Dimethyl Sulfoxide Has an Impact on Epigenetic Profile in Mouse Embryoid Body - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:59 |只看该作者 |倒序浏览 |打印

作者：Misa Iwatani, Kohta Ikegami, Yuliya Kremenska, Naka Hattori, Satoshi Tanaka, Shintaro Yagi, Kunio Shiota作者单位：Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, Tokyo, Japan


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      【摘要】% |8 g2 U  W& q' r" s+ l) g
   Dimethyl sulfoxide (DMSO), an amphipathic molecule, is widely used not only as a solvent for water-insoluble substances but also as a cryopreservant for various types of cells. Exposure to DMSO sometimes causes unexpected changes in cell fates. Because mammalian development and cellular differentiation are controlled epigenetically by DNA methylation and histone modifications, DMSO likely affects the epigenetic system. The effects of DMSO on transcription of three major DNA methyltransferases (Dnmts) and five well-studied histone modification enzymes were examined in mouse embryonic stem cells and embryoid bodies (EBs) by reverse transcription-polymerase chain reaction. Addition of DMSO (0.02%¨C1.0%) to EBs in culture induced an increase in Dnmt3a mRNA levels with increasing dosage. Increased expression of two subtypes of Dnmt3a in protein levels was confirmed by Western blotting. Southern blot analysis revealed that DMSO caused hypermethylation of two kinds of repetitive sequences in EBs. Furthermore, restriction landmark genomic scanning, by which DNA methylation status can be analyzed on thousands of loci in genic regions, revealed that DMSO affected DNA methylation status at multiple loci, inducing hypomethylation as well as hypermethylation depending on the genomic loci. In conclusion, DMSO has an impact on the epigenetic profile: upregulation of Dnmt3a expression and alteration of genome-wide DNA methylation profiles with phenotypic changes in EBs. 8 O6 |' @7 Z6 g! g
      【关键词】 Dimethyl sulfoxide DNA methylation Epigenetics Differentiation DNA methyltransferase- u3 j7 l- O) n! n6 ?
   INTRODUCTION$ S9 f0 F" c6 e! U- _! s
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Dimethyl sulfoxide (DMSO), an amphipathic molecule, is one of the most commonly used chemicals in the biological and medical sciences as a solvent for water-insoluble substances and a cryopreservant for various cell lines. It has multiple effects on cellular functions (e.g., metabolism and enzymatic activity) and on cell growth by affecting cell cycle and apoptosis .& l1 T, v! ~: I! t* E+ r
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Mammalian development and cellular differentiation are controlled by DNA methylation; the developmentally essential genes Oct-4 and Sry are controlled to be expressed during a limited period of development by DNA methylation .

In the establishment and maintenance of the proper DNA methylation patterns in the mammalian genome, DNA methyltransferases (Dnmts) play critical roles. To date, five Dnmts, Dnmt1, 2, 3a, 3b, and 3L, have been identified. Because in vitro enzymatic activity is lacking in Dnmt2 and 3L . These enzymes and Dnmts coordinate the epigenetic systems.
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Mutations in genes of epigenetic factors have been implicated as the causes of various diseases. Mutations in DNMT3b are associated with ICF syndrome (immunodeficiency, centromere instability, and facial abnormalities) .
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Based on these observations, we hypothesized that the effects on cell fate by DMSO should be interpreted by its effects on the epigenetic systems. We examined this hypothesis by using mouse embryonic stem cells (ESCs) and embryoid bodies (EBs). Differentiation of ESCs into EBs has been used as a model of normal and abnormal mammalian development . In this study, mRNA levels of epigenetic regulators, including Dnmts and histone modification enzymes, were analyzed. We also investigated a genome-wide DNA methylation profile in EBs to examine the effects of quantitative changes in an enzyme's expression.

MATERIALS AND METHODS( K) i4 Y0 a" V, |
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Culture of ESCs, EBs, and DMSO Treatment

The ESC lines (MS12) derived from C57BL/6 strain mice  were cultured on embryonic fibroblast feeder cells with ESC medium: Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 15% fetal bovine serum and 1,000 U/ml of leukemia inhibitory factor (LIF; ESGRO, Chemicon International, Temecula, CA, http://www.chemicon.com). At passage 18, ESCs were treated with or without 0.1% (vol/vol) DMSO (Wako Pure Chemicals, Osaka, Japan, http://www.wako-chem.co.jp/english) for 4 days and harvested. EBs were induced by culturing ESCs at passage 16 without a feeder layer and LIF in bacteriological Petri dishes and simultaneously treated with or without DMSO. They were cultured under DMSO treatment for 4 days in EB medium:DMEM (Invitrogen) supplemented with 10% fetal bovine serum and then collected for nucleic acids and protein extractions.  ~% w: \* ]( t2 A3 L

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction! T( Z& A6 A+ B. O# T$ _. y2 f

Total RNA was purified from cultured ESCs and EBs using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For reverse transcription-polymerase chain reaction (RT-PCR), first-strand cDNA was synthesized from 1.0 µg of total RNA using a Superscript first-strand synthesis system with random hexamer primers (Invitrogen). RT-PCR was performed with rTaq polymerase (TOYOBO, Tokyo, http://www.toyobo.co.jp/e) except in the case of Dnmt3a, which was amplified with Immolase (BIOLINE, London, http://www.bioline.com). Sets of primer sequences for RT-PCR were as follows: Dnmt1: 5'-CAGGAGTGTGTGAGGGAG-3' and 5'-GGTGTCACTGTCCGACTTGC-3', Dnmt3a: 5'-ACCCATGCCAAGACTCACCTTC-3' and 5'-TCCACCTTCTGAGACTCTCCAG-3', Dnmt3b: 5'-TCAGACACGAAGGATGCTCC-3' and 5'-ACAGGGTACTCCTGCACATG-3', G9a: 5'-TTTGGCCATGAGGCTGTT-3' and 5'-CCAGATGCATGTCATCACTCA-3', Suv39h1: 5'-GGAGAAAGATGGCGGAAA-3' and 5'-GACAAGAAAGCTTGGCTAGT-3', Suv39h2: 5'-TCTTTGGCGACGAGTGTG-3' and 5'-AGAATCTGGCCATCCTTTCC-3', Sir2: 5'-CTGACGACTTCGACGACGAC-3' and 5'-TGCTGAACAAAAGTATATGGACCTATC-3', mDot1: 5'-AACTATGTCCTGATCGACTACG-3' and 5'-TCCTCTGTCATCTTGATCTCATC-3', and ß-actin: 5'-TTCTACAATGAGCTGCGTGTGG-3' and 5'-ATGGCTGGGGTGTTGAAGGT-3'. The thermocycling program used with rTaq polymerase was an initial cycle of 95¡ãC for 1 minute, followed by 30 cycles of 94¡ãC for 30 seconds, and 30 seconds at the following annealing temperatures: 58¡ãC for ß-actin, 60¡ãC for Suv39h1, 62¡ãC for Dnmt1 and mDot1, and 65¡ãC for Dnmt3b, G9a, Suv39h2, and Sir2 and then 72¡ãC for 1 minute. RT-PCR for Dnmt3a using Immolase was performed with an initial cycle of 95¡ãC for 10 minutes, followed by 30 cycles of 94¡ãC for 30 seconds, 65¡ãC for 30 seconds, and 72¡ãC for 1 minute. A digitized image of ethidium bromide-stained gel was analyzed by densitometry with NIH Image 1.61 software (National Institutes of Health, Bethesda, MD, http://www.nih.gov).  T, b1 b$ C1 k# w. M

Real-Time Quantitative RT-PCR! }' L& p) T  ?; v

Expression of Dnmt3a was monitored by SYBR Green I in SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) on a ABI PRISM 7500 or 7900 HT sequence detection system (Applied Biosystems) according to the manufacturer's protocol. Nine nanomolar each of forward and reverse primers described above for Dnmt3a or ß-actin and 1 µl and 0.5 µl of cDNAs were used for Dnmt3a and ß-actin, respectively, in 20 µl. A standard curve was established by a dilution series of cDNA to estimate mRNA levels. Correlation values (r2) of the standard curve were 0.98 and 0.99 for Dnm3a and ß-actin, respectively. The slope of the standard curve was determined to calculate PCR efficiency using the equation: PCR efficiency = 10¨C1/slope ¨C 1. The values for Dnmt3a and ß-actin were 1.04 and 1.00, respectively. Quantitative expression level was calculated using the following equation: the value = 1/(1 PCR efficiency)CT. The slope of the standard curve and cycle thresholds (CTs) were analyzed using ABI PRISM 7500 SDS software. Expression of Dnmt3a was normalized to ß-actin as an internal control. At least three independent PCRs were performed in duplicate for all samples.
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Protein Extraction and Western Blotting
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Mouse EBs and ESCs were lysed in RIPA buffer (50 mM Tris-HCl . The chemiluminescence signals, which were obtained with SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical), were visualized by Chemi-Smart 3000 (Vilber Lourmat, Marne-la-Vall¨¦e, France, http://www.vilber.com). For reprobing, the blotted membrane was rinsed with Restore Western Blot Stripping Buffer (Pierce Chemical).- d4 M2 f8 M3 J. P

Preparation of Genomic DNA
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Genomic DNA was extracted as previously described ).
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Restriction Landmark Genomic Scanning& r$ F0 b% y3 z& y% P- f
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The restriction landmark genomic scanning (RLGS) was performed using the restriction enzyme combination of Not I, Pvu II, and Pst I as described previously dGTP (GE Healthcare). Labeled DNA was digested by Pvu II (Nippon Gene) after first dimensional electrophoresis in 0.9% agarose disc gel. After in-gel digestion with Pst I (Nippon Gene), the DNA fragments were separated by a second dimensional electrophoresis in a polyacrylamide slab gel. The gel was then dried and exposed to x-ray film (Kodak, XAR 5; Eastman Kodak, Rochester, NY, http://www.kodak.com) for 2¨C3 weeks at ¨C80¡ãC.

Methylation-Sensitive Quantitative Real-Time PCR
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DNA methylation status at Sall3 locus was evaluated using methylation-sensitive quantitative real-time PCR as previously described . Twenty nanograms of DNA treated with or without Not I was subjected to PCR with a primer pair amplifying a genomic fragment containing the Not I site. The methylation ratio was determined as the proportion of undigested DNA in Not I-treated DNA to that in Not I-untreated DNA. The amplification was monitored with SYBR green on an ABI Prism 7500 Sequence Detection System (Applied Biosystems) following the manufacturer's protocol. Initial DNA amount in the reaction mix was normalized with the value obtained with the primer pair of Xist1 that was designed to amplify fragments without the Not I site. More than three independent PCRs in triplicate were performed. The primers of Sall3 and Xist1 are as follows: Sall3: 5'-TTATACAACCTCGAACTAGCTGGG-3' and 5'-GCATCCTGAATCCATGAACCCT-3', Xist1: 5'-CACACACACCCTGCCCAATC-3' and 5'-GGGATTCGCCTTGATTTGTGGT-3'.
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Southern Blot Hybridization

Genomic DNA that was digested with Msp I (Takara) or Hap II (Takara) was electrophoresed on a 0.8% agarose gel. After being hydrolyzed with 0.25 N HCl and denatured with 1.5 M NaCl in 0.5 N NaOH, DNA was transferred to a nylon membrane. The membrane was hybridized with pMO for endogenous C-type retrovirus (MoMuLV) (GenBank accession: NC_001501) or pMR150 for minor satellite repeats (X14469  and X07949), which was labeled with Gene Images random prime labeling module (GE Healthcare). The bound probes were detected by using Gene Images CDP-star detection module (GE Healthcare) with x-ray film (RX-U; Fuji, Kanagawa, Japan, http://www.fujifilm.com)., f3 U8 e, Y8 i1 u) G
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RESULTS0 N: p9 w( M6 {0 h3 w

DMSO Increases Expression of Dnmt3as in ESCs and EBs  J7 U- J! Z5 s. @" b$ s. h
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ESCs, which were maintained without DMSO treatment, were cultured under differentiation conditions to form 4-day EBs in the absence or presence of various concentrations (0.02%¨C1%) of DMSO (Fig. 1A). In contrast to uniform spheres of EBs cultured without DMSO, EBs with irregular shapes and increased sizes appeared in high concentrations (0.5%, 1%) of DMSO (Fig. 1B). In these phenotype changes of EBs induced by DMSO, we presume that epigenetic systems should be involved.% |: D5 E/ x# P, p$ x5 \3 v( W

Figure 1. Culture of ESCs and of EBs with or without DMSO. (A): Cultivation scheme of mouse ESCs (MS12 line) and EBs. ESCs were cultured for 4 days in the presence of LIF and feeder layer with or without DMSO, or were induced to form EBs by removal of LIF and feeder layer, and were cultured for another 4 days with various concentrations of DMSO. (B): Micrographs of EBs cultured in medium containing DMSO. Concentrations are indicated above the images. Scale bars = 200 µm. Abbreviations: DMSO, dimethyl sulfoxide; EB, embryoid body; ESC, embryonic stem cell; LIF, leukemia inhibitory factor.) E4 F7 G* f" z/ e6 ]  r6 o
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By semiquantitative RT-PCR, we examined expression of genes related to epigenetic systems such as Dnmts and histone modification enzymes. mRNA levels of Dnmt1 and Dnmt3b, which were expressed in ESCs and EBs, were not affected by DMSO treatment as judged by the densitometry of RT-PCR products (Fig. 2A). The fold changes of intensities of bands between the control and 1% DMSO-treated EBs showed 1.10 and 0.93 for Dnmt1 and Dnmt3b, respectively. In spite of the report that mDot1 mRNA was increased by DMSO treatment in mIMCD cells , mRNAs for histone methyltransferases (G9a, Suv39h1, Suv39h2, and mDot1), and a histone deacetylase, Sir2, were expressed equally in ESCs as well as EBs at different DMSO concentrations. On the contrary, intensities of Dnmt3a in EBs, treated with 0.5% and 1% DMSO, increased almost double.
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Figure 2. The effects of DMSO on expression of epigenetic factors in ESCs and EBs. (A): Expression of epigenetic factors in ESCs and EBs was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Reactions were carried out with ). (C): Dnmt3a protein expression was analyzed by Western blotting. The positions of proteins (left side) and molecular weights of the markers (right side) are shown. Abbreviations: DMSO, dimethyl sulfoxide; Dnmt, DNA methyltransferase; EB, embryoid body; ESC, embryonic stem cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.* I: E7 i. J: N
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Real-time quantitative RT-PCR confirmed the increase of Dnmt3a mRNA (Fig. 2B). In ESCs, 0.1% of DMSO treatment caused a slight increase in the level of Dnmt3a mRNA. An approximately twofold increase in Dnmt3a mRNA was observed by differentiation of ESCs to EBs. In EBs, statistically significant increases in levels of Dnmt3a mRNA corresponded to increases in DMSO dosage.

To address the question whether Dnmt3a protein would increase coordinately with transcripts of Dnmt3a by DMSO treatment, Dnmt3a proteins in EBs were analyzed by Western blotting. Two distinct bands, which were detected by anti-Dnmt3a monoclonal antibody, indicated the expression of Dnmt3a and Dnmt3a2 in 4-day EBs. Both types of Dnmt3a protein levels in EBs treated with 0.5% and 1% DMSO increased to two times those in nontreated EBs (Fig. 2C). From this finding, taken together with upregulated mRNA level by DMSO treatment, it was clear that DMSO increased expression of Dnmt3as (Dnmt3a and Dnmt3a2).
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DMSO Affects DNA Methylation Status of Repetitive Sequences in EBs! ^! Z) Y: O1 T0 i& T' ^
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The mammalian genome consists of genic and nongenic regions, such as repetitive sequences, and the latter occupy a large part of the genome. Both regions are methylated de novo by Dnmt3as . Minor satellite repeats, which are located in the centromeric regions, and endogenous retroviruses, which are interspersed in the mouse genome, are families of repetitive sequences. To assess the effects of DMSO (0.1%) on methylation levels in EBs, Southern blot was performed using probes for minor satellite repeats (pMR150) and endogenous C-type retroviruses (pMO) (Fig. 3). The differences in amounts of small fragments between lanes I, DNA digested by a DNA methylation-insensitive restriction enzyme, Msp I, and lanes II, digested by a methylation-sensitive enzyme, Hap II, indicate that these repetitive sequences were hypermethylated in EBs. The 0.1% DMSO treatment caused the disappearance of the small fragments of minor satellite and C-type retrovirus repeats in the EB genome (lanes III). These data indicated that DMSO prompted DNA methylation of these nongenic regions in the EBs.( F4 R1 r1 C1 t! w) V9 F+ G0 z

Figure 3. Effect of dimethyl sulfoxide (DMSO) on DNA methylation status of repetitive sequences. Msp I-digested genomic DNA from untreated EBs (lane I) and Hap II-digested genomic DNA from untreated (lane II) and 0.1% (vol/vol) DMSO-treated EBs (lane III) were subjected to Southern blot hybridization using probes for minor satellite repeats (pMR150) and endogenous C-type retrovirus repeats (pMO). Molecular weights are indicated on the right panel.
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DMSO Affects Genome-Wide DNA Methylation Profiles of Genic Areas in EBs1 s* S$ y+ _: Z; U; Q( [2 X
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Next, we focused on the effect of DMSO on DNA methylation at the genic region using the RLGS method. In the RLGS profile, the spot is visible when the corresponding cutting site of Not I, a methylation-sensitive restriction enzyme, is hypomethylated, whereas it is invisible when the site is hypermethylated. Most Not I sites are in CpG islands . Therefore, RLGS enables us to simultaneously analyze thousands of loci in genic regions.
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The RLGS profiles, consisting of approximately 1,500 spots, were compared between control and 0.1% DMSO-treated EBs (Fig. 4A). In RLGS profiles of DMSO-treated EBs, 11 unique spots (T-DMR  12¨C15) disappeared in DMSO-treated EBs, indicating that DMSO caused hypermethylation of these four sites. Thus, 15 genomic loci were epigenetically affected by DMSO treatment, whereas thousands of loci remained unchanged (Fig. 4A, 4B).  p% g, `# J; @2 f. b! _7 L$ w

Figure 4. Analysis of genome-wide DNA methylation status in mouse EBs untreated or treated with DMSO and methylation status of Sall3 locus. (A): RLGS profile obtained with genomic DNA of EBs treated with 0.1% (vol/vol) of DMSO. The 15 spots, which differentially appeared by treatment with DMSO, are marked with numbered circles. (B): Higher magnification of the RLGS profile. The 11 RLGS spots that emerged (i.e., hypomethylated) and the four spots that disappeared (i.e., hypermethylated) by DMSO treatment are indicated with white and black arrowheads, respectively. (C): Methylation-sensitive quantitative real-time PCR analysis for Sall3 locus. Amplification curves with asterisks, circles, and squares represent Not I-treated genomic DNA of ESCs and of EBs with or without DMSO treatment, respectively (left panel). Methylation levels of Sall3 locus were estimated as described in Materials and Methods. Differences between samples were statistically analyzed by t test (*p

We compared these 15 spots (T-DMRs . By methylation-sensitive PCR, DNA methylation status of this locus in DMSO-treated EBs was estimated to be approximately 3.5 times lower than that in EBs without DMSO (Fig. 4C).
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Figure 5. Matching of T-DMRs (DMSO) to previously identified T-DMRs. T-DMRs (DMSO) 1¨C15 (Fig. 4) were compared with previously identified T-DMRs by matching RLGS profiles. T-DMRs (DMSO) 2 and 12 correspond to T-DMRs 148 and 253 (which were previously identified), respectively. The 13 novel RLGS spots¡ªT-DMRs (DMSO) 1, 3¨C11, and 13¨C15¡ªwere designated as T-DMRs 582 and 701¨C712, serially. Abbreviations: DMSO, dimethyl sulfoxide; EB, embryoid body; ESC, embryonic stem cell; RLGS, restriction landmark genomic scanning; T-DMR, tissue-dependent and differentially methylated region." z8 n% C/ A3 @- c/ h. d
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DISCUSSION

The present study clearly demonstrates that DMSO has an impact on the epigenetic regulatory system, changes the genome-wide DNA methylation status, and induces formation of structurally abnormal EBs. Irreversible phenotypic changes in Friend cells were induced by DMSO . Because DNA methylation plays a critical role in mouse development and the definition of cell properties, change in genome-wide DNA methylation profiles induced by DMSO treatment may be responsible for these phenomena.* @8 F( Y4 }- ^) d+ x  B0 [/ C0 ]+ _

The human and mouse genomes contain 30,000¨C40,000 genes; however, genes occupy only a small percentage of the approximately 3 x 109 bp haploid genome. In contrast, 41%¨C48% of the mammalian genome is composed of nongenic repetitive elements, including satellites interspersed repeats such as retroviruses , which suggests that the methylation status of many loci must be affected by DMSO.2 @$ p5 I, D' a1 ~
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Differentiation of ESCs to EBs causes both hypermethylation and hypomethylation at various loci genome-wide in mammals . The effects of DMSO on DNA methylation status of CpG islands during differentiation are summarized in Figure 6. When ESCs differentiate to EBs, 34 loci were hypermethylated, and 30 loci were hypomethylated, whereas 203 spots were unchanged. Of 34 loci that typically became hypermethylated during differentiation of ESCs to EBs, 11 remained hypomethylated after DMSO treatment. Similarly, the DNA methylation status of three out of the 30 hypomethylated loci, and one out of the 203 unchanged loci was affected by DMSO treatment. Thus, DMSO induces alteration of DNA methylation status at selected loci and generates a unique DNA methylation profile of EBs, accompanying the abnormal phenotypes.

Figure 6. The impact of dimethyl sulfoxide (DMSO) on tissue-dependent and differentially methylated region (T-DMRs). This figure illustrates the T-DMRs that were hypermethylated, hypomethylated, or unchanged during the differentiation of ESCs into EBs. T-DMRs with methylation status affected by DMSO are in bold italics. Numbers with white letters in shaded square boxes and numbers with black letters in the shaded balloon represent hypomethylated and hypermethylated loci by DMSO, respectively. Abbreviations: Dnmt, DNA methyltransferase; EB, embryoid body; ESC, embryonic stem cell.

We demonstrated the upregulation of mRNA and protein expression in Dnmt3as by DMSO. Overexpression of Dnmt3as causes de novo hypermethylation of both genic and nongenic regions in vivo . Therefore, DMSO-induced increased expression of Dnmt3as should be involved in hypermethylation occurring at nongenic regions and selected gene loci of EB genome.

Despite increased expression of Dnmt3as and hypermethylation of the repetitive sequences and the selected loci, a number of hypomethylated loci (11 spots) are greater than that of hypermethylated loci (four spots) in genic regions (Fig. 4). In many cancer cells with epigenetic abnormalities, genomic DNA has shown to be globally hypomethylated with hypermethylation at selected genes. Such locus-specific DNA methylation status on genomic loci is contributed by complex combinations of Dnmts and other epigenetic regulators. Dnmt1 and Dnmt3s functionally cooperate with each other during methylation of genomic DNA . Therefore, genome-wide alteration of the DNA methylation profile by DMSO should not be explained simply by the increased levels of Dnmt3as.! K" C' a3 w# b$ |
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CONCLUSION

We conclude that DMSO upregulates expression of Dnmt3as and affects DNA methylation status at restricted loci accompanied with abnormal EB formation. Physiological and toxicological assessment of chemical agents at epigenetic levels is important, and analysis of genome-wide DNA methylation profiles will be useful in evaluating epimutagens.$ l- g% K+ t3 l; \/ M2 d0 w
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DISCLOSURES
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The authors indicate no potential conflicts of interest.. D5 o* C. ^( d* y

ACKNOWLEDGMENTS8 P" ^" f! F1 w* n) Y
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We thank Dr. Maddy Roberts and Chiaki Maeda for proofreading the original manuscript. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences, Health Science Research Grant from the Ministry of Health, Labor and Welfare of Japan, and the Grant-in-aid for Scientific Research, Ministry of Education, Culture, Sports, Science and Technology, Japan (15208027, 15080202) (to K.S.).
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