Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks

Genome-wide dynamic changes in DNA methylation are indispensable for germline development and genomic imprinting in mammals. Here, we report single-base resolution DNA methylome and transcriptome maps of mouse germ cells, generated using whole-genome shotgun bisulfite sequencing and cDNA sequencing (mRNA-seq). Oocyte genomes showed a significant positive correlation between mRNA transcript levels and methylation of the transcribed region. Sperm genomes had nearly complete coverage of methylation, except in the CpG-rich regions, and showed a significant negative correlation between gene expression and promoter methylation. Thus, these methylome maps revealed that oocytes and sperms are widely different in the extent and distribution of DNA methylation. Furthermore, a comparison of oocyte and sperm methylomes identified more than 1,600 CpG islands differentially methylated in oocytes and sperm (germline differentially methylated regions, gDMRs), in addition to the known imprinting control regions (ICRs). About half of these differentially methylated DNA sequences appear to be at least partially resistant to the global DNA demethylation that occurs during preimplantation development. In the absence of Dnmt3L, neither methylation of most oocyte-methylated gDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independent methylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells.


Introduction
Throughout mammalian gametogenesis, dynamic DNA methylation changes occur in a sex-and sequence-specific manner. These changes result in the establishment of oocyte-and spermspecific genomic imprints and unique methylation patterns of repetitive elements via DNA methyltransferase activity [1][2][3][4]. This process is indispensable for functional gamete and embryo development. For example, sex-specific methylation imprints are maintained throughout cell division after fertilization, despite genome-wide demethylation and de novo methylation during embryogenesis. These imprints control parent-of-origin specific monoallelic expression of a subset of genes, which are known as imprinted genes [5][6][7][8][9]. In addition, DNA methylation during spermatogenesis plays a crucial role in meiotic progression and retrotransposon silencing [10][11][12][13][14]. However, little is known about the profile and functional role of DNA methylation during oogenesis, except for the establishment of genomic imprints.
Shotgun bisulfite sequencing (SBS) may be able to overcome this limitation and enable the determination of the cytosine methylation status of individual CpG sites at a whole-genome level without a bias toward CpG-rich regions [22,23,26] and with only relatively small-scale DNA samples [24,27]. As a result, in this study, an improved SBS method for small-scale DNA samples was used to analyze the DNA methylome of mouse germ cells. In addition, the mouse germ cell transcriptome was investigated using high-throughput cDNA sequencing (mRNA-seq) to reveal relationships between DNA methylation and gene transcription in both male and female germ cells.

Genome sequencing
We performed SBS analysis by using MethylC-seq [22] and a new SBS method called ''whole bisulfitome-amplified DNA sequencing'' (WBA-seq). The MethylC-seq and WBA-seq libraries were generated as shown in Figure S1. The MethylC-seq method generated 1010 and 1085 million tags (reads) from germinal vesicle (GV) stage oocytes and epididymal sperm, respectively. Oocyte DNA libraries generated by MethylC-seq showed higher redundancies than sperm DNA libraries. For example, 33.0% and 81.7% of the 21 million cytosines of CpGs in the mouse genome were covered by at least 1 sequence read from GV oocytes and sperm, respectively; whereas the average read depth (i.e., the number of hits of reads that were mapped to a given position) was over 106 for both germ cells ( Figure S2). The WBA-seq method generated 307 and 397 million tags from GV oocytes obtained from wild-type and Dnmt3L-deficient (Dnmt3L 2/2 ) mice, respectively. WBA-seq libraries for GV oocytes showed higher genome coverage (60% of genomic CpGs were covered by at least 1 read) but with smaller average read depth (7.46) than MethylC-seq library. Some reads from the oocyte libraries strongly matched mitochondrial DNA (mtDNA), satellite, low complexity, or simple repeat sequences ( Figure S3), which might have been due to a distinct genomic copy number bias in the mitochondria of germ cells or an overamplification bias. Thus, SBS results were simplified by removing the redundancy information (only mtDNA was separately examined for DNA methylation) and combining MethylC-seq and WBA-seq results for wild-type oocytes. Consequently, the average read depth was 18.86, 4.46, and 12.56for wild-type and Dnmt3L 2/2 oocytes, and sperm, respectively, and 70.8%, 45.6%, and 79.9% of genomic CpGs were covered by at least 1 sequence read from each cell type (Table 1 and Figure S3). Furthermore, the average read depths of MethylC-seq of mouse blastocysts and embryonic stem cells (ESCs), which served as zygote and stem cell controls, were 12.86and 6.16, respectively ( Table 1).

Methylome of mouse germ cells
The average methylation level of wild-type oocytes (40.0%) was less than half that of sperm (89.4%) ( Figure S4). This difference in global DNA methylation between male and female germ cells was consistent with results from the previous studies [28,29]. The Dnmt3L 2/2 oocyte genome was observed to be hypomethylated, exhibiting a methylation level of only 5.5%. Furthermore, blastocysts showed a lesser extent of methylation (21.3%) than did wild-type oocytes; ESCs, on the other hand, showed relatively high levels of methylation (70.6%). To elucidate the distribution of methylation levels on CpG sites, on regional and genome-wide scales, we created dot plots of CpG methylation for individual chromosomes and histograms of the methylation levels for all CpGs. These graphs revealed that hypermethylated CpGs in oocytes tended to cluster in transcribed regions of particular genes (e.g., Kcnq1 or Rlim genes, known to be expressed in oocytes [30,31]); the sperm genome was almost entirely hypermethylated, except at most CpG-rich regions ( Figure 1 and Figure S5). Specifically, 55.7% of the CpGs in the oocyte genome exhibited ,10% methylation, whereas another 32.0% of CpGs exhibited $90% methylation (Figure 2A). The Dnmt3L 2/2 oocyte genome was also hypomethylated in almost all chromosomal regions ( Figure S6). The methylation level of the mtDNA genome in

Author Summary
In mammals, germ-cell-specific methylation patterns and genomic imprints are established throughout large-scale de novo DNA methylation in oogenesis and spermatogenesis. These steps are required for normal germline differentiation and embryonic development; however, current DNA methylation analyses only provide us a partial picture of germ cell methylome. To the best of our knowledge, this is the first study to generate comprehensive maps of DNA methylomes and transcriptomes at single base resolution for mouse germ cells. These methylome maps revealed genome-wide opposing DNA methylation patterns and differential correlation between methylation and gene expression levels in oocyte and sperm genomes. In addition, our results indicate the presence of 2 types of methylation patterns in the oocytes: (i) methylation across the transcribed regions, which might be required for the establishment of maternal methylation imprints and normal embryogenesis, and (ii) retroviral methylation, which might be essential for silencing of retrotransposons and normal oogenesis. We believe that an extension of this work would lead to a better understanding of the epigenetic reprogramming in germline cells and of the role for gene regulations. Dnmt3L 2/2 oocytes (4.4%) was lower than that observed in wildtype oocytes (6.6%). Sperm methylation levels, by comparison, were relatively high (14.7%), whereas those of the blastocysts and ESCs were quite low (1.3% and 2.1%, respectively) ( Figure S4). Since previous studies revealed a significant correlation between CpG frequency and methylation within intra-and intergenic regions in somatic cells [32,33], the CpG density and methylation levels were compared to identify genome-wide differential methylation patterns in germ cells. CpG density was defined as the number of CpG dinucleotides in 200 nucleotide (nt) windows (e.g., 1 CpG dinucleotide per 200 nt corresponds to a density of 0.005). At low CpG densities (range, 0.005-0.05), the oocyte genome was about 50% methylated, whereas the sperm genome was 80-90% methylated. At moderate to high CpG densities (range, 0.05-0.2), both male and female germ cells were hypomethylated ( Figure 2B). Furthermore, 4 families of transposable elements (long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), long terminal repeats (LTRs), and DNA transposons) were moderately methylated in oocyte genomes but were hypermethylated in sperm. In addition, a general trend towards higher methylation levels at higher CpG densities in the oocyte genome occurred in LTRs. Conversely, a trend toward lower CpG methylation levels at higher CpG densities in the wild-type oocyte and sperm genomes was observed in SINEs and DNA transposons. In contrast, all of these transposable elements were hypomethylated in Dnmt3L 2/2 oocytes. Interestingly, however, there was partial CpG methylation in LINEs and LTRs at relatively high CpG densities (range, 0.03-0.1). These complete or partial undermethylations were confirmed by bisulfite sequencing in L1 LINEs, B1/Alu SINEs, and intracisternal A particle (IAP) LTRs ( Figure  S7). These results suggested that each germ cell has a unique sequence-and CpG-density-dependent methylation pattern. In addition, oocyte CpG methylation, except in a subset of retrotransposons, appears to be Dnmt3L dependent.
We also characterized the methylation patterns of 15 germlinedifferentially methylated regions (gDMRs). The differential (between oocyte and sperm) methylation occurs at imprinted gene loci (also called imprinting control regions (ICRs)). The ICRs of maternally methylated imprinted genes (e.g., Nespas-Gnas) were shown to be hypermethylated in oocytes but hypomethylated in sperm, while the converse was true in ICRs of paternally-methylated imprinted genes (e.g., H19) ( Figure 3 and Figure S8). Interestingly, only the Snrpn Open boxes represent the location of CpG islands (CGIs). Red, purple, blue, and green boxes represent the methylation levels at individual CGIs in wild-type oocyte, Dnmt3L 2/2 oocyte, sperm, and blastocyst genomes, respectively, determined by our results from shotgun bisulfite sequencing (SBS) method and Smallwood's results from reduced representation bisulfite sequencing (RRBS) method [38]. doi:10.1371/journal.pgen.1002440.g001 gDMR was partially methylated (35.7%), whereas all other maternal ICRs were hypomethylated in Dnmt3L 2/2 oocytes (Table 2). This residual methylation might result in the stochastic acquisition of the maternal imprint in the progeny of Dnmt3L 2/2 females [34]. These results strongly suggested that the methylation level of individual CpGs can be determined from DNA methylome maps with a high degree of accuracy.
The study of mammalian DNA methylation patterns has previously suggested that methylation predominantly occurs at CpG sites; however, more recent studies, based on SBS methods, have indicated that methylation at non-CpG sites also occurs in human ESCs [22,23]. Detection of non-CpG methylation is one of the applications of the bisulfite-based methylation analysis but is problematic due to the incomplete conversion of cytosine, and overestimates of such cytosine by PCR amplification, which cannot be discriminated from true methylation. In order to evaluate the methylation status of non-CpG sites and avoid these problems, additional SBS analysis of mouse GV oocytes, sperm, blastocysts, and ESCs was performed by a non-amplification technique, termed Post-Bisulfite Adapter Tagging (PBAT) [Miura F. & Ito T, personal communication]. All C (originally methylated cytosine) and T (originally unmethylated cytosine) that mapped to genomic CpG and CpH sites (H = A, T, or C) were counted. The PBAT results showed CpG methylation ratios (C ratios = 0.395, 0.748, 0.137, 0.615 in oocytes, sperm. blastocysts, and ESCs) which are similar to the average methylation levels of individual DNA methylome maps obtained by MethylC-seq and WBA-seq among all examined cells. Interestingly, a relatively high fold enrichment of non-CpG methylation was observed in GV oocytes (C ratio = 0.034-0.038), but not in the other cell types, including mouse ESCs (C ratio ,0.01) ( Figure S11).

Relationship between the DNA methylome and transcriptome of mouse germ cells
To elucidate the interaction between intragenic DNA methylation and gene transcription, the correlation between promoter and gene-body methylation and expression levels for 20,854 different genes was examined. The mRNA-seq profiles for germ cells and ESCs are shown in Table S1. The results showed that mRNA transcript levels in oocytes were strongly correlated to gene-body methylation levels (Spearman's r.0.5, p,1610 29 ) but were not significantly correlated to promoter methylation levels (|r|,0.1) ( Figure 4A). For example, the regions +2 to +5 kb from the transcription start site (TSS) and 0 to 25 kb from the transcription termination site (TTS) were hypermethylated (60-90% methylation) for the top 20% of expressed genes but were hypomethylated (10-30% methylation) for the bottom 20% of expressed genes. However, areas near the TSS (6500 base pairs (bp)) were hypomethylated (10-20% methylation) in all genes, regardless of their expression level. In contrast, in the Dnmt3L 2/2 oocyte genome, the correlation between gene expression and genebody methylation was very weak (|r|,0.1) ( Figure 4B). In the sperm genome, promoter methylation was negatively correlated (Spearman's r = 20.36, p,1610 29 ) with gene expression, whereas gene-body methylation was positively correlated (Spearman's r = 0.14-0.16, p,1610 29 ) to gene expression; the latter correlation was weaker than that observed in the oocyte genome ( Figure 4C).

Role of Dnmt3L in the DNA methylome/transcriptome relationship
Further investigation of gene expression patterns in oocyte genomes revealed that the mRNA transcript levels between wildtype and Dnmt3L 2/2 oocytes were very highly correlated (R 2 = 0.9611) ( Figure 5A). In fact, there were no significant differences in the expression levels of representative oocyte-specific genes (e.g., Gdf9, Bmp15, Bcl2l10, Zp1, Zp2, Zp3, Zar1, Npm2, Nlrp5, and Dppa3, which are responsible for ovarian follicle formation, reproduction, and early development [35]) and DNA methyltransferase genes (e.g., Dnmt1, a maintenance methyltransferase, and Dnmt3a and Dnmt3b de novo methyltransferases); the expected difference in the expression level of Dnmt3L between wild-type and Dnmt3L 2/2 oocytes was observed ( Figure 5B, 5C). These results suggested that changes in gene expression did not occur during oogenesis, despite global intragenic hypomethylation in Dnmt3L 2/2 oocytes. Furthermore, the expression levels and exon patterns of maternally-methylated imprinted genes across each ICR were not altered in Dnmt3L 2/2 oocytes ( Figure 3 and Figure 5D). This result suggested that the disruption of maternal methylation imprints in the Dnmt3L 2/2 oocyte genome was not due to the lack of their transcription [36]. On the other hand, maternal methylation imprints at ICRs (and many other hypermethyla- Surprisingly, gene expression in ESC genomes was negatively correlated with promoter methylation and was not positively correlated with gene-body methylation ( Figure S12). Meanwhile, these ESCs showed the apparent expression of all DNA methyltransferase gene families including Dnmt3L ( Figure S13). Previous studies indicated that the zygotic and somatic functioning of Dnmt3L is not essential for global methylation in ESCs in mice [6]. Thus, unlike oocytes, the functional role of Dnmt3L in genebody methylation after fertilization is unclear. However, the expression of pluripotency-associated genes, Pou5f1, Klf4, Sox2, Myc, Nanog, and Lin28a, was clearly observed in ESCs. The expression of Pou5f1, Lin28a, and Glis1, recently identified as maternal reprogramming factors, were also observed in oocytes ( Figure S14). While differential expression of the pluripotency genes among germ and stem cells was observed, the promoter regions of these genes demonstrated low-level methylation in almost all of the examined cells. In sperm cells, only the Nanog promoter was hypermethylated (this result was similar to a previous study [29]).

Identification and characterization of germline differentially methylated regions
To identify gDMRs, the average CpG methylation levels of individual CpG islands (CGIs), which are CpG-rich genomic regions often lacking DNA methylation, were calculated. Recently, Illingworth et al. determined the number of CGIs by deep sequencing of isolated, unmethylated DNA clusters [37]. Among the 23,021 mouse CGIs (22,974 CGIs were informative in both oocytes and sperm), 2014 were highly methylated ($80% methylation) in oocytes, 818 were highly methylated in sperm, and 377 were highly methylated in both germ cells ( Figure 6A). Furthermore, we also identified 1678 gDMRs ($80% methylation in 1 gamete and #20% in the other), 1329 of which were oocytespecific methylated CGIs, while the remaining 349 were spermspecific methylated CGIs ( Figure 6A, Figure S6, and Table S2). Among these gDMRs, 646 gDMRs were confirmed to show a differential methylation status between GV oocytes and sperm (by similar criteria: $75% methylation in 1 gamete and #25% in the other); the methylation status was previously examined by performing large-scale bisulfite sequencing of CpG-rich regions of the genome (reduced representation bisulfite sequencing: RRBS) (Table S3) [38]. Additionally, almost all known ICRs except Zdbf2 DMRs (which do not have any CGIs) were reidentified from our gDMR list (Table S2).
A total of 78% oocyte-methylated gDMRs (n = 1045) were located within the intragenic regions. Approximately 25% of the oocyte-methylated gDMRs (n = 322) overlap with either the first exon or the proximal promoter regions of the genes, as has been observed with most of the described maternal ICRs [39]; only 5% of the sperm-methylated gDMR (n = 18) showed such overlap. Alternatively, 34% of sperm-methylated gDMRs (n = 120) overlap with intergenic regions, as in all known paternal ICRs ( Figure 6B). Interestingly, oocyte-methylated gDMRs in transcribed regions tended to be more abundant within highly expressed genes, but such a trend was not observed in the sperm genome ( Figure 6C). Oocyte-methylated gDMRs were also identified in non-imprinted genes, such as the DNA methyltransferase genes (e.g., Dnmt1 and Dnmt3b) and some male germline-specific genes (e.g., Piwil1, Spag1, Ggnbp2, Tbpl1, Spata16, Ggn, Acrbp, and Cd46). The oocytemethylated gDMR in Dnmt1 was located in spermatocyte-and somatic-specific exons, while oocyte-specific exons were hypomethylated in oocytes ( Figure S9). Dnmt3L 2/2 oocytes also showed hypomethylation in most of these gDMRs. Significant changes in the expression levels of genes with alternative splicing patterns were not observed in the Dnmt3L 2/2 oocyte genome ( Figure 3, Figure 5E, and Figure S9). These results indicate that these oocytespecific methylated gDMRs do not regulate gene expression or alternative splicing during the oocyte stage. To determine whether or not these germ cell-specific methylations are maintained after fertilization, when the genomes undergo global demethylation, the individual CGI methylation levels in blastocyst genomes were calculated. In blastocysts, all ICRs demonstrated low to moderate methylation (25.1-64.3%), whereas many gDMRs were demethylated (0-20%) ( Figure 6D). Furthermore, 817 oocyte-methylated gDMRs (including Piwil1, despite being a non-imprinted gene locus) and 34 sperm-specific gDMRs were resistant to demethylation during early embryogenesis ($20% methylation in blastocysts) ( Figure 6D and Table S2).

Discussion
To the best of our knowledge, this is the first study to generate single-base resolution maps of DNA methylomes spanning the entire genome of mouse germ cells. The oocyte maps are particularly valuable and informative because, in the past, such an analysis was prohibitive due to the need for large quantities of DNA. Recently, Smallwood et al. [38] reported large-scale DNA methylation patterns in mouse germ cells by using the RRBS method, which targets only CpG-rich regions. However, our more comprehensive results provide strong evidence that gene expression was positively correlated to Dnmt3L-dependent intragenic methylation in oocytes, and that methylation patterns in oocytes differed from those in sperm and non-germline cells.
The functional role of gene-body methylation has been an enigma despite its conservation in plants and animals [41][42][43]. Maunakea et al. [44] suggested that gene-body methylation is involved in the regulation of alternative splicing events. Although methylated gDMRs were detected in the alternative exons of Dnmt1 and Gnas in mouse oocytes, loss of oocyte-specific methylation marks in the Dnmt3L 2/2 oocytes did not affect the expression patterns of alternatively spliced transcripts. Therefore, our results indicate that gene-body methylation is not involved in alternative splicing in oocytes.
Previously, Chotalia et al. [36] showed that transcription during the oocyte stage is required for the establishment of maternal methylation marks on an imprinted gene. The present results show that Dnmt3L 2/2 oocytes lost almost all of their maternal methylation imprints while maintaining a constant amount of mRNA through each ICR despite the global loss of intragenic methylation. Thus, these results strongly suggest that the establishment of genomic imprints via transcription is mediated by Dnmt3L-dependent intragenic methylation.
A possible mechanism for gene-body methylation involves the exposure of intragenic regions to DNA methyltransferases, considering that RNA polymerase disrupts the chromatin structure during transcription. However, not all transcripts across gDMRs corresponded to highly expressed genes in oocytes ( Figure 6C). Therefore, other epigenetic marks with an open chromatin structure might also be important for DNA methylation in oocytes. For instance, a recent knockout study showed that Kdm1b, which encodes histone H3K4 demethylase, is required for the establishment of some maternal methylation imprints [45]. Thus, several factors, including transcriptional and epigenetic modifica- tions, might be involved in Dnmt3L-mediated intragenic methylation.
The results of this study show that gene-body methylation was correlated to gene expression in sperm. However, the extent of that correlation is much less than in oocytes due to genome-wide hypermethylation, including in low-CpG-density regions. In male germline cells, global methylation acquisition begins during late embryonic development and before birth [3]. To more clearly show this correlation, analysis of early-stage germ cells in fetal or neonatal animals might be required. Surprisingly, a positive correlation between mRNA expression and gene-body methylation was not observed in mouse ESCs. In addition, the accumulation of non-CpG methylation was not observed in mouse ESCs. These results contradict the results of another study, which showed that active transcription was associated with intragenic DNA methylation with non-CpG methylation in human ESCs [22,23]. This discrepancy might reflect the differences between human and mouse ESCs, the precise cell derivations or culture conditions [46,47]. However, further comparative studies on germ cell epigenomes from other species are required to further elucidate the functional role of epigenetic marking systems.
In this study, a large number of heritable oocyte-specific methylation marks were identified within a set of novel CpG islands [37]. The difference in the number of oocyte-and spermspecific gDMRs reflects the fact that only 3 or 4 paternallymethylated imprinted loci were observed, as compared to approximately 20 maternally-methylated imprinted loci. The reason for the relative abundance of oocyte-specific methylated CGIs might be related to the intragenic methylation of CpG-rich regions, which are hypomethylated in sperm. The results show that most of the oocyte-specific marks are Dnmt3L-dependent, similar to results recently obtained by RRBS-based analysis [38]. However, whether all of these CpG-rich regions serve as imprinting methylation marks is unclear. For instance, although many genes with oocyte-specific methylation marks were identified ( Figure 6B), the evidence that these genes were imprinted was lacking (e.g., Piwil1 and Dnmt1). These methylation marks might not be involved in the formation of a fertile oocyte but might play crucial roles in gene expression after fertilization. Furthermore, ESC methylomes showed that many gDMRs, especially spermspecific gDMRs, acquired new methylation patterns after implantation. Methylation of these CGIs might control tissuespecific gene expression [48,49]. Partial alternation of imprinted methylation patterns in ESCs were observed in the present study, potentially caused by significant differences in the extent of the ICRs during embryo development [39]. A fuller understanding of epigenetic stability will require further methylome profiling during early embryogenesis and stem cell differentiation. The present study also identified a gDMR as a novel ICR candidate in the intron of the imprinted Gpr1 gene. Thus, traditional promoter arrays may not identify all ICRs. However, further analyses are needed to determine which gDMRs, identified in the CpG methylome maps, are true ICRs at the imprinted Gpr1-Zdbf2 locus [40,50].
mRNA-seq results showed that the expression levels of most genes in the wild-type and Dnmt3L 2/2 oocytes were similar. For instance, the expression level of almost all oocyte-specific genes, which regulate ovarian follicle formation, reproduction, and early development, were not significantly altered ( Figure 5B and Table  S1). These results are consistent with the findings of previous studies, which showed that Dnmt3L 2/2 female mice were capable of producing fertile oocytes (however, their offspring were not viable due to the lack of imprinting) [5,6]. Thus, regulation of oocyte-specific genes must be beyond the control of Dnmt3Ldependent cytosine methylation.
Although Dnmt3L 2/2 oocytes showed global hypomethylation at low to high CpG densities, some families of retrotransposons, such as LINEs and LTRs, were partially methylated at moderate to high CpG densities. Therefore, Dnmt3L-independent methylation might be involved in the silencing of retrotransposons and completion of oocyte meiosis. Previously, De La Fuente et al. [51] showed that Hells (also known as Lsh), which encodes a member of the sucrose non-fermenter 2 (SNF2) family of chromatin remodeling proteins, is required for DNA methylation of IAP and pericentromeric satellite repeats as well as repression of IAP retrotransposition in pachytene oocytes. Unfortunately, measurement of the methylation levels of satellite DNA, which is abundant in the pericentromeric regions, was not possible because these sequences were excluded from our analysis. However, a previous sequencing study showed that methylation levels of satellite DNA did not differ between the wild-type and Dnmt3L 2/2 oocytes [52]. Combined, these results suggest the presence of 2 types of oocyte methylation patterns: (i) Dnmt3Lmediated intragenic methylation that is essential for early embryogenesis and (ii) Dnmt3L-independent retroviral and pericentromeric methylation, which may be mediated by Hells activity, is crucial for oocyte meiosis [51]. Further studies on Hells-mediated oocyte methylation are required to elucidate the details of this mechanism.
Previous studies on the cytosine methylation of mtDNA have been highly controversial. A recent study by Shock et al. [53] reported cytosine methylation and hydroxymethylation in mammalian mitochondria. Our results indicated that mtDNA is unmethylated in blastocysts and ESCs, but is partially methylated in germ cells. Whether or not 5-hydroxymethylcytosine (5-hmC) exists in mitochondrial or genomic chromosomes of germ cells remains unclear. Meanwhile, rapid hydroxylation of 5-methylcytosine (5-mC) in the paternal pronucleus during zygotic development was also recently reported [54,55]. Currently, it is difficult to assess hydroxymethylation profiles in oocyte genomes due to the limited DNA recovery. Further investigation of cytosine modification during germ cell and zygote development will be required in the future to better understand this process.
The DNA methylome maps of mouse germ cells, in this study, were derived from SBS data and, therefore, accurately represent methylation levels of individual CpGs on a whole-genome level. The adaptation of the SBS method for small-scale DNA analysis, described in the present report, has the potential to enable further analyses of germline lineages. The current work examined SBS library construction using 3 methods, MethylC-seq, WBA-seq, and PBAT. MethylC-seq basically required only microograms of DNA [22,23,56], thus over amplification might cause redundancy in oocyte libraries. The latter methods allow comprehensive methylome analysis in samples with low amounts of starting DNA by avoiding DNA damage due to sodium bisulfite treatment (after adapter ligation, in the case of MethylC-Seq). Recent studies using BS sequencing have shown that methylated cytosine is abundant in the non-CpG regions of human pluripotent stem cells and mouse oocytes [22,23,39,56]; however, the function of non-CpG methylation in mammalian genomes remains unclear. The PBAT results also showed an abundance of non-CpG methylation in oocytes, with results similar to a previous sequencing study on imprinted loci [39]. However, accurate assessment of non-CpG methylation is required using increased sequencing depths because methylation levels of the non-CpG sites were much lower than those of the CpG sites. SBS library construction was conducted by WBA-seq from 2000 fully matured (metaphase II stage) oocytes; sufficient quantities for sequencing were not obtained. During oogenesis, most of the oocyte specific imprinted methylation marks were established during the GV stage. This contrasted to a previous study where a continuous increase in methylation levels was observed [38]. Further improvement of SBS methods, requiring smaller amounts of DNA, is needed to provide complete germ cell methylome maps and to elucidate the exact function of non-CpG methylation in germ cells.
In conclusion, we constructed the first extensive, high-resolution maps of DNA methylomes of mouse oocytes and sperm. These maps described the epigenetic properties of these DNA methylomes. Our data could serve as a platform for future studies to elucidate the role of epigenetic modifications in the development and functioning of germ and stem cells. Such studies are anticipated to improve our understanding of epigenetic reprogramming.

Preparation of MethylC-seq libraries
Five thousand germinal vesicle (GV)-stage oocytes were collected from the ovarian follicles of adult (7-to 9-week-old) female C57BL/6N mice (Clea Japan, Tokyo, Japan) 44-48 h after they were injected with equine chorionic gonadotropin. Three hundred blastocysts at embryonic day 3.5 were obtained from superovulated adult female C57BL/6N mice by flushing the uterus. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA). Sperm were released from the cauda epididymises of adult male C57BL/6N mice. Sperm DNA was isolated by a standard phenol-chloroform extraction procedure with dithiothreitol (DTT). Genomic DNA from 2 lines of ESCs derived from C57BL/6J mice (Clea Japan) was extracted using the DNeasy Blood & Tissue Kit (Qiagen). DNA samples were sheared into 100-bp fragments in oocytes and 200-bp fragments in other samples using the Covaris S2 focused acoustic system (Covaris, Woburn, MA). Cytosine-methylated adapters (Illumina, San Diego, CA) were ligated to DNA by using the Paired-End DNA Sample Prep Kit or ChIP-Seq DNA Sample Prep Kit (Illumina). DNA fragments were isolated by 2-3% agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit (Qiagen). Sodium bisulfite conversion was performed using the Epitect Bisulfite Kit (Qiagen).
All bisulfite-converted DNA molecules were polymerase chain reaction (PCR)-amplified as follows: 2.5 U of Hot Start Taq polymerase (TaKaRa, Tokyo, Japan), 5 mL 106 PCR buffer, 25 mM dNTPs, 1 mL of each PCR Primer PE 1.0 and 2.0 (Illumina) (50 mL final). Thermocycling parameters were: initial denaturation at 94uC for 1 min, 15-25 cycles of denaturation at 94uC for 30 s, annealing at 65uC for 30 s, and extension at 72uC for 30 s, followed by a final extension at 72uC for 5 min. PCR reaction products were purified using the QIAquick kit (Qiagen).

Preparation of whole WBA-seq libraries
Two thousand GV-stage oocytes were collected from 7-to 9week-old female C57BL/6N mice (Clea Japan) and, 2300 GVstage oocytes were collected from 7-15-week-old Dnmt3L 2/2 female mice (129SvJae6C57BL/6N hybrid genetic background) [6,57]. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen), and then bisulfite-treated with Epitect Bisulfite Kit (Qiagen). Subsequently, the bisulfite-converted DNA was amplified using Epitect Whole Bisulfitome Kit (Qiagen). The collected DNA was sheared into 200-bp fragments using Covaris S2. Unmodified Paired-End adapters (Illumina) were ligated to the DNA by using the Paired-End DNA Sample Prep Kit (Illumina). DNA fragments were isolated by 2% agarose gel electrophoresis and purified using the QIAquick Kit (Qiagen). All DNA was PCR amplified and purified in the same manner as the MethylC-seq method, except the number of PCR cycles was reduced to 7.

Preparation of mRNA sequencing libraries
Total RNA from 1000 wild-type GV oocytes, 500 Dnmt3L 2/2 GV oocytes, sperm, and ESCs was extracted using the RNeasy Mini Kit (Qiagen) and treated with DNase I (Promega, Madison, WI). RNA-Seq libraries were constructed using the mRNA-Seq Sample Preparation Kit (Illumina).

Sequencing
The MethylC-seq for blastocysts, WBA-seq, and PBAT libraries were sequenced on a HiSeq 2000 sequencing system (Illumina); the other MethylC-seq and mRNA-seq libraries were sequenced on a Genome Analyzer II (Illumina). Sample preparation, cluster generation, and sequencing were performed using the Paired-End Cluster Generation Kit-HS and the TruSeq SBS Kit-HS for the HiSeq 2000. Similarly, the Paired-End Cluster Generation Kits v2 and v4 and 18-and 36-Cycle Sequencing Kits v3 and v4 were used for the Genome Analyzer II. All kits were from Illumina.

Gene mapping
All sequenced reads were processed using the standard Illumina base-calling pipeline (v1.4-1.7). Generated sequence tags were mapped onto the mouse genome (mm9, UCSC Genome Browser, July 2007, Build 37.1) by using the Illumina ELAND program.
MethylC-seq tags (36 or 76 nt) were mapped with a custom Perl program, as described previously [17,22]. Briefly, all cytosines in the tags were replaced by thymines. Next, these tags were aligned to 2 mouse genome reference sequences (mm9), such that the antisense strand had cytosines replaced by thymines and the sense strand had guanines replaced by adenines. Finally, all tags (32-76 nt) that mapped uniquely without any mismatches to both strands were compiled and used for further analyses.
The 76 nt WBA-seq tags were mapped as follows. All tags were converted to 2 types of reads; in 1 read (''For'' read), cytosines were replaced by thymines and in the other read (''Rev'' read), guanines were replaced by adenines. Both ''For'' and ''Rev'' reads were aligned to sense and antisense mm9 strands. A total of 793, 397, 948, 480, and 238 million tags were aligned in wild-type oocytes, Dnmt3L 2/2 oocytes, sperm, blastocysts, and ESC genomes, respectively. To avoid bias, tags mapped with multiple hits or matched chromosome M (mitochondria), chromosome Y, or 3 types of repetitive sequences (simple repeat, low complexity repeat, and satellite DNA sequences) were omitted from further analyses.
The 47 nt PBAT tags (trimmed first 4 nt and last 1 nt) were mapped as follows. All guanidines in the tags were replaced by adenines, and these tags were aligned to sense and antisense strands mm9.
For gene-level analysis, the concentrations of the perfectly matching 35 nt (trimmed first nt) mRNA-seq tags from wild-type oocytes, Dnmt3L 2/2 oocytes, sperm, and ESCs were calculated for the genomic regions corresponding to those covered by the RefSeq transcript models. The expression level of 20,854 unique genes was ranked by expression levels (calculated as RPKM values) in each library (Table S1). A total of 33, 28, 23, and 25 tags were aligned in 4 mRNA-seq libraries, respectively. mRNA-seq data analysis was performed and visualized using GenomeStudio Data Analysis software (Illumina).

Methylation analysis
The percentage of individual cytosines methylated at all CpG sites covered by at least 1 read was calculated as 1006(number of aligned cytosines (methylated cytosines))/(total number of aligned cytosines and thymines (originally unmethylated cytosines)). All genomic CpG methylation data are available on our website (http://www.nodai-genome.org/mouse_en.html). The CpG and non-CpG (CpH) methylation levels determined by PBAT results were calculated as the ratio between the total read C and the total read T mapped to genomic cytosines. Bisulfite conversion failure rates were calculated by read C:T ratios from lambda DNA mapping data. The failure rates were as follows: GV oocyte, 0.009; sperm, 0.008; blastocysts, 0.011; and ESCs, 0.006. Locations of transposable elements in the mouse genome (mm9) were obtained from the UCSC Genome Browser, and the average methylation levels of the whole genome and each transposable element were recalculated from the ratio of the aligned cytosines and thymines in each sequence. Lists of 23,021 CGIs were obtained from a previous report [37]. Around the TSS and TTS (65 kb), genomic regions were divided into 20-bp bins. For each bin, the average methylation value was calculated for each gene. The expression level of 20,854 genes was divided into 5 percentile groups ranked by RPKM values, and the average methylation level for each group was mapped onto the gene structure model. These computational analyses were performed using a custom Perl program. Supercomputing resources were provided by the Human Genome Center, Institute of Medical Science, University of Tokyo.

Statistical analysis
Correlations between gene expression ranks and average methylation levels in the promoter (6500 bp from the TTS) or gene-body regions (gene-body 1: +2 to +5 kb from the TSS; genebody 2: 0 to 25 kb from the TTS) were calculated using Spearman's rank correlation coefficient (r). An R-squared value (R 2 ) was calculated to evaluate the correlation of RPKM values between wild-type and Dnmt3L 2/2 oocytes. Statistical analysis was performed using the R statistical package.

Bisulfite sequencing
To analyze the methylation of the three transposable elements (L1 LINE, B1/Alu SINE, and IAP LTR), 20 wild-type GV oocytes were obtained from adult female C57BL/6N mice. Bisulfite sequencing conditions and primer sets for the three transposable elements were described, previously [52]. To analyze the methylation of the Gpr1 locus, 10 blastocysts were obtained from BJF1 (C57BL/6N6JF1) and Dnmt3L mat2/2 (Dnmt3L 2/2 6JF1) mice [6,57]. Genomic DNA from blastocysts was isolated using the QIAamp DNA Mini Kit (Qiagen) and treated with sodium bisulfite with the Epitect Bisulfite Kit (Qiagen). The Gpr1 gDMR sequence was amplified with 2 rounds of nested PCR. The first-round PCR reaction contained 1 U of Hot Start Taq polymerase (TaKaRa), 16 PCR buffer, 200 mM dNTPs, 1 mM forward primer, and 1 mM reverse primer (20 mL final). Thermocycling parameters were as follows: initial denaturation at 94uC for 1 min, 35 cycles of denaturation at 94uC for 30 s, annealing at 50uC for 30 s, and extension at 72uC for 30 s, followed by a final extension at 72uC for 5 min. Subsequently, 2 mL of the product was used as the input for the second-round PCR, which was performed in the same manner. Primer sets for the nested PCR were as follows: Gpr1-BSF1 (59-GATTAGATTAGGTTAG-TTTGGAA-39) and Gpr1-BSR1 (59-ACTAAAACACTAAT-CACCAAATA-39) for the first round; Gpr1-BSF2 (59-AGAT-TAGGTTAGTTTGGAATT-39) and Gpr1-BSR2 (59-AACAC-TAATCACCAAATAATTC-39) for the second round. The second-round PCR product was subcloned and sequenced, as described previously [50]. The percentage methylation was calculated as 1006(number of methylated CpG dinucleotides)/ (total number of CpGs). At least 10 clones from each parental allele were sequenced. Sequence data were analyzed using the QUMA quantification tool for methylation analysis [58].

Accession number
The MethylC-seq, WBA-seq, PBAT, and mRNA-seq data in this study have been deposited in the DNA Data Bank of Japan (DDBJ) under accession number DRA000484. Figure S1 Schematic of the SBS library construction procedure. MethylC-Seq libraries were generated by ligation of methylated sequencing adapters to fragmented genomic DNA followed by gel purification, sodium bisulfite conversion, and PCR amplification (left). WBA-seq libraries were generated by ligation of unmodified sequencing adapters to bisulfite-modified (amplified using EpiTect Whole Bisulfitome Kits) and fragmented genomic DNA followed by gel purification and PCR amplification (middle). PBAT libraries were generated by double-stranded DNA synthesis from bisulfitetreated (single-stranded) DNA with random primers containing sequencing adapters (right). (TIF) Figure S2 The percent of the oocyte and sperm genomes covered by differing minimum numbers of MethylC-seq and WBA-seq reads. (TIF)  Figure S5 High-resolution DNA methylome map on mouse X inactivation center region in chromosome X (100,200,000-101,200,000). GenomeStudio view of Refseq's positions, repetitive element, CpG methylation map, CpG densities, CGI positions, and CGI methylation map were shown. Red, purple, blue, green, and khaki dots and boxes represent the methylation levels at individual CpGs and CGIs in wild-type oocyte, Dnmt3L 2/2 oocyte, sperm, blastocyst, and ESC genomes, respectively, as shown in Figure 1. (TIF) Figure S6 DNA methylome maps of each chromosome of mouse germ cells. The methylation levels of each chromosome in wild-type oocytes, Dnmt3L 2/2 oocytes, and sperm in 10 kb windows (excluding mitochondrial chromosome, chromosome Y, and unplaced contigs). Red, purple, and blue lines represent the methylation levels in wild-type oocytes, Dnmt3L 2/2 oocytes, and sperm, respectively. Red and blue boxes represent oocytemethylated and sperm-methylated gDMRs, and red and blue pins indicate maternal and maternal ICRs, respectively. (TIF)