Animals and Embryo Cloning

All animal experiments were approved by the Institutional Review Board of the University of Yamanashi (permit number A 24-50) and Tokyo University of Agriculture (permit number 100553). BDF1 mice (C57BL/6NJcl × DBA/2JJcl; CLEA Japan, Tokyo, Japan) were used in all experiments. Mature sperm was isolated from 10- to 12-wk-old mice.

Tail-tip fibroblast cell cultures were established at the time of sperm collection to obtain somatic donor nuclei for generating CL mice [17]. The donor nucleus was injected into enucleated oocytes from BDF1 mice according to our original protocol, in which reconstructed oocytes were treated with tricostatin A after oocyte activation. Four-cell-stage embryos were transferred into the oviducts of recipient females and 11 males in four litters developed to full term.

Sperm Collection

Sperm were collected from CL mice exhibiting no phenotypic abnormalities, their descendants, and wild-type (WT) mice (10–12 wks old). After incubation in 200 μl of TYH medium for 1 h, the sperm were collected by the swim-up method and used for DNA methylation analysis.

DNA preparation

Sperm DNA was isolated by a standard phenol-chloroform extraction procedure with dithiothreitol, and 1 μg of DNA was dissolved in 130 μl of 10 mM Tris-HCl (pH 8.0) and sheared with an S220 focused ultrasonicator (Covaris, Woburn, MA), yielding 500-bp fragments. The AMPure XP system (Agilent Technologies, Santa Clara, CA) was used to purify the fragmented DNA as follows. Sheared DNA (130 μl) was mixed with 1.8× volume (234 μl) of AMPure XP reagent and allowed to stand for 15 min at room temperature. The beads were collected using a magnetic stand, the supernatant was removed, and pelleted beads were rinsed with 70% ethanol and dried by incubation at 37°C for 5 min. DNA was then eluted from the beads with 20 μl of RNase-free water. The eluted DNA solution was dried under vacuum and dissolved in 7 μl of RNase-free water.

Target Enrichment

Targets' enrichment by liquid-phase hybridization capture was performed using the SureSelect Mouse Methyl-Seq kit (Agilent Technologies) [22]. Genomic DNA (7 μl) that was fragmented and purified as described above was supplemented with 3 μl of formamide (biochemistry grade; Wako Pure Chemical Industries, Osaka, Japan) and overlaid with 80 μl of mineral oil (Sigma-Aldrich, St. Louis, MO). The DNA was completely denatured by incubating the tube at 99°C for 10 min; the tube was then cooled to and maintained at 65°C for at least 5 min before adding the following reagents. Hybridization buffer and capture probe mix were prepared according to the manufacturer's protocol, and they were each overlaid with 80 μl of mineral oil and incubated at 65°C for 10 min. The two solutions were then combined and mixed thoroughly by pipetting. The combined solution was transferred to a tube containing the denatured input DNA (maintained at 65°C as described above), and the solution was thoroughly mixed by pipetting. The tube was incubated at 65°C for 24 h to allow hybridization between probes and targets. A 50-μl volume of well-suspended DynaBeads MyOne Streptavidin T1 solution (Life Technologies, Carlsbad, CA) was placed in a 1.5-ml tube, and the beads were washed twice with 200 μl of binding buffer. The hybridization reaction supplemented with 200 μl of binding buffer was added to the pelleted beads with thorough mixing. After incubation at room temperature for 30 min with agitation, the beads were collected using a magnetic stand and were washed with 500 μl of wash buffer 1, and then subjected to three rounds of washing and resuspension in prewarmed buffer 2, followed by incubation at 65°C for 10 min. After removing the washing solution from the tube, enriched DNA was eluted by incubating the beads in 20 μl of elution buffer at room temperature for 20 min. The eluate was immediately subjected to bisulfite treatment.

Bisulfite Treatment

The EZ DNA Methylation-Gold kit (Zymo Research, Irvine, CA) was used for bisulfite treatment of target-enriched DNA according to the manufacturer's instructions. The enriched DNA solution (20 μl) was mixed with 130 μl of freshly prepared CT conversion reagent, and the mixture was incubated at 64°C for 2.5 h. The 10-min incubation step at 98°C was omitted because the target-enriched DNA was already denatured. After purification and desulfonation, bisulfite-treated DNA was eluted with 20 μl of M-elution buffer.

PBAT Library Construction and Illumina Sequencing

We used bisulfite-treated DNA for library preparation according to the PBAT protocol [22] (also available from  http://crest-ihec.jp/english/epigenome/index.html), except that the following primers were used. The primer used for first-strand synthesis was 5′-biotin ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT WWW WNN NN-3′ (W = A or T). The indexed primer used for second-strand synthesis was 5′-CAA GCA GAA GAC GGC ATA CGA GAT XXX XXX GTA AAA CGA CGG CCA GCA GGA AAC AGC TAT GAC WWW WNN NN-3′, where XXX XXX represents the index sequence of each primer. The constructed SSM-PBAT libraries were sequenced as previously described [234–5] using the Illumina HiSeq2500 system (San Diego, CA).

Target Methylome Sequence Alignment and Statistical Analysis

SSM-PBAT reads were aligned to the mouse genome (mm10; Genome Reference Consortium Mouse Build 38) using the Bismark tool (v.0.10.0;  http://www.bioinformatics.babraham.ac.uk/projects/bismark/), with the specific options: −q −n 2 –l 93 –pbat. Statistical significance of DNA methylation at each CpG site and CGI (CpG islands) was evaluated by the Fisher exact test and the Mann-Whitney U test. Composite profiles were generated using SeqMonk ( http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/).

Summary of SSM-PBAT Library

Somatic cell nuclear transfer (SCNT) clones used in these experiments had normal phenotype, and their sperm were active and were obtained at high concentrations. Sequence analysis of sperm samples from five wild-type (WT) and eight SCNT mice yielded 25.4 million to 60.7 million reads (29.1%–47.5% of total reads) that mapped to the mm10 reference (Table 1). The average depth of each sample was 15.2–37.6, which covered 68.8%–77.9% of target regions at five depths. This indicated that obtained sequencing data were sufficient for further analysis of clone-specific methylation errors.

TABLE 1

Summary of SSM-PBAT libraries in cloned mouse sperm.

We also determined the correlation coefficient of DNA methylation data between individuals. The correlation coefficients showed similarly high values (i.e., 0.97–0.99; Supplemental Fig. S1A; Supplemental Data are available online at  www.biolreprod.org). Values for CGI and its shores (CGIsh: ±2 kb of CGI), and germ line differentially methylated regions (DMRs; Supplemental Fig. S1, B–D) were also similar. A violin plot analysis showed that all samples analyzed belonged to the same group (Supplemental Fig. S2). These results demonstrate that overall methylation profiling does not detect latent methylation errors in the sperm of CL mice, although they may be present, albeit at a low rate and with a high degree of variability.

Profiling of Differentially Methylated Sites and Islands

To identify differentially methylated sites (DMS) in individual sperm genome libraries, the methylation level of each CpG site was compared to the average level in WT, and significance was assessed with Fisher exact test (P < 0.01). Both hypermethylated and hypomethylated DMS were detected, ranging from 154 to 875 and 106 to 1874, respectively (Fig. 1A), out of a total of 2 454 646–2 840 983 tested CpG sites (Supplemental Table S1). Remarkably, two of eight CL mice (CL1 and CL7) had significantly higher numbers of DMS (Grubbs test for outliers; P = 0.0045 in CL1, P = 0.0116 in CL7)—which were predominantly hypomethylated (Grubbs test for outliers; P = 0.0393 in CL1, P = 0.0004 in CL7)—than the others. The DMS were widespread in the sperm genome and were potentially associated with genome-wide DNA methylation and/or demethylation events through germ line reprogramming (Fig. 1B and Supplemental Table S2). Interestingly, DMS were frequently located on chromosome 13 in CL7 (hyper: 0.089%; hypo: 0.419%). Differences at hypermethylated and hypomethylated DMS in CL1 and CL7 were mostly in the ±15% to ±50% range (Fig. 1C). Differentially methylated sites were detected in promoter and exon regions, although many were located in intergenic and intronic regions (Fig. 2). These results demonstrate that aberrant CpG loci are present in the CL mouse sperm genome, consistent with the error-prone signature of epigenetic reprogramming in somatic cell cloning.

FIG. 1

Differentially methylated CpG sites in CL mouse sperm. A) Number of hypermethylated and hypomethylated DMS in the sperm of WT (n = 5) and CL (n = 7) mice. DNA methylation levels at each CpG site across targeted regions (2 881 255 CpG sites) were compared between each individual and the average of WT mice, which served as the normal sperm methylation control. Fisher exact test was used to assess DMS (P < 0.01). B) DMS distribution on each chromosome of CL1 and CL7 mouse sperm. Hypermethylated (red) and hypomethylated (blue) DMS are shown as a chromosome-scale distribution. C) Differential methylation levels at DMS in CL1 and CL7 mouse sperm. Histograms of differences between CL and the average WT methylation level (AWT) were constructed based on hypermethylated (CL − AWT) and hypomethylated (AWT − CL) DMS.

FIG. 2

Distribution of DMS in specific genomic regions in CL1 and CL7. Pie charts show the frequency of DMS in genic, intronic, and intergenic regions.

We then assessed the contribution of clustered DMS to CGI and CGIsh in the sperm genome. CGI and CGIsh that included >15% DMS of total CpGs and were significantly different by the Mann-Whitney U test (P < 0.01) were defined as differentially methylated CGIs (DMI) and CGIsh (DMIsh), respectively (Fig. 3, A and B, and Supplemental Table S3). We classified CGI and CGIsh (23 021 both upstream and downstream) in each CL mouse as DMI or DMIsh. In total, 27 DMI (hyper: 11; hypo: 16) and 9 DMIsh (hyper: 6; hypo: 3) were identified; consistent with the DMS analysis, many DMI were observed in the sperm libraries of CL1 and CL7. Although further studies are required to discriminate between cloning-associated errors and occurring variations, these data provide experimental evidence that CGI methylation in CL mice is distinct from that observed in WT mice.

FIG. 3

Differentially methylated CGI in CL mouse sperm. A) Number of hypermethylated and hypomethylated DMI and DMIsh in the sperm of individual CL mice. B) Methylation level of common DMI (CGI IDs: 4464 and 11496) and DMIsh (CGI IDs: 4458 and 21008) in individual WT and CL mice. Red and blue squares represent individuals exhibiting significant differences (P < 0.01). C) A motif of DMIsh that binds to the reprogramming factors POU domain/class 5/transcription factor 1 (OCT4) and sex-determining region Y-box 17 (SOX17). The motif that shows 60% homology to target sequences was detected at a significant frequency (P < 0.01) by HOMER ( http://homer.salk.edu/homer/).

Interestingly, some DMI and DMIsh were common to multiple CL mouse sperm libraries. CGIsh reportedly discriminate between different cell types, including somatic [23] and reprogrammed [24] cells, and are therefore potential targets for DNA methylation errors. For example, a hypermethylated DMI (CGI4464) was observed in three of the seven CL mice (Fig. 3B and Supplemental Fig. S3), and two of the mice had the same hypomethylated DMI (CGI11496). These DMI may be loci that are susceptible to changes in methylation caused by erroneous germ line reprogramming. Similarly, some DMIsh were consistently hypermethylated and hypomethylated in the cloned mice (Fig. 3B). To investigate the possible effect of DMI and DMIsh on gene expression, we investigated the neighboring genes (±5 kb from DMI or DMIsh; Table 2). The DMI existed in the promoter of small G-protein-signaling modulator 1, complexin 3, WNK lysine-deficient protein kinase 4, Arhgap, PC esterase domain-containing 1B, and histocompatibility 2 O region α genes, whereas DMIsh were also located in the promoter of gap junction protein α4. Interestingly, a motif (CCATTGTATGCAAAT) that binds to the reprogramming factors POU domain/class 5/transcription factor 1 and sex-determining region Y-box 17 (also known as OCT4 and SOX17, respectively) was detected in the DMIs (Fig. 3C).

TABLE 2

The neighboring genes of DMI and DMIsh in sperm of cloned mice.

Transmission of CL DMI to Offspring

To determine whether DNA methylation errors in the sperm of SCNT cloned mice are transmitted to the genome of descendants' sperm, we analyzed sperm derived from CL1 and CL7 offspring (Supplemental Table S4). There was no evidence that DNA methylation errors in the sperm of CL mice were present in the sperm of their offspring (Fig. 4 and Supplemental Tables S5–S7). Although the DMI (CGI ID 4462) of CL7 was, in fact, transmitted to offspring No. 3 (Fig. 4, B and C, and Supplemental Tables S6 and S7), this likely arose by stochastic occurrence of DMS and DMI.

FIG. 4

DNA methylation status in sperm from offspring of CL1 and CL7. Results are shown as methylation change breadth in a two-dimensional coordinate graph. Methylation levels of each DMI (A: CL1/CL1OF; B: CL7/CL7OF) and DMIsh (C: CL7/CL7OF) are represented by different colors, and each offspring is represented by a different symbol (○, △, and □). DMI and DMIsh marked by asterisks are significantly different (P < 0.01). Statistical significance was evaluated in the same way as for CL mouse data sets (see text).