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¹ Cellular Biochemistry, Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
² The Institute for Biogenesis Research, Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96822, USA
³ Bioresource Centre, Riken, 3-1-1, Koyadai, Tsukuba, 305-0074, Ibaraki, Japan

	Abstract

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DNA methylation controls various developmental processes by silencing, switching and stabilizing genes as well as remodeling chromatin. Among various symptoms in cloned animals, placental hypertrophy is commonly observed. We identified the Spalt-like gene3 (Sall3) locus as a hypermethylated region in the placental genome of cloned mice. The Sall3 locus has a CpG island containing a tissue-dependent differentially methylated region (T-DMR) specific to the trophoblast cell lineage. The T-DMR sequence is also conserved in the human genome at the SALL3 locus of chromosome 18q23, which has been suggested to be involved in the 18q deletion syndrome. Intriguingly, larger placentas were more heavily methylated at the Sall3 locus in cloned mice. This epigenetic error was found in all cloned mice examined regardless of sex, mouse strain and the type of donor cells. In contrast, the placentas of in vitro fertilized (IVF) and intracytoplasmic sperm injected (ICSI) mice did not show such hypermethylation, suggesting that aberrant hypermethylation at the Sall3 locus is associated with abnormal placental development caused by nuclear transfer of somatic cells. We concluded that the Sall3 locus is the area with frequent epigenetic errors in cloned mice. These data suggest that there exists at least genetic locus that is highly susceptible to epigenetic error caused by nuclear transfer.

	Introduction

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Most cells of higher eukaryotes differentiate without changing DNA sequence. Cells differentiate into specific types by activation and inactivation of particular sets of genes. DNA methylation is involved in various biological phenomena (Bird 2002; Li 2002) such as cell differentiation (Takizawa et al. 2001), X chromosome inactivation (Norris et al. 1991), genomic imprinting (Stoger et al. 1993), heterochromatin formation (Jones et al. 1998) and tumorgenesis (Issa et al. 1994).

Mammalian cloning using adult somatic cells has been successful in several species (Renard et al. 2002; Wilmut et al. 2002). Cloned offspring develop a variety of abnormal phenotypes such as increased body weight (large fetus syndrome), pulmonary hypertension, placental overgrowth, respiratory problems and early death (Lanza et al. 2000; Hill et al. 2000; Tamashiro et al. 2000; Tanaka et al. 2001; Ogonuki et al. 2002). This suggests a disruption of the normal developmental program. On this basis we expected and have identified several aberrantly methylated loci in the tissues of full-term cloned fetuses (Ohgane et al. 2001). Interestingly, each cloned animal has a different DNA methylation pattern and the extent of hyper- or hypo-methylation varies among the individuals. Cloned embryos at blastocyst or earlier developmental stages were reported to have unusual DNA methylation patterns at both repetitive and single copy gene regions (Santos et al. 2002; Bourc’his et al. 2001; Kang et al. 2001, 2002). Cloned fetuses of later developmental stages also showed aberrant DNA methylation at loci of imprinted and X-chromosomal genes compared with control fetuses (Humpherys et al. 2001; Xue et al. 2002). These findings suggest that cloned animals produced by somatic nuclear transfer have different methylation patterns from normal animals. At present, however, it remains to be seen whether aberrant DNA methylation at certain loci is related to the phenotypes specific to cloned animals.

Overgrowth of the placenta is one of the commonly observed symptoms in all cloned mice regardless of the sex and strain of animal and the type of donor cell (Wakayama & Yanagimachi 2001; Ogura et al. 2000a). Abnormal gene expression has been detected in term placentas of cloned mice (Humpherys et al. 2002; Suemizu et al. 2003). Thus, there may be genomic loci associated with the abnormal placental development in cloned mice. By using restriction landmark genomic scanning (RLGS) method, we have previously investigated genome-wide DNA methylation of CpG islands in mouse embryonic stem (ES) cells, embryonic germ (EG) cells and trophoblast stem (TS) cells before and after differentiation (Shiota et al. 2002). We have also investigated CpG islands of terminally differentiated germ cells and several somatic tissues using the same technique. There are many CpG islands with tissue-dependent differentially methylated regions (T-DMRs) (Imamura et al. 2001; Shiota et al. 2002). Here, we report that a T-DMR within the Sall3 locus at the telomeric E3 subregion of mouse chromosome 18 is hypermethylated in the placentas of all cloned mice examined.

	Results

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Hypermethylation of the Sall3 locus in placentas of cloned mice

Based on our previous studies, we prepared a T-DMR panel consisting of 247 loci detected by RLGS, one of which, locus #148, was methylated only in TS cells (Shiota et al. 2002). Intriguingly, locus #148 was matched to one of the aberrantly methylated loci in the placenta of cloned mice reported in the previous paper (spot 8 in Ohgane et al. 2001). It is also mapped to the Sall3 locus at the telomeric E3 subregion of mouse chromosome 18 (Fig. 1A).

View larger version (45K): [in this window] [in a new window]   Figure 1  Aberrant DNA hypermethylation at the Sall3 locus in placentas of cloned mice. (A) The genomic structure of the Sall3 locus with the position of the probe and expected bands in Southern blotting. The Sall3 locus is at the telomeric E3 subregion of chromosome 18. NotI is a methylation-sensitive restriction enzyme. The NotI site, aberrantly methylated in the placenta of the cloned mouse, is located approximately 1.1 kb downstream of Sall3 exon 1. Closed boxes represent three exons of the Sall3 gene. Numbers below the boxes are nucleotide numbers, designating the translation-starting site as number 1. An open box indicates the position of the probe. Methylation status of the Sall3 locus was analysed by Southern blotting using NotI and PstI. The bands expected in Southern blotting in Figs 1 and 3 are depicted as unmethylated (U, 350 bp) or methylated (M, 910 bp). N, NotI; P, PstI. (B) Aberrant hypermethylation of the Sall3 locus in placentas of fetuses cloned with female cumulus cells of B6D2F1 mice. The methylation status of placentas of cloned fetuses (n = 10) was compared with that of placentas of control B6D2F1 fetuses produced by natural mating of C57BL/6 and DBA2 mice (NM, n = 4). The value (%) under each lane denotes the methylation rate of the NotI site (M/M +U). The difference in methylation rate was statistically significant between the cloned and control mice (68 ± 8% and 54 ± 7%, respectively, P < 0.01). A NM placenta of B6D2F1 mouse was dissected into the labyrinth zone (La) and the junctional zone (Ju), and their methylation status at the Sall3 locus was analysed. The placental labyrinth zone and the junctional zone were methylated at almost the same rates (52% and 50%, respectively). (C) Aberrant hypermethylation of the Sall3 locus in the placentas of fetuses cloned with fibroblast cells. #18, one B6D2F1 female cloned with a foetal fibroblast. #19, one (B6 x JF1)F1 male cloned with a foetal fibroblast. #20, one (B6 x JF1)F1 female cloned with a tail tip fibroblast. Two controls are fetuses obtained by IVF of C57BL/B6 and JF1 mice. (D) The methylation status of the NotI site in control placentas of B6D2F1 fetuses produced by ICSI and IVF. Five placentas each of ICSI fetuses and IVF fetuses were subjected to Southern blotting under the same conditions as in Fig. 1B. (E) The methylation degree of the NotI site evaluated by real time genomic PCR. The methylation levels evaluated by Southern blotting in Fig. 1B,D were confirmed by real time PCR using three of the placentas of cloned (closed circle) and control fetuses (open circle). For the controls (NM, IVF and ICSI), average methylation degrees of three placentas are shown. (F) Correlation between placental weight and methylation rate of 10 B6D2F1 fetuses cloned with cumulus cells. The placental weight and methylation rate of each cloned mouse are plotted. The methylation rate and placental weight show a positive correlation (coefficient r-value of 0.61, P < 0.1).

Mouse placenta consists of junctional and labyrinth zones (Cross et al. 1994). Morphological examination of placentas revealed that an expanded spongiotrophoblast layer in the junctional zone is the major cause of placentomegaly in cloned mice (Tanaka et al. 2001). DNA methylation of the Sall3 locus was not different between the labyrinth and junctional zones (52% and 50%, respectively) (Fig. 1B). This implies that the hypermethylation of the Sall3 locus is not the result of change in the proportion of certain trophoblast subtypes in the placentas of cloned mice.

Methylation status of the Sall3 locus in placentas of cloned mice with various donor cells

Since placentomegaly is observed regardless of the types of donor cell, we next investigated whether DNA hypermethylation at the Sall3 locus occurs in the placenta of mice cloned with other types of cells (Fig. 1C). In the placenta of a female-fibroblast clone (#18, B6D2F1), the locus was hypermethylated compared with NM controls (72% vs. 54%). Similarly, in (B6xJF1)F1 mice, the placentas of a male-fibloblast clone (#19) and a female-fibroblast clone (#20) showed hypermethylation compared with controls (65%, 58% vs. 45% and 43%, respectively). Thus, the Sall3 locus is hypermethylated regardless of the genetic background, sex and type of the nuclear donor cells.

Oocyte manipulation and in vitro culture do not affect the methylation status of the Sall3 locus

To control for the possibility that the in vitro culture of oocytes and embryos by themselves may trigger abnormal DNA methylation at gene loci (Doherty et al. 2000), we examined the methylation status of the placentas of fetuses produced by ICSI and IVF. We found that the degrees of methylation of the Sall3 locus in placentas of ICSI and IVF fetuses were 53 ± 4% and 52 ± 6%, respectively (Fig. 1D). Thus, the placentas of mice produced after in vitro manipulation were not significantly different from those of NM controls as far as the methylation status of the Sall3 locus is concerned.

The extent of DNA methylation was confirmed by quantitative genomic PCR (Fig. 1E). The placental genome of the NM, IVF and ICSI controls showed 56, 54, and 58% methylation, respectively, at the NotI site of the Sall3 locus. Similarly, clone #4, which showed moderate hypermethylation by Southern blotting was 59% methylated. In contrast, clones #3 and #6 showed hypermethylation (69%) compared with the NM, IVF and ICSI controls.

DNA methylation level of the Sall3 locus correlates with placental weight in cloned mice

To evaluate the relationship between methylation aberration and placental phenotype, methylation rate and placental weight of each cloned placenta are plotted in Fig. 1(F). There was a positive correlation between the extent of hypermethylation of the Sall3 locus and placental weight in cloned mice (r = 0.61, P < 0.1).

Methylation status of CpGs within the Sall3 T-DMR

We analysed the methylation status of the Sall3 locus in ES and TS cells by bisulfite sequencing to determine the size of the Sall3 T-DMR. The T-DMR was 904 bp in length and located in a region just 5' of the NotI site extending to 3' region that is highly homologous with human (Fig. 2A). The T-DMR containing 31 CpGs was hypermethylated in TS cells compared with ES cells, as previously reported (Shiota et al. 2002). Bisulfite sequencing analysis of the Sall3 T-DMR revealed hypermethylation in the placenta of cloned mouse (#15) throughout the T-DMR (Fig. 2B). Comparison between the human and mouse SALL3/Sall3 locus showed the sequence homology in T-DMR as well as promoter and exon 1 (Fig. 2C). Although the methylation status of the NotI site was about 50% in the control placentas (Fig. 1B), the methylation pattern of the whole T-DMR was unlike the pattern of typical imprinted genes. In imprinted genes, differentially methylated regions would show about 50% methylation within whole regions.

View larger version (28K): [in this window] [in a new window]   Figure 2  DNA methylation status of T-DMR at the Sall3 locus in the placenta of cloned mouse. (A) Genomic structure of a 5 kb region that includes Sall3 exon1, the CpG island, T-DMR and the trophoblast-specifically methylated NotI site. Moving averages of GC content (jagged line) and CpG frequency (black bar) are plotted on the graph. Below the graph are marked the Sall3 exon1 (closed box) and the differentially methylated NotI site. The CpG island was formulated by an average GC content greater than 50% and that of CpG frequency greater than 0.6 (Gardiner-Garden & Frommer 1987). Both Sall3 exon 1 and the trophoblast-specifically methylated NotI site are within the CpG island. Methylation status around the differentially methylated NotI site in ES and TS cells is shown below the genomic structure of the Sall3 locus. The CpG sites analysed by sodium bisulfite sequencing are shown as open circles at the very top. Comparison of the methylation status between ES and TS cells indicated that the Sall3 T-DMR included the differentially methylated NotI site and the region highly homologous with human sequence. Each line represents one DNA fragment sequenced. Only methylated CpGs are shown as closed circles. (B) Aberrant hypermethylation throughout the T-DMR in the placenta of a cloned mouse. There are 31 CpGs within the Sall3 T-DMR, and the CpG sites are shown as open circles at the very top. The positions of two CpGs affecting NotI digestion are marked with a box, and their methylation rates are shown by percentage. The overall percentage of methylated CpGs is shown in the right side (methyl-CpGs/all CpGs). In cloned mouse #15, aberrant hypermethylation occurred in the region highly homologous with human sequence. (C) Genomic sequence conservation at and around the Sall3 locus in human and mouse. (Left panel) Comparison of the nucleotide sequences of the mouse and human Sall3/SALL3 5' region. Within the 5 kb orthologous regions, nucleotide sequences of a putative promoter with exon1 and T-DMR are conserved. (Right panel) Gene map of the telomeric region of mouse and human chromosome 18. The order of genes in this region is conserved in the mouse and in the human but in the reversed order.

The Sall3 gene was suggested to play important physiological roles in various tissues (Ott et al. 1996). To address the question whether the epigenetic error at the Sall3 locus of cloned mice may occur in other tissues, we investigated the methylation status of the Sall3 locus in the brain and liver of cloned fetuses at term by Southern blotting (Fig. 3). The Sall3 locus was not methylated in the brain and liver in the control as reported previously (Shiota et al. 2002). The Sall3 T-DMR was hypomethylated in these tissues of the clones as in the control, although the liver of clones was slightly methylated. This shows that the Sall3 locus is methylated specifically in the placenta, perhaps in the trophoblast cell lineage, as previously reported (Ohgane et al. 2001; Shiota et al. 2002) and that the aberrant hypermethylation occurs uniquely in the placenta of cloned mice.

View larger version (45K): [in this window] [in a new window]   Figure 3  Methylation status at the Sall3 locus in the brain and liver of cloned fetuses. Methylation status of the NotI site in foetal brain (Br) and liver (Li) of cloned and control fetuses were analysed under the same conditions as in Figure 1. Cloned mouse fetuses #1 and #2 are from the same B6D2F1 conceptuses analysed in Figure 1. M and U indicate methylated and unmethylated bands, respectively. The Sall3 locus is barely methylated both in cloned and control fetuses.

	Discussion

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Mouse interspecific hybrids have been reported to show a hypertrophic placental phenotype quite similar to that of cloned placentas (Zechner et al. 1996). As found in cloned placentas, these interspecific placentas also exhibited a wide range of placental weight, enlarged spongiotrophoblast layer, increased incidence of glycogen cell differentiation and obscure borders at spongiotrophoblast and labyrinth layers (Zechner et al. 1996; Tanaka et al. 2001). Since the methylation rate of the Sall3 T-DMR correlated with the typical placental phenotype (placentomegaly) in cloned mice, it will be interesting to investigate DNA methylation status of the Sall3 locus in the interspecific hybrid mice.

The production rate of cloned animals is low. Only 2–3% or less, of all reconstituted oocytes, develop into live offspring (Solter 2000; Renard et al. 2002; Wilmut et al. 2002). Most embryos/fetuses die during development, and improper placental development may be, in part, responsible for this. Establishment of cell- and tissue-specific DNA methylation is important for normal embryonic development (Bird 2002; Li 2002; Shiota & Yanagimachi 2002). Therefore, genomic loci frequently associated with the epigenetic error have been explored in the cloned animals. The finding of the Sall3 locus showing frequent abnormal DNA methylation is crucial. From the data, we concluded that there is a genomic locus highly susceptible to epigenetic error caused by nuclear transfer.

	Experimental procedures

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Animals

Placentas, brains and livers of cloned and control fetuses at 19.5 dpc were collected and analysed for DNA methylation. One term placenta of naturally mated mouse was mechanically dissected into the junctional and labyrinth zones after removal of the embryo. Donor cells for cloning were adult cumulus cells (Wakayama et al. 1998), adult tail tip fibroblast (Wakayama & Yanagimachi 1999) and foetal fibroblast (Ogura et al. 2000b). Control fetuses were obtained by natural mating, in vitro fertilization (Toyoda et al. 1971) or intracytoplasmic sperm injection (Kimura & Yanagimachi 1995). Strains of males and females used for the production of fetuses are described in figure legends.

Database search and RLGS spot cloning

DNA of an RLGS spot showing aberrant methylation in one cloned mouse (spot 8 in Ohgane et al. 2001) was purified by the NotI trapper method as described elsewhere (Ohgane et al. 1998, 2002). The purified DNA was initially ligated into the NotI and PstI sites of pBluescript II (Stratagene, CA). The inserted fragments were amplified by PCR with the M4-RV primer set and cloned into the pGEM-T vector (Promega, WI). Cloned DNA was sequenced by using a Shimadzu autosequencer system (Shimadzu, Kyoto, Japan) following the manufacturer's instructions. The nucleotide sequence obtained from spot DNA cloning was compared with NCBI (http://www.ncbi.nlm.nih.gov) and Ensemble (http://www.ensembl.org) mouse and human genome sequence databases by the BLAST search program. Human genes on chromosome 18 responsible for inherited diseases were ascertained by searching the OMIM database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = OMIM).

Southern blotting

Genomic DNAs (5 µg each) from cloned and control mouse tissues (placenta, brain and liver) were double-digested with NotI and PstI, and separated on a 1.4% agarose gel followed by blotting on to nylon membrane. A probe specific to spot 8 (the Sall3 locus) sequence was labelled with a DIG-dUTP using a random primer labelling kit (Roche Diagnostics, Mannheim, Germany). Hybridization and washing were performed under stringent conditions as described elsewhere (Hirosawa et al. 1994). Signals were detected with a DIG luminescent nucleotide detection kit (Roche Diagnostics) containing alkaline phosphatase (AP)-conjugated anti-DIG antibody and AP substrate, and visualized on X-ray film (Fuji Film, Tokyo, Japan).

Statistical analysis

The intensities of the methylated and unmethylated bands were measured by NIH image software provided by the National Institutes of Health (ftp://rsbweb.nih.gov/pub/nih-image/nih-image161_fat.hqx). Methylation rate of the NotI site of the Sall3 locus was calculated by the formula: (intensity of methylated band)/(intensity of methylated band) + (intensity of unmethylated band) and presented by mean ± standard deviation. The difference in methylation rate between cloned and control mice was evaluated by the independent Student's t-test. The relationship between placental weight and the Sall3 methylation rate of cloned placentas was tested with Pearson's correlation coefficient, and its P-value was evaluated from the coefficient r-value.

Bisulfite sequencing

Bisulfite sequencing was performed following previously reported procedure (Imamura et al. 2001). In brief, 5 µg each of EcoRI-digested genomic DNA were modified with sodium metabisulfite, and one-tenth of each modified DNA was amplified with AmpliTaq Gold (Perkin Elmer, Norwalk, CT, USA) and the following primer sets: BisF1, 5'-GGGAAGTAAATGTTTTTTGGTT-3'; BisR1, 5'-AACTAACTAAAAAAACTCTATATC-3'; BisF2, 5'-GTTAGGGTTTTTTTAGGGTATTAGT-3'; BisR2, 5'-CCCTAATCTACCCAACATATACAAA-3'; BisF3, 5'-GATTAAATGAATGGATTTATTTTTGT-3'; and BisR3, 5'-ATTAACTATCTAAAATTTTCAACAC-3'. Amplified fragments were cloned into pGEM-T vector (Promega), and 10 or 12 independent clones for each primer set were sequenced to determine methylation status.

Real time PCR

Real time genomic PCR was performed using the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, CA) following the manufacturer's instructions. Placental genomic DNAs (40 ng), with or without NotI-digestion (methylated DNA or total DNA, respectively) were analysed by real time PCR using F5 (5'-TACCAGCACGGAGGCCGAGGA-3') and R3 (5'-GATCATAAAAAGTTGGCTTTTAAGG-3') primer pair flanking the differentially methylated NotI site. TaqMan Rodent GAPDH Control Reagent (Applied Biosystems) was used for normalization of the template DNA amount. Methylation degree of the NotI site was calculated by the formula: (quantity of methylated DNA)/(quantity of total DNA).

	Acknowledgements

 
We thank Dr Steven Ward for reading the original manuscript and valuable suggestions and Dr Jody Haigh for helpful comments. We also thank Ms. Naoko Sato, Mr Masahiro Kujiraoka, Mr Tetsuya Abe and Mr Stephen Black for their help. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences and the Grant-in-aid for Scientific Research, Ministry of Education, Culture, Sports, Science and Technology, Japan (11794010) (K.S.), the Harold Castle Foundation and the Victoria and Geist Foundation (R.Y.).

	Footnotes

 
Communicated by: Shinichi Aizawa

^aPresent address: Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-Ku, Kobe 650-0047, Japan.

^* Correspondence: E-mail: ashiota{at}mail.ecc.u-tokyo.ac.jp

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Received: 27 November 2003
Accepted: 19 December 2003

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