 was involved in skeletal muscle development; however, the molecular function and regulatory mechanism of CRABP2 in myogenesis remained unclear. In this study, we found that the expression of the CRABP2 gene was upregulated during C2C12 differentiation. An over-expression assay revealed that CRABP2 promotes myogenic transformation by regulating the cell cycle during C2C12 differentiation. The region from −459 to −4 bp was identified as the core promoter and contains a TATA box, a GC box and binding sites for the transcription factors MyoD and Sp1. Over-expression, site-directed mutagenesis and EMSA assays indicated that the transcription factors MyoD and Sp1 regulate CRABP2 expression and promote myoblast differentiation in C2C12 cells.


Introduction
Myogenesis can be regarded as a two-step process consisting of the determination, in which the satellite cells commit to the mature muscle lineage, and subsequent differentiation of mononuclear myoblasts to multinuclear myotubes [1]. A previous study documented that several myogenesis factors (MyoD, Myf5, MRF4 and MyoG as well as members of the MyoD gene family) are critical for the determination and terminal differentiation of skeletal muscle cells [2]. In cultured non-muscle cells, the exogenous expression of the MyoD gene family can induce myogenic differentiation [3,4,5,6]. The MyoD gene family proteins bind to the CANNTG sequence (also known as the Ebox sequence), which is present in the promoters and enhancers of many muscle-specific genes [7,8]. Through the DNA-protein interaction, the MyoD gene family induces a conformational change and promotes muscle cell-specific gene transcription. Additionally, the MyoD gene family also promotes cell differentiation and inhibits the cell cycle [9]. Sp1 belongs to a family of zinc-finger transcription factors involved in the early development of an organism [10,11]. It contains 3 zinc finger motifs, which bind to GC-rich sequences. Specifically, the Sp1 protein recognizes a motif of GGGCGG or other related GC-rich sequences [12]. The Sp1 protein regulates the expression of several genes involved in cell differentiation and embryonic development, enhancing or repressing gene activity [13,14,15,16].
Vitamin A and its related molecules (retinol, retinaldehyde and retinoic acid) specifically bind many distinct cytoplasmic proteins and play essential roles in vision, growth, reproduction and cell differentiation [17]. The cellular retinoic acid binding proteins (CRABPs) are well-characterised members of a large family of small proteins that specifically bind retinoic acid [18] and mainly exert their biological function within cells. The CRABPs are abundantly expressed in numerous developing tissues [19], which suggests that CRABPs may perform specific functions during morphogenesis [20,21]. CRABP2 is a low molecular mass (15 kDa) protein that belongs to the multi-gene family of cellular retinoic acid binding proteins [22]. During mouse embryonic development, CRABP2 was expressed in many organs [21], we used the GNF SymAtlas expression datasets ( http://symatlas.gnf.org/ SymAtlas/), which demonstrated that CRABP2 mRNA was upregulated from day 6.5 to day 10.5. These data indicate that the CRABP2 gene may play a vital role during embryonic development.
The precise biological function and regulatory mechanism of the CRABP2 gene was unknown. Our recent LongSAGE analysis indicated that the CRABP2 gene contributes to prenatal skeletal muscle development in pigs [23]. Myoblast differentiation is a critical molecular event during foetal muscle development. We hypothesised that the CRABP2 gene plays an important role in myogenesis. In this study, we analysed the biological function and regulatory mechanism of the CRABP2 gene in C2C12 cells.

Results
The CRABP2 gene was upregulated during C2C12 differentiation We analysed the expression of the genes (CRABP1, CRABP2, CRBP1, CRBP2) expressing carrier proteins for Vitamin A and related molecules by RT-PCR during C2C12 differentiation. The results shown reveal expression levels of CRABP1, CRABP2, CRBP1, CRBP2 observed. We found that the CRABP1 and CRBP2 genes were not detected and the mRNA expression of the CRBP1 gene was unchanged, but the CRABP2 gene was markedly upregulated (Figure 1). To further test the expression change of the CRABP2 gene during C2C12 differentiation, quantitative realtime PCR (qRT-PCR) was performed. The results suggested that CRABP2 was upregulated from day 0 to day 4 during myogenic differentiation in C2C12 cells ( Figure 2).

CRABP2 promotes C2C12 differentiation
To study the effects of CRABP2 in C2C12 cells, we constructed CRABP2 lentivirus vector for transit gene expression. Analysis by western blotting verified that CRABP2 was expressed in 293T cells ( Figure 3) and the titer of the lentivirus particle was 2E +9 TU/ml.
After transfecting C2C12 cells with CRABP2 expression vector, we observed cell morphology by fluorescence microscopy and studied a mixture of cell populations with various CRABP2 expression levels by qRT-PCR. We found that the expression levels of CRABP2 significantly increased ( Figure 4) and that the cell morphology was characteristic of differentiation ( Figure 5) in overexpression (OE) group compared with Blank control (CON) and Negative control (NC) groups.
In contrast with the CON and NC groups, FACS cell sorting results of the OE group showed no significant changes in the percentage of cells in the G1 phase, but the percentage of cells in the G2/M phase decreased and the percentage of cells of S phase increased (p,0.05) ( Figure 6). It has been reported that the number of cells in G2/M phase decreases and more cells remain in S phase during the early differentiation of C2C12 cells [24]. Because the percentage of S phase cells was higher and the percentage of G2/M phase cells was lower in OE group than control group, indicating that S phase arrested and the number of cells entered into G2 phase decreased. These data demonstrated that DNA synthesis reduced, C2C12 cell no longer continue to proliferate, may enter the cell differentiation.

Identification of the core promoter region
To determine the functional fragment of the CRABP2 promoter, we inserted a 2.4 kb fragment into the promoter-less vector pGL 3basic. The resulting plasmid (pGL 3 -A) was transfected into C2C12 cells, and the luciferase activity measured. Compared with cells transfected with pGL 3 -basic, the cells transfected with the pGL 3 -A plasmid had significantly increased luciferase activity, revealing that the 2.4 kb fragment was a functional promoter fragment. To further identify the precise promoter region, the four promoter luciferase reporter plasmids (pGL 3 -B, pGL 3 -C, pGL 3 -D and pGL 3 -E) were constructed by further deletion using pMD18 T-A as a template. As shown by transient transfection results, the pGL 3 -E plasmid contained the basal promoter activity (Figure 7).
Four promoter luciferase reporter plasmids (pGL 3 -a, pGL 3 -b, pGL 3 -c and pGL 3 -d) were constructed by further deletion of pGL 3 -E. The results indicated that the pGL 3 -E plasmid with the 2459 bp to 24 bp fragment contained the minimal core promoter region ( Figure 8).
Identification of MyoD and Sp1 as transcription factors of the CRABP2 gene Bioinformatic analyses using TFSEARCH, TESS, Web Promoter Scan Service and MatInspector programs indicated that the CRABP2 promoter contains a TATA box, a GC box and binding sites for the transcription factors MyoD and Sp1. The core promoter region contains the TATA box and the GC box, as well as MyoD and Sp1 binding sites.
To determine experimentally that MyoD and Sp1 are regulators of the CRABP2 gene, an over-expression vector of MyoD and a site-directed mutation vector of Sp1 were constructed. Cotransfection with the minimal core promoter vector was performed to verify the transcription factors MyoD and Sp1, respectively. The results showed that the MyoD and Sp1 sites in the promoter region were necessary and functional to drive the basal reporter expression in transient transfection assays in C2C12 cells ( Figure 9).

Validation of MyoD and Sp1 transcription factors as regulators for the CRABP2 gene
To further confirm the function of MyoD and Sp1, electrophoretic mobility shift assays (EMSA) were carried out to analyse binding capabilities in cell nuclear extracts.
Oligonucleotides were synthesized and BIO-labelled and then incubated with nuclear extracts from C2C12 cells. Specific DNAprotein complexes were identified from C2C12 nuclear extracts ( Figure 10). The binding activities were different between C2C12 myoblasts and C2C12 myotubes. In C2C12 myotubes, the binding activity of MyoD was decreased; however, the binding activity of Sp1 was enhanced. These results indicate that MyoD and Sp1 specifically bind to promoter elements to regulate CRABP2 expression in C2C12 cells at different times during differentiation.

Discussion
Our previous LongSAGE analysis suggested that CRABPs were involved in skeletal muscle development in the pig throughout embryonic development. 23 Therefore, the aim of this study was to investigate the function of CRABPs during the process of differentiation of C2C12 cells into myotubes. The RT-PCR and real time PCR analyses suggested that CRABP2 was increasingly expressed during C2C12 cell differentiation. Interestingly, we found that CRABP2 over-expression accelerated the process of C2C12 differentiation into myotubes in vitro. These observations suggest that CRABP2 contributes to the process of C2C12 myoblast differentiation into myotubes.
We then carried out a promoter analysis through a step-by-step deletion process to identify the core promoter region. Bioinformatics analyses showed that the promoter region contained a TATA box, a GC box, and MyoD and Sp1 transcription factor binding sites. We constructed a MyoD over-expression vector and an Sp1 site-directed mutation vector. Co-transfection was performed to demonstrate the effects of MyoD and Sp1 transcription factors on the CRABP2 gene. The EMSA results indicated that the predicted MyoD and Sp1 binding sites bound to the MyoD and Sp1 proteins, respectively, in nuclear extracts from C2C12 cells. Additionally, excess amounts of unlabelled oligonucleotides inhibited the binding activities in C2C12 cells. This result further proved that the MyoD and Sp1 binding sites were functional regulators for the CRABP2 gene. These results demonstrated that the putative MyoD and Sp1 binding sites in the core promoter of CRABP2 may be responsible for transcriptional regulation in C2C12 cells.
Transcription factors are trans-regulatory factors that exert biological function by interaction with cis-regulatory elements of structural genes. The MyoD family recognizes and binds the consensus sequence CANNTG (N for any nucleotide), and the promoter region of CRABP2 contains a CANNTG sequence. The MyoD gene family forms a very complex regulatory network by interacting with a number of positive and negative factors to regulate CRABP2 gene transcription, controlling cell growth and cell differentiation. The Sp1 transcription factor was involved in regulating cell growth and controlling morphogenetic pathways both in mammals and in invertebrates [25,26]. In our study, Sp1 regulated gene transcription by binding the GGGCGG sequence in specific regulatory sites of the CRABP2 gene. Therefore, MyoD and Sp1 are important regulators for the CRABP2 gene and directly bind to the promoter region to influence CRABP2 expression. CRABP2 is in turn involved in regulating C2C12 cell differentiation.
In summary, CRABP2 expression was upregulated from day 0 to day 4 at the mRNA level in differentiating C2C12 cells. The overexpression of CRABP2 led to cell cycle changes in C2C12 cells in vitro. We have identified a biological function for CRABP2 in C2C12 differentiation and found that MyoD and Sp1 play a regulatory role in CRABP2 gene transcription by binding in the core promoter region between 2459 bp and 24 bp upstream of the CRABP2 gene. These results give new insight into the biological function and regulatory mechanism of the CRABP2 gene.

Materials and Methods
Cell culture and RNA extraction   maintained at 37uC in 5% CO 2 . The C2C12 cells were seeded in six-well plates. After approximately 12-16 h, when cell confluence reached approximately 60%-70%, the differentiation of C2C12 myoblasts into myotubes was induced by the addition of differentiation medium (DMEM containing 2% horse serum instead of 10% FBS).
Starting at the beginning of differentiation, the C2C12 cells were cultured in six-well plates and harvested for RNA extraction at 0, 1, 2, 3 and 4 days. Total RNA samples were extracted using TRIZOL (Invitrogen, USA) according to the manufacturer's instructions. The cDNA samples were obtained by reverse transcription from 1 mg RNA using oligo(dT) and M-MLV reverse transcriptase (Promega, USA).

RT-PCR analysis
The RT-PCR (Reverse Transcription-Polymerase Chain Reaction) was performed in a volume of 10 ml containing 1 ml of 106 PCR buffer (plus Mg 2+ ), 100 mmol/L of each dNTP, 0.5 mmol/L of each PCR primer, 1.0 U Taq DNA polymerase (Takara, Japan) and 0.5 ml of cDNA template. The primers were designed and synthesized as shown in Table 1. The PCR reaction was 5 min at 95uC followed by 27 cycles of 30 s at 95uC, 30 s at the specific melting temperatures of each primer pair, 30 s at 72uC and a final extension time of 5 min at 72uC.

Real-time PCR analysis
Real-time PCR amplifications were carried out using the Light Cycler Real Time PCR instrument (model 7500; Applied Biosystems, Inc., Foster City, CA). The PCR reaction contained 506 ROX Reference DyeII, 26 SYBR Green Realtime PCR Master Mix (Takara, Japan), 10 mM primers and 1.0 ml of cDNA template. Pure water was added for a total volume of 20.0 ml. Gene amplification was performed under the following conditions: 95uC for 30 s, followed by 40 cycles of 95uC for 5 s, 60uC for 20 s and 72uC for 34 s. The PCR was performed in biological triplicate for each condition. The target gene data was analysed and normalized to GAPDH mRNA levels.

Construction and transfection of the CRABP2 lentivirus vector
The vector overexpressing CRABP2 was constructed to study its function in C2C12 cells. The cDNA fragment was isolated and ligated into the pGC-FU vector linearized by cutting with Age I restriction enzyme, purchased from GeneChem Co., Ltd, Shanghai, China. The expression plasmid containing the fusion protein was extracted using ultrapure endotoxin-free extraction kits (Omega, USA). The transfection of 293T cells was carried out using Lipofectamine 2000 according to the manufacturer's instructions. After a 48 h incubation, the cell supernatant containing virus-like particles was collected. After concentration, the viral titres were determined in the 293T cells.
The C2C12 cells were plated on 6-well plates and cultured overnight at 37uC in 5% CO 2 . When cell confluence reached approximately 60-70%, lentivirus of a specific quantity was added to cells. After 12 h, the transfection medium was removed and replaced with normal growth medium.     After 3-4 days, the GFP expression was tested by fluorescence microscopy to determine transfection efficiency. The cells were then harvested and used for the cell cycle assay and RNA extraction. All transfections were performed in triplicate for each plasmid.

Flow cytometry analysis (FCM)
The C2C12 cells were collected and centrifuged at 1200 rpm for 5 min. The cells were washed twice in 4uC pre-chilled PBS (phosphate buffered saline, pH = 7.2-7.4), fixed with 4uC pre-chilled 70% ethanol, centrifuged at 1500 rpm for 5 min to remove stationary phase cells and resuspended in PBS. The cells were filtered once through 400 mesh and centrifuged at 1200 rpm for 5 min, and after the PBS supernatant was removed, the cells were then stained with 1 ml of PI at 4uC in darkness for 30 min. Finally, the cells were analysed by flow cytometry (FACSCalibur, BD, USA).

Construction of the luciferase reporter plasmids
The 59-upstream 2.4 kb promoter fragment of the CRABP2 gene was amplified from mouse genomic DNA and then inserted into the pMD18-T vector. It was named pMD18 T-A.
The construction of the luciferase reporter gene plasmid was carried out as follows: the pMD18 T-A vector was cut using MluI and NcoI restriction enzymes and then ligated into linearized basic vector (named pGL 3 -A). Fragments of different lengths from the 59-upstream sequence were amplified using pMD18T-A as a template and then ligated into linearized pGL 3 -basic vector (named pGL 3 -B, pGL 3 -C, pGL 3 -D and pGL 3 -E). The plasmids pGL 3 -A, pGL 3 -B, pGL 3 -C, pGL 3 -D and pGL 3 -E were constructed to contain 22292 bp to +115 bp, 21646 bp to +115 bp, 21111 bp to +115 bp, 2719 bp to +115 bp and 2459 bp to +115 bp of the CRABP2 gene, respectively.
To identify more precisely the core promoter region, we performed further deletions on the promoter fragment pGL 3 -E: four luciferase reporter gene plasmids, pGL 3 -a (2290 bp to +115 bp), pGL 3 -b (2178 bp to +115 bp), pGL 3 -c (24 bp to +115 bp) and pGL 3 -d (+44 bp to +115 bp), were constructed using the above methods. Each promoter luciferase plasmid was constructed using a sequence-specific forward primer and a common reverse primer (shown in Table 2). The sequences were analysed by Invitrogen.
All experiments were performed in triplicate, and the data were presented as mean 6 SD. Student's t-test was used to determine statistical significance.  Luciferase assay The C2C12 cells were plated on 24-well plates and cultured overnight at 37uC to ensure approximately 60%-70% confluence. Co-transfections were performed using 2.0 ml of Lipofectamine 2000 reagent (Invitrogen, USA), 0.8 mg of the firefly luciferase plasmid DNA and 0.008 mg of pRL-TK plasmid DNA (Promega, USA) as an internal control. The transfection medium was removed and replaced with growth medium after 5 h.
The Firefly and Renilla luciferase activities were measured at 24 h after transient transfection using the Dual-Glo Luciferase assay system (Promega, USA) and a TD20/20 luminometer (Turner Designs) according to the manufacturer's instructions. Each plasmid was tested in three independent experiments. The luciferase activity was normalized using the Renilla luciferase activity levels and expressed as relative luciferase unites (RLU) to reflect the gene promoter activity.

Construction of the MyoD expression vector
The cDNA sequence of the MyoD gene was amplified using mouse muscle mRNA with the primers listed in Table 1. The PCR product was cloned into the pMD18-T vector (Invitrogen, USA) and then inserted into the EcoRV/NotI sites in the linearized pcDNA3.1(+) vector (Invitrogen, USA). The recombinant plasmid was sequenced by Invitrogen.

Construction of the Sp1 site-directed mutation vector
To identify Sp1 as a regulatory factor for the CRABP2 gene in C2C12 cells, we constructed a site-directed mutation vector of Sp1. The site-directed mutagenesis of the Sp1 binding site was obtained by a two-step PCR protocol using pMD18 T-A as a template. The original GGC site was mutated to TTA. The mutagenic primers were 59 AGGTGGTGTGGAAGGCGGT-TAGGGGGCGGGGCCGCCTCATGCACCAGCT 39 (forward primer) and 59 CCTAACCGCCTTCCACACCACCT 39 (reverse primer). Thermocycler parameters were set at 95uC for 5 min followed by 27 cycles of 95uC for 30 s, 60uC for 30 s and 72uC for 30 s with a final extension time of 5 min at 72uC. The final PCR product was cloned into pGL 3 -basic and named pGL 3 -muE.

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from C2C12 myoblasts and myotubes were prepared according to the instruction manual of the NProtein Extraction kit (Exprogen, Beijing, China). The proteins were measured using a BCA Protein Assay Kit (Exprogen, Beijing, China). The probes for MyoD and Sp1 were synthesized by Integrated DNA Technologies (IDT, USA).
The binding reactions were performed in a 10 ml mixture containing 1.0 ml of 106 Binding Buffer, 1.0 ml of Poly (dI:dC), 2 mg of nuclear extract and 0.5 ml of Bio-labelled probes. For the competition experiments, unlabelled probes were added to the binding reaction mixture and co-incubated. Subsequently, DNAprotein complexes were separated by 6.5% non-denaturing polyacrylamide gel electrophoresis in Tris-Boric acid (TBE) buffer. Electroblotting and chemiluminescence detection were performed according to the instructions of the BiotinLight TM EMSA Kit (Exprogen, Beijing, China). Results were observed by chemiluminescence imager.