Comparison of the methylomes in NGOs, GVOs, and Dnmt-KO.

<p>(A) Overall levels of CG methylation and non-CG methylation in NGOs, GVOs, Dnmt1-KO, Dnmt3a-KO, Dnmt3b-KO, and Dnmt3L-KO. The data were not corrected for the bisulfite non-conversion rate (0.43–0.87, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003439#pgen-1003439-t001" target="_blank">Table 1</a>). Thus, NGOs, Dnmt3a-KO, and Dmt3L-KO may actually have no or very little non-CG methylation. (B) Profiles of non-CG methylation along chromosome 4 determined using a non-overlapping sliding window of 50 kb in all samples. The data for the two strands are shown separately above and below the horizontal line.</p

Genome-Scale Assessment of Age-Related DNA Methylation Changes in Mouse Spermatozoa

<div><p>DNA methylation plays important roles in the production and functioning of spermatozoa. Recent studies have suggested that DNA methylation patterns in spermatozoa can change with age, but the regions susceptible to age-related methylation changes remain to be fully elucidated. In this study, we conducted genome-scale DNA methylation profiling of spermatozoa obtained from C57BL/6N mice at 8 weeks (8w), 18 weeks (18w) and 17 months of age (17m). There was no substantial difference in the global DNA methylation patterns between 18w and 17m samples except for a slight increase of methylation levels in long interspersed nuclear elements in the 17m samples. We found that maternally methylated imprinting control regions (mICRs) and spermatogenesis-related gene promoters had 5–10% higher methylation levels in 8w samples than in 18w or 17m samples. Analysis of individual sequence reads suggested that these regions were fully methylated (80–100%) in a subset of 8w spermatozoa. These regions are also known to be highly methylated in a subset of postnatal spermatogonia, which might be the source of the increased DNA methylation in 8w spermatozoa. Another possible source was contamination by somatic cells. Although we carefully purified the spermatozoa, it was difficult to completely exclude the possibility of somatic cell contamination. Further studies are needed to clarify the source of the small increase in DNA methylation in the 8w samples. Overall, our findings suggest that DNA methylation patterns in mouse spermatozoa are relatively stable throughout reproductive life.</p></div

Mouse Oocyte Methylomes at Base Resolution Reveal Genome-Wide Accumulation of Non-CpG Methylation and Role of DNA Methyltransferases

<div><p>DNA methylation is an epigenetic modification that plays a crucial role in normal mammalian development, retrotransposon silencing, and cellular reprogramming. Although methylation mainly occurs on the cytosine in a CG site, non-CG methylation is prevalent in pluripotent stem cells, brain, and oocytes. We previously identified non-CG methylation in several CG-rich regions in mouse germinal vesicle oocytes (GVOs), but the overall distribution of non-CG methylation and the enzymes responsible for this modification are unknown. Using amplification-free whole-genome bisulfite sequencing, which can be used with minute amounts of DNA, we constructed the base-resolution methylome maps of GVOs, non-growing oocytes (NGOs), and mutant GVOs lacking the DNA methyltransferase Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3L. We found that nearly two-thirds of all methylcytosines occur in a non-CG context in GVOs. The distribution of non-CG methylation closely resembled that of CG methylation throughout the genome and showed clear enrichment in gene bodies. Compared to NGOs, GVOs were over four times more methylated at non-CG sites, indicating that non-CG methylation accumulates during oocyte growth. Lack of Dnmt3a or Dnmt3L resulted in a global reduction in both CG and non-CG methylation, showing that non-CG methylation depends on the Dnmt3a-Dnmt3L complex. Dnmt3b was dispensable. Of note, lack of Dnmt1 resulted in a slight decrease in CG methylation, suggesting that this maintenance enzyme plays a role in non-dividing oocytes. Dnmt1 may act on CG sites that remain hemimethylated in the <i>de novo</i> methylation process. Our results provide a basis for understanding the mechanisms and significance of non-CG methylation in mammalian oocytes.</p></div

Stability of DNA methylation levels of various genomic features.

<p>Mean methylation levels (%) of CpG cytosines in promoter, exon, intron, intergenic regions LINE, LTR, SINE and L1 elements. The evolutionary ages of the L1 elements [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167127#pone.0167127.ref035" target="_blank">35</a>] are indicated. Data are shown as mean ± standard error (SE). Different letters indicate statistically significantly methylation differences (P < 0.05).</p

Identification of differentially methylated promoters.

<p>(A) Hierarchical clustering of differentially methylated promoters. Z-scored methylation levels are color-coded as shown. CGI and non-CGI promoters are also indicated. (B) Chromosome distribution of the differentially methylated promoters. (C) GO analysis of the promoters in Cluster I. Statistically significant (<i>P</i> < 0.05) GO terms are indicated with BH-corrected <i>P</i>-values. (D) Methylation levels of the promoters in Cluster I. Data are shown as mean ± SE. Different letters indicate statistically significantly methylation differences (<i>P</i> < 0.05). (E) Methylation pattern of the <i>Mael</i> promoter. The vertical axis indicates the methylation levels (%).</p

Sequencing and mapping summary.

<p>Sequencing and mapping summary.</p

Relationship between CG methylation and non-CG methylation in GVOs.

<p>(A) Levels of CG methylation and non-CG methylation across the entire chromosome 4 in a non-overlapping sliding window of 50 kb. The two strands were separately analyzed for non-CG methylation. (B) Correlation between the levels of CG methylation and non-CG methylation in 10-kb non-overlapping sliding windows across the genome was indicated using Spearman's rank correlation coefficient. (C) Effect of CG on non-CG methylation at positions immediately upstream and downstream of mC sites. Blue, orange, and gray bars indicate the levels of non-CG methylation around CG sites with at least 10× coverage with methylation levels of 80–100%, 40–60%, and 0–20%, respectively. (D) Levels of CG methylation and non-CG methylation relative to gene structure. The upstream and downstream regions (10-kb each) were split into 10 non-overlapping windows to determine the methylation levels. The intragenic or coding regions were divided into 20 small windows for methylation analysis.</p

CG and non-CG methylation levels in different genomic elements in NGOs, GVOs, Dnmt3a-KO, and Dnmt3L-KO.

<p>(A) Levels of CG methylation and non-CG methylation in intragenic and intergenic regions. Intragenic regions are further divided into 5′UTRs, exons, introns, and 3′UTRs according to the RefSeq annotations. (B) Levels of CG methylation and non-CG methylation in different types of repetitive elements. Annotation for repetitive elements was obtained from the UCSC Genome Browser.</p

Strand asymmetry in methylation at CG and non-CG sites.

<p>(A) Fractions of CG and CAG/CTG sites showing strand asymmetry in methylation (hemimethylation) in GVOs, Dnmt1-KO, and Dnmt3b-KO. The data were taken from CG and CAG/CTG sites with methylation levels of ≥70% and ≥40%, respectively, on at least one strand, with a read depth of at least 10× for both strands (left). Bar charts show the hemimethylated fractions (right). Statistical differences in methylation between the strands were calculated using Fisher's exact test (p<0.05). Note that most CAG/CTG sites are hemimethylated in all samples and that CG sites show increased hemimethylation in Dnmt1-KO. (B) CG sites hemimethylated in Dnmt1-KO but not in GVOs. Dnmt1-KO demonstrated an increase in hemimethylated CG sites (orange, left), most of which were fully methylated in GVOs.</p

Abundant non-CG methylation in GVOs.

<p>(A) Proportions of mCs in contexts of CG, CHG, and CHH. Data for mCs at positions covered at least 4× on the same strand were used, and those with more than 100× coverage were excluded. (B) Levels of methylation at CG and non-CG sites. Non-CG sites were further divided into different tri- (CHG and CHH) and di-nucleotide sequences (CA, CT, and CC). (C) Bases neighboring the highly methylated (≥30%) non-CG sites.</p