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Nature Genetics | Article

日本語要約

Epigenetic memory at embryonic enhancers identified in DNA methylation maps from adult mouse tissues

Journal name:
Nature Genetics
Volume:
45,
Pages:
1198–1206
Year published:
DOI:
doi:10.1038/ng.2746
Received
Accepted
Published online

Abstract

Mammalian development requires cytosine methylation, a heritable epigenetic mark of cellular memory believed to maintain a cell's unique gene expression pattern. However, it remains unclear how dynamic DNA methylation relates to cell type–specific gene expression and animal development. Here, by mapping base-resolution methylomes in 17 adult mouse tissues at shallow coverage, we identify 302,864 tissue-specific differentially methylated regions (tsDMRs) and estimate that >6.7% of the mouse genome is variably methylated. Supporting a prominent role for DNA methylation in gene regulation, most tsDMRs occur at distal cis-regulatory elements. Unexpectedly, some tsDMRs mark enhancers that are dormant in adult tissues but active in embryonic development. These 'vestigial' enhancers are hypomethylated and lack active histone modifications in adult tissues but nevertheless exhibit activity during embryonic development. Our results provide new insights into the role of DNA methylation at tissue-specific enhancers and suggest that epigenetic memory of embryonic development may be retained in adult tissues.

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  1. Distinct tissue-specific methylomes.
    Figure 1: Distinct tissue-specific methylomes.

    (a) UCSC Genome Browser snapshot of DNA methylomes derived from tissues of a single mouse near the Hoxa locus. Each track spans percent mCG (%mCG) values between 0% and 100%. Tracks are colored by tissue type, as determined by the clustering in c. (b) Box plots of the percent mCG distributions for each tissue, calculated from non-overlapping 10-kb bins spanning the mouse genome. Box-plot edges indicate the 25th and 75th percentiles, central bars indicate medians and whiskers indicate non-outlier extremes. Dashed lines indicate 80% and 90% mCG. (c) Dendrogram constructed from 1-kb regions exhibiting significant tissue-specific methylation (P = 0.001, χ2 test). Distance is measured as 1 – Pearson's correlation coefficient. (d) Global abundance of methylation in the non-CG context, expressed as the difference between the fraction of non-CG cytosines that are methylated and the bisulfite non-conversion rate. (e) Abundance of large domains bearing low (L), medium (M) or high (H) methylation, as determined by HMMs independently trained on each tissue.

  2. Identification of tissue-specific methylated regions.
    Figure 2: Identification of tissue-specific methylated regions.

    (a) UCSC Genome Browser snapshot of tsDMRs identified by HMM segmentation of the χ2 statistic for methylation variation (top two tracks). The abundance of DNA methylation in each tissue is indicated below. The tissue specificity of highlighted tsDMRs is indicated above. (b) Quantification of DNA methylation abundance at tsDMRs from a. (c) An HMM was used to segment the mouse genome into regions of low (L, gray), medium (M, white) and high (H, red) tissue specificity for DNA methylation. Shown is the fraction of the genome spanned by these regions, with the high-specificity group denoting tsDMRs. (d) Size distribution of tsDMRs (H) and non-tsDMRs (L). Box plot edges indicate the 25th and 75th percentiles, and whiskers indicate non-outlier extremes. (e) Distributions of average values (left) and standard deviations (right) of percent mCG for tsDMRs (H) and non-tsDMRs (L). Dashed lines indicate the median, m.

  3. Tissue-specific methylated regions are predominantly regulatory elements.
    Figure 3: Tissue-specific methylated regions are predominantly regulatory elements.

    (a) Enrichment of H3K4me1, H3K4me3, H3K27ac and DNA methylation in liver cells for liver tsDMRs and tsDMRs identified in other tissues. Bone marrow is shown immediately below liver in the H3K4me3 plot. (b) Average PhastCons conservation scores relative to tsDMRs. Higher values indicate greater conservation. (c) Left, heatmap representing the abundance of DNA methylation for tsDMRs in all tissues. Each row represents a tsDMR, and rows are grouped into horizontal blocks that represent the tsDMRs of a particular tissue, with the number of elements in the block shown to the right. A given tsDMR may appear in more than one block. Right, the percentage of tsDMRs in each block within 500 bp of promoters, distal regulatory elements (enhancers and CTCF-binding sites), genic regions and intergenic regions. The genome-wide average for each element is included as the bottom row.

  4. Tissue-specific conservation of regulatory elements.
    Figure 4: Tissue-specific conservation of regulatory elements.

    (a,b) Average PhastCons conservation scores relative to tsDMRs that are proximal (within 2.5 kb) (a) or distal (beyond 2.5 kb) (b) to annotated transcription start sites (TSSs). Higher values indicate greater conservation.

  5. Transcription factor binding motif enrichment near tsDMRs.
    Figure 5: Transcription factor binding motif enrichment near tsDMRs.

    (a) Heatmap representing the enrichment of transcription factor binding motifs for the tsDMRs identified in each tissue. Each row represents a motif, and the corresponding transcription factors for selected motifs are labeled on the right. (b) Predicted regulatory elements in heart (p300-binding sites (gray), chromatin-predicted enhancers (blue), tsDMRs (red)) were overlapped, and the density of known tissue-specific motifs (ETS, FOXO1, GATA4, JUN, MEF2A, NF1, SOX6, STAT3, TEAD4) relative to these aligned intersected sites is shown. (c) Cumulative density of graphs in b with respect to the absolute distance to the predicted regulatory element. The horizontal dashed line indicates 50% cumulative density, and vertical dotted lines indicate the resolution in base pairs of each set of predicted regulatory elements to achieve 50% cumulative density.

  6. AD-I tsDMRs demonstrate features of dormant enhancers.
    Figure 6: AD-I tsDMRs demonstrate features of dormant enhancers.

    (a) Heatmap representing the density of H3K4me1 and H3K27ac marks relative to all tsDMRs identified in heart, kidney and olfactory bulb. Also indicated are AD-A tsDMRs and AD-I tsDMRs. (b) AD-A and AD-I tsDMRs were identified for ten tissues where histone modification data are publicly available28 (spleen, thymus, bone marrow, intestine, liver, heart, kidney, cerebellum, cortex, olfactory bulb). Shown are the average profiles of H3K4me1, H3K27ac and DNA methylation. Each line represents the average over a tissue or replicate pair. (c) Heatmap representing the enrichment of transcription factor binding motifs for the AD-I and AD-A tsDMRs of each tissue. Each row represents a motif, and several rows are labeled on the right. The numbers of AD-A and AD-I tsDMRs are indicated in parentheses above. (d) Heatmap representing the enrichment of GO biological process terms obtained by running the GREAT tool35 on AD-I tsDMRs. (e) Average PhastCons conservation scores for AD-A tsDMRs and AD-I tsDMRs identified in cerebellum, kidney and heart. Higher values indicate greater conservation.

  7. AD-I tsDMRs are active during development.
    Figure 7: AD-I tsDMRs are active during development.

    (a) For AD-I tsDMRs identified in adult cerebellum, adult cortex and adult heart, shown is the average enrichment of H3K4me1 and H3K27ac marks in adult tissue and E14.5 tissue. Two biological replicates for each sample are shown. (b) AD-A and AD-I tsDMRs were associated with genes within 50 kb. Shown is the distribution of expression for genes associated with AD-A tsDMRs and AD-I tsDMRs in respective adult tissue. For AD-I tsDMRs for which RNA sequencing data exist at earlier developmental time points (cerebellum, cortex, olfactory bulb: brain E14.5; heart: heart E14.5; liver: liver E14.5), also shown is the distribution of expression of AD-I tsDMR–associated genes in developmental tissue. P values, Wilcoxon. (c) Heatmap of the overlap enrichment of AD-I tsDMRs with enhancers predicted in developing embryos (left) and adult cells (right). Enrichment is determined relative to sets of random AD-I tsDMRs. (d) Box plots of overlap enrichment from c of AD-I tsDMRs with enhancers predicted in developing or adult tissues. (e) Overlap of p300-binding sites34 identified in embryonic forebrain, midbrain and limb with AD-I tsDMRs (red) identified in ectodermal tissue (cerebellum, cortex, olfactory bulb) and all other tissues. As a comparison, overlap was also performed against random sets of AD-I tsDMRs (gray), shown as mean ± s.d. (f) In vivo reporter assays of enhancer activity for VISTA enhancers mm447 and mm414, as obtained from the VISTA enhancer browser39. (g) UCSC Genome Browser snapshot of AD-I tsDMRs identified in cerebellum (highlighted), which overlap with an in vivo–validated enhancer active in developing mouse midbrain. Also shown are active histone modifications (H3K4me1, H3K4me3, H3K27ac) and DNase I hypersensitivity sites (DHSs) in developing mouse brains (E14.5, E18.5) and adult cerebellum. For all box plots, edges indicate the 25th and 75th percentiles, and whiskers indicate non-outlier extremes.

  8. Vestigial enhancers across development and strains.
    Figure 8: Vestigial enhancers across development and strains.

    (a) Heatmaps representing the enrichment of chromatin (me1, H3K4me1; ac, H3K27ac) at predicted enhancers with active chromatin in E14.5 mouse brain that consistently retain or lose active chromatin marks in all adult brain tissues (cerebellum, cortex, olfactory bulb). The DNA methylation status in adult tissues is indicated on the right. (b) Percentage of E14.5 mouse brain enhancers belonging to various epigenetic states in adult brain tissue (cer, cerebellum; cor, cortex; olf, olfactory bulb). E14.5 enhancers were overlapped with adult enhancers (A), and the remainder were overlapped with adult tsDMRs (D). The remaining fraction is labeled as inactive (I). (c) Heatmap comparing the enrichment of transcription factor binding motifs for vestigial enhancers in brain tissue T1 with those in brain tissue T2. Each row represents a motif, and several rows are labeled on the right. (d) Top, heatmap of DNA methylation status for various tissues and mouse strains, centered on vestigial enhancers identified in C57BL/6 mouse cortex. Bottom, box plots comparing C57BL/6 cortex vestigial enhancer methylation with that of each tissue or strain. Distance is measured as the absolute difference in methylation of each vestigial enhancer between two tissues. Box plot edges indicate the 25th and 75th percentiles, and whiskers indicate non-outlier extremes. *P < 1 × 10−3, Wilcoxon. (e) Average profiles of DNA methylation for cortical tissue from C57BL/6, C/129 and 129/C mice centered at C57BL/6 cortex vestigial enhancers.

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Author information

Affiliations

  1. Ludwig Institute for Cancer Research, La Jolla, California, USA.

    • Gary C Hon,
    • Yin Shen,
    • David F McCleary,
    • Feng Yue,
    • My D Dang &
    • Bing Ren

  2. Bioinformatics and Systems Biology Program, University of California, San Diego, La Jolla, California, USA.

    • Nisha Rajagopal &
    • Bing Ren

  3. Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, La Jolla, California, USA.

    • Bing Ren

  4. Institute of Genomic Medicine, University of California, San Diego School of Medicine, La Jolla, California, USA.

    • Bing Ren

  5. Moores Cancer Center, University of California, San Diego School of Medicine, La Jolla, California, USA.

    • Bing Ren

Contributions

G.C.H., N.R. and F.Y. performed bioinformatics analysis. G.C.H., Y.S., D.F.M. and M.D.D. performed experiments. G.C.H. and B.R. prepared the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (1,785 KB)

    Supplementary Figures 1–8 and Supplementary Note

Excel files

  1. Supplementary Table 1 (17,230 KB)

    tsDMRs in mouse tissues

  2. Supplementary Table 2 (21 KB)

    Motif analysis of tsDMRs not recovered by known regulatory elements

  3. Supplementary Table 3 (13,208 KB)

    AD-A and AD-I tsDMRs in mouse tissues

  4. Supplementary Table 4 (11 KB)

    Overlap of embryonic enhancers with adult AD-I tsDMRs

  5. Supplementary Table 5 (17,387 KB)

    Predicted enhancers

  6. Supplementary Table 6 (16 KB)

    Known motifs used in analysis

Additional data