A unique regulatory phase of DNA methylation in the early mammalian embryo

Journal name:
Nature
Volume:
484,
Pages:
339–344
Date published:
DOI:
doi:10.1038/nature10960
Received
Accepted
Published online

Abstract

DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methylcytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and so far no base-resolution maps exist to support and refine it. Here we generate genome-scale DNA methylation maps in mouse gametes and from the zygote through post-implantation. We find that the oocyte already exhibits global hypomethylation, particularly at specific families of long interspersed element 1 and long terminal repeat retroelements, which are disparately methylated between gametes and have lower methylation values in the zygote than in sperm. Surprisingly, the oocyte contributes a unique set of differentially methylated regions (DMRs)—including many CpG island promoters—that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and become hypermethylated after the blastocyst stage. Our data provide a genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.

At a glance

Figures

  1. Global CpG methylation dynamics across early murine embryogenesis.
    Figure 1: Global CpG methylation dynamics across early murine embryogenesis.

    a, Samples isolated for methylation analysis with replicate number (n) highlighted. d.p.f., days post fertilization; h.p.f., hours post fertilization. b, Fraction of 100-bp tiles with high (≥0.8, red), intermediate (inter, >0.2 and <0.8, green) and low (≤0.2, blue) methylation values. Brain, heart and liver tissue are shown for adult comparisons. c, Histogram of methylation values across 100-bp tiles. n is the number of tiles for each stage. d, Box plots of methylation values across local CpG densities highlight the difference between hypomethylated pre-implantation tissues and the somatic pattern seen in sperm, post-implantation and adult samples. Circle indicates the median, edges the 25th/75th percentile and whiskers the 2.5th/97.5th percentile. e, CpG density distribution for >0.2 methylation (left panel) and≤0.2 methylation (right panel) tiles in stages that display somatic versus embryonic patterning (red and blue lines, respectively).

  2. Major transitions in DNA methylation levels during early development.
    Figure 2: Major transitions in DNA methylation levels during early development.

    a, 100-bp tiles available for pairwise comparison across consecutive embryonic stages. Tiles that remain unchanged (stable) at the indicated transitions are shown in light blue. Tiles that change by greater than 0.2 and are significant by t-test are highlighted in dark blue. b, 100-bp tiles with increasing (red) or decreasing (green) methylation levels at each consecutive transition show that major transitions are largely unidirectional. c, Box plot of methylation levels for sperm-specific DMRs (n = 134,038 tiles). Red line indicates the median, edges the 25th/75th percentile and whiskers the 2.5th/97.5th percentile. d, Box plot of methylation levels for oocyte-specific DMRs (n = 6,394 tiles) as in c. e, Seventy-four CpGs within sperm-specific DMR tiles (c) could be ascribed to paternal or maternal alleles and tracked across stages. Paternal CpG methylation values (blue line, median; coloured space, 25th/75th percentile) decrease by the zygote stage whereas maternal CpG methylation (red line, median; coloured space, 25th/75th percentile) remains unchanged. If untracked, these CpGs have an intermediate methylation value between those ascribed to a parent of origin (black line).

  3. Specific families of LINE and LTR retroelements exhibit the most dramatic methylation changes in the sperm to zygote transition.
    Figure 3: Specific families of LINE and LTR retroelements exhibit the most dramatic methylation changes in the sperm to zygote transition.

    a, Histogram of the difference in methylation levels, where negative values represent tiles decreasing from sperm to zygote, within LINE retroelement features that are captured by RRBS. 85% of the elements have a significant difference (P<0.04, FDR <0.05; t-test). The distribution is bimodal with 18% of elements displaying a change in methylation status ≥0.45 as highlighted in red. b, Differences in methylation between sperm and zygote within annotated LTR retroelements. Compared to LINEs, a smaller fraction of elements appear to be regulated by DNA demethylation (61% significant, 10% of those sampled exhibiting changes ≥0.45 as highlighted in red). ce, Box plots of methylation levels in oocyte, sperm and zygote (top panels) as well as the distributions of change in methylation levels between sperm and zygote (bottom panels) for specific LINE-1 families, including those that are (c, d) or are not dynamic (e). Top panels: the red line indicates the median, edges the 25th/75th percentile and whiskers the 2.5th/97.5th percentile. Bottom panels: members of each family that are demethylated by greater than 0.45 are highlighted in red. fh, Box plots of methylation levels in oocyte, sperm and zygote (top panels) and the distributions of change in methylation levels between sperm and zygote (bottom panels) for specific families of LTR retroelements, including MMERGLN (f), RLTR10C (g) and IAP elements (h). Top and bottom panels as in ce. i, Mean methylation level for all elements of the L1Md_A LINE (solid blue line) and IAP LTR class (solid red line) that do not markedly change contrasted by LINEs (dashed blue line) and LTR elements (dashed red line) that show the greatest loss at fertilization. SINE elements (green line) are less methylated in sperm than other repeat elements and appear to decrease to oocyte levels.

  4. Differentially methylated regions represent discrete gamete-specific feature classes.
    Figure 4: Differentially methylated regions represent discrete gamete-specific feature classes.

    a, Heat map of methylation levels (black, 0; red, 1; grey, missing value) in 376 identified 100-bp tiles (rows) that behave as oocyte-contributed DMRs in the zygote. Tiles are sorted by functional classes and clustered within each class. Fifteen known ICRs, shown at the bottom, behave similarly in the early embryo and retain intermediate methylation through implantation. Other includes both Other and No annotation. b, Genomic features (top) and promoters of different CpG densities (bottom) in oocyte-contributed DMRs. Top: oocyte DMRs are enriched for promoters. Bottom: most of the 105 promoters that overlap oocyte-contributed DMR tiles are high CpG density promoters containing CpG islands (HCPs, light blue). c, Heat map of methylation levels (black, 0; red, 1; grey, missing value) in 4,894 identified 100-bp tiles (rows) that behave as sperm-contributed DMRs in pre-implantation embryos. Tiles are sorted and highlighted as in a. d, Genomic features in sperm-contributed DMRs are generally intergenic.

  5. DMRs resolve after cleavage to univalent hyper- or hypomethylated values in a gamete-of-origin-specific fashion.
    Figure 5: DMRs resolve after cleavage to univalent hyper- or hypomethylated values in a gamete-of-origin-specific fashion.

    a, Single CpG resolution methylation within 2 kilobases (kb) of the Cpne7 promoter in gametes and across embryonic development (rows). Dark grey bar highlights the CpG island. A CpG proximal to the island can be tracked to a phase resolving SNP and is highlighted in light grey, with paternal (X1) and maternal (C57) methylation values included as an inset for each trackable phase. Values for SNP methylation in ‘cleavage’ correspond exactly to those captured in the zygote. Blue bars, CpG methylation; red bars, CpA methylation. b, Composite plot of CpG (blue) and CpA (red) methylation for all HCPs (left) and for promoters that are specifically hypermethylated in oocytes (transcription start site (TSS) DMRs, right). The region ±2kb of the TSS is marked in grey. Identified promoter DMRs contributed by the oocyte are hypermethylated around the periphery of the TSS and resolve to intermediate values throughout cleavage. An expected HCP methylation architecture is re-acquired for these DMRs around implantation. c, Mean methylation levels across stages for oocyte-contributed DMRs in promoters (red, dashed line) versus the complete set (red, solid line). d, Sperm-contributed DMRs (blue line) generally resolve to hypermethylation.

Accession codes

Primary accessions

Gene Expression Omnibus

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Author information

  1. These authors contributed equally to this work.

    • Zachary D. Smith &
    • Michelle M. Chan

Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • Zachary D. Smith,
    • Michelle M. Chan,
    • Tarjei S. Mikkelsen,
    • Hongcang Gu,
    • Andreas Gnirke,
    • Aviv Regev &
    • Alexander Meissner
  2. Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA

    • Zachary D. Smith,
    • Tarjei S. Mikkelsen &
    • Alexander Meissner
  3. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Zachary D. Smith &
    • Alexander Meissner
  4. Computational and Systems Biology Program, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Michelle M. Chan
  5. Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Aviv Regev

Contributions

Z.D.S. and A.M. conceived the study and Z.D.S., M.M.C. and A.M. facilitated its design. Z.D.S. collected samples and performed methylation profiling, M.M.C. performed all analysis with assistance from T.S.M. and Z.D.S. H.G. and A.G. provided critical technical assistance and expertise. Z.D.S., M.M.C., T.S.M., A.R. and A.M. interpreted the data. Z.D.S., M.M.C. and A.M. wrote the paper with the assistance of the other authors.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

RRBS data is deposited at the Gene Expression Omnibus under accession number GSE34864.

Author details

Supplementary information

PDF files

  1. Supplementary Information (1.5M)

    This file contains Supplementary Figures 1-11 and full legends for Supplementary Tables 1- 4 and Supplementary Movie 1.

Excel files

  1. Supplementary Table 1 (3.6M)

    This file contains promoter methylation levels at fertilization and across early embryonic development - see legend in Supplementary Information file.

  2. Supplementary Table 2 (28K)

    This file contains Long Interspersed Element (LINE) retrotransposon feature methylation across early embryonic development - see legend in Supplementary Information file.

  3. Supplementary Table 3 (42K)

    This file contains Long Terminal Repeat (LTR) retrotransposon feature methylation across early embryonic development - see legend in Supplementary Information file.

  4. Supplementary Table 4 (149K)

    This file contains feature designation and methylation status for identified oocyte-contributed Differentially Methylated Regions (DMRs) across early embryonic development - see legend in Supplementary Information file.

Movies

  1. Supplementary Movie 1 (6.9M)

    This file contains a movie of a representative polar body biopsy for zygotes and cleavage stage embryos collected in this study - see legend in Supplementary Information file.

Additional data