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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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Unique regulation of tandem repeat-containing regions.A, DNA methylation dynamics of transposable elements. Mean methylation levels of CpGs in various classes of SINEs, LINEs, LTRs and DNA repeats and SVA subfamilies are shown. SVA_A showed an especially high methylation level in blastocysts (59.2%). B, Proportions of repeat copies overlapping>70% methylated windows in human blastocysts. We analyzed only SINEs, LINEs, LTRs, DNA repeats, SVAs and satellites with>100 copies in the human genome. The top ten repeat names with the highest proportions are shown. The raw data are shown in S4 Table. C, Relationships between methylation levels and CpG densities. Mean methylation levels of CpGs in SVA_A are plotted against CpG densities. D, MER34C2 copies overlapping>70% methylated windows in human blastocysts. 39 MER34C2 copies are all tandemly repeated within the PTPRN2 gene locus. E, Proportions of maternal and paternal gDMRs containing VNTRs. Counts of gDMRs with VNTRs and total gDMRs are indicated. F, Proportions of mean methylation levels of CGIs with and without VNTRs in human blastocysts. Only autosomal CGIs hypermethylated in both gametes were analyzed. 118 of 499 CGIs with VNTRs and 31 of 2,222 CGIs without VNTRs showed>70% methylation (P = 0, chi-square test). G, Characteristics of VNTRs highly methylated in blastocysts. Using Tandem Repeats Finder [41], the size of the consensus pattern, the number of tandemly aligned copies and the alignment score were compared between VNTRs of <50% methylated CGIs and>70% methylated CGIs shown in (F). The alignment score calculated by Tandem Repeat Finder reflects the degree of similarity between repeat copies. When several VNTRs were found in a CGI, the VNTR with the highest alignment score was analyzed. 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 Mann-Whitney U test was used to calculate P-values. No sequence motif was found among the consensus patterns of the>70% methylated CGIs using DREME [42]. H, Mean methylation levels of CpGs in ALR. Oocytes showed the highest methylation level (80.6%).
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pgen-1004868-g004: Unique regulation of tandem repeat-containing regions.A, DNA methylation dynamics of transposable elements. Mean methylation levels of CpGs in various classes of SINEs, LINEs, LTRs and DNA repeats and SVA subfamilies are shown. SVA_A showed an especially high methylation level in blastocysts (59.2%). B, Proportions of repeat copies overlapping>70% methylated windows in human blastocysts. We analyzed only SINEs, LINEs, LTRs, DNA repeats, SVAs and satellites with>100 copies in the human genome. The top ten repeat names with the highest proportions are shown. The raw data are shown in S4 Table. C, Relationships between methylation levels and CpG densities. Mean methylation levels of CpGs in SVA_A are plotted against CpG densities. D, MER34C2 copies overlapping>70% methylated windows in human blastocysts. 39 MER34C2 copies are all tandemly repeated within the PTPRN2 gene locus. E, Proportions of maternal and paternal gDMRs containing VNTRs. Counts of gDMRs with VNTRs and total gDMRs are indicated. F, Proportions of mean methylation levels of CGIs with and without VNTRs in human blastocysts. Only autosomal CGIs hypermethylated in both gametes were analyzed. 118 of 499 CGIs with VNTRs and 31 of 2,222 CGIs without VNTRs showed>70% methylation (P = 0, chi-square test). G, Characteristics of VNTRs highly methylated in blastocysts. Using Tandem Repeats Finder [41], the size of the consensus pattern, the number of tandemly aligned copies and the alignment score were compared between VNTRs of <50% methylated CGIs and>70% methylated CGIs shown in (F). The alignment score calculated by Tandem Repeat Finder reflects the degree of similarity between repeat copies. When several VNTRs were found in a CGI, the VNTR with the highest alignment score was analyzed. 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 Mann-Whitney U test was used to calculate P-values. No sequence motif was found among the consensus patterns of the>70% methylated CGIs using DREME [42]. H, Mean methylation levels of CpGs in ALR. Oocytes showed the highest methylation level (80.6%).

Mentions: As described above, global methylation changes of SINEs, LINEs, LTRs and DNA repeats were very similar to other genomic regions in early human embryos (S3 Figure). We further analyzed mean methylation levels of CpGs in various classes of these transposable elements (Fig. 4A, see also S3 Table for details). These repeat classes showed similar methylation changes: ∼60% methylated in oocytes, ∼80% methylated in sperm, ES and blood cells and ∼30% methylated in blastocysts. These data suggested that SINEs, LINEs, LTRs and DNA repeats were essentially not resistant to genome-wide demethylation after fertilization. Mouse IAPs are known to be protected from demethylation during preimplantation development [5], [21]. To identify transposable elements specifically protected from demethylation during human preimplantation development, we screened repeat copies overlapping windows showing>70% methylation in blastocysts (0.3% of all windows) (S4 Table). We found that SINE-VNTR-Alu (SVA) subfamilies, especially SVA_A, frequently overlapped the>70% methylated windows (Fig. 4B). SVA_A also showed the highest methylation level in blastocysts (59.2%) whereas the other repeat sequences were <50% methylated (Fig. 4A and S3 Table). SVA is a hominid-specific repeat family that remains active in the human genome [22]. Similar to mouse LTRs [5], methylation levels of CpGs within SVAs are positively correlated with CpG density in human oocytes and blastocysts (Fig. 4C and S5 Figure). LTR12 subfamilies, which are LTRs of HERV9, also tended to overlap the>70% methylated windows (Fig. 4B). Interestingly, both SVA and LTR12 subfamilies contain CpG-rich variable number tandem repeats (VNTRs) [22], [23]. We also noticed that whereas the MER34C2 consensus sequence does not contain VNTRs, MER34C2 copies overlapping the>70% methylated windows were all tandemly repeated in a single genomic locus (Fig. 4D). VNTRs were also found in the two paternal gDMRs (Fig. 4E). VNTRs were not a common feature of the maternal gDMRs, but a significantly higher proportion of the maternal gDMRs did contain VNTRs as compared with all CGIs (gDMRs: 11/44, CGIs: 1763/27718, chi-square P = 4.1×10−7). Therefore, we focused on CGIs hypermethylated in both gametes and found that CGIs containing VNTRs were preferentially protected from demethylation in blastocysts (Fig. 4F). A comparison between VNTRs of>70% and <50% methylated CGIs in blastocysts revealed that VNTRs with more repeats tended to be protected from demethylation, whereas no sequence motif was found (Fig. 4G). These data suggested that VNTRs might underlie silencing of specific transposable elements and the protection of paternal gDMRs.


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)

Unique regulation of tandem repeat-containing regions.A, DNA methylation dynamics of transposable elements. Mean methylation levels of CpGs in various classes of SINEs, LINEs, LTRs and DNA repeats and SVA subfamilies are shown. SVA_A showed an especially high methylation level in blastocysts (59.2%). B, Proportions of repeat copies overlapping>70% methylated windows in human blastocysts. We analyzed only SINEs, LINEs, LTRs, DNA repeats, SVAs and satellites with>100 copies in the human genome. The top ten repeat names with the highest proportions are shown. The raw data are shown in S4 Table. C, Relationships between methylation levels and CpG densities. Mean methylation levels of CpGs in SVA_A are plotted against CpG densities. D, MER34C2 copies overlapping>70% methylated windows in human blastocysts. 39 MER34C2 copies are all tandemly repeated within the PTPRN2 gene locus. E, Proportions of maternal and paternal gDMRs containing VNTRs. Counts of gDMRs with VNTRs and total gDMRs are indicated. F, Proportions of mean methylation levels of CGIs with and without VNTRs in human blastocysts. Only autosomal CGIs hypermethylated in both gametes were analyzed. 118 of 499 CGIs with VNTRs and 31 of 2,222 CGIs without VNTRs showed>70% methylation (P = 0, chi-square test). G, Characteristics of VNTRs highly methylated in blastocysts. Using Tandem Repeats Finder [41], the size of the consensus pattern, the number of tandemly aligned copies and the alignment score were compared between VNTRs of <50% methylated CGIs and>70% methylated CGIs shown in (F). The alignment score calculated by Tandem Repeat Finder reflects the degree of similarity between repeat copies. When several VNTRs were found in a CGI, the VNTR with the highest alignment score was analyzed. 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 Mann-Whitney U test was used to calculate P-values. No sequence motif was found among the consensus patterns of the>70% methylated CGIs using DREME [42]. H, Mean methylation levels of CpGs in ALR. Oocytes showed the highest methylation level (80.6%).
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Show All Figures
getmorefigures.php?uid=PMC4263407&req=5

pgen-1004868-g004: Unique regulation of tandem repeat-containing regions.A, DNA methylation dynamics of transposable elements. Mean methylation levels of CpGs in various classes of SINEs, LINEs, LTRs and DNA repeats and SVA subfamilies are shown. SVA_A showed an especially high methylation level in blastocysts (59.2%). B, Proportions of repeat copies overlapping>70% methylated windows in human blastocysts. We analyzed only SINEs, LINEs, LTRs, DNA repeats, SVAs and satellites with>100 copies in the human genome. The top ten repeat names with the highest proportions are shown. The raw data are shown in S4 Table. C, Relationships between methylation levels and CpG densities. Mean methylation levels of CpGs in SVA_A are plotted against CpG densities. D, MER34C2 copies overlapping>70% methylated windows in human blastocysts. 39 MER34C2 copies are all tandemly repeated within the PTPRN2 gene locus. E, Proportions of maternal and paternal gDMRs containing VNTRs. Counts of gDMRs with VNTRs and total gDMRs are indicated. F, Proportions of mean methylation levels of CGIs with and without VNTRs in human blastocysts. Only autosomal CGIs hypermethylated in both gametes were analyzed. 118 of 499 CGIs with VNTRs and 31 of 2,222 CGIs without VNTRs showed>70% methylation (P = 0, chi-square test). G, Characteristics of VNTRs highly methylated in blastocysts. Using Tandem Repeats Finder [41], the size of the consensus pattern, the number of tandemly aligned copies and the alignment score were compared between VNTRs of <50% methylated CGIs and>70% methylated CGIs shown in (F). The alignment score calculated by Tandem Repeat Finder reflects the degree of similarity between repeat copies. When several VNTRs were found in a CGI, the VNTR with the highest alignment score was analyzed. 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 Mann-Whitney U test was used to calculate P-values. No sequence motif was found among the consensus patterns of the>70% methylated CGIs using DREME [42]. H, Mean methylation levels of CpGs in ALR. Oocytes showed the highest methylation level (80.6%).
Mentions: As described above, global methylation changes of SINEs, LINEs, LTRs and DNA repeats were very similar to other genomic regions in early human embryos (S3 Figure). We further analyzed mean methylation levels of CpGs in various classes of these transposable elements (Fig. 4A, see also S3 Table for details). These repeat classes showed similar methylation changes: ∼60% methylated in oocytes, ∼80% methylated in sperm, ES and blood cells and ∼30% methylated in blastocysts. These data suggested that SINEs, LINEs, LTRs and DNA repeats were essentially not resistant to genome-wide demethylation after fertilization. Mouse IAPs are known to be protected from demethylation during preimplantation development [5], [21]. To identify transposable elements specifically protected from demethylation during human preimplantation development, we screened repeat copies overlapping windows showing>70% methylation in blastocysts (0.3% of all windows) (S4 Table). We found that SINE-VNTR-Alu (SVA) subfamilies, especially SVA_A, frequently overlapped the>70% methylated windows (Fig. 4B). SVA_A also showed the highest methylation level in blastocysts (59.2%) whereas the other repeat sequences were <50% methylated (Fig. 4A and S3 Table). SVA is a hominid-specific repeat family that remains active in the human genome [22]. Similar to mouse LTRs [5], methylation levels of CpGs within SVAs are positively correlated with CpG density in human oocytes and blastocysts (Fig. 4C and S5 Figure). LTR12 subfamilies, which are LTRs of HERV9, also tended to overlap the>70% methylated windows (Fig. 4B). Interestingly, both SVA and LTR12 subfamilies contain CpG-rich variable number tandem repeats (VNTRs) [22], [23]. We also noticed that whereas the MER34C2 consensus sequence does not contain VNTRs, MER34C2 copies overlapping the>70% methylated windows were all tandemly repeated in a single genomic locus (Fig. 4D). VNTRs were also found in the two paternal gDMRs (Fig. 4E). VNTRs were not a common feature of the maternal gDMRs, but a significantly higher proportion of the maternal gDMRs did contain VNTRs as compared with all CGIs (gDMRs: 11/44, CGIs: 1763/27718, chi-square P = 4.1×10−7). Therefore, we focused on CGIs hypermethylated in both gametes and found that CGIs containing VNTRs were preferentially protected from demethylation in blastocysts (Fig. 4F). A comparison between VNTRs of>70% and <50% methylated CGIs in blastocysts revealed that VNTRs with more repeats tended to be protected from demethylation, whereas no sequence motif was found (Fig. 4G). These data suggested that VNTRs might underlie silencing of specific transposable elements and the protection of paternal gDMRs.

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
Related in: MedlinePlus