Role of Tet1 in erasure of genomic imprinting (original) (raw)

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Gene Expression Omnibus

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The accession number for the RNA-seq and RRBS data presented in this study is available from the Gene Expression Omnibus (GEO) database under accession GSE49764.

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Acknowledgements

We thank W. Jiang for help with FACS sorting of PGCs; A. Inoue, S. Matoba and D. Cai for critical reading of the manuscript. This project is supported by NIH U01DK089565 (to Y.Z.). S.Y. is supported by a postdoctoral fellowship from the Japan Society for the Promotion of Science (JSPS). Y.Z. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute, Boston Children's Hospital, Boston, 02115, Massachusetts, USA
    Shinpei Yamaguchi, Li Shen, Yuting Liu, Damian Sendler & Yi Zhang
  2. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, 02115, Massachusetts, USA
    Shinpei Yamaguchi, Yuting Liu & Yi Zhang
  3. Division of Hematology/Oncology, Department of Pediatrics, Boston Children’s Hospital, Boston, 02115, Massachusetts, USA
    Shinpei Yamaguchi, Yuting Liu & Yi Zhang
  4. Department of Genetics, Harvard Medical School, Boston, 02115, Massachusetts, USA
    Yi Zhang
  5. Harvard Stem Cell Institute, WAB-149G, 200 Longwood Avenue, Boston, Massachusetts 02115, USA ,
    Yi Zhang

Authors

  1. Shinpei Yamaguchi
  2. Li Shen
  3. Yuting Liu
  4. Damian Sendler
  5. Yi Zhang

Contributions

S.Y. and Y.Z. conceived the project, designed the experiments and wrote the manuscript; S.Y., L.S., Y.L. and D.S. performed experiments and analysed the data.

Corresponding author

Correspondence toYi Zhang.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Tet1 paternal knockout mice exhibit various phenotypes, including fetal and postnatal growth defects, and neonatal and embryonic lethality.

a, Scatter plot of body and placental sizes for each pup. b, Average placental weight of E13.5 and E16.5 embryos. Error bars indicate s.e.m. **P < 0.01. c, Percentage of pups that die within 3 days of birth. **P < 0.01. d, Box plot presentation of body weights of 4- and 8-week-old control and paternalKO mice. Note that paternalKO mice exhibited a larger variation and smaller body weight compared to the control mice. Middle lines in the coloured boxes indicate the median, box edges indicate the 25th/75th percentiles, and whiskers the 0th/100th percentiles. e, Mating scores of Tet1+/− male × wild-type female (×Het) and _Tet1_−/− male × wild-type female (×KO) mice. Black dots indicate the numbers of pups for each litter recovered by Caesarean section at E19.5. Red lines indicate the mean litter size. f, The embryos and placentae at E13.5 recovered from four litters of Tet1 paternalKO mice. The dotted ovals indicate the residues of absorbed embryos. g, E10.5 paternalKO embryos recovered from five litters. Dotted oval indicates morphologically abnormal embryos. h, Top: E9.5 paternalKO embryos recovered from two litters. Dotted ovals indicate small embryos whose placentae are morphologically abnormal. Bottom: placentae corresponding to the embryos on the top panels. Red rectangles indicate morphologically abnormal placentae. i, Representative images of haematoxylin and eosin staining of E9.5 placentae. Red rectangles indicate the enlarged regions shown at the right panels. Ch, chorionic plate; Gi, trophoblast giant cells; La, labyrinthine zone; Sp, spongioblast. j, Schematic representation of Tet1 paternalKO phenotypes. Each bar represents an individual mouse. About 40% of paternalKO mice exhibit early embryonic lethality. About half of the remaining mice die neonatally, and about half of the surviving mice show postnatal growth defects. In contrast, abnormality is rarely observed in control, except at the neonatal stage (less than 10%).

Extended Data Figure 2 Dysregulation of imprinted genes in Tet1 paternal knockout embryos.

a, Relative expression levels of imprinted genes in E9.5 embryos analysed by RNA-seq. The averaged FPKM (fragments per kb of exon per million fragments mapped) value of three control embryos is set as 1. Arrows indicate dysregulated embryos. The location of each gene is indicated in the diagram on top of the panel. b, Relative average expression change of maternally and paternally imprinted genes. The imprinted genes that are highly expressed in E9.5 embryos (FPKM >1.0) are used in this analysis. Error bars indicate s.e.m. *P < 0.05. c, Summary of imprinted genes dysregulated in paternalKO embryos. N, normal size; S, small size; +, normal expression; −, downregulated.

Extended Data Figure 3 Perturbation of gene expression in Tet1 paternal knockout embryos.

a, Heat-map representation of markedly downregulated genes (FC >1.5) in at least two E9.5 paternalKO embryos. Only those genes with significant change in at least two paternalKO embryos were shown. b, GO analysis of the 905 downregulated genes. c, Heat-map representation of markedly upregulated genes (FC >1.5) in at least E9.5 paternalKO embryos. d, GO analysis of the 635 upregulated genes.

Extended Data Figure 4 Hypermethylation in germline differentially methylated regions in Tet1 paternal knockout embryos and placentae, and Tet1 knockout sperm.

a, RT–qPCR analysis of Igf2r and Air in E9.5 embryos. The average value of control embryos (n = 13) is set as 1. Error bars indicate s.e.m. b, Bisulphite sequencing analysis of Air_–_Igf2r DMR of E9.5 embryos. The analysed region is indicated by a red line at the top of the diagram. Each CpG is represented by a circle with methylation and non-methylation represented by open and filled circles, respectively. The percentages of DNA methylation are indicated. c, qRT–PCR analysis of Impact in E9.5 embryos. The average value of control embryos (n = 13) is set as 1. Error bar indicates s.e.m. d, Bisulphite sequencing analysis of Impact DMR of E9.5 embryos. The analysed region is indicated by a red line at the top of the diagram. e, The weight of individual E19.5 paternalKO placentae (top) and qRT–PCR analysis of several imprinted gene expression patterns in each placenta. The average value of control placentae (n = 19) is set as 1. Asterisks indicate small placentae. Arrows indicate the samples showing a marked change in expression levels. Error bars indicate s.e.m. f, Bisulphite sequencing analysis of Peg3 DMR of E19.5 placentae. The analysed region is indicated at the top of the diagram. The percentages of DNA methylation are indicated. g, Bisulphite sequencing analysis of the Peg10 DMR in Tet1-heterozygous and knockout sperm. The analysed region is indicated by a red line at the top of the diagram.

Extended Data Figure 5 Summary of RRBS analysis.

a, b, Distribution of the level of methylation change for changed sites and promoters in E13.5 PGCs (a) and sperm (b) (cutoff, 10%). c, Cumulative distribution curve of methylation change in E13.5 PGCs for the sites showing methylation change in Tet1 knockout sperm. Note that methylation levels in E13.5 Tet1 knockout PGCs of both 422 hypermethylated sites in sperm (red) and 1,484 hypomethylated sites in sperm (blue) are higher than that of non-changed sites.

Extended Data Figure 6 Hypermethylation in Tet1 knockout E13.5 PGCs at imprinted genes and germline genes.

a, GO analysis of the genes with hypermethylated promoters in Tet1 knockout E13.5 PGCs. b, c, Representative DNA methylation profiles of selected imprinting genes (b) and germline genes (c) in E13.5 PGCs analysed by RRBS. Vertical grey lines represent the sequence read depth for each cytosine scored, and 30 reads were shown at most. The vertical red lines represent percentage of methylation for each cytosine scored, which range from 0% to 50%. For each gene, RefSeq exon organization (blue) and location of CGIs (green) are shown at the bottom.

Extended Data Figure 7 Hypermethylation in Tet1 knockout sperm at imprinted genes.

a, b, Mouse phenotype ontology enrichment of hypermethylated sites in Tet1 knockout sperm (a) and hypermethylated sites in both Tet1 knockout E13.5 PGCs and sperm (b). c, Representative DNA methylation profiles of selected imprinting genes in sperm analysed by RRBS. Vertical grey lines represent the sequence read depth for each cytosine scored, and 30 reads are shown at most. The vertical red lines represent percentage of methylation for each cytosine scored, which range from 0% to 50%. For each gene, RefSeq exon organization (blue) and location of CGIs (green) are shown at the bottom.

Extended Data Figure 8 Tet1 knockout affected loci are enriched in late demethylated regions in PGCs.

a, Methylation dynamics of the loci that are hypermethylated in Tet1 knockout E13.5 PGCs. Methylation levels were obtained from a previous study15. Middle lines in the coloured boxes indicate the median, box edges indicate the 25th/75th percentiles, and whiskers the 2.5th/97.5th percentiles. b, Percentages of hypermethylated promoters in Tet1 knockout PGCs among late demethylated promoters. The RRBS-covered promoters with >25% methylation in E9.5, E10.5 and E11.5 PGCs (ref. 14) were analysed. ***P < 1.0 × 10−15, **P < 0.01.

Extended Data Figure 9 Early embryonic lethality and dysregulation of imprinted gene expression in Tet1 paternal knockout mice generated by ref. 17 .

a, Representative image of paternalKO pups from the same litter showing big variation in body and placenta sizes. Embryos and their placentae were recovered by Caesarean section at the day of birth (E19.5). b, Average placental weights at E19.5. Error bars indicate s.e.m. **P < 0.01. c, Mating scores of Tet1+/− male × wild-type female (×Het) and _Tet1_−/− male × wild-type female (×KO) mice. Black dots indicate the number of pups for each litter recovered by Caesarean section at E19.5. Red lines indicate the mean litter size. d, Representative image of E10.5 Tet1 paternalKO embryos from the mating of wild-type female with Tet1 knockout male mice. The dotted oval indicates morphologically abnormal embryos. e, Left: E9.5 paternalKO embryos recovered from a single litter. Dotted oval indicates small embryos whose placentae are morphologically abnormal. Right: placentae of the corresponding embryos shown in the left panel. The red rectangle indicates morphologically abnormal placentae. f, Summary of embryonic abnormality. Asterisk indicates the numbers of morphologically abnormal embryos (E10.5) or placentae (E9.5). g, qRT–PCR analysis of selected imprinted genes in each of the E9.5 embryos from two litters (paternalKO 1 and paternalKO 2) are shown. Arrows indicate the samples showing marked change in expression level. The Ct values are normalized to the expression level of Gapdh.

Extended Data Figure 10 Defects in embryonic development and genomic imprinting in E19.5 Tet1 maternal knockout embryos and placentae.

a, Representative image of embryos and placentae from a single litter of E19.5 Tet1 maternalKO mice. Arrowheads indicate dead embryos whose placentae were morphologically normal. b, The ratio of dead embryos with relatively normal placentae. c, d, Box blot representation of the weight of embryos (c) and placentae (d). Middle lines in the coloured boxes indicate the medians, box edges indicate the 25th/75th percentiles, and whiskers the 0th/100th percentiles. e, qRT–PCR analysis of paternally imprinted genes in Tet1 maternalKO placentae. Arrows indicate the placentae whose embryos were found dead in utero, as indicated by arrowheads in a. The average value of control embryos (n = 19) is set as 1. Error bars indicate s.e.m. f, BS-seq revealed hypermethylation in Tet1 maternalKO mice in the IG-DMR locus. Note that methylation level in Tet1 maternalKO mice was significantly higher than in control mice, although IG-DMR gained methylation during placental development. Each dot represents the methylation level of each placenta. The numbers next to each dot indicate the number of clones sequenced. The average methylation levels are indicated by horizontal lines. The analysed region is indicated by a red line at the top of the diagram. g, BS-seq analysis of Rasgrf1 DMR of E19.5 placentae. The analysed region is indicated by a red line at the top of the diagram. Each CpG is represented by a circle with methylation and non-methylation represented by open and filled circles, respectively. The percentages of DNA methylation are indicated. *P < 0.05. **P < 0.01.

Extended Data Figure 11 Model explaining Tet1 paternal knockout phenotypes, using one maternal germline DMR as an example.

Black and white bars represent methylated and unmethylated state, respectively. In the control, the maternal allele is completely demethylated through Tet1-mediated imprinting erasure during PGC reprogramming. However, in Tet1 knockout PGCs, DMRs fail to be demethylated. The remaining methylation in the maternal allele is inherited to the next generation, which leads to imprinting defects.

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Yamaguchi, S., Shen, L., Liu, Y. et al. Role of Tet1 in erasure of genomic imprinting.Nature 504, 460–464 (2013). https://doi.org/10.1038/nature12805

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