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. 2016 Nov 3;99(5):1045-1058.
doi: 10.1016/j.ajhg.2016.08.021. Epub 2016 Oct 27.

Allele-Specific Methylome and Transcriptome Analysis Reveals Widespread Imprinting in the Human Placenta

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Free PMC article

Allele-Specific Methylome and Transcriptome Analysis Reveals Widespread Imprinting in the Human Placenta

Hirotaka Hamada et al. Am J Hum Genet. .
Free PMC article

Abstract

DNA methylation is globally reprogrammed after fertilization, and as a result, the parental genomes have similar DNA-methylation profiles after implantation except at the germline differentially methylated regions (gDMRs). We and others have previously shown that human blastocysts might contain thousands of transient maternally methylated gDMRs (transient mDMRs), whose maternal methylation is lost in embryonic tissues after implantation. In this study, we performed genome-wide allelic DNA methylation analyses of purified trophoblast cells from human placentas and, surprisingly, found that more than one-quarter of the transient-in-embryo mDMRs maintained their maternally biased DNA methylation. RNA-sequencing-based allelic expression analyses revealed that some of the placenta-specific mDMRs were associated with expression of imprinted genes (e.g., TIGAR, SLC4A7, PROSER2-AS1, and KLHDC10), and three imprinted gene clusters were identified. This approach also identified some X-linked gDMRs. Comparisons of the data with those from other mammals revealed that genomic imprinting in the placenta is highly variable. These findings highlight the incomplete erasure of germline DNA methylation in the human placenta; understanding this erasure is important for understanding normal placental development and the pathogenesis of developmental disorders with imprinting effects.

Keywords: DNA methylation; RNA sequencing; X-chromosome inactivation; genomic imprinting; germline differentially methylated region; human placenta; whole-genome bisulfite sequencing.

Figures

Figure 1
Figure 1
Genome-wide Profiling of Allelic DNA Methylation (A) Violin plots of methylation levels of windows hypermethylated (≥80%) or hypomethylated (≤20%) in one or both gametes (window size = 20 CpGs, step size = 10 CpGs). We compared the methylation profile of the CT cells (1st-CT #6 [♀]) with those of human gametes, blastocysts, cord blood cells (DRA003802), and ES cells (GSM706059). Oo-specific (Sp-specific) methylated windows are defined as windows hypermethylated in oocytes (sperm) and hypomethylated in sperm (oocytes). Thin and thick lines are boxplots, and white dots indicate the median. (B) Chromosomal distribution of maternally and paternally methylated windows in 1st-CT #6. The x and y axes show chromosome numbers and the maternal methylation level minus the paternal methylation level ([M − P] level), respectively. Windows showing ≥30% [M − P] levels and statistically significant allelic methylation differences (BH-corrected p < 0.05) are shown in red, and those with ≤ −30% [M − P] levels are in blue. The other windows are in gray. A histogram of the distribution of the [M − P] levels and the proportions of maternally and paternally methylated windows are also shown. (C) Boxplots of methylation levels of the maternal (M) and paternal (P) alleles of windows in 1st-CT #6. 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. Histograms of the distribution of the [M − P] levels are also shown (colored as defined in [B]).
Figure 2
Figure 2
Identification of gDMRs (A) Allelic DNA methylation patterns of candidate DMRs (c-DMRs) obtained from ten 1st-CT samples. Boxplots of methylation levels of the maternal (M) and paternal (P) alleles, chromosomal distribution of mean [M − P] values, and histograms of the distribution of the [M − P] values are shown. In the chromosome maps, red circles indicate c-DMRs showing ≥30% [M − P] levels and statistically significant allelic methylation differences (BH-corrected p < 0.05). Similarly, blue circles indicate those with ≤ −30% [M − P] values. The other c-DMRs are shown as gray circles. Each x indicates a known gDMR. (B) Box plots of allelic DNA methylation levels of candidate gDMRs covered by both 1st-CT and 2nd/term-CT samples. (C) Classification of confirmed mDMRs according to their mean [M − P] values and location. The data of the 1st-CT samples were used. Known ubiquitous and placenta-specific mDMRs are also shown for comparison. (D) Variations in allelic methylation between samples. We calculated the SD value for each confirmed mDMR by using the data of the 1st-CT samples. Only mDMRs for which there were data from three or more samples were analyzed. Box plots with individual data points are shown. Known ubiquitous and placenta-specific mDMRs are also included for comparison. (E) Violin plots of methylation levels of confirmed mDMRs. 1st-CT #6 and #9 were analyzed by WGBS. Additional CT samples and stromal cells obtained from two first-trimester placentas were analyzed via targeted bisulfite sequencing. External WGBS data were used for human gametes, blastocysts, cord blood cells (DRA003802), a term placenta analyzed without a cell purification step (GSM1134682), and ES cells (GSM706059). Only confirmed mDMRs covered by all the samples were analyzed (n = 404). (F) DNA methylation patterns of a confirmed pDMR located downstream of GRHL1 (MIM: 609786). Each vertical bar represents a CpG site. The pDMR was hypomethylated in oocytes, hypermethylated in sperm, and paternally methylated in CT cells.
Figure 3
Figure 3
Transcriptome-wide Screening of Imprinted Genes (A) Chromosomal distribution of candidate imprinted genes. The x axis and y axis show chromosome numbers and the maternal expression ratio ([M-expression] ratio), respectively. Genes showing >65% [M-expression] ratios and statistically significant allelic expression differences (BH-corrected p < 0.05) are shown in red, and those with <35% [M-expression] ratios are in blue. Each x indicates a known imprinted gene with an [M-expression] ratio > 65% or < 35%. The other genes are in gray. (B) Counts of candidate and known imprinted genes. (C) DNA methylation patterns of the PROSER2-AS1 DMR. [M-expression] ratios of associated genes are shown in parentheses. (D) The DNMT1 and ACCS imprinted gene clusters. Genes with [M-expression] ratios > 65% and < 35% are shown in red and blue, respectively. Genes with 35%–65% [M-expression] ratios are in gray. Genes without available allelic expression ratios are shown as white boxes without gene symbols. An asterisk indicates that it was unclear whether C11orf96 was really maternally expressed because the [contamination] rate of C11orf96 was not available.
Figure 4
Figure 4
Allelic Regulation of X-Linked Genes and gDMRs (A) Boxplots of maternal expression ratios ([M-expression] ratios) of X-linked genes. (Left) 18 1st-CT samples were analyzed, and samples with >65% and <35% median [M-expression] ratios are shown in red and blue, respectively. (Right) Summary of allelic expression of X-linked and autosomal genes from the 18 1st-CT samples. X-linked genes had higher [M-expression] ratios than autosomal genes (p < 2.2 × 10−16, Mann Whitney U-test). (B) A chromosome map of [M-expression] ratios of X-linked genes (1st-CT #3). Genes with >65% [M-expression] ratios are shown in red, and those with 35%–65% [M-expression] ratios are in gray with gene symbols. The [M-expression] ratio of XIST was 14.4% (shown in blue). (C) DNA methylation patterns of the LOC389906-ds DMR. LOC389906 is an escape gene as shown in (B). (D) Bisulfite sequencing analysis of the LOC389906-ds DMR. Black and white circles indicate methylated and unmethylated residues, respectively. The percentages of methylated CpG sites are indicated. (E) DNA methylation patterns of the NUDT10 DMR. (F) Bisulfite sequencing analysis of the NUDT10 DMR. (G) A high correlation between NUDT10 expression levels and the median [M-expression] ratios of X-linked genes. 18 female and three male 1st-CT samples were analyzed. Pearson’s r was 0.93.
Figure 5
Figure 5
Schematic Illustration of Allelic DNA Methylation Levels of Oocyte- and Sperm-Specific Methylated Regions during Human Development For oocyte-specific methylated regions, the maternal alleles maintain high methylation levels throughout human development. The paternal alleles are predominantly hypermethylated in the embryonic lineage after implantation, but the de novo methylation and/or methylation maintenance of the paternal alleles are incomplete in the placenta. Consequently, many oocyte-specific methylated regions maintain maternally biased DNA methylation in the placenta. In contrast, sperm-specific methylated regions are demethylated during preimplantation development, and very few regions maintain paternally biased DNA methylation after implantation.

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