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Genome-wide analysis of DNA methylation dynamics during early human development.

Okae H, Chiba H, Hiura H, Hamada H, Sato A, Utsunomiya T, Kikuchi H, Yoshida H, Tanaka A, Suyama M, Arima T - PLoS Genet. (2014)

Bottom Line: Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation.These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development.Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.

View Article: PubMed Central - PubMed

Affiliation: Department of Informative Genetics, Environment and Genome Research Center, Tohoku University Graduate School of Medicine, Sendai, Japan; JST, CREST, Saitama, Japan.

ABSTRACT
DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5-10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.

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Establishment and maintenance of imprinted DMRs.A, A heatmap of mean methylation levels of imprinted DMRs. We classified the 67 known human imprinted DMRs [17], and found that 44 were maternal germline DMRs (M-gDMRs), 2 were paternal germline DMRs (P-gDMRs) and 21 were secondary DMRs (sDMRs). 15 M-gDMRs are reported to be maintained only in the placenta and shown as “Pla-specific gDMRs”. gDMRs other than placenta-specific ones showed 35–65% methylation levels in blood cells but the intermediate methylation levels were not well maintained in ES cells (11/31 showed>75% methylation). Methylation levels are color coded as indicated. The raw data are shown in S1 Table. B, Methylation patterns at the human GNAS locus. The vertical axis indicates the methylation level (%). In this locus, there were two gDMRs and two sDMRs. All DMRs overlap promoter regions. C, Methylation patterns at the human DNMT1 locus. The promoter region of the somatic isoform of DNMT1 (DNMT1s) is known to show maternal allele-specific methylation in the placenta [45]. The DNMT1 DMR was hypomethylated in both ES and blood cells, suggesting placenta-specific protection of the maternal allele from demethylation. D, Box plots of mean methylation levels of gDMRs and oocyte-specific methylated CGIs in blastocysts. Boxes represent lower and upper quartiles and horizontal lines indicate the median. Whiskers extend to the most extreme data points within 1.5 times the interquartile range from the boxes. The open circles indicate the data points outside the whiskers. Methylation levels of mouse gDMRs and oocyte-specific methylated CGIs [5] are shown for comparison. E, Methylation patterns of an oocyte-specific methylated CGI. A single blastocyst was used for the analysis. Black and white circles indicate methylated and unmethylated residues, respectively. The percentages of methylated CpG sites are indicated. F, Bisulfite sequencing analyses of X-linked CGIs hypermethylated in oocytes. A single blastocyst was used for each bisulfite sequencing analysis.
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pgen-1004868-g002: Establishment and maintenance of imprinted DMRs.A, A heatmap of mean methylation levels of imprinted DMRs. We classified the 67 known human imprinted DMRs [17], and found that 44 were maternal germline DMRs (M-gDMRs), 2 were paternal germline DMRs (P-gDMRs) and 21 were secondary DMRs (sDMRs). 15 M-gDMRs are reported to be maintained only in the placenta and shown as “Pla-specific gDMRs”. gDMRs other than placenta-specific ones showed 35–65% methylation levels in blood cells but the intermediate methylation levels were not well maintained in ES cells (11/31 showed>75% methylation). Methylation levels are color coded as indicated. The raw data are shown in S1 Table. B, Methylation patterns at the human GNAS locus. The vertical axis indicates the methylation level (%). In this locus, there were two gDMRs and two sDMRs. All DMRs overlap promoter regions. C, Methylation patterns at the human DNMT1 locus. The promoter region of the somatic isoform of DNMT1 (DNMT1s) is known to show maternal allele-specific methylation in the placenta [45]. The DNMT1 DMR was hypomethylated in both ES and blood cells, suggesting placenta-specific protection of the maternal allele from demethylation. D, Box plots of mean methylation levels of gDMRs and oocyte-specific methylated CGIs in blastocysts. Boxes represent lower and upper quartiles and horizontal lines indicate the median. Whiskers extend to the most extreme data points within 1.5 times the interquartile range from the boxes. The open circles indicate the data points outside the whiskers. Methylation levels of mouse gDMRs and oocyte-specific methylated CGIs [5] are shown for comparison. E, Methylation patterns of an oocyte-specific methylated CGI. A single blastocyst was used for the analysis. Black and white circles indicate methylated and unmethylated residues, respectively. The percentages of methylated CpG sites are indicated. F, Bisulfite sequencing analyses of X-linked CGIs hypermethylated in oocytes. A single blastocyst was used for each bisulfite sequencing analysis.

Mentions: Germline DMRs (gDMRs) frequently serve as imprinting control regions [16] and we were interested in how many gDMRs exist in the human genome. Among the 67 known imprinted DMRs [17], 46 DMRs were classified as gDMRs according to the following definition: DMRs hypermethylated in one gamete and hypomethylated in the other (Fig. 2A, B and S1 Table). Of these, 15 reportedly placenta-specific DMRs were lost in blood cells (Fig. 2A, C). The other 31 gDMRs showed intermediate methylation levels in blood cells, but about one-third of these gDMRs were not maintained in ES cells (H9 ES cells: Fig. 2A, H1 and HUES6 ES cells: S4A Figure), indicating the instability of gDMRs in human ES cells. Importantly, oocyte-specific methylated autosomal CGIs showed methylation levels very similar (median  = 37.5%) to gDMRs (median  = 39.2%) in human blastocysts (Fig. 2D). We confirmed monoallelic methylation of four autosomal CGIs in human blastocysts by using conventional bisulfite sequencing (Fig. 2E and S4B Figure). We also analyzed two X-linked CGIs hypermethylated in oocytes and found that these CGIs showed high methylation levels in male blastocysts (the X chromosome of male blastocysts is derived from oocytes) and monoallelic methylation in female blastocysts (Fig. 2F). Consistently, X-linked CGIs with oocyte-specific methylation showed higher methylation levels than autosomal ones in blastocysts (the WGBS data were derived from a pool of blastocysts) (Fig. 2D). A similar tendency was also observed in the sliding window-based analyses (S2C Figure). These data suggested that a substantial number of oocyte-specific methylated CGIs may maintain maternal allele-specific methylation in human blastocysts. In contrast, most oocyte-specific methylated CGIs were significantly demethylated compared with gDMRs in mouse blastocysts (Fig. 2D).


Genome-wide analysis of DNA methylation dynamics during early human development.

Okae H, Chiba H, Hiura H, Hamada H, Sato A, Utsunomiya T, Kikuchi H, Yoshida H, Tanaka A, Suyama M, Arima T - PLoS Genet. (2014)

Establishment and maintenance of imprinted DMRs.A, A heatmap of mean methylation levels of imprinted DMRs. We classified the 67 known human imprinted DMRs [17], and found that 44 were maternal germline DMRs (M-gDMRs), 2 were paternal germline DMRs (P-gDMRs) and 21 were secondary DMRs (sDMRs). 15 M-gDMRs are reported to be maintained only in the placenta and shown as “Pla-specific gDMRs”. gDMRs other than placenta-specific ones showed 35–65% methylation levels in blood cells but the intermediate methylation levels were not well maintained in ES cells (11/31 showed>75% methylation). Methylation levels are color coded as indicated. The raw data are shown in S1 Table. B, Methylation patterns at the human GNAS locus. The vertical axis indicates the methylation level (%). In this locus, there were two gDMRs and two sDMRs. All DMRs overlap promoter regions. C, Methylation patterns at the human DNMT1 locus. The promoter region of the somatic isoform of DNMT1 (DNMT1s) is known to show maternal allele-specific methylation in the placenta [45]. The DNMT1 DMR was hypomethylated in both ES and blood cells, suggesting placenta-specific protection of the maternal allele from demethylation. D, Box plots of mean methylation levels of gDMRs and oocyte-specific methylated CGIs in blastocysts. Boxes represent lower and upper quartiles and horizontal lines indicate the median. Whiskers extend to the most extreme data points within 1.5 times the interquartile range from the boxes. The open circles indicate the data points outside the whiskers. Methylation levels of mouse gDMRs and oocyte-specific methylated CGIs [5] are shown for comparison. E, Methylation patterns of an oocyte-specific methylated CGI. A single blastocyst was used for the analysis. Black and white circles indicate methylated and unmethylated residues, respectively. The percentages of methylated CpG sites are indicated. F, Bisulfite sequencing analyses of X-linked CGIs hypermethylated in oocytes. A single blastocyst was used for each bisulfite sequencing analysis.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263407&req=5

pgen-1004868-g002: Establishment and maintenance of imprinted DMRs.A, A heatmap of mean methylation levels of imprinted DMRs. We classified the 67 known human imprinted DMRs [17], and found that 44 were maternal germline DMRs (M-gDMRs), 2 were paternal germline DMRs (P-gDMRs) and 21 were secondary DMRs (sDMRs). 15 M-gDMRs are reported to be maintained only in the placenta and shown as “Pla-specific gDMRs”. gDMRs other than placenta-specific ones showed 35–65% methylation levels in blood cells but the intermediate methylation levels were not well maintained in ES cells (11/31 showed>75% methylation). Methylation levels are color coded as indicated. The raw data are shown in S1 Table. B, Methylation patterns at the human GNAS locus. The vertical axis indicates the methylation level (%). In this locus, there were two gDMRs and two sDMRs. All DMRs overlap promoter regions. C, Methylation patterns at the human DNMT1 locus. The promoter region of the somatic isoform of DNMT1 (DNMT1s) is known to show maternal allele-specific methylation in the placenta [45]. The DNMT1 DMR was hypomethylated in both ES and blood cells, suggesting placenta-specific protection of the maternal allele from demethylation. D, Box plots of mean methylation levels of gDMRs and oocyte-specific methylated CGIs in blastocysts. Boxes represent lower and upper quartiles and horizontal lines indicate the median. Whiskers extend to the most extreme data points within 1.5 times the interquartile range from the boxes. The open circles indicate the data points outside the whiskers. Methylation levels of mouse gDMRs and oocyte-specific methylated CGIs [5] are shown for comparison. E, Methylation patterns of an oocyte-specific methylated CGI. A single blastocyst was used for the analysis. Black and white circles indicate methylated and unmethylated residues, respectively. The percentages of methylated CpG sites are indicated. F, Bisulfite sequencing analyses of X-linked CGIs hypermethylated in oocytes. A single blastocyst was used for each bisulfite sequencing analysis.
Mentions: Germline DMRs (gDMRs) frequently serve as imprinting control regions [16] and we were interested in how many gDMRs exist in the human genome. Among the 67 known imprinted DMRs [17], 46 DMRs were classified as gDMRs according to the following definition: DMRs hypermethylated in one gamete and hypomethylated in the other (Fig. 2A, B and S1 Table). Of these, 15 reportedly placenta-specific DMRs were lost in blood cells (Fig. 2A, C). The other 31 gDMRs showed intermediate methylation levels in blood cells, but about one-third of these gDMRs were not maintained in ES cells (H9 ES cells: Fig. 2A, H1 and HUES6 ES cells: S4A Figure), indicating the instability of gDMRs in human ES cells. Importantly, oocyte-specific methylated autosomal CGIs showed methylation levels very similar (median  = 37.5%) to gDMRs (median  = 39.2%) in human blastocysts (Fig. 2D). We confirmed monoallelic methylation of four autosomal CGIs in human blastocysts by using conventional bisulfite sequencing (Fig. 2E and S4B Figure). We also analyzed two X-linked CGIs hypermethylated in oocytes and found that these CGIs showed high methylation levels in male blastocysts (the X chromosome of male blastocysts is derived from oocytes) and monoallelic methylation in female blastocysts (Fig. 2F). Consistently, X-linked CGIs with oocyte-specific methylation showed higher methylation levels than autosomal ones in blastocysts (the WGBS data were derived from a pool of blastocysts) (Fig. 2D). A similar tendency was also observed in the sliding window-based analyses (S2C Figure). These data suggested that a substantial number of oocyte-specific methylated CGIs may maintain maternal allele-specific methylation in human blastocysts. In contrast, most oocyte-specific methylated CGIs were significantly demethylated compared with gDMRs in mouse blastocysts (Fig. 2D).

Bottom Line: Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation.These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development.Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.

View Article: PubMed Central - PubMed

Affiliation: Department of Informative Genetics, Environment and Genome Research Center, Tohoku University Graduate School of Medicine, Sendai, Japan; JST, CREST, Saitama, Japan.

ABSTRACT
DNA methylation is globally reprogrammed during mammalian preimplantation development, which is critical for normal development. Recent reduced representation bisulfite sequencing (RRBS) studies suggest that the methylome dynamics are essentially conserved between human and mouse early embryos. RRBS is known to cover 5-10% of all genomic CpGs, favoring those contained within CpG-rich regions. To obtain an unbiased and more complete representation of the methylome during early human development, we performed whole genome bisulfite sequencing of human gametes and blastocysts that covered>70% of all genomic CpGs. We found that the maternal genome was demethylated to a much lesser extent in human blastocysts than in mouse blastocysts, which could contribute to an increased number of imprinted differentially methylated regions in the human genome. Global demethylation of the paternal genome was confirmed, but SINE-VNTR-Alu elements and some other tandem repeat-containing regions were found to be specifically protected from this global demethylation. Furthermore, centromeric satellite repeats were hypermethylated in human oocytes but not in mouse oocytes, which might be explained by differential expression of de novo DNA methyltransferases. These data highlight both conserved and species-specific regulation of DNA methylation during early mammalian development. Our work provides further information critical for understanding the epigenetic processes underlying differentiation and pluripotency during early human development.

Show MeSH