Overexpression of DNA Methyltransferase in Myoblast Cells Accelerates Myotube Formation

Eur. J. Biochem. 231,282-291 (1995) 0 FEBS 1995

Overexpression of DNA methyltransferase in myoblast cells accelerates myotube formation Hidekazu TAKAGI, Shoji TAJIMA and Akira ASANO

Institute for Protein Research, Osaka University, Japan

(Received 28 MarcM5 May 1995) - EJB 95 048612

We overexpressed mouse DNA methyltransferase in murine C2C12 myoblast cells and tested the isolated clones for their ability to differentiate. Significant numbers of the clones showed distinct myotubes 24 h after the isolated transformants had been induced to differentiate, whereas the parent C2C12 cells did not form myotubes at this time point. Transfection of the vacant vector or the plasmid containing the reverse-oriented DNA methyltransferase cDNA did not provide significant numbers of transformants with the accelerated differentiation phenotype, suggesting that the effect is caused by the expression of DNA methyltransferase. The expressions of skeletal muscle myosin and creatine kinase in clones that showed the accelerated differentiation-phenotype were also induced about 24 h earlier and at higher levels relative to the parent C2C12 or the control cells, indicating that the entire process of myogenesis had been accelerated. All the methyltransferase-transfected clones, regardless of their phenotypes, demonstrated about threefold higher DNA methyltransferase activity and higher methylation levels than those of the clones transfected with vector alone or the reverse-oriented plasmid. At the early stage of transfection of the sense-oriented plasmid, high de novo methylation activities were detected. We consider it likely that this high de novo methylation activity is the reason for the high methylation levels and the accelerated myotube formation of the clones transfected with the sense-oriented plasmid. In some transformants which showed the accelerated differentiation phenotype, MyoDl was already fully expressed under the growth conditions while, in control cells, MyoDl was expressed at low levels. This elevated level of MyoDl transcription could account for the accelerated myotube formation observed in the transformants. The methylation state of the HpaTI sites in exon 1 through exon 2 of the MyoDl gene and the expression of the MyoDl transcript are positively correlated.

Keywords. DNA methyltransferase ; DNA methylation; MyoDl ; myogenesis.

In vertebrates, genomic DNA is often methylated at the 5 position of cytosine in the sequence CpG, and this methylation is thought to affect gene expression (Cedar, 1988; Bird, 1992). The promoter regions of genes that are actively transcribed are usually undermethylated, while those of silent genes are heavily methylated (Cedar, 1988; Bird, 1992; Razin and Cedar, 1991). In contrast to this general rule, the expression of the class-I multihistocompatable gene, H-2K, is positively correlated with the extent of methylation (Tanaka et al. 1983) and, similarly, undermethylation of specific sites in associated with the suppres- sion of the expression of insulin-like growth factor 2 (Zgf2) and its receptor (Zgf2r) (Sasaki et al., 1992; Stoger et al., 1993; Li et al., 1993). DNA methylation is thought to affect transcription by (a) inhibiting the binding of proteins such as enhancers or suppressors to DNA or (b) promoting the binding of proteins that specifically recognize the methylated DNA. Examples of the first scenario are the enhancers E2F (Kovesdi, 1987), MLTF (Watt, 1988) and c-Myc (Prendergast and Ziff, 1991), where binding to specific binding sites is inhibited by methylation. Ex-

Correspondence to S . Tajima, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, Japan 565

Phone: +81 6 879 8628. Fax: +81 6 879 8629. Abbreviations. CK, creatine kmase ; DMEM, Dulbecco’s modified

Eagle’s medium ; GraPDH, glyceraldehyde-3-phosphate dehydrogenase ; MeFdse, DNA methyltransferase; S-AdoMet, S-adenosylmethionine.

Enzyme. DNA methyltransferase (EC 2.1.1.37).

amples of the latter scenario are MeCPl (Boyes and Bird, 1992), MeCP2 (Meehan et al., 1992) and MDBP2 (Pawlak et al., 1991), which bind specifically to methylated DNA and inhibit transcription. At least in one case, structural features of the chromatin may also be required for inhibition of expression of a methylated gene (Bushhausen et al., 1985).

In mice, the methylation pattern of genomic DNA begins to be established at the implantation stage of embryogenesis (Monk, 1990) by a so-called de novo methylation activity that recognizes and transfers methyl groups to unmethylated DNA. Once the methylation pattern has been formed, it is maintained in somatic cells by a ‘maintenance’ methylase (MeTase; Razin and Cedar, 1991 ; Toth et al., 1990) that functions during DNA replication to copy the methylation pattern onto the newly synthesized DNA strand. Consistent with this function, the MeTase is localized at the replication foci during the S phase (Leonhardt et al., 1992). In vitro, MeTase also has de novo methylation activity (Griinwald and Drahovsky, 1984; Bolden et al., 1986), however, it is not yet known whether MeTase works as a de novo methylase in vivo, and thus plays a role in creating the DNA methylation pattern.

The methylation pattern is dynamic and changes during cell differentiation. When tissue-specific genes begin to be expressed, the promoter regions of many of those genes are demethylated (Cedar, 1988; Bird, 1992; Razin and Cedar, 1991). The chromatin context appears to be important for this regula-

Takagi et al. (Eur: J. Biochem. 231) 283

tion, although the primary signal for methylation and demethylation is thought to reside in the nucleotide sequence itself (Paroush et al., 1990; Hasse and Schulz, 1994; Lichtenstein et al., 1994). Recently, DNA demethylation activity was detected in vitro (Jest, 1993; Vairapandi and Duker, 1993) and shown to be active during myogenesis through a mechanism resembling excision repair (Jost and Jost 1994).

Experimental manipulation of MeTase activity allows the consequence of perturbing DNA methylation to be observed in viva cDNA clones of mammalian MeTase have been isolated from mouse (Bestor et al., 1988) and human (Yen et al., 1992). When DNA methylation activity was modulated in vivo using MeTase cDNA and its gene as the experimental tools, imprinting, terminal differentiation and cell fate were affected. Com- plete disruption of the MeTase gene results in lethality of the homozygous mutant embryo at midgestation (Li et al., 1992) and cancels the imprinting of the genes I$, Igf2r and H19 (Li et al., 1993). The expression of the antisense strand of MeTase cDNA in C3H10T1/2 cells induces their differentiation into myotubes (Szyf et al., 1992), similar to the effect of 5-azacytidine (Jones and Taylor, 1980), a demethylating reagent. Overex- pression of the MeTase cDNA increases the methylation levels and induces tumorigenic transformation in NIH3T3 cells (Wu et al., 1993).

As mentioned above, cell differentiation correlates with DNA demethylation (Razin et al., 1986; Jost and Jost, 1994). Indeed, there appears to be a causal connection ; demethylation in C3HIOTU2 fibroblasts induced by 5-azacytidine triggers a preferential differentiation into myotubes (:ones and Taylor, 1980). Therefore, we expected that the terminal differentiation of the cells into myotubes could be suppressed if MeTase activity is overexpressed in myoblasts. In the present study, we overexpressed MeTase in C2C12 myoblasts and monitored the effect on terminal differentiation. Unexpectedly, we found that terminal differentiation was significantly promoted rather than suppressed. Our results predict the existence of specific genes that play important roles in myogenesis and that are susceptible to methylation which, in turn, positively affects myogenesis. We suggest that one of these target genes could be MyoDl.

MATERIALS AND METHODS Cells. Mouse myoblast cells, C2C12 (Blau et al., 1985),

were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum, 100 unitdm1 penicillin and 100 pg/ml streptomycin. Murine erythroleukemia cells, MELI 1A2 (Watanabe and Oishi, 1987), were maintained in ES medium (Nissui), supplemented with 2% fetal bovine serum. The cells were cultured at 37 "C in a 5 % CO, atmosphere in plastic dishes that were coated with 1 % gelatin. Differentiation of C2C12 myoblast cells into myotubes was induced by changing the medium to Cosmedium (CosmoBio) when the cells reached near confluency (Ichikawa et al., 1993).

Construction of the MeTase expression vector. Mouse MeTase cDNAs were cloned from the cDNA library of murine erythroleukemia cells (Clontec) using the plasmid pR2K as a probe, which encodes 2 kb of the 3' region of the MeTase cDNA. pR2K was kindly provided by Dr Bestor (Harvard Uni- versity, Medical School, USA; Bestor et al., 1988). Clones covering the entire MeTase-coding sequence were ligated into a full- length clone and subcloned into the expression vector pKCRH2PL (Mishina et al., 1984), which was generously provided by Dr Morimoto (Central Laboratory of Mitsubishi Kasei Co. Ltd).

Transfection and isolation of stable transformants. The calcium-phosphate method (Chen and Okayama, 1987) was used

for all transfection experiments, except for experiment 2 (Ta- ble 1) where electroporation (Gene Pulser, BioRad) was used according to the manufacturer's manual. A mixture of 20 pg pKCRH2PL or the vector containing the full-length MeTase cDNA (in the sense or the reverse orientation) and 0.5 pg pSV2neo was transfected. Stably transformed cells were se- lected with geneticin (G418) and independent clones were isolated (Southern and Berg, 1982). For transient transfections, pSV2neo was omitted from the DNA mixture, and the cells were harvested after 24 h culture in growth medium.

MeTase activity. The nuclear extract was prepared as described (Bestor and Ingram, 1983) and used as the source of the enzyme. To measure the total methylation activity, poly[d(I-C) . poly[d(I-C)] (Pedrali-Noy and Weissbach, 1986 ; Pfeifer and Drahovsky, 1986) and a synthetic 33-bp oligonucleotide, containing one methylated strand (hemimethylated) at CpG sequences (Szyf et al., 1991) were used as substrates. To measure de novo methylation activity, poly[d(G-C)] . poly[d(G-C)] and the synthetic 33-bp oligonucleotide without methylated deoxy- cytidylic acid were used. The reaction mixture contained 0.5- 1.0 pg nuclear protein, 0.1 pg DNA and 1 pCi 3H-labeled S- adenosylmethionine ([3H]S-AdoMet; 55 - 85 Ci/mmol, NEN) in a volume of 25 p1 reaction buffer (Bestor and Ingram). After 1 h incubation at 37 "C, the mixture was chilled on ice and unlabeled S-AdoMet was added to a final concentration of 1.5 mM. The mixture was applied to DE8l filter disks (Whatman) which were washed as described (Sambrook et al., 1989). The radioactivity bound to the filter was determined in a scintillation counter. The protein concentration was determined using a bicinchoninic acid Protein Assay Reagent (Pierce) and bovine serum albumin as a standard.

Determination of methylation levels in the genomic DNA. Methylation levels in genomic DNA were determined using commercially available cytosine methylases. Sonicated DNA (200 ng) was methylated with [3H]S-AdoMet (1 pCi, 55-85 Ci/ mmol, NEN) and 3-4 units M.SssI or M.HpaI1 in a total volume of 20 pl reaction buffer at 37°C for 2 h. The radioactivity incorporated into DNA was determined as described above.

Creatine kinase (CK) activity. Cultured cells in a 6-cm plastic dish were washed with Dulbecco's phosphate-buffered saline (NaCVP,; Dulbecco and Vogt, 1954) and lysed with I ml lysis buffer (Delaporte et al., 1986). The cell lysate was briefly sonicated, then centrifuged for 20min at 20000Xg and 4°C. The supernatant was used to measure the enzyme activity as described (Ennor and Stocken, 1953 ; Hughes, 1962).

Antibodies. Anti-(skeletal muscle myosin) serum was raised against purified chick gizzard myosin, which was generously provided by Dr Inoue (Osaka University, Japan). An antiserum reactive with mouse MeTase was raised against a glutathione S- transferase fusion protein expressed in Escherichia coli. To this end, an approximately l-kb DNA [2979(BarnHI)-4028(EcoRI), encoding amino acids 918-1268] of mouse MeTase cDNA (Bestor et al., 1988), was ligated into pGEX2T (Pharmacia) and expressed in E. coli strain NM522 in the presence of isopropyl- P-D-thiogalactoside. The expressed gluthatione S-transferase fusion protein accumulated in inclusion bodies. Inclusion bodies were purified (Sambrook et al., 1989) and the glutathione S- transferase fusion protein was further purified by electroelution from an SDS/polyacrylamide gel. The antibodies raised in a rabbit were immunoselected using antigen-coupled Sepharose C14B as an affinity matrix.

Western blotting. Cells were lysed and electrophoresed in a 7% SDS/polyacrylamide gel according to the method described by Laemmli (Laemmli, 1970). After electrophoresis, protein bands were electrophoretically transferred to nylon membranes (Gene Screen). The binding sites on the membrane

284 Takagi et al. (Em J. Biochem. 231)

sheet were blocked with 5 % skimmed milk, 0.02 % sodium azide, then incubated with anti-(skeletal muscle myosin) serum in antibody buffer (1 % bovine serum albumin, 0.1 % Tween- 20, and 0.02% sodium azide in NaCIP,). After incubation, the membrane sheets were incubated with goat anti-rabbit IgC serum conjugated to alkaline phosphatase. Bound secondary antibodies were visualized using a color reaction buffer containing 0.05 mg/ml indoyl phosphate, 1 mg/ml p-nitroblue tetrazolium chloride, 4 mM magnesium acetate in 50 mM glycineNaOH, pH 9.6. To detect MeTase, the membrane sheets were incubated with immunoselected anti-MeTase serum, followed by incubation with lZ5I-labeled protein A. The membrane sheets were exposed to X-ray film at -70°C using intensifying screens.

Immunoprecipitation of MeTase. To label cell proteins, cells were cultured in methionine-free DMEM (Sigma), for 30 min, then 50 pCi [35S]methionine (EXPRE35S35S, New England Nuclear) was added, and the incubation was continued for 5 h. After this incubation, a nuclear extract was prepared as described above. The extract was precipitated with 10% trichlo- roacetic acid washed with cold acetone, then dried. A 1-ml aliquot of 0.1 M NaCI, 2 mM EDTA and 0.2% SDS in 50 mM Tris/HCI, pH 7.4, was added to the precipitate, which was then briefly sonicated. The mixture was boiled for 2 min, cooled, then resuspended in a 100-yl aliquot 20% Triton X-100, 7.5 units/ ml Trasylol, 50 mM iodoacetic acid, and immunoselected anti- MeTase IgG was added. After a overnight incubation, protein- A-Sepharose (Pharmacia) was added to precipitate the MeTase- antibody complex. The beads were washed with 0.1 M NaCl, 5 mM EDTA, 0.1 % Triton X-100 and 0.75 unit/ml Trasylol in 50 mM Tris/HCI, pH 7.4. Proteins were solubilized in sample buffer and were then electrophoresed in a 7 % SDS/polyacrylamide gel. Labeled bands were detected by fluorography (Bonner and Laskey, 1974).

Blotting of RNA and DNA. RNA was isolated as described (Chomzynski and Sacchi, 1987) from cells proliferating in growth medium and in differentiation medium before they were confluent. Isolated RNA was denatured with glyoxal and dimethylsulfoxide, electrophoresed on a 1.5 % agarose gel (Menju et al., 1989), and blotted onto Hybond N+ (Amersham) with 50 mM NaOH. The blotted sheet was prehybridized with 30% formamide, 7% SDS, 1 % bovine serum albumin, 1 mM EDTA and 0.2 M sodium phosphate, pH 7.2, at 65°C for 30- 60 min, then hybridized overnight with 32P-labeled mouse MyoDl, mouse myogenin, mouse Id, and human glyceraldehyde-3-phosphate dehydrogenase (GraPDH) cDNAs in the same buffer at 65°C. The labeling of cDNA was performed by the hexadeoxynucleotide multi-priming method (Feinberg and Vo- gelstein, 1984). Mouse MyoDl and Id cDNAs were generously provided by Dr Weintraub (Fred Hutchinson Cancer Research Center, Seattle, USA), and mouse myogenin cDNA was kindly provided by Dr Wright (University of Texas South Westem Medical Center, Dallas, USA). The hybridized sheet was washed at 65°C for 30min each with: (a) 2XNaCVCit (IXNaCVCit contains 0.15 M sodium chloride and 15 mM sodium citrate, pH 7.4) and 1 % SDS, (bj 0.2XNaCKit and 1 % SDS; (cj 0.2X NaCVCit, 30% formamide and 0.1 % SDS.

Southern-blotting analysis was performed by a modification (Church and Gilbert, 1984) of the standard procedure (Sambrook et al., 1989). The blotted sheet was prehybridized with 30% formamide, 7% SDS, 1 % bovine serum albumin, 1 mM EDTA and 0.2 M sodium phosphate, pH 7.2, at 42°C for 1 h and hybridized overnight at 42°C in the same buffer with a 32P-labeled MyoDl probe. To prepare this probe, EcoRI fragments of genomic DNA prepared €ram MEL11A2 cells were ligated into a 2EMBL4 vector, and clones with an approximately 16-kb insert containing the MyoDl gene were isohted. A fragment of the

Table 1. Classification of the transformants and their MeTase activity. The clones that showed distind myotubes after 24 h incubation in the differentiation medium were expressed as myotube formation (+), At that time, if the myotube was not observed or apparently was the same as that of the parent C2C12 cells the myotube formation was expressed as (-). The transfected plasmids were vacant vector (pKCR, mock), MeTase cDNA in sense (pKCR-MTR) and in reverse (pKCR- RTM) orientations, respectively. Hemi-rnethylated synthetic 33-bp ohgo-- nucleotide and poly[d(I-C)] poly[d(I-C)] were used as substrates for experiment 1 and experiment 2, respectively. The numbers in parentheses show the number of clones analyzed. Values are means i S.D.

Plasmid Myo- Number MeTase activity tube

% pmol . h-' . mg protein-'

pKCR-MTR + 22 (71) 15.5 Ir 2.2 (3) 13.5 2 1.9 (3)

- 16 (100) 5.1 Ir 1 . 1 (3)

pKCR-MTR + 16 (28) 29.1 27.3 (8) - 41 (72) 25.2 +- 4.8 (6)

- 58 (95) 8.0 I 4.1 (8)

Experiment 1

- 9 (29) -

pKCR (mock) + 0 (0)

Experiment 2

pKCR-RTM + 3 ( 5 ) 7.3 Ir 3.7 (3)

gene was '*P-labeled (Feinberg and Vogelstein, 1984) and used as the probe. Membrane sheets were washed at 65 "C for 30 m i n each with 2XNaCVCit and 1. % SDS, 0.2XNaCKit and 1 % SDS. and 0.2XNaCKit and 0.1 % SDS.

RESULTS

Overexpression of the MeTase in C2C12 cells promotes myogenesis. To determine whether or not aberrant methylation of genomic DNA affects myogenic differentiation, we overexpressed MeTase in C2C12 myoblast cells. To this end, we transfected MeTase and neomycin cDNAs, both under the control of the simian virus 40 promoter, into C2C12 cells, isolated 31 geneticin-resistant clones, and tested their ability to differentiate. Previous reports (Jones and Taylor, 1980; Yisraeli et al., 1986; Szyf et al., 1992) suggested that overexpression of the MeTase would inhibit terminal differentiation of these cells. Surprisingly, however, 22 clones (71 %) formed distinct myotubes after a 24-h incubation (Table 1, experiment 1 ; Fig. I), when the isolated transformants were induced to differentiate, whereas the parent C2C12 cells did not form myotubes at this time point (Fig. 1). Myotube formation of the remaining nine clones did not occur, even 48 h after the medium had been changed (Fig. 1 ; clone MTR3g). Similarly, none of the clones that were mock transfected with the pKCR vector alone showed myotube formation under these conditions. We, therefore, conclude that the effect of accelerated myotube formation is insert dependent.

In a second experiment, we transfected, by electroporation, sense and antisense MeTase cDNA (pKCR-RTM). From both transfections, we isolated stable clones and classified them as in experiment 1 (Table 1, experiment 2). Of all the clones transfected with the sense-oriented plasmids, 16 out of 57 clones (28 %) again displayed the ability to form myotubes in our assay (Table 1, experiment 2). In contrast, only three out of 61 clones (5 %) transfected with the antisense-oriented plasmid showed this phenotype. This result suggests that the effect is not due to

Takagi et al. (Eur: J. Biochem. 231)

c2c12 KCR4 (-1 MTR4c (+) MTR3g (-)

285

Oh

24h

48h

-~ ___ - . I _.

Fig. 1. Phase-contrast micrographs of the myotube formation of the transformants. The parent C2C12 cells, the isolated transformants of KCR4(mock), MTR4c. and MTR3g cells were cultured in the growth medium to near confluency, then changed to the differentiation medium The symbols (+) and (-) are the phenotypes for the myotube formahon, as descrlbed in Table 1. The phase-contrast mcrographs were taken after 0,24 and 48 h after the medium change. The bar indicates 100 pm.

Table 2. Induction of skeletal musde myosin during myotube formation. The expression of myosin was determined densitometrically at appropriate time intervals after the medium had been changed to the differentiation medium and summarized. The symbols (+) and (-) are the phenotypes for the myotube formation, as described in Table 1. The values were normalized to the amount of myosin in C2C12 cells after 48 h incubation in the differentiation medium.

Clone Myotube Myosin expression after

Table 3. Induction of CK activity during myotube formation. The activity was determined at appropriate time intervals after the medium had been changed to the differentiation medium as described in Materials and Methods and summarized. The symbols (+) and (-) are the phenotypes for the myotube formation, as described in Table l .

Clone Myotube Specific activity after

Oh 1 2 b 24h 36h 48h

Oh 1 2 h 2 4 h 3 6 h 4 8 h unitdpg protein

c2c12 KCR4(mock) -

KCR3b(mock) -

MTR4c + MTRl a + MTR3 1 + MTRld MTR2h

MTR3g -

-

-

0 2 23 0 2 15 0 1 2 0 1 66 0 1 51 0 2 38 0 2 10 0 1 26 0 1 8

69 60 70

140 124 95 61 77 35

100 115 89

170 165 152 98

115 I 5

c 2 c 1 2 KCR4(mock) - KCR3b(mock) - MTR4c + MTRla + MTR31 + MTR3g - MTRld -

MTR2h -

0 2 22 51 82 0 3 25 55 78 0 1 15 46 76 0 2 49 81 102 0 2 42 73 106 0 1 39 70 102 0 2 20 52 65 0 0 19 44 82 0 1 11 21 57

recombination of the MeTase cDNA at random sites in the ge- nome, but caused by the expression of MeTase. The reason for the accelerated myotube formation in three clones transfected with the antisense MeTase cDNA will be discussed later.

To determine if the myotube formation in the positive transformants reflects the onset of the myogenic process, we measured the expression profiles of skeletal muscle myosin and CK, which are biochemical markers for terminal differentiation, during the differentiation of the transformants. In MTR4c cells, myosin was detected 18 h after the medium was changed (data not shown). After 24 h incubation in the differentiation medium, the amount of myosin expressed in MTR4c cells was 2.5-times higher than that in parent C2C12, KCR4 (mock-transfected) or MTR3g cells (Table 2), neither of which formed typical myotubes after 24 h incubation in the differentiation medium. The expression profiles of myosin in all other independently isolated clones that scored positive for accelerated myotube formation

were similar to each other (Table 2). Similar results were obtained for CK. CK activity in MTR4c cells was detected after 18 h incubation in the differentiation medium (data not shown). After 24 h incubation in the differentiation medium, the CK level was twofold higher than that in the parent C2C12, KCR4 (mock-transfected), or MTR3g cells (Table 3). As for myosin, the expression of CK activity in all other clones that scored positive for accelerated myotube formation showed similar patterns (Table 3). In addition, CK activities in all the isolated clones after 24 h incubation in the differentiation medium were determined. Without exception, CK activities of the clones that showed accelerated myotube formation were higher than those in the parent C2C12 cells, mock-transfected cells, or the clones that showed non-accelerated myotube formation. Thus, we can conclude that the accelerated myotube formation observed in the transformants is accompanied by a quick induction of both myosin and CK activity.

286 Takagi et al. ( E m J. Biochem. 231)

Table 4. Ability to incorporate methyl group into the genomic DNA by cytosine methylses. The genomic DNA prepared from the cells cultured in the growth medium was used as the methyl-group acceptor. The radioactivities were determined as described in Materials and Methods. The symbols (+) and (-) are the phenotypes for the myotube formation, as described in Table 1. The clones KCR(mock), MTR and RTM were obtained from the plasmid transfections of vacant vector, MeTise cDNA in sense and in reverse orientations, respectively. Values respresent means ? S. I). The numbers in parenthesis show the number of clones analyzed.

Clone Myotube MSssI M.HpnII

pmolipg DNA c2c12 5.72 L 0.16 (4)

6.06 I: 0.73 (4) KCR(mock) - MTR + 4.10 rt 0.42 (7) MTR - 3.88 5 0.70 (5) RTM + 5.66 i 0.24 (3)

7.32 i 0.74 (5) RTM -

~ ~~ ~

a The relative activity was normalized to the value of that obtained in parent C2C12 cells.

The DNA methylation levels in the transformants. Although the sense-oriented MeTase cDNA accelerated myotube formation of only some of the transformants, all the tested clones, regardless of their phenotypes, demonstrated about threefold higher MeTase activity than those of the clones transfected with vector alone or the reverse-oriented plasmid (Table 1).

We, therefore, considered the possibility that, although the MeTase activity was similar, the 5-methyldeoxycytidine content of the genomic DNA might be different in the transformants transfected with the sense-oriented MeTase plasmid, resulting in the difference in the ability to form myotubes. To evaluate the DNA methylation levels in the genomic DNA of the clones, we measured the ability to incorporate radioactivity from t3H]S- AdoMet into the genomic DNA by the methylases SssI (which recognizes the sequence CG) and HpaII (which recognizes the sequence CCGG). The transformants transfected with the sense- oriented MeTase cDNA incorporated 30% (about 2 pmol/pg DNA) less radioactivity using SssI and 50% less using HpaII than that of the parent C2C12 cells or the mock clones (Table 4). Statistic differences in the ability to incorporate the methyl group into the genomic DNA by methylase SssI were detected between KCR(mock) and MTR(+) (P < 0.01), and KCR(mock) and MTR(-) (P<O.Ol), but not between MTR(+) and MTR(-). The genomic DNA of the transformants transfected with the sense-oriented MeTase cDNA were significantly more methylated than that of the mock-plasmid-transfected cells. Among the transformants that were transfected with the sense-oriented plasmid, however, no difference was observed between the clones that demonstrated the accelerated myotube formation and those that did not. Thus, we concluded that the acceleration of myotube formation was not simply caused by the methylation levels of the genomic DNA.

MeTase levels at an early stage of the transfection. We no- ticed that once the transformants acquired the phenotype of accelerated or non-accelerated myotube formation, it was stable, and that cells retained the phenotype through several rounds of passages, while the cells continued to express threefold higher MeTase activity than parent C2C12 cells. Thus, the persistent expression of MeTase at high levels, was not the cause for the establishment of the phenotype. Accordingly, we considered it reasonable to assume that transient and high level expressions of MeTase induced an aberrant pattern of methylation in the early stage of the transfection, and that this caused the different phenotypes.

Therefore, to evaluate MeTase in the early stage of the transfection, the abundance and the activity of the transiently ex-

pressed MeTase in C2C12 cells were measured directly. When the sense-orientated plasmid was transfected and transiently expressed in C2C12 cells, a MeTase band that moved faster (about 20-kDa smaller) than the endogenous MeTase band in C2C12 or in MEL cells was the major band detected by Western blotting (Fig. 2A, lanes 1-3, 9 and 11). Since the faint high-mobility band was also observed in MEL cells (Fig. 2A, lane l), it might be a degradation product of intact MeTase (lanes 3 and 11). To determine if the newly synthesized MeTase had an identical mobility to that of the endogenous enzyme, the cells transfected transiently with the sense-oriented plasmid were pulse labeled with ["Slmethionine and immunoprecipitated with anti-MeTase serum. The major MeTase band in the immunoprecipitates again moved faster in a SDS/polyacrylarnide gel, with a mobility identical to that detected by Western blotting and clearly distinct from that of the major form of endogenous MeTase (Fig. 2A, lanes 5-7 and 13-15). A high-mobility MeTase was previously reported when mouse MeTase cDNA was transiently expressed in COS cells (Czank et al., 1991 ; Carlson et al., 1992). In contrast to these transient expressions, however, the stable transformants that were transfected with the sense-oriented plasmid showed a major band with a normal-mobility MeTase band. either by irnmunoblotting or by immunoprecipitation of the protein with specific antibodies (Fig. 2 B). Intriguingly, after several passages during the cloning to isolate the stable transformants, the high-mobility population of MeTase disappeared (or, in some cases, became a minor species).

We next measured the MeTase activities of nuclear extracts prepared from cells that overexpressed the MeTase cDNA, either transiently or stably. Poly[d(G-C) . poly[d(G-C)] and non-methylated synthetic 33-bp oligonucleotide, as well as poly[d(I-C) J . poly[d(I-C)] and the hemi-methylated synthetic oligonucleotide, were used as methyl-group acceptors. The activity required to methylate the substrates poly[d(G-C)] . poly[d(G-C)] and non- methylated synthetic oligonucleotide is a measure of the poten- tial de novo methylation activity of MeTase. All nuclear extracts, except for that prepared from the cells that were transiently transfected with the sense-oriented plasmid, showed activities of less than 2 pmol . h-' . mg protein-' when poly[d(G-C)] . poly[d(G-C)] was used as a substrate (Table 5). In striking contrast, the cells that transiently expressed the sense-oriented plasmid and showed a major high-mobility MeTase band, showed more than 10-fold higher de now methylation activity (26.7 pmol . h . mg protein-') than the other cells (Table 5). Similar tendencies in the activities were observed when non-methylated and hemi- methylated synthetic oligonucleotides were used as methyl- group acceptors (Table 5). We consider it likely that this high de now methylation activity at the early stage of the transfection

Takagi et al. (Eur. J. Biochern. 231) 287

Table 5. Methylation activities of transiently and stably expressed exogenous MeTase. MeTase activity was measured as described in Ma- terials and Methods. Total activity was measured using poly[d(I-C)] . poly[d(I-C)] [d(I-C)], 'maintenance' methylation activity was determined using synthetic hemi-methylated 33-bp oligonucleotide (hemi- SmC-oligo), and de novo methylation activity was evaluated using poly[d(G-C)] . poly[d(G-C)] [d(G-C)] and synthetic non-methylated synthetic 33-bp oligonucleotide as substrates, respectively. The symbols (+) and (-) are the phenotypes for the myotube formation, as described in Table 1.

9 10 11 12 M,, 13 14 15 16 x lo3

B

myotube + - - + + - -

1 2 3 4 Mr 5 6 7 8 9 x 1 0 ' ~

Fig. 2. Immunodetections of transiently or stably expressed MeTase. (A) The plasmids of pKCR-MTR (sense-oriented, lanes 3, 7, 11 and IS) or pKCR-RTM (reverse-oriented, lanes 4, 8, 12 and 16) were transfected into C2C12 cells and the transient expression of MeTase was detected. After the recovery of the cells in the growth medium for 24 h, nuclei were prepared for analysis by Western blotting (lanes 1-4 and 9-12), or incubated further with EXPFE3SS35S for the immunoprecipitation (lanes 5-8 and 13-16). The membranes were exposed overnight (lanes 1-8) or for 2 h (lanes 9-16). (B) The parent C2C12 and the stable- transformant cells either transfected with sense-oriented (M11-3, M2-1 and MTR4c), or with reverse-oriented (RS-3) plasmid were analyzed by Western blotting (lanes 1-4) or immunoprecipitation of the 35S-labeled MeTase as in (A) (lanes 5-9). 30 pg of transiently expressed and 100 pg stably expressed nuclear proteins were applied for the Western blotting. The arrowheads indicate the position of the endogenous MeTase and the arrows indicate the positions of the MeTase that showed high mobility. The symbols (+) and (-) are the phenotypes as shown in Table 1. The MEL cells have five copies of MeTase genes and express high levels of MeTase (Bestor et al., 1988). Molecular-mass standards (M,XlO. ') are indicated.

Clone/ Myo- Methylation activity plasmid tube for methyl group acceptor

d(1-C) d(G-C) hemi- oligonu- SmC- cleotide oligonucleotide

pmol . h-' . mg protein-'

c2c12

Transient pKCR(mock) pKCR-MTr pKCR-RTM

Stable M3-1 Mll-4 Rll-2 R10-5

12.3 0.8 9.8 1.6

9.9 1.2 9.2 1.9 131.8 26.7 67.0 20.9

10.9 1.4 8.5 1.1

+ 28.3 2.0 15.5 2.0 25.4 1.7 17.3 2.2

+ 4.7 0.6 6.8 0.9 9.2 0.8 7.2 1.2

-

-

the cytomegalovirus promoter into C2C12 cells. Five out of SS clones (9 %) demonstrated the phenotype of accelerated myogenesis, and none of 33 control transformants bearing the mock plasmid showed this phenotype. MeTase activities of the nuclear extract prepared from transiently transfected cells with the sense-oriented plasmid under the control of cytomegalovirus promoter were 22.2 pmol . h-' . mg protein-' and 6.7 pmol . h-' . mg protein-', respectively, when poly[d(I-C)] . poly[d(I-C)] and poly[d(G-C)] . poly[d(G-C)] were used as substrates. When MeTase cDNA in pKCRH2PL was transfected, 22 out of 31 clones (71 %) demonstrated the phenotype of accelerated myogenesis (Table 1) and the transiently expressed MeTase activities were 131.8 pmol . h-' . mg protein-' and 26.7 pmol . h-' . mg protein-', respectively, when poly[d(I-C)] . poly[d(I-C)] and poly[d(G-C)] . poly[d(G-C)] were used as substrates (Table 5) . Thus, as expected, the frequency to acquire the accelerated myotube formation phenotype correlated with MeTase activity in the early stage of the transfection.

Possible target gene(s) of DNA methylation. As described above (Tables 4 and S), the methylation levels of the DNA transfected with the sense-oriented plasmid were in the same range, regardless of the differences in the cell phenotype. This suggested that not the absolute amount of methylation, but the methylation of specific sites in the genoinic DNA, were important for the ability to differentiate rapidly.

We, therefore, tested whether or not the expressions of myogenic factors might be altered (Weintraub, 1993; Olson and Klein, 1994). In MTR4c cells, which showed the phenotype of accelerated myotube formation (Fig. 3A), MyoDl was already fully expressed under the conditions of growth while, in MTR3g cells, which showed non-accelerated myotube formation, and the parent C2C12 cells, MyoDl was expressed at low levels under these conditions. The expression of MyoDl is at a low level

is the reason for the high methylation levels and the accelerated myotube formation of the stable transformants transfected with the sense-oriented plasmid.

If this were the case, the frequency of the transformants that give accelerated myotube formation should depend on the MeTase activity at the early stage of transfection. To address this issue, we transfected MeTase cDNA under the control of

288

A

Takagi et al. (EUK J. Biochem. 231)

C2C12 ,MTR3g (-! MTR4c (4-1 A -

1 2 3 4 5 6 7 8 Fig.3. Expression of MyoDl, myogenin and Id in C2C12 and the transformant cells. (A) RNA was prepared from C2C12 cells (lanes 1 - 5) and MTR3g cells (lanes 6-10) that showed non-accelerated myotube formation, and from MTR4c cells (lanes 11-16) that showed accelerated myotube formation, before the medium was changed to the differentiation medium (lanes 1, 6 and I l ) , and 12 (lanes 2, 7 and 12), 18 (lane 13), 24 (lanes 3, 8 and 14), 36 (lanes 4, 9 and 15), and 48 h (lanes 5 , 10 and 16) after the medium change. Northem-blotting analysis was performed as described in Materials and Methods. MyoD1, myogenin, Id and GraPDH (internal standard) were sequentially.probed on the same sheet. B. RNA was prepared from C2C12 cells and the indicated transformants cultured under the growth conditions. MyoDl and GraPDH probes were hybridized on the same sheet. The symbols (+) and (-) are the phenotypes for the myotube formation, as described in Table 1. The standards of 18s and 28s ribosomal RNAs are indicated.

Hpall Mspl

1 2 3 4 5 6 7 8 9 10111213141516

when C2C12 cells are proliferating, and is induced in confluent cells and/or in the differentiation medium (Ichikawa et al., 1993). The induction of myogenin in MTR4c cells was significantly more rapid compared to induction in MTR3g cells and the parent C2C12 cells (Fig. 3A). As myogenin acts downstream of MyoDl and is induced only after the terminal differentiation program starts (Olson and Klein, 1994), a quick expression of myogenin in MTR4C cells seems to be the result of accelerated terminal differentiation of the cells. The down regulation of the expression of Id, a negative factor for myogenesis (Benzera et al., 1990), was similar in MTR4c, MTR3g and C2C12 cells. MRF4 was not detected and Myf5 was faintly detected after differentiation in C2C12 cells (Ichikawa, K. and Takagi, H., un- published observation) and the transformants showed similar expression profiles (data not shown). The enhanced expression of MyoDl, even in growing cells, was also observed in three addi- tional clones that showed accelerated myotube formation and was not observed in two clones that showed the non-accelerated phenotype (Fig. 3 B).

The methylation state of the MyoDl gene in the transformants was evaluated by HpaII and MspI digestion. All four clones that showed accelerated myotube formation (Fig. 4) de-

I -: i B r n i Hindlil 1 2 3 4 5 6 7

Smal Hindlll smal

probe 1 probe 2

probe 3 b- 1 IHpall E x a ” I site MyoDl gene 0.5Kb

Fig. 4. Southern-blotting analysis of the MyoDl gene by methylation- sensitive HpuII. The genomic DNA (10 pg) from C2C12 and indicated clones was digested first with HindIII, then with HpaII or MspI. The membrane sheet was probed with the 5‘ HindIII-SmaI fragment [probe 1, (A)] the SmaI-Smal fragment [probe 2, (B)] or the 3’ SmaI-Hind111 fragment [probe 3, (C)] shown in the figure. The arrowheads indicate the position of the 2.6-kb HindIII-Hind111 fragment of MyoD1. The symbols (+) and (-) are the phenotypes for the myotube formation, as described in Table 1. The standards (kb) of the HindIII-digested fragments of lDNA and the HaeIII-digested fragments of 4x174 DNA are indicated.

monstrated resistance to HpaII digestion and all three clones that showed the non-accelerated phenotype demonstrated a rather high HpaII susceptibility. The 2.4-kb and 1.8-kb HpaII fragments detected with probe 2 (the SmaI-SmaI fragment; Fig. 4B) were observed selectively in the clones that showed accelerated myotube formation. The 2.4-kb fragment was also detected with probe 1 (the 5’ HindIII-SmaI fragment; Fig. 4A) and with probe 3 (the SmaII-Hind111 fragment; Fig. 4C), and the 1.8-kb fragment was also detected with probe 3 but not with the probe

Takagi et al. ( E m J. Biochem. 231) 289

Table 6. Expression and methylation level of MyoDl gene in the transformants. The expression levels of the MyoDl transcript in the transformants shown in Fig. 5 were normalized to that of GraPDH in the same sheet. The amount of 1.8-kb and 2.4-kb fragments, the HpaII digests shown in Fig. 4, were determined by image analyzer (BAS 2000, Fuji Photo Film) and normalized to the sum total of the digests. The symbols (+) and (-) are the phenotypes for the myotube formation, as described in Table 1. Normalized ratios of MyoDl/GraPDH expressions are shown in parentheses.

Clone/ Myo- MyoDl/GraPDH Content of cell tube 1.8 kb + 2.4 kb

Exp. 1 Exp. 2 Exp. I Exp. 2

c2c12 MTR4c MTRla MTR3 1 MTR3f

MTR7-4 MTR 10- 1 MTRld MTR2h MTR3g

MTR5-4

MTR1-2

MTR4-3

MTR6-2

0.14 (1) + 0.49 (3.5) + 0.42 (3.0) + 0.42 (3.0) + 0.34 (2.4) + + + - 0.09 (0.6) - 0.09 (0.6)

0.11 (0.8) - - -

-

0.22 (1) 21 0.72 (3.3) 73

74 76 78

1.42 (6.6) 0.43 (2.0) 0.61 (2.8)

27 10

5 0.01 (0.1) 0.26 (1.2) 0.27 (1.3)

%

25 68

55 40 50

6 26 35

1. Accordingly, the 2.4-kb fragment was produced by digestion at the most 3’ HpaII site (site 8), and the 1.8-kb HpaII band was produced by digestion at both the most 3’ HpaII site and the most 5’ SmaI site (sites 1 and S), respectively. The remaining seven or six HpaII sites (sites 1-7 or 2-7) were resistant to the digestion and hence allowed the production of 2.4-kb or 1.8-kb fragments, respectively. We conclude that the HpaII sites in exon 1 through exon 2 (sites 2-7) are heavily methylated in the clones that show accelerated myotube formation.

Results (Table 6) indicate that the methylation levels of sites 2-7 and the expression level of the MyoDl transcript are positively correlated.

DISCUSSION The methylation of genomic DNA contributes to the control

of tissue-specific gene expression (Razin and Cedar, 1991 ; Bird, 1992). When the cells start to differentiate, many of the genes that are induced to express become demethylated. The skeletal- muscle-type a-actin gene, for example, has to be demethylated in its promoter region to allow expression (Yisraeli et al., 1986). When the antisense strand of MeTase is forced to express in C3HlOT1/2 cells, the cells start to differentiate into myoblasts and myotubes (Szyf et al., 1992), as when the cells are treated with 5-azacytidine, the demethylating reagent (Jones and Taylor, 1980). These reports suggest that the genomic DNA must be demethylated for myogenesis to occur. Therefore, we expected that if MeTase cDNA was overexpressed in myoblast C2C12 cells, the terminal differentiation of the cells to form myotubes would be inhibited. However, unexpectedly, many of the transformants isolated by transfecting the sense-oriented plasmid- containing MeTase cDNA had the ability to form myotubes quickly compared to the parent C2C12 cells. This effect was neither due to the vector-containing simian virus 40 promoter by itself, nor was it the result of recombination of the transfected

MeTase cDNA, but to the expression of the MeTase. Since the frequency of appearance of the phenotype of rapid myotube formation depended on the strength of the promoter to express MeTase, the DNA methylation appeared to directly affect the phenolype. However, once the phenotypes had been fixed, the continuous expression of the threefold higher MeTase activity in the stable transformants than that in the control cells had no further influence on the phenotype. This high MeTase activity affected neither the methylation level of the DNA nor the phenotypes of the transformants. The MeTase expressed in the early stage of the transfection had very high de novo methylation activity. It was strongly suggested that this high de novo methylation activity was the cause of the effect.

It is still difficult to explain, however, how different phenotypes arise after identical transfection of the MeTase cDNA, in particular as all clones exhibit an identical level of DNA methylation and MeTase activity. The only explanation for this is that the genes targeted for methylation were different, thus resulting in different phenotypes. In some of the isolated transformants that showed accelerated myotube formation, MyoDl transcripts were expressed at high levels, even under the conditions of growth, where expression of MyoDl in the parent C2C12 cells is low (Ichikawa et al., 1993). This overexpression of MyoDl could be the reason for the accelerated myotube formation, i.e. the cells were ready to differentiate. The enhancement of the transcript level of MyoDl was strongly correlated with the methylation of HpaII sites from the first through the second ex- ons of the gene (sites 2-7). In a second transfection experiment, three out of 61 clones transfected with antisense-oriented plasmid showed accelerated myotube formation. The positive phenotype in the three clones, RTM1-4, RTM9-4 and RTM11-2, was accompanied by a quick induction of CK activity. However, none of the clones showed high MeTase activity (Table 1), nor were more clones methylated than in C2C12 cells (Table 4). RTMl-4 cells neither expressed high MyoDl transcripts under the growing condition, nor were heavily methylated at the HpaII sitse 2-7. RTM9-4 cells showed a slightly high level of the MyoDl transcript under the growth condition but, in contrast, was unmethylated at the HpaII sites. The positive phenotype for these cells may be due to a factor other than methylation. Interestingly, RTMl l-2 cells expressed a threefold higher MyoDl transcript under the growt conditions than C2C12 cells, and HpaII sites 2-7 were heavily methylated (data not shown). Electroporation by itself might directly affect the methylation state of the MyoDl gene in this case. This, although is not proved, supports the idea that the methylation at the specific site(s) in the MyoDl gene can directly promote its expression.

It is reported that the methylation levels of the entire MyoDl gene in the cell lines established by 5-azacytidine treatment of C3HlOT1/2 cells do not correlate with MyoDl gene expression (Michalowsky and Jones, 1989). Moreover, both in vivo and in vitro, MyoDl transcription are inhibited by methylation of the gene at all CpG sites (Zingg et al., 1991). HpaII sites 1-4 of the MyoDl gene are unmethylated in muscle as well as in liver, kidney and heart (Zingg et al., 1994); sites 5 and 6 are partially methylated in muscle and spleen but not in testis and C3HlOTlI 2 cells (Jones et al., 1990; Zingg et al., 1994). The result is not compatible with our present result that the methylation of HpaII sites 2-7 in the MyoDl gene positively correlate to the high level of its expression. However, it should be noted that all reports describe the relationship between MyoDl gene methylation and expression when the cells or tissues are already dif- ferentiated into myotubes or under the conditions of differentiation. Under these conditions, even the clones that showed an accelerated differentiation phenotype with the methylated HpaII sites did not show any differences in MyoDl expression with

290 Takagi et al. ( E m J. Biochem. 231)

the control cells (see Fig. 3). The methylated HpaII sites were not demethylated during myotube formation (data not shown), indicating that methylation does not affect MyoDl expression under the conditions of differentiation. The methylation and expression of the MyoDl gene positively correlated only in C2C12 cells under the growth conditions. At present, we do not know if this methylation of the gene directly affected the transcription of MyoDl. We cannot, of course, exclude the fact that genes other than the MyoDl gene, which were not tested, are also targets of methylation, and affect myotube formation.

Generally, the methylation of a gene suppresses its expression (Razin and Cedar, 1991). However, the expression of the class-I multihistocompatibility gene, H-2K, is positively correlated with the extent of methylation (Tanaka et al., 1983), and a recent report revealed that the complete lack of methylation suppresses the expression of the imprinted genes Igf2 and Igf2r (Li et a]., 1993), the genes that were specifically methylated in the expressing chromosomes (Sasaki et al., 1992; Stoger et al., 1993). These reports indicate that some of the genes are induced by methylation. We consider it likely that methylation of the MyoDl gene directly activates the transcription or inhibits the binding of a suppressor.

The transient transfection of MeTase into C2C12 cells caused expression of the high-mobility MeTase protein. This molecular species was the major component detected by pulse labeling of the protein. An identical phenomenon was observed when the cDNA was expressed in COS cells (Czank et al., 1991 ; Carlson et al., 1992). The mouse MeTase in the oocyte and in the early stage embryo also moves faster in SDS/polyacrylamide gel electrophoresis than MeTase of somatic cells (Carlson et al., 1992). MeTase in the early stage embryo does not translocate to the nucleus (Carlson et al., 1992), while that expressed transiently in C2C12 cells completely localized to the nucleus (data not shown). The high-mobility population of MeTase could be the degradation product of the newly synthesized molecule (Bes- tor and Ingram, 1985) or arise because it lacks a post-transla- tional modification(s) (Carlson et al., 1992). At present, we have no direct evidence, however, that the high mobility of MeTase is responsible for the high de now methylation activity at the early stage of transfection.

We thank Peter Walter at University of California, at San Francisco, USA, for critical reading of the manuscript and many helpful discus- sions. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan to ST.

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