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Cellular Signalling
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TEAD1 controls C2C12 cell proliferation and differentiation and regulates three noveltarget genes
Fengli Wang 1, Hongyang Wang 1, Hao Wu, Haifang Qiu, Cuiping Zeng, Ling Sun, Bang Liu ⁎Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan,Hubei, China
Laboratory of Agricultural Animal Genetics, Breeding anEducation, College of Animal Science and Technology, HuNo.1 Shizishan Street, Hongshan District, Wuhan, Hubei P
E-mail address: [email protected] (B. Liu).1 These authors contributed equally to this work.
TEAD1 is a transcription factor involved in activation of muscle specific genes, such as the cardiac muscletroponin T gene, skeletal muscle actin, myosin heavy chains genes. Here, we reported that TEAD1 wasexpressed ubiquitously in different mouse tissues and was up-regulated in differentiation process of themouse myoblast cell line C2C12. Functional assay revealed that overexpression of TEAD1 gene can arrestthe C2C12 cell cycle and promote C2C12 cell differentiation. To understand the physiological role ofTEAD1 in muscle development, three new regulated genes of TEAD1, Mrpl21, Ndufa6 and Ccne1, were iden-tified by expression analysis, promoter activity measurement assay. The expression patterns of target geneswere detected in the cell differentiation process. The Mrpl21 and Ndufa6 genes were up-regulated incell differentiation while Ccne1 gene was significantly down-regulated. Overexpression of Mrpl21 andNdufa6 in C2C12 can up-regulate Myh4 gene expression thus promote C2C12 differentiation, but did notaffect cell cycle. Co-overexpression of Ccne1 with Ndufa6 resulted in Myh4 expression decrease and thenumber of S-phase cells slight increase. Together, our results suggested that TEAD1 may mediate muscledevelopment through its target genes, Mrpl21, Ndufa6 and Ccne1.
Myogenesis is a complicated process, which was regulated by anumber of transcription factors, including myogenic determinationfactors Myf5 and MyoD, and differentiation factors myogenin, Myf4and MEF2 [1]. In addition to these well-known transcription factors,recently, a new transcription factor TEAD1, has been attracting muchattention. TEAD1 belongs to the TEA domain family, which shares aconserved DNA binding domain and regulates a considerable num-ber of biological process [2–4]. Previous studies have indicated thatTEAD1 constitutively expresses in cardiac and skeletal muscles inhumans, mice and pigs [4,5], and its disruption leads to heart de-fection and embryonic lethality in mice [6]. TEAD1 regulates the ex-pression of many muscle-specific genes through binding to theM-CAT motif (TEAD1 protein binding site) in the gene promoter or
gy and Animal Breeding, Keyd Reproduction of Ministry ofazhong Agricultural University,rovince 430070, China.
12 Published by Elsevier Inc. All righ
enhancer regions [7,8]. The transcriptional regulation of TEAD1 tomuscle-specific genes is implemented by co-operating with numerousco-factors, including MEF2 [7], vestigial like 2 [9], vestigial like 4 [10].
TEAD1 has emerged as a transcription factor involved in the specificactivation of several muscle genes, and was originally described as a fac-tor binding to non-muscle specific cis-elements of the SV40 enhancer.TEAD1 has also been found to bind to the M-CAT (5-CATTCCT-3) se-quence [11,12]. There are four TEAD genes in vertebrates, which expressin a complex pattern and can produce several isoforms [4,13,14]. Notably,theM-CAT sequence that binds TEAD1 is present in the regulatory regionsof several mammalian and avian cardiac and skeletal muscle-specificgenes, including those encoding cardiac muscle troponin T and skeletalmuscle actin, myosin heavy chains, and acetylcholine receptors [15–17].
The M-CAT sequence has also been connected to the regulation ofthe mammalian smooth muscle-actin gene in a complex mechanisminvolving several single-strand DNA-binding proteins [18,19].
There is significant difference between the transcriptional regula-tion mechanism of TEAD1 in various cells [20]. In cardiovasculardevelopment, MEF2, TEAD and the FoxO family of proteins are themain regulators of Myocardin expression [21]. The C2C12 cell differ-entiation process can be induced by adding horse serum to theDMEM medium. In the process of C2C12 cell differentiation, theexpression of differentiation-related genes, such as myosin, increasedsignificantly. The Myh4 gene, skeletal muscle myosin heavy chain 4,also known as myosin 2b, is up-regulated in myoblast differentiationand can be used as a sign of C2C12 differentiation.
The restriction enzyme recognition sites aremarkedwith lowercase letters. The protectivebases of restriction enzyme recognition are italicized.
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In different tissues, TEAD1 plays multiple roles via regulating differ-ent targeting genes. Current research shows that TEAD1 overexpressioncan promote age-dependent cardiac dysfunction in mice [22]. TheTEAD1 gene function in C2C12 myoblast has not been reported. In thisstudy, the expression of the TEAD1 gene was significantly up-regulatedduring C2C12 differentiation, which implies that the TEAD1 gene mayplay a role in C2C12 cell differentiation. TEAD1 overexpression cansignificantly up-regulate Myh4 gene expression, which indicates thatTEAD1 can promote myoblast differentiation. We validated three newTEAD1-dependent targets: Mrpl21, Ndufa6 and Ccne1 in C2C12 myo-blasts and myotubes.
2. Materials and methods
2.1. Tissue samples collection and cell culture
The kunming mice used in this study were purchased from theWuhan Center for Disease Control. Ten different tissues, includingheart, liver, spleen, lung, kidney, skeletal muscle, fat, stomach, smallintestine and brain tissues, of three adult mice were collected.
The mouse skeletal myoblast cell line C2C12 was purchasedfrom the China Center for Type Culture Collection and maintainedin Dulbecco's modified Eagle's medium (DMEM, Thermo Scientific)supplemented with 10% fetal bovine serum (FBS, Invitrogen Life Tech-nologies), 100 units/ml penicillin–100 μg/ml streptomycin (InvitrogenLife Technologies) and cultured in a 5% CO2-humidified incubator at37 °C. For proliferation, C2C12 myoblasts were cultured in a growthmedium of DMEM supplemented with 10% FBS. For differentiating tomyotubes, when grown to approximately 90% confluence, C2C12 myo-blasts were transferred into a differentiationmedium of DMEMwith 2%horse serum (HS, Sigma) for up to 7 days.
2.2. Gene expression analysis by RT-PCR and Q-PCR
Total RNA of mouse tissues was extracted from 200 mg of eachsample using the Tissue RNA Extraction Kit (Tiangen) following stan-dard manual. The C2C12 cells were cultured in 6-well plates. Cells inproliferation and in various periods of differentiation (1 d, 2 d, 4 d,6 d, 8 d) were harvested for RNA extraction to analyze the gene ex-pression in differentiated C2C12 cells. Total RNA was isolated usingthe Trizol Reagent (Invitrogen) according to the manufacturer's in-structions. The cDNA of each sample was reverse-transcribed fromnormalized RNA using the First Strand cDNA Synthesis Kit (FermentasScientific). cDNA was diluted with H2O at the rate of 1:4 for Q-PCR.
The gene expression of the three samples in the same treatmentwasdetected and Q-PCR was performed in triplicate. The gene expressionlevels were quantified relative to the expression of mouse actin geneusing the Lightcycler 480 (Roche) software, and employing an opti-mized comparative Ct (ΔΔCt) value method. ΔCt is the Ct subtractedfrom the average Ct of reference from each gene Ct value: −ΔΔCt=ΔCt (sample)−ΔCt (calibrator). This is followed by transformingthese values to absolute values, using the formula: relative expressionlevel=2−ΔΔCT. The primers designed for target genes and internalcontrol actin are shown in Table 1.
2.3. Vector construction and cell transfection
The CDS (coding sequence) of the mouse TEAD1 gene was obtainedfrom GenBank (NM_009346) and cloned into pcDNA3.1(+) to con-struct the TEAD1 expression vector pcDNA3.1(+)-TEAD1. The putativepromoter sequences of theMrpl21, Ndufa6, and Ccne1 genes containingthe TEAD1 protein binding site were inserted into the pGL3-Basic(Promega) to construct a promoter–reporter vector. The CDS regionsof Mrpl21, Ndufa6 and Ccne1 were also inserted into pcDNA3.1(+)vector to form the overexpressed vector pcDNA3.1(+)-Mrpl21,pcDNA3.1(+)-Ndufa6, pcDNA3.1(+)-Ccne1.
The overexpression and RNA interference of the TEAD1 gene inC2C12 cells were carried on in 6-wells plates while the target genepromoter activity in C2C12 cells was tested in 48-wells plates.Transfections of the pcDNA3.1(+)-TEAD1 and pcDNA3.1(+) vectorswere performed using 10 μl Lipofectamine 2000 reagent (Invitrogen)with 4 μg vector for eachwell. Transfections of synthetic siRNAmoleculeswere performed using a 5 μl Lipofectamine 2000 reagent (Invitrogen)with a 20 nmol siRNA for eachwell.We performed the promoter activitymeasurement in 48-well plates. For eachwell, we used 0.19 μg promotervector firefly luciferase plasmid DNA, 0.02 μg of pRL-TK plasmid DNA(Promega) as an internal control and 0.19 μg pcDNA3.1(+) vectoror pcDNA3.1(+)-TEAD1. The co-transfection of the promoter vector,pRL-TK vector and pcDNA3.1(+) group was used as the control group.The co-transfection of pGL3-Basic with the pRL-TK group (Promega)was used as negative control while the pGL3-control with pRL-TK wasused as the positive control.
2.4. Dual-luciferase reporter assay
Promoter activity of targets was measured by Dual luciferasereporter assay. After co-transfection of the vectors for 24 h, the fireflyluciferase and renilla luciferase activities were measured respectivelyusing the dual luciferase reporter assay system(Promega, Cat. NO.E1910),while the relative promoter activity was measured using the followingformula: relative promoter activity=(the firefly luciferase activity)/(renilla luciferase activity). Promoter activity measurement of each treat-ment was repeated three times independently.
2.5. Western blot
After indicated treatments, cells were washed with ice-cold PBStwice and lysed with lysis buffer containing 0.5% NP40 and 100 μMPMSF. 10 μg protein with a loading buffer was then electrophoresedon 12% SDS-PAGE for 120 min at 90 V, then immediately transferredto polyvinylidene fluoride membranes. The membranes were blockedwith TBS containing 0.1% Tween-20 and 5% nonfat milk for 2 h at
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Fig. 1. Expression pattern of the TEAD1 gene in adult mouse tissues and differentiatedC2C12 cells. (A) Semi-RT-PCR was used to detect the expression level of the TEAD1gene in mouse tissues: heart, liver, spleen, lung, kidney, skeletal muscle, fat, stomach,small intestine and brain. (B) The expression of TEAD1 in different differentiationperiods of C2C12 cells. 0 d means cells were in proliferating stage. Gene expressionlevel of 0 d cells was normalized to 1. Values represent the mean±SD of three inde-pendent experiments. * pb0.05.
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room temperature, then immunoblotted with the respective primaryantibodies at 4 °C overnight. Secondary antibody conjugated withhorseradish peroxidase (HRP) was incubated in 37 °C for 90 min and
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Fig. 2. Themorphological changes of C2C12 cells during differentiation The C2C12 cells were cu90% confluence, differentiation medium of DMEMwith 2% horse serumwas added in C2C12 mshould be changed everyday. The word under every image indicates days of cell differentiationmicroscope in a ×200 view.
We detected the cell cycle of two groups of cells, one transfectedwith pcDNA3.1(+) vector and the other transfected with TEAD1overexpression vector pcDNA3.1(+)-TEAD1. Cells were washed withice-cold PBS twice, digested with 0.25% trypsin containing 0.02%EDTA, centrifuged at 2000 ×g for 2 min, and fixed with 70% ethanol at−20 °C overnight. After that, cells were washed with ice-cold PBStwice and dyed with 500 μl PBS containing 50 μg/μl PI, 100 μg/μlRNase A and 0.2% Triton X-100, and incubated in 4 °C for 45 min. Even-tually, the cell cycle was measured with Flow Cytometer.
2.7. Statistical analysis
The DNA sequenceswere analyzed for the presence of putative tran-scription factor binding sites using the TESS (Transcription ElementSearch System) web tool available from the Computational Biologyand Informatics Laboratory at the University of Pennsylvania. Data arepresented as themeans±the standard deviations. The statistical signif-icance was determined with the Student's t-tests usingMicrosoft Excel.p values of b0.05 were considered significant, while p values of b0.01were considered extremely significant.
3. Results
3.1. TEAD1mRNA level is up-regulated in the process of C2C12 differentiation
In order to understand the expression pattern of the TEAD1 gene,weemployed RT-PCR to detect the expression level of mouse TEAD1 in dif-ferentmouse tissues.We found that TEAD1was expressed ubiquitously
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in various tissues including heart, liver, spleen, lung, kidney, skeletalmuscle, fat, stomach, small intestine and brain. To understand the ex-pression profile in myogenesis, TEAD1 mRNA level was detected withQ-PCR in different C2C12 differentiation periods. The expression levelof the TEAD1 gene in proliferating myoblasts was relatively low, butincreased significantly during the differentiation process (Fig. 1B). Theshape of cells was recorded by CCD in a Olympusmicroscope. The mor-phological changes were shown in Fig. 2. From the first differentiationday, cells extension and elongation can be observed in differentiationmedium. Until day 4, we observed multinucleated myotubes formed.Up to day 7, multiple muscle tube finally fused and formed muscle fi-bers. The morphological changes confirmed that the cells are exactlyin differentiation state.
3.2. TEAD1 arrests cell cycle and promotes C2C12 myoblast differentiation
To explore the function of TEAD1 in C2C12 differentiation, Wetransfected pcDNA3.1(+)-TEAD1 into C2C12 cells to overexpressthe TEAD1 gene, while pcDNA3.1(+) was used as a control. Q-PCRand western blot were employed to determine the effective TEAD1overexpression (Fig. 3A and C). The cell cycle was measured to testthe effect of TEAD1 on cell proliferation, while the expression ofthe myogenic differentiation marker gene Myh4 was detected byQ-PCR to validate the influence on cell differentiation. Comparedwith the control group, cells transfected with pcDNA3.1(+)-TEAD1
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Fig. 3. TEAD1 regulates Myh4 gene expression thus promotes C2C12 differentiation and arrtransfected with pcDNA3.1(+)-TEAD1 vector while pcDNA3.1(+) vector was used as the cogroup indicates cells transfected with synthetic control oligonucleotide. (C) Results of the Tcell transfected with TEAD1 siRNA control and siRNA. (E) Myh4 gene expression in C2C12 cecell cycle by flow cytometry analysis of C2C12 transfected with pcDNA3.1(+)-TEAD1 and pcthree identical experiments. * pb0.05; ** pb0.01.
were observed a significant increase of the cells in the G2/M phaseand a significant decrease of cells in the S phase (Fig. 3F). Due to theoverexpression of TEAD1 in C2C12 cells, the relative expression levelof the Myh4 gene was significantly higher than the control group(Fig. 3E), while TEAD1 knockdown caused a decrease in the Myh4gene expression (Fig. 3D). These results suggested that TEAD1 mayarrest cell cycle and promote C2C12myoblast myogenic differentiation.
3.3. Ccne1 gene is regulated by TEAD1 during transition of cell proliferationand differentiation
In our previous study, CHIP-on-chip approach was performed tosearch genome-wide TEAD1 targets in mouse skeletal muscle. Wefound an essential cell cycle regulation gene Ccne1 in CHIP-on-chipdata [23]. To evaluate whether TEAD1 arrests the cell cycle throughregulating Ccnel, we designed a series of experiments to confirmedour propose. To evaluate the functional importance of TEAD1 inmediating the transcriptional activity of the Ccne1 gene, promot-er activity Ccne1 was tested. We found that co-transfection withpcDNA3.1(+)-TEAD1decreased the reporter activity by 50% comparingwith control (Fig. 4A). Similarly, transfecting pcDNA3.1(+)-TEAD1in C2C12 decreased the mRNA level of the Ccne1 gene to 50% ofpcDNA3.1(+) group (Fig. 4B). However, surprisingly, we did not seeany change of gene expression while knockdown of TEAD1 by siRNA(Fig. 4C). Western blot was adopted to check whether TEAD1 affects
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est cell cycle. (A) The TEAD1 gene was effectively overexpressed in C2C12. Cells werentrol. (B) TEAD1 was knocked down by specific siRNA in C2C12 cells. Negative controlEAD1 protein in C2C12 cells by Western blot. (D) Myh4 gene expression level in C2C12lls with different treatments, pcDNA3.1(+) group as the control. (F) Statistical results ofDNA3.1(+). Results shown are the mean±SD of triplicate determinations from one of
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Cyclin E protein level, and the result was consistent with the mRNAlevel, that is, overexpression of TEAD1 decreased the protein abundanceand depression of TEAD1 had no significant influence on cyclinE proteinlevel (Fig. 4D). To further investigate whether Ccne1 was involved inC2C12 cell differentiation or was regulated by other factors in this pro-cess, we detected the expression pattern in different differentiationperiods of C2C12 cells using Q-PCR. Results showed a sharp reductionof gene expression between 0 d and 3 d of differentiation, and theexpression level maintained to 8 d of differentiation (Fig. 4E). Theseresults indicated that Ccne1 gene expression was negatively regulatedby TEAD1 and the expression level of Ccne1 was much more sensitiveto TEAD1 overexpression than to TEAD1 knockdown.
3.4. The transcription of Mrpl21 and Ndufa6 is regulated by TEAD1
From the CHIP-on-chip data, we found another two novel genesMrpl21 and Ndufa6. In order to confirm whether TEAD1 actually regu-lates the transcription of the Mrpl21 and Ndufa6, we isolated the pro-moter region of Mrpl21 and Ndufa6, measured promoter activity,checked the effect of the TEAD1 on gene expression level. TESS softwarepredicted that a putative TEAD1 protein binding site was located in-1927 to -1935 of Mrpl21 gene and -1054 to -1062 of the Ndufa6gene. Thebinding sequenceswere 5′-ACATTCCT-3′ and5′-TAGAATGT-3,respectively.
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Fig. 4. TEAD1 regulates Ccne1 in mRNA and protein level. (A) The promoter activity of the Ccnassaywas performedwith the target gene promoter-luciferase reporter transfected into C2C12of Ccne1was regulated by TEAD1 in C2C12 cells. pcDNA3.1(+) vector or pcDNA-TEAD1 vectorwby TEAD1 in C2C12 cells. Control siRNA or TEAD1 specific siRNAwas transiently transfected in ton Western blots, knockdown of TEAD1 did not affect Ccne1 protein level and TEAD1 overedifferentiation periods of C2C12 cells. 0 d means cells were proliferating. Gene expression leve
To evaluate the functional importance of TEAD1 in mediating thetranscriptional activity of the Mrpl21 and Ndufa6 gene, promoteractivity of Mrpl21 and Ndufa6 was tested. Results are shown inFig. 3A. As expected, TEAD1 overexpression affected Mrpl21 andNdufa6 promoter activity: a 2-fold increase of promoter activity ofthe Mrpl21 gene was observed while Ndufa6 promoter activity inpcDNA3.1(+)-TEAD1 group was 2-fold lower than when pcDNA3.1(+)(Fig. 5A). Thus, it can be stated that the TEAD1 protein can positivelyregulate the transcription activity of the Mrpl21 gene and negativelycontrol the Ndufa6 gene. To determine if TEAD1 influenced the mRNAlevel of the Mrpl21 and Ndufa6 genes, expression levels of Mrpl21 andNdufa6 were measured by Q-PCR. The results showed that the mRNAlevel of Mrpl21 was 1.5-fold of the control group while the expressionof Ndufa6 decreased to 40% of the control (Fig. 5B).
To further investigate the functional consequences of aberrantexpression of TEAD1, we knocked down TEAD1 gene using the siRNAexperiment. We first determined the optimal siRNA sequence throughtransfection into C2C12 and examined the mRNA and protein levels.Data in Fig. 3B show that TEAD1 mRNA was significantly knockeddown and the TEAD1 protein level was evidently lower than siRNA con-trol in siRNA-1 and siRNA-2 (Fig. 3C). Expression levels of the Mrpl21and Ndufa6 gene were assessed by Q-PCR. Mrpl21 expression wasdown-regulated by about 40% and Ndufa6 was up-regulated by about30% (Fig. 5C). The expression data of Mrpl21 and Ndufa6 supported
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he C2C12 cells. (D) Results of Cyclin E protein level in C2C12 cell bywestern blot. As shownxpression can induce Ccne1 decrease. (E) The expression of the Ccne1 gene in differentl of 0 d cells was normalized to 1. * pb0.05; ** pb0.01.
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Fig. 5. The transcription ofMrpl21 and Ndufa6 is regulated by TEAD1. (A) The promoter activity ofMrpl21 and Ndufa6was regulated by TEAD1 overexpression in C2C12 cells. A transienttransfection assay was performedwith the target gene promoter-luciferase reporter transfected into C2C12 cells in the presence of a pcDNA3.1(+)-TEAD1 vector or the pCDNA3.1 vectoras a control. (B) The expression of Mrpl21 and Ndufa6 was regulated by TEAD1 in C2C12 cells. pcDNA3.1(+) vector or pcDNA3.1(+)-TEAD1 vector was transiently transfected by theC2C12 cells. Q-PCR was used to detect themRNA level of Mrp21 and Ndufa6 in two groups. The experiment was repeated three times.(C) The expression of Mrpl21 and Ndufa6was reg-ulated by TEAD1 in C2C12 cells. Control siRNA or TEAD1 specific siRNA was transiently transfected in the C2C12 cells. The expression of Mrpl21 and Ndufa6 was detected by Q-PCR. Theexperimentwas repeated three times. (D) The expression pattern of theMrpl21 and Ndufa6 gene in different differentiation periods of C2C12 cells. 0 dmeans cells are proliferating. Geneexpression level of 0 d cells was normalized to 1. Statistical significance were analyzed by the Student's t-tests * pb0.05; ** pb0.01.
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the conclusion that TEAD1 regulated themRNA level of theMrpl21 andNdufa6 gene. In order to understand the expression patterns of Mrpl21and Ndufa6 in C2C12 differentiation state, Q-PCR was performed todetermine the mRNA level of both genes during myoblast differentia-tion. We found that Mrpl21 and Ndufa6 mRNA levels were both lowat the myoblast stage. Ndufa6 mRNA expression increased markedlyduring the last two days of differentiation, especially in 8 d, where theexpression levels were more than 6-fold elevated comparing with 0 d(Fig. 5D). The speed of the increase of Mrpl21 was relatively low, andshowed about a 3-fold increase when compared with 0 d (Fig. 5D).
3.5. The function of Mrpl21, Ndufa6 and Ccne1 in C2C12 cell proliferationand differentiation
After verification of Mrpl21, Ndufa6 and Ccne1 as target genes ofTEAD1, we wondered the preliminary function of these three genes inC2C12 cell proliferation and differentiation. We constructed the CDS re-gions of three genes into pcDNA3.1(+) vector to form the overexpressedvector respectively. The control vector and gene overexpression vectorpcDNA3.1(+)-Mrpl21, pcDNA3.1(+)-Ndufa6, pcDNA3.1(+)-Ccne1,the combination of pcDNA3.1(+)-Ndufa6 and pcDNA3.1(+)-Ccne1was transfected into C2C12 cells. ThemRNA level of C2C12 cell differen-tiationmarkerMyh4was then detected byQ-PCR,meanwhile, we testedthe C2C12 cell cycle usingflowcytometry.We found that overexpressionof Mrpl21 and Ndufa6 resulted in elevated Myh4 expression and Ccne1did not affect Myh4 expression in C2C12 cells. Co-overexpression ofNdufa6 and Ccne1 significantly decreased the Myh4 expression level(Fig. 6A). The cell cycle was not affected by Mrpl21 and Ndufa6overexpression, but overexpressed Ccne1 alone or co-overexpressedwith Ndufa6 can significantly increase the number of cells in S-phase(Fig. 6B–E).
4. Discussion
TEAD1 (TEA domain family member 1), also known as TEF-1(transcriptional enhancer factor-1), belongs to the TEA domain familywhich contains a conserved domain that bind M-CAT (muscle cytidine-adenosine-thymidine) element in gene promoter regions to promoteor inhibit gene expression. This interactionwas first found in the chickencardiac troponin T (Cardiac Troponin T) gene promoter [24]. The TEAD1family plays a crucial role in cardiac development, cardiac hypertrophy,arrhythmia, muscle development, formation of myoblasts, and regener-ation and hypertrophy of skeletal muscle and neural crest during devel-opment. Studies in mice have shown that the expression of the TEAD1gene can be detected in 10.5 days of embryonic development, and thatTEAD1 knockout in mice leads to increased pericardial cavity, bradycar-dia, fourth ventricle increase and embryonic death in 12.5 days [6].These abnormal phenotypes predict that TEAD1 functions in the devel-opment of embryonic heart and other organs, and the other membersof this family cannot compensate for its function. The TEAD1 familyplays a crucial role in regulating the expression of Cardiac troponin T,β-myosin heavy chain, smooth muscle α-actin and skeletal α-actin,etc. [20]. Although TEAD1 plays a essential role in regulating muscledevelopment, no study has yet examined its exact function.
We analyzed TEAD1 gene expression in different tissues of adultmice, as well as in various differentiation periods of C2C12. The expres-sion pattern of TEAD1 in mouse tissues showed that TEAD1 wasexpressed abundantly in each tissue, which indicates that the TEAD1gene has multiple functions and its target genes are widely expressed.In C2C12 cells, TEAD1 was up-regulated during C2C12 myoblast differ-entiation, which implies that TEAD1 may participate in, and promote,myoblast differentiation. Functional assay in C2C12 cells was carriedout to explore TEAD1 function in cell proliferation and differentiation.The results showed that TEAD1 gene overexpression in C2C12 can
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0%
10%
20%
30%
40%
50%
60%
G1 G2 S
pcDNA3.1(+)
pcDNA3.1(+)-Ccne1
*
*
Per
cent
age
of t
otal
cel
ls
Cell cylce phase
0%
10%
20%
30%
40%
50%
60%
G1 G2 S
pcDNA3.1(+)
pcDNA3.1(+)-Mrpl21
Per
cent
age
of t
otal
cel
ls
Cell cylce phase
0%
10%
20%
30%
40%
50%
60%
G1 G2 S
pcDNA3.1(+)
pcDNA3.1(+)-Ndufa6pcDNA3.1(+)-Ccne1
*
*
Per
cent
age
of t
otal
cel
ls
Cell cylce phase
A
B C
D E
0
1
2
3
4
pcDNA3.1(+) Ndufa6 Mrpl21 Ccne1 Ndufa6+Ccne1
Gen
e ex
pres
sion
fol
dch
ange
* *
*
Fig. 6. Mrpl21, Ndufa6 and Ccne1 gene function in C2C12 cell proliferation and differentiation. (A) The mRNA level of Myh4 was detected by Q-PCR in Mrpl21, Ndufa6, Ccne1overexpressed and Ccne1-Ndufa6 co-overexpressed C2C12 cells. (B–E) Statistical results of cell cycle by flow cytometry analysis of C2C12 transfected with pcDNA3.1(+) and in-dicated gene overexpression vector. Results shown are the mean±SD of triplicate determinations from one of three identical experiments. * pb0.05; ** pb0.01.
680 F. Wang et al. / Cellular Signalling 25 (2013) 674–681
promote the expression of the cell differentiation marker geneMyh4, which indicated that the TEAD1 gene promoted C2C12 celldifferentiation, while TEAD1 knockdown down-regulated the ex-pression of the Myh4 gene and represses C2C12 cell differentiation.Meanwhile, overexpression TEAD1 in C2C12 cells can result in Sphase cell number decrease and arrest cell cycle.
In our former study, ChIP-on-chip assay was used to rapidly exam-ine TEAD1 direct targets in the whole genome. However, large-scaleand high-throughput experiments may result in some false positiveresults, thus, it was necessary to revalidate the target genes indepth in further experiments. Thus, we used expression patternsanalysis, luciferase reporter system to verify the genuine regulatedgenes in the CHIP-on-chip data. We detected putative Mrpl21,Ndufa6 and Cccne1 gene expression by Q-PCR, and data showedthat TEAD1 down-regulated the expression of Ndufa6 and Ccne1,while up-regulated Mrpl21 gene expression. We then isolated thepromoter regions of mouse Mrpl21, Ndufa6 and Ccne1 and predictedthe TAED1 protein binding site in the promoter regions.Dual-luciferase reporter system showed that activity of the Mrpl21promoter was positively regulated, while activity of Ndufa6 andCcne1 was negatively regulated by TEAD1. Functional analysis re-vealed that Mrpl21 and Ndufa6 can promote Myh4 gene expressionlevel and did not affect C2C12 cell cycle.
From gene expression and promoter activity results, we can drawthe conclusion that TEAD1 can regulate the expression of Mrpl21,
Ndufa6 and Ccne1. This raised the question whether TEAD1 playeda role in regulating these target genes in the process of C2C12 cell dif-ferentiation. Thus, we detected the mRNA level in different time pe-riods of C2C12 cell differentiation, and data showed that Mrpl21and Ndufa6 were up-regulated and Ccne1 was down-regulated. Theexpression patterns of the Mrpl21 and Ccne1 genes were consistentwith the regulation pattern of TEAD1. Because TEAD1 up-regulatesexpression patterns in the process of C2C12 cell differentiation(positive-regulation of Mrpl21 and negative-regulation of Ccne1),Mrpl21 gradually increased, while the mRNA level of Ccne1 sharplydecreased. However, the expression pattern of Ndufa6 appeared toshow a contradictory result. That was because other factors may beinvolved in the regulation of the Ndufa6 gene during C2C12differentiation.
The Ccne1 gene plays an important role in cell cycle regulation,and its abnormal expression is associated with various tumors. It be-gins to express in G1 phase and forms an active complex with cyclindependent kinase 2 (CDK2), pulling the cell cycle from G1-S phasetransition [25]. The Ccne1 function in C2C12 cells was also tested inour study, we showed that overexpression of Ccne1 resulted a slightincrease of cell number in S-phase, but did not affect Myh4 gene ex-pression, which confirmed that the regulation of TEAD1 to Ccne1gene expression is a very important event. Since TEAD1 inhibits themRNA level in the Ccen1 gene and protein abundance in C2C12 cell,the expression level of this gene significantly decreases during
681F. Wang et al. / Cellular Signalling 25 (2013) 674–681
differentiation transition. In cell cycle detection assay, we found thatoverexpression TEAD1 resulted in C2C12 cell decrease in S phase,while overexpression Ccne1 caused partial increase of cell number inS phase. The results mentioned to us that TEAD1 regulated cell cyclepartially through suppressing Ccne1 gene expression, but other down-stream targets of TEAD1 also participated in cell cycle regulation. Thismay explainwhyTEAD1 can repress cell cycle and promote C2C12myo-blast differentiation.
In summary, we identified the function of the TEAD1 gene in C2C12cells, which implicated that TEAD1 suppressed C2C12 proliferation andpromoted cell differentiation. The novel TEAD1 candidate target genesMrpl21, Ndufa6 and Ccne1 were found, and the expression patternsof these target genes were analyzed. Our data initially conclude thatTEAD1 regulates C2C12 differentiation through negatively regulatingthe expression of Ccne1, which can explain the transition between pro-liferation and differentiation. These findings provide important cluesthat will enable further function analysis of the TEAD1 gene.
Acknowledgments
Thisworkwas supported by theNationalNatural Science Foundationof China (31072012), the National High Science and TechnologyFoundation of China (2011AA100304).
References
[1] M. Buckingham, L. Bajard, T. Chang, P. Daubas, J. Hadchouel, S.Meilhac, D.Montarras,D. Rocancourt, F. Relaix, Journal of Anatomy 202 (1) (2003) 59–68.
[2] T.R. Burglin, Cell 66 (1) (1991) 11–12.[3] A. Andrianopoulos, W.E. Timberlake, The Plant Cell 3 (8) (1991) 747–748.[4] A.F. Stewart, S.B. Larkin, I.K. Farrance, J.H. Mar, D.E. Hall, C.P. Ordahl, Journal of
Biological Chemistry 269 (5) (1994) 3147–3150.
[5] X. Xu, S. Xing, Z.Q. Du, M.F. Rothschild, M. Yerle, B. Liu, Comparative Biochemistryand Physiology. Part B, Biochemistry & Molecular Biology 150 (4) (2008) 447–453.
[6] Z. Chen, G.A. Friedrich, P. Soriano, Genes & Development 8 (19) (1994) 2293–2301.[7] T. Maeda, M.P. Gupta, A.F. Stewart, Biochemical and Biophysical Research Com-
[19] E.A. Swartz, A.D. Johnson, G.K. Owens, American Journal of Physiology 275 (2 Pt 1)(1998) C608–618.
[20] T. Yoshida, Arteriosclerosis, Thrombosis, and Vascular Biology 28 (1) (2008) 8–17.[21] E.E. Creemers, L.B. Sutherland, J. McAnally, J.A. Richardson, E.N. Olson, Develop-
ment 133 (21) (2006) 4245–4256.[22] R.W. Tsika, L. Ma, I. Kehat, C. Schramm, G. Simmer, B. Morgan, D.M. Fine, L.M.
Hanft, K.S. McDonald, J.D. Molkentin, M. Krenz, S. Yang, J. Ji, Journal of BiologicalChemistry 285 (18) (2010) 13721–13735.
[23] H. Qiu, F. Wang, C. Liu, X. Xu, B. Liu, BMC Molecular Biology 12 (2011) 1.[24] T.A. Cooper, C.P. Ordahl, Journal of Biological Chemistry 260 (20) (1985) 11140–11148.[25] M. Ohtsubo, A.M. Theodoras, J. Schumacher, J.M. Roberts, M. Pagano, Molecular
