First published online January 10, 2008
Stem Cells
Vol. 26 No.
3
March 2008, pp.
783
-788
doi:10.1634/stemcells.2007-0907; www.StemCells.com
© 2008 AlphaMed Press
STEM CELL GENOMICS AND PROTEOMICS |
Nuclear Transfer Alters the DNA Methylation Status of Specific Genes in Fertilized and Parthenogenetically Activated Mouse Embryonic Stem Cells
Takafusa Hikichia,
Takashi Kohdab,
Sayaka Wakayamaa,
Fumitoshi Ishinob,
Teruhiko Wakayamaa
aLaboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN Kobe Institute, Kobe, Japan;
bDepartment of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Key Words. Parthenogenesis • Nuclear transfer • Reprogramming • Embryonic stem cells
Correspondence:
Correspondence: Takafusa Hikichi, Ph.D., 2-2-3 Minatojima-minamimachi Chuo-ku, Kobe 650-0047, Japan. Telephone: 81-78-306-3049; Fax: 81-78-306-3095; e-mail: hikichi{at}cdb.riken.jp
Received on October 28, 2007;
accepted for publication on December 27, 2007.
First published online in STEM CELLS EXPRESS January 10, 2008.
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ABSTRACT
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Recent cloning technology has been demonstrated successfully using nuclear transfer (NT) techniques to generate embryonic stem (ES) cells. Mice can be cloned from adult somatic cells or ES cells by NT, and such cloned embryos can be used to establish new NT-ES cell lines. However, ES cells derived from parthenogenetic embryos show epigenetic disorders and low potential for normal differentiation unless used to produce subsequent generations of NT-ES lines. Thus, enucleated oocytes can initialize epigenetic modification, but the extent and efficacy of this remain unclear. In this study, our goal was to clarify why the contribution rate of ES cells derived from parthenogenetic embryos (pES) cells appears to improve after NT. We compared gene expression profiles between pES and NT-pES cell lines using DNA microarray analysis and allele-specific DNA methylation analysis. Although changes in expression level were observed for 4% of 34,967 genes, only 81 (0.2%) showed common changes across multiple cell lines. In particular, the expression level of a paternally expressed gene, U2af1-rs1, was significantly increased in all NT-pES cell lines investigated. The methylation status at the upstream differentially methylated region of U2af1-rs1 was also changed significantly after NT. This was observed in NT-pES cells, but also in conventionally produced NT-ES cells, which has never been reported previously. These results suggest that NT affects the epigenetic status of a few gene regions in common and that a change in the methylation status of U2af1-rs1 could be used as a genetic marker to investigate the effects of NT.
Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION
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Somatically derived nuclear transfer (NT) embryonic stem (ES) cells and ES cells derived from fertilized embryos are transcriptionally and functionally indistinguishable [1, 2], and NT-ES cell technology is developing rapidly in terms of its application to regenerative medicine [3–5]. Results to date indicate that NT affects epigenetic modification, and several studies have shown that it affects the expression pattern of several genes [6–8]. Moreover, microarray analysis has shown abnormal gene expression levels after NT [9–11]. However, it has not been demonstrated whether any particular genes or genomic domains are strongly influenced by NT. Because embryonic resources are limited, it is difficult to compare gene expression profiles exhaustively. Therefore, to perform reliable analyses, it is important to prepare sufficient numbers of cell lines.
To study the effects of NT on epigenetic modification, in this study we used parthenogenetic embryonic stem (pES) cell as donors, as these uniparental lines show inherent epigenetic flaws that are normally lethal. Such parthenogenetic embryos have only maternal alleles, and the embryos lack the expression of some critical imprinted genes [12, 13]. As these epigenetic disorders cause developmental abnormalities [14], genomic imprinting clearly has important roles in normal mammalian development. However, although parthenogenetic embryos lack full developmental potential because of missing male alleles, pES cell lines can be established from them [15] and can contribute to chimeric mice, albeit at a low rate [16]. Moreover, parthenogenetic gene expression profiles were maintained even after the pES cells developed into somatic cells in chimeras [16]. Because such pES cells can maintain their abnormal epigenetic state and can be prepared in large numbers for analysis, we believe that they will prove very suitable to analyze the influences of NT on epigenetic modifications.
We have already established NT-pES cell lines from our original pES cell lines and demonstrated that their potential for both in vivo and in vitro differentiation was significantly (two to five times) better than that of the original pES cells [17]. Thus, the NT procedure clearly affected the epigenetic modification of parthenogenetic cells. To clarify this process, we compared the gene expression profile of pES/NT-pES cell lines using DNA microarray analysis. This showed that only a few genes were affected in common across multiple NT-pES cell lines. We also noted that the expression level of a paternally expressed gene, U2af1-rs1, derived from the nuclear donor cells, was changed significantly by the NT procedure, and we analyzed its DNA methylation status in both NT-pES and conventional NT-ES cells.
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MATERIALS AND METHODS
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Background of pES and NT-pES Cell Lines
In this study, we created original pES cell lines by combining two genetically different strains of mouse oocytes, C57BL/6-GFP Tg [18] and 129/Sv, instead of the usual method of producing parthenogenetic embryos from single oocytes [17]. Genetically, the resulting constructs are gynogenetic F1 hybrids. An F1 genetic background is known to enhance the somatic cell nuclear transfer (SCNT) cloning success rate [19, 20]. This approach also allowed us to introduce the green fluorescence protein (GFP) marker into pES cells at the same time.
NT-pES cell lines were used as described [21, 22]. Briefly, cloned embryos were generated from pES cell nuclei (original and first-generation), and these were used for the establishment of second-generation NT-pES cell lines. We repeated this process to establish third-generation NT-pES cell lines. All the established cell lines were examined for the production of GFP to demonstrate the success of NT. Even if some parthenogenetic embryos had been produced accidentally, they could have been detected because they would not express the gene for GFP. All pES and NT-pES cells were collected for study within six passages from the establishment of each cell line. NT-ES cell lines derived from cumulus cells and tail-tip fibroblasts [3] were used as controls.
DNA Microarray Analysis
The CodeLink system (Mouse Whole Genome Array; Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) was used to determine and compare the expression levels of 36,000 genes in three normally fertilized ES cell lines, one pES cell line, two second-generation NT-pES cell lines, and two third-generation NT-pES cell lines. Total RNA was purified using RNeasy Kits (Qiagen, Hilden, Germany, http://www1.qiagen.com). The cRNA preparation and CodeLink hybridization were conducted according to the manufacturers' protocols. The expression levels of the different genes were assessed using GenePix Pro software. The signals were normalized using the "qspline" algorithm as implemented in the Bioconductor package of the R statistics program (http://www.r-project.org). The data from the individual microarrays are accessible for download through the National Center for Biotechnology Information's Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) via series accession number GSE3128.
Quantitative Reverse Transcription-Polymerase Chain Reaction Amplification
The cDNA was synthesized from 1 µg of total RNA using Superscript II reverse transcriptase (Life Technologies, Rockville, MD, http://www.lifetech.com) with an oligo(dT) primer. For reverse transcription (RT)-polymerase chain reaction (PCR), 1 ng of cDNA in a 100-µl reaction mixture containing 1x ExTaq buffer (Takara Bio, Shiga, Japan, http://www.takara-bio.com), 2.5 mM dNTP mixture, primers, and 2.5 U of ExTaq enzyme (Takara) were subjected to 30 PCR cycles at 96°C for 15 seconds, 65°C for 30 seconds, and 72°C for 30 seconds on a PerkinElmer GeneAmp PCR system 9600 (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com). Target cDNA fragments were cloned into plasmids to use as standards in the quantitative analysis of gene expression. The primers used for quantification were as follows: Snrpn forward (F), 5'-CCC TTC TCT TCC CCT ACA ATG C-3', and reverse (R), 5'-AAG CTT GCA GGT ACA CAA TTT CAC-3'; U2af1-rs1 F, 5'-GCA GTC CAG GTC CAC AAA GC-3', and R, 5'-GCC TTA GCT GGG CTC AGG TT-3'; and Gapd F, 5'-CAC TCT TCC ACC TTC GAT GC-3', and R, 5'-CAC TCT TCC ACC TTC GAT GC-3'. Gene expression levels were measured using an ABI Prism 7700 detection system and SYBR Green PCR Core Reagents (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). No amplification of RT-PCR products was detected in the controls lacking reverse transcriptase.
Allele-Specific DNA Methylation Analysis
Genomic DNA was extracted from each set of cultured ES cells using DNeasy Tissue Kits (Qiagen). The DNA was subjected to bisulfite modification, PCR amplification, subcloning, and sequencing as described [23], with some modifications. The mutagenized DNA was resuspended in 5 µl of TE buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and 1 µl was used for PCR amplification. All mutagenized DNAs were subjected to multiple independent PCR amplifications to ensure the recovery of different strands of DNA. The primers used for amplification of each differentially methylated region (DMR) were as follows: Snrpn-bi F, 5'-AAT TTG TGT GAT GTT TGT AAT TAT TTG G-3', and R, 5'-ATA AAA TAC ACT TTC ACT ACT AAA ATC C-3'; U2af1-bi F, 5'-TTA TTG TAG GAG TAT TTT TAT TGT AGG G-3', and R, 5'-CCA TAA TAC AAC TAA AAC AAT AAT TAT CTA CC-3'; and intracisternal A particle (IAP)-bi F, 5'-TTG ATA GTT GTG TTT TAA GTG GTA AAT AAA-3', and R, 5'-AAA ACA CCA CAA ACC AAA ATC TTC TAC-3'.
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RESULTS
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Effects of NT on Gene Expression Profiles of pES Cell Lines
We examined the gene expression profile of the pES/NT-pES cell lines using a DNA CodeLink microarray system to determine and compare the expression levels of 36,000 genes. We prepared two second-generation and two third-generation NT-pES cell lines and compared their gene expression profiles with those of the original pES cells. At first, we selected those genes that more than doubled or halved their expression levels after NT (Table 1). The expression levels of between 212 and 882 genes were changed in each NT-pES cell line. We then looked for gene expression levels that changed across multiple NT-pES cell lines. If NT affects the epigenetic regulation of any particular gene region constantly, the same quantitative alternation should have been detected in all the cell lines. Although the expression levels of 775 genes decreased in four of the NT-pES cell lines, 729 were observed in only one or two lines (Table 2). Moreover, 538 gene expression levels were increased in NT-pES cells, but 507 of these were observed in only one or two lines (Table 2). We assumed that these genes had been affected by the NT procedure randomly and excluded them from the subsequent assay objectives. We then checked the reliability of the data using normally fertilized ES cells. We found that the expression levels of several genes were unstable even in fertilized ES cells, so we excluded these from those candidate genes showing common changed expression levels in three or more cell lines.
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Table 2. Numbers of genes showing changed expression level across multiple nuclear transfer-parthenogenetic embryonic stem cell lines
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Overall, only seven genes showed changed expression levels in all the NT-pES cell lines examined (Table 3). From a consideration of the known genetic functions and specificity of gene regulation, we concentrated on two imprinted genes, U2af1-rs1 and Snrpn. These genes are paternally expressed and not detected in parthenogenetic embryos. Although several other paternally expressed genes, such as Igf2 and Peg1, were contained in the CodeLink microarray plate, their expression levels were not increased in all NT-pES cells. Thus, the NT procedure appears to have affected the gene expression of U2af1-rs1 and Snrpn. The expression levels of the two genes were then determined more precisely by real-time PCR analysis. For U2af1-rs1, the expression level in the original pES cells was approximately 25% of the level in normally fertilized ES cells. After NT, all the NT-pES cell lines expressed this gene at levels equivalent to that of the controls (Fig. 1A). On the other hand, Snrpn in pES cells was expressed at only 5% of the level of controls (Fig. 1B). Although we expected that the expression level would increase in all NT-pES cell lines, changes in this gene were observed in only two cell lines (Fig. 1B), and the expression level in all cells was lower than that in the control ES cells.
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Figure 1. Gene expression levels in pES and NT-pES cells. Gene expression levels were determined using quantitative polymerase chain reaction in triplicate and normalized using the detected expression level of Gap. Each original parthenogenetic ES cell gene expression level is expressed as 1.00. (A): Expression levels of U2af1-rs1 in the pES and NT-pES cells. We examined three independent first-generation pES cell lines and eight NT-pES cell lines. Yellow columns show the original pES cells, green columns show second-generation NT-pES cells, and dark green columns show third-generation NT-pES cell lines. Blue columns show levels in Cont ES cells. After NT, the expression levels of the U2af1-rs1 gene were significantly increased in all NT-pES cell lines. (B): The expression levels of Snrpn in the pES and NT-pES cells. We examined one pES cell line and four NT-pES cell lines (second and third generations). In one of the second-generation NT-pES cell lines, the expression level of Snrpn was increased, but in one other it was unchanged. Using repeated NT procedures, the expression level increased after the second generation. Abbreviations: Cont, control; ES, embryonic stem; NT, nuclear transfer; pES, parthenogenetic embryonic stem.
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DNA Methylation Status in NT-pES Cells
To test for clear evidence of epigenetic changes after NT, we next examined the DNA methylation state of the U2af1-rs1 and Snrpn promoter regions. DNA methylation is an important epigenetic modification, especially in genomic imprinting. If NT affects the regulation of imprinted genes, obvious changes in the methylation status of the promoter regions would be expected. We constructed a primer set for the DMR of the U2af1-rs1 promoter region and confirmed that half (56.0%) of the alleles in the U2af1-rs1 promoter region were in a hypermethylated state in ES cells (data not shown). All alleles were hypermethylated in the pES cells (Fig. 2A). We compared the methylation status of pES and NT-pES cells in two independent pES cell lines. In both lines, 42.9% to 46.7% of alleles were hypomethylated after NT (Fig. 2A). However, although there was a change of DNA methylation status in the Snrpn DMR after nuclear transfer, hypomethylation was found in only 7% (2 of 27) of all genomes (Fig. 2B). To determine whether cell culture and cell passage would affect DNA methylation status in these regions, we examined methylation status in 30 passaged pES cell lines. Both alleles of U2af1-rs1 and Snrpn maintained their hypermethylated status in pES cells even after 30 passages (data not shown). We also investigated the methylation status of the IAP repetitive region as a negative control. All alleles of this region were hypermethylated in pES cells, and their status was unchanged in NT-pES cells (Fig. 2C).
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Figure 2. DNA methylation status in NT-pES cells. Shown are the methylation patterns of the DMR of U2af1-rs1 (A), Snrpn (B), and IAP (C) using bisulfite sequencing (filled circles, methylated; open circles, unmethylated). Cytosines are shown for a number of independently sequenced templates (horizontal lines). These DMRs of two imprinted genes are methylated in maternal alleles only, whereas the repetitive region of IAP is hypermethylated in all alleles. In normally fertilized cells, approximately half of the sequenced templates were highly methylated, presumably the paternal allele. By contrast, in the parthenogenetic cells, all sequenced templates were methylated, because they only possessed maternal alleles. After NT, some alleles were significantly demethylated. Thus, NT affected the methylation status of the U2af1-rs1 promoter region. Abbreviations: DMR, differentially methylated region; IAP, intracisternal A particle; NT, nuclear transfer; pES, parthenogenetic embryonic stem.
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Effects of NT on the DNA Methylation Status of the U2af1-rs1 Promoter Region in SCNT-ES Cells
Because parthenogenetic cell lines are abnormal, there is a possibility that the demethylation observed in the U2af1-rs1 promoter region was an artifact. To investigate whether this could be observed in SCNT-ES cells as well as NT-pES cells, we investigated DNA methylation status in NT-ES cells derived from two types of somatic cells (cumulus cells and tail-tip fibroblasts) and in normally fertilized ES cells. If the NT procedure affects epigenetic modifications in common, demethylation would also be observed following SCNT. Because all these somatic and ES cells have both paternal and maternal alleles, approximately half (47%–55%) the alleles were hypermethylated in the U2af1-rs1 promoter region (Fig. 3A). However, the ratio of hypermethylated alleles was decreased in all NT-ES cell lines (Fig. 3A, 3B) derived from cumulus cells (27.3% and 15.4%) and from fibroblasts (36.4% and 33.3%) and in normally fertilized NT-ES cells (27.3% and 33.3%). Clearly, the demethylation effect of NT was observed in several types of NT-ES cells, as well as in NT-pES cells. For the Snrpn DMR, the methylation ratio ranged from 46.7% to 54.5% in NT-ES cells established from all types of cells, and no significant changes were observed (Fig. 3B).
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Figure 3. DNA methylation status of U2af1-rs1 in ntES cells. (A): The methylation patterns of the differentially methylated region of U2af1-rs1 were observed using bisulfite sequencing in several types of ntES cells established by somatic cell nuclear transfer. Both somatic cells (cum and tail fib cells) and ES cells maintained their differential methylation status. After nuclear transfer, the ratio of hypermethylated alleles was decreased compared with whole alleles. (B): The ratio of hypermethylated alleles in ntES cells. We defined a hypermethylated allele as one in which 80% or more of the cytosines were methylated. Abbreviations: cum, cumulus; DMR, differentially methylated region; ES, embryonic stem; fib, fibroblast; ntES, nuclear transfer embryonic stem.
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DISCUSSION
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Epigenetic modification through NT is an important area for the potential improvement of cloning techniques. However, because conventionally cloned embryos are so rare, the effect of NT has been unclear. To address this problem, we used parthenogenetically derived ES cells, which are much more readily available. Previously, we produced NT-pES cells by using pES cells as nuclear donors and evaluated their potential for differentiation [17]. We found that NT significantly affects several cell characteristics, such as the potential for differentiation in chimeric mice. These data indicated that NT could indeed affect gene regulation. Previous reports have shown that NT does not affect the characteristics of original embryonic carcinoma (EC) or fertilized ES cells [24, 25]. Although we cannot exclude the possibility of cell-based selection because pES cells lack differentiation potential compared with EC or ES cells, this significant effect of NT for developmental improvement might have been observed clearly. To determine the cause, we performed comprehensive analysis of the expression levels of 36,000 genes using DNA microarrays. The expression levels of 1,313 genes were increased or decreased in four of the NT-pES cell lines (Table 2). However, 93.7% of the changes were observed only in one or two cell lines, and these changes were not seen in any specific genomic domain. Although abnormal expression levels of the Oct3/4 gene have been observed in NT embryos [7], this disorder was observed in only one NT-pES cell line in our study. Thus, the effect of NT is not constant, and such changes in gene expression may occur randomly in each procedure.
From comparing the gene expression levels of four NT-pES cell lines with those of pES cells, only seven genes were picked up as candidates that were affected by NT. In particular, we noted a paternally expressed gene, U2af1-rs1 (U2 small nuclear ribonucleoprotein auxiliary factor small subunit-related). This gene is an essential mammalian gene splicing factor, and its expression is related to allele-specific DNA methylation [26, 27]. In parthenogenetic embryos, the gene promoter region remained hypermethylated (Fig. 2A), and no gene expression was observed (Fig. 1A). Moreover, this DNA methylation was erased on some alleles after NT; this typical methylation change to the U2af1-rs1 promoter was also observed in NT-ES cell lines produced by using somatic cells, cumulus cells, or tail-tip fibroblasts as nucleus donors (Fig. 3). Because normal somatic cells contain both paternal and maternal alleles, half of the genomes are hypermethylated and half are hypomethylated in this region. After NT, the proportions of hypomethylated alleles were significantly increased in all cell lines examined: four SCNT-ES lines and two NT-ES lines derived using ES cell nuclei. Thus, the effect of the NT procedure on the U2af1-rs1 genomic region is not an artifact of parthenogenetic ES cells, as it can be observed in conventional SCNT-ES cells.
Sotomaru et al. reported that U2af1-rs1 expression was not subject to DNA methylation [28]. By contrast, we found that the expression of U2af1-rs1 was related to DNA methylation level in the promoter region. One explanation of these different results is that they might arise from differences in the methods and sites checked in each study. We examined the DNA methylation status around the promoter region, which contains DNase I-hypersensitive sites (HSS). Recently, Andollo et al. examined the expression level and DNase I sensitivity of the U2af1-rs1 gene using differentiated ES cells and suggested that expression might be regulated by an open chromatin conformation in an upstream region that contains an HSS of U2af1-rs1 [29]. DNA methylation status is known to be unstable under particular culture conditions [30]. However, no clear methylation changes were observed in other DMRs or repetitive sequences (Fig. 2B, 2C), and we observed no significant alteration of methylation status after 30 passages in both DMRs of U2af1-rs1 and Snrpn (data not shown). Moreover, differentiation potential was also not improved by prolonged culture [17]. Thus, pES and NT-pES cells were not affected under our culture conditions. Previously, we showed that NT-pES maintains the parthenogenetic methylation status in two DMRs: H19-DMR and IG-DMR [17]. Although parthenogenetic mice were born following NT using a combination of H19-DMR-knockout mice and IG-DMR-knockout mice [31, 32], our results indicate that these DMRs are not related to the improvement of developmental potential of NT-pES cells.
In this study, we found seven candidate genes by microarray analysis and investigated the imprinted gene U2af1-rs1 to detect the effect of NT. Although there was a significant effect on the methylation status of this epigenetic marker, it is possible that the other six candidate genes also have important roles in determining developmental potential. Further research is necessary to clarify this, especially if we are to apply the findings to reproductive medicine. In conclusion, although there is no direct evidence that U2af1-rs1 is a key factor for the improvement of developmental potential in NT-ES cells, this gene is an important candidate that can change cell characteristics. We suggest that U2af1-rs1 is a strong candidate with which to investigate the effects of NT on gene regulation mechanisms.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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T.W. was supported by Grant 15080211, and T.W. and Shin-ichi Nishikawa were supported by a project for the realization of regenerative medicine from the Ministry of Education, Science, Sports, Culture and Technology of Japan.
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Abstract
Introduction
