Stem Cells http://www.stemcellsportal.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online July 13, 2006
Stem Cells Vol. 24 No. 11 November 2006, pp. 2549 -2556
doi:10.1634/stemcells.2005-0427; www.StemCells.com
© 2006 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0427v1
24/11/2549    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iwatani, M.
Right arrow Articles by Shiota, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iwatani, M.
Right arrow Articles by Shiota, K.

STEM CELL GENETICS AND GENOMICS

Dimethyl Sulfoxide Has an Impact on Epigenetic Profile in Mouse Embryoid Body

Misa Iwatani, Kohta Ikegami, Yuliya Kremenska, Naka Hattori, Satoshi Tanaka, Shintaro Yagi, Kunio Shiota

Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, Tokyo, Japan

Key Words. Dimethyl sulfoxide • DNA methylation • Epigenetics • Differentiation • DNA methyltransferase

Correspondence: Kunio Shiota, D.V.M, Ph.D., Laboratory of Cellular Biochemistry, Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan. Telephone: +81-3-5841-5472; Fax: +81-3-5841-8189; e-mail: ashiota{at}mail.ecc.u-tokyo.ac.jp

Received August 31, 2005; accepted for publication July 1, 2006.
First published online in STEM CELLS EXPRESS   July 13, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
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%–1.0%) 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
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 [1]. It also changes cell fates by inducing differentiation of various types of cells [1–4] and promoting blastocyst formation in animal cloning [5].

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 [6, 7]. Methylation is the sole modification of the vertebrate genome and occurs mainly at the 5-position of cytosines in cytosine-guanine (CpG) dinucleotides [8]. The modification is involved in epigenetic regulation of gene function, which is "mitotically and/or meiotically heritable, and can not be explained by changes in DNA sequences" [9]. Epigenetic systems regulate various genetic functions, including chromosomal stability, repression of transposable elements, and gene silencing [10–12] of developmentally regulated genes and genes expressed in a tissue-specific manner [13, 14]. Tissue-dependent and differentially methylated regions (T-DMRs) have been found in CpG-rich, unique sequences, including tissue-specific genes. The changes in genome-wide DNA methylation status of such T-DMRs provide every cell or tissue a unique DNA methylation profile consisting of methylated and unmethylated T-DMRs [15–17].

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 [18, 19], only Dnmt1, Dnmt3a, and Dnmt3b have been intensively studied [20, 21]. DNA methylation is associated with histone modifications in epigenetically regulated regions [20, 22–24]. Histone modifications were mediated by the following enzymes: G9a, Suv39h1, and Suv39h2 are histone H3 lysine nine (H3-K9) methyltransferases [12]; mDot1 is an H3-K79 methyltransferase [25]; and Sir2{alpha} is a class III histone deacetylase [26]. These enzymes and Dnmts coordinate the epigenetic systems.

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) [27], and those in MeCP2 are associated with Rett syndrome [28]. Epigenetic abnormalities have been reported in cloned animals [29–31] and cancerous cells [32] and are also caused by chemicals that are called "epimutagens" [33].

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 [34]. It is also a suitable model for monitoring epigenetic modifications because, during differentiation from ESCs, EBs establish a specific DNA methylation profile associated with both hypermethylation and hypomethylation at multiple loci [16]. 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
Culture of ESCs, EBs, and DMSO Treatment
The ESC lines (MS12) derived from C57BL/6 strain mice [35] 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.

RNA Extraction and Reverse Transcription-Polymerase Chain Reaction
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{alpha}: 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°C for 1 minute, followed by 30 cycles of 94°C for 30 seconds, and 30 seconds at the following annealing temperatures: 58°C for ß-actin, 60°C for Suv39h1, 62°C for Dnmt1 and mDot1, and 65°C for Dnmt3b, G9a, Suv39h2, and Sir2{alpha} and then 72°C for 1 minute. RT-PCR for Dnmt3a using Immolase was performed with an initial cycle of 95°C for 10 minutes, followed by 30 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 72°C 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).

Real-Time Quantitative RT-PCR
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 = 10–1/slope – 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.

Protein Extraction and Western Blotting
Mouse EBs and ESCs were lysed in RIPA buffer (50 mM Tris-HCl [pH 8.0], 400 mM NaCl, 1% Nonident P-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1 µg/ml aprotinin, 5 µg/ml pepstatin, 0.5 mM EDTA, and 0.5 mM NaF). The lysates were clarified by centrifugation at 15,000 rpm for 30 minutes at 4°C, and 5-µg aliquots of the lysates were subjected to SDS polyacrylamide gel electrophoresis on 7.5% gel. Protein concentration was determined by using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL, http://www.piercenet.com) according to the manufacturer's instructions. The proteins were transferred to polyvinylidene difluoride membrane and probed with 1:125 diluted anti-Dnmt3a monoclonal antibody (clone 64B1446; Imgenex, San Diego, http://www.Imgenex.com) or 1:1,000 diluted anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody (Imgenex) as the first antibody and 1:5,000 diluted anti-mouse immunoglobulins conjugated with horseradish peroxidase (Wako Pure Chemicals) as the second antibody. The anti-Dnmt3a monoclonal antibody recognizes two Dnmt3a subtypes, Dnmt3a and Dnmt3a2 [36]. The chemiluminescence signals, which were obtained with SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical), were visualized by Chemi-Smart 3000 (Vilber Lourmat, Marne-la-Vallée, France, http://www.vilber.com). For reprobing, the blotted membrane was rinsed with Restore Western Blot Stripping Buffer (Pierce Chemical).

Preparation of Genomic DNA
Genomic DNA was extracted as previously described [37]. Briefly, cells were suspended in lysis buffer (150 mM EDTA, 10 mM Tris-HCl [pH 8.0], and 1% SDS) containing 10 mg/ml proteinase K (Merck, Darmstadt, Germany, http://www.merck.com). The mixture was incubated at 55°C for 20 minutes. After two phenol/chloroform/isoamyl alcohol (50:49:1) extractions, genomic DNA was precipitated in ethanol and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]).

Restriction Landmark Genomic Scanning
The restriction landmark genomic scanning (RLGS) was performed using the restriction enzyme combination of Not I, Pvu II, and Pst I as described previously [38]. Genomic DNA was treated with Klenow fragment (Takara, Kyoto, Japan, http://www.takara-bio.com) in the presence of dGTP{alpha}S, dCTP{alpha}S (GE Healthcare, Little Chalfont, Buckinghamshire, UK, http://www.gehealthcare.com), ddATP, and ddTTP (Takara) to prevent nonspecific labeling. After digestion by Not I as a landmark enzyme (Nippon Gene, Toyama, Japan, http://www.nippongene.com), cohesive ends were isotopically labeled with Sequenase Ver 2.0 (USB Corporation, Cleveland, http://www.usbweb.com) in the presence of [{alpha}-32P]dCTP and [{alpha}-32P]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 2–3 weeks at –80°C.

Methylation-Sensitive Quantitative Real-Time PCR
DNA methylation status at Sall3 locus was evaluated using methylation-sensitive quantitative real-time PCR as previously described [39]. 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'.

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 [GenBank] 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).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
DMSO Increases Expression of Dnmt3as in ESCs and EBs
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%–1%) 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.


Figure 1
View larger version (47K):
[in this window]
[in a new window]
  		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.

 
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 [25], mRNAs for histone methyltransferases (G9a, Suv39h1, Suv39h2, and mDot1), and a histone deacetylase, Sir2{alpha}, 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.


Figure 2
View larger version (39K):
[in this window]
[in a new window]
  		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 [RT(+)] or without [RT(-)] reverse transcriptase. Primer sets are indicated to the left of the gel images. Concentrations (vol/vol) of DMSO were 0% (lane 1), 0.02% (lane 2), 0.1% (lane 3), 0.5% (lane 4), and 1% (lane 5). (B): Dnmt3a expression was analyzed by real-time quantitative RT-PCR. The value obtained in ESCs without DMSO was set to 1. Shaded blocks represent mean values, and standard error is represented by vertical bars. Differences between samples were analyzed by t test (*p < .1, **p < .05, ***p < .005 [n = 3]). (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.

 
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).

DMSO Affects DNA Methylation Status of Repetitive Sequences in EBs
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 [40]. 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.


Figure 3
View larger version (76K):
[in this window]
[in a new window]
  		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.

 
DMSO Affects Genome-Wide DNA Methylation Profiles of Genic Areas in EBs
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 [41], which are characterized by high CG content and frequent CpG appearance [42], and are often localized at promoter regions of housekeeping genes and many tissue-specific genes [42–44]. Therefore, RLGS enables us to simultaneously analyze thousands of loci in genic regions.

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 [DMSO] 1–11) emerged, indicating that DMSO induced hypomethylation of these 11 sites in EBs (Fig. 4B). In contrast, four spots (T-DMRs [DMSO] 12–15) 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).


Figure 4
View larger version (62K):
[in this window]
[in a new window]
  		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 < .001, [ESCs: n = 3, EBs: n = 4]) (right panel). Abbreviations: DMSO, dimethyl sulfoxide; {Delta}Rn, delta normalized reporter; EB, embryoid body; ESC, embryonic stem cell; PCR, polymerase chain reaction; RLGS, restriction landmark genomic scanning; T-DMR, tissue-dependent and differentially methylated region.

 
We compared these 15 spots (T-DMRs [DMSO] 1–15) with T-DMRs that we previously identified [15, 16] (Fig. 5). The two spots, T-DMRs (DMSO) 2 and 12, were matched with T-DMRs 148 and 253, respectively, whereas the other 13 spots were novel. We defined them as T-DMRs 582 and 701–712, serially (Fig. 5). One of the matched spots is located in Sall3 gene (T-DMR [DMSO] 2), which is specifically hypermethylated in the trophoblast lineage but hypomethylated in ESCs [31]. 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).


Figure 5
View larger version (11K):
[in this window]
[in a new window]
  		Figure 5. Matching of T-DMRs (DMSO) to previously identified T-DMRs. T-DMRs (DMSO) 1–15 (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—T-DMRs (DMSO) 1, 3–11, and 13–15—were designated as T-DMRs 582 and 701–712, 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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosures
 Acknowledgments
 References
 
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 [2, 3]. In animal cloning technology, DMSO improved the frequency of development to blastocyst stage and full term [5]. 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.

The human and mouse genomes contain 30,000–40,000 genes; however, genes occupy only a small percentage of the approximately 3 x 109 bp haploid genome. In contrast, 41%–48% of the mammalian genome is composed of nongenic repetitive elements, including satellites interspersed repeats such as retroviruses [45, 46]. A recent database analysis suggested that approximately half of all promoter regions are located in CpG islands [44]. We have identified many regions in CpG islands that have different methylation statuses depending on cell types and tissues, indicating that dynamic change in DNA methylation occurs during differentiation [15–17]. The present study demonstrated that DMSO impacts DNA methylation status genome-wide, including genic and nongenic regions of EBs differentiated from ESCs. Furthermore, hypermethylation as well as hypomethylation occurred at 15 independent genomic loci after DMSO treatment. However, note that there are approximately 15,500 CpG islands in the mouse genome [46], which suggests that the methylation status of many loci must be affected by DMSO.

Differentiation of ESCs to EBs causes both hypermethylation and hypomethylation at various loci genome-wide in mammals [16]. 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
View larger version (34K):
[in this window]
[in a new window]
  		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 [40]. Abnormal expression of Dnmts has been observed in cancerous cells in which aberrant DNA methylation occurred; such expression includes an increase in DNMT1 and DNMT3b expression in various cancer cells [47] and a higher level of DNMT3a expression in leukemia [48, 49]. The preference of Dnmts for target loci within CpG islands was observed in ESCs [39]. Catalytic activities of Dnmt3a showed preference for nucleotide composition around the CpG sites [50]. 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 [51, 52]. Methylation of DNA, however, was not solely regulated by Dnmts; chromatin configuration affects DNA methylation status and vice versa [20, 22–24]. Therefore, genome-wide alteration of the DNA methylation profile by DMSO should not be explained simply by the increased levels of Dnmt3as.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosures
 Acknowledgments
 References
 
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.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
References
 
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.).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 

  1. Santos NC, Figueira-Coelho J, Martins-Silva J et al. Multidisciplinary utilization of dimethyl sulfoxide: Pharmacological, cellular, and molecular aspects. Biochem Pharmacol 2003;65:1035–1041.[CrossRef][Medline]

  2. Preisler HD, Giladi M. Differentiation of erythroleukemic cells in vitro: Irreversible induction by dimethyl sulfoxide (DMSO). J Cell Physiol 1975;85:537–546.[CrossRef][Medline]

  3. Gusella J, Geller R, Clarke B et al. Commitment to erythroid differentiation by friend erythroleukemia cells: A stochastic analysis. Cell 1976;9:221–229.[CrossRef][Medline]

  4. Edwards MK, Harris JF, McBurney MW. Induced muscle differentiation in an embryonal carcinoma cell line. Mol Cell Biol 1983;3:2280–2286.[Abstract/Free Full Text]

  5. Wakayama T, Yanagimachi R. Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei. Reproduction 2001;122:49–60.[Abstract]

  6. Hattori N, Nishino K, Ko YG et al. Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J Biol Chem 2004;279:17063–17069.[Abstract/Free Full Text]

  7. Nishino K, Hattori N, Tanaka S et al. DNA methylation-mediated control of Sry gene expression in mouse gonadal development. J Biol Chem 2004;279:22306–22313.[Abstract/Free Full Text]

  8. Sinsheimer RL. The action of pancreatic deoxyribonuclease. II. Isomeric dinucleotides. J Biol Chem 1955;215:579–583.[Free Full Text]

  9. Russo VEA, Martienssen RA, Riggs AD. Introduction. In: Russo VEA, Martienssen RA, Riggs AD, eds. Epigenetic Mechanisms of Gene Regulation.Woodbury, NY: Cold Spring Harbor Laboratory Press,1996;1–4.

  10. Bird AP, Wolffe AP. Methylation-induced repression–belts, braces, and chromatin. Cell 1999;99:451–454.[CrossRef][Medline]

  11. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002;16:6–21.[Free Full Text]

  12. Lachner M, Jenuwein T. The many faces of histone lysine methylation. Curr Opin Cell Biol 2002;14:286–298.[CrossRef][Medline]

  13. Imamura T, Ohgane J, Ito S et al. CpG island of rat sphingosine kinase-1 gene: Tissue-dependent DNA methylation status and multiple alternative first exons. Genomics 2001;76:117–125.[CrossRef][Medline]

  14. Futscher BW, Oshiro MM, Wozniak RJ et al. Role for DNA methylation in the control of cell type specific maspin expression. Nat Genet 2002;31:175–179.[CrossRef][Medline]

  15. Shiota K, Kogo Y, Ohgane J et al. Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice. Genes Cells 2002;7:961–969.[Abstract]

  16. Kremenskoy M, Kremenska Y, Ohgane J et al. Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses. Biochem Biophys Res Commun 2003;311:884–890.[CrossRef][Medline]

  17. Shiota K. DNA methylation profiles of CpG islands for cellular differentiation and development in mammals. Cytogenet Genome Res 2004;105:325–334.[CrossRef][Medline]

  18. Okano M, Xie S, Li E. Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells. Nucleic Acids Res 1998;26:2536–2540.[Abstract/Free Full Text]

  19. Hata K, Okano M, Lei H et al. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development 2002;129:1983–1993.[Medline]

  20. Li E. Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 2002;3:662–673.[CrossRef][Medline]

  21. Chen T, Li E. Structure and function of eukaryotic DNA methyltransferases. Curr Top Dev Biol 2004;60:55–89.[Medline]

  22. Xin Z, Tachibana M, Guggiari M et al. Role of histone methyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center. J Biol Chem 2003;278:14996–15000.[Abstract/Free Full Text]

  23. Lehnertz B, Ueda Y, Derijck AA et al. Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin. Curr Biol 2003;13:1192–1200.[CrossRef][Medline]

  24. Espada J, Ballestar E, Fraga MF et al. Human DNA methyltransferase 1 is required for maintenance of the histone H3 modification pattern. J Biol Chem 2004;279:37175–37184.[Abstract/Free Full Text]

  25. Zhang W, Hayashizaki Y, Kone BC. Structure and regulation of the mDot1 gene, a mouse histone H3 methyltransferase. Biochem J 2004;377:641–651.[Medline]

  26. Vaquero A, Scher M, Lee D et al. Human SirT1 interacts with histone H1 and promotes formation of facultative heterochromatin. Mol Cell 2004;16:93–105.[CrossRef][Medline]

  27. Hansen RS, Wijmenga C, Luo P et al. The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc Natl Acad Sci U S A 1999;96:14412–14417.[Abstract/Free Full Text]

  28. Shahbazian MD, Zoghbi HY. Rett syndrome and MeCP2: Linking epigenetics and neuronal function. Am J Hum Genet 2002;71:1259–1272.[CrossRef][Medline]

  29. Ohgane J, Wakayama T, Kogo Y et al. DNA methylation variation in cloned mice. Genesis 2001;30:45–50.[CrossRef][Medline]

  30. Humpherys D, Eggan K, Akutsu H et al. Epigenetic instability in ES cells and cloned mice. Science 2001;293:95–97.[Abstract/Free Full Text]

  31. Ohgane J, Wakayama T, Senda S et al. The Sall3 locus is an epigenetic hotspot of aberrant DNA methylation associated with placentomegaly of cloned mice. Genes Cells 2004;9:253–260.[Abstract/Free Full Text]

  32. Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3:415–428.[Medline]

  33. Holliday R. Mutations and epimutations in mammalian cells. Mutat Res 1991;250:351–363.[CrossRef][Medline]

  34. O'Shea KS. Embryonic stem cell models of development. Anat Rec 1999;257:32–41.[CrossRef][Medline]

  35. Kawase E, Suemori H, Takahashi N et al. Strain difference in establishment of mouse embryonic stem (ES) cell lines. Int J Dev Biol 1994;38:385–390.[Medline]

  36. Chen T, Ueda Y, Xie S et al. A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J Biol Chem 2002;277:38746–38754.[Abstract/Free Full Text]

  37. Ohgane J, Aikawa J, Ogura A et al. Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning. Dev Genet 1998;22:132–140.[CrossRef][Medline]

  38. Ohgane J, Hattori N, Shiota K. Analysis of tissue-specific DNA methylation during development. Methods Mol Biol 2005;289:371–382.[Medline]

  39. Hattori N, Abe T, Hattori N et al. Preference of DNA methyltransferases for CpG islands in mouse embryonic stem cells. Genome Res 2004;14:1733–1740.[Abstract/Free Full Text]

  40. Chen T, Ueda Y, Dodge JE et al. Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol Cell Biol 2003;23:5594–5605.[Abstract/Free Full Text]

  41. Fazzari MJ, Greally JM. Epigenomics: Beyond CpG islands. Nat Rev Genet 2004;5:446–455.[CrossRef][Medline]

  42. Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J Mol Biol 1987;196:261–282.[CrossRef][Medline]

  43. Larsen F, Gundersen G, Lopez R et al. CpG islands as gene markers in the human genome. Genomics 1992;13:1095–1107.[CrossRef][Medline]

  44. Suzuki Y, Tsunoda T, Sese J et al. Identification and characterization of the potential promoter regions of 1031 kinds of human genes. Genome Res 2001;11:677–684.[Abstract/Free Full Text]

  45. Lander ES, Linton LM, Birren B et al. Initial sequencing and analysis of the human genome. Nature 2001;409:860–921.[CrossRef][Medline]

  46. Waterston RH, Lindblad-Toh K, Birney E et al. Initial sequencing and comparative analysis of the mouse genome. Nature 2002;420:520–562.[CrossRef][Medline]

  47. Kanai Y, Ushijima S, Kondo Y et al. DNA methyltransferase expression and DNA methylation of CPG islands and peri-centromeric satellite regions in human colorectal and stomach cancers. Int J Cancer 2001;91:205–212.[CrossRef][Medline]

  48. Mizuno S, Chijiwa T, Okamura T et al. Expression of DNA methyltransferases DNMT1, 3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood 2001;97:1172–1179.

  49. Tessema M, Langer F, Dingemann J et al. Aberrant methylation and impaired expression of the p15(INK4b) cell cycle regulatory gene in chronic myelomonocytic leukemia (CMML). Leukemia 2003;17:910–918.[CrossRef][Medline]

  50. Lin IG, Han L, Taghva A et al. Murine de novo methyltransferase Dnmt3a demonstrates strand asymmetry and site preference in the methylation of DNA in vitro. Mol Cell Biol 2002;22:704–723.[Abstract/Free Full Text]

  51. Liang G, Chan MF, Tomigahara Y et al. Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol Cell Biol 2002;22:480–491.[Abstract/Free Full Text]

  52. Kim GD, Ni J, Kelesoglu N et al. Co-operation and communication between the human maintenance and de novo DNA (cytosine-5) methyltransferases. Embo J 2002;21:4183–4195.[CrossRef][Medline]




This article has been cited by other articles:


Home page
 Hum ReprodHome page
B. Balaban, B. Urman, B. Ata, A. Isiklar, M.G. Larman, R. Hamilton, and D.K. Gardner
A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation
Hum. Reprod., June 10, 2008; (2008) den222v1.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0427v1
24/11/2549    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Iwatani, M.
Right arrow Articles by Shiota, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Iwatani, M.
Right arrow Articles by Shiota, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.peprotech.com/