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The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity

Abstract

The RNA modification N6-methyladenosine (m6A) has critical roles in many biological processes1,2. However, the function of m6A in the early phase of mammalian development remains poorly understood. Here we show that the m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m6A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m6A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.

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Fig. 1: YTHDC1 depletion induces 2C-like state transition.
Fig. 2: YTHDC1 mediates Setdb1 H3K9me3-dependent retrotransposon repression.
Fig. 3: m6A-YTHDC1 regulates retrotransposon repression and Dux-dependent 2C-like transition.

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Data availability

The data supporting the conclusions of this Article, including H3K9me3–YTHDC1 ChIP–seq, m6A–YTHDC1 RIP-seq, ChIRP-seq, 4sUDRB-seq and RNA-seq data are available at GEO under accession GSE146467. The m6A RIP-seq data were from GSE5266219, GSE619988, GSE14531520 and GSE13359914. The H3K9me3 ChIP-seq data of Setdb1-KO mouse ES cells was obtained from the BioProject accession PRJNA54454018. The raw uncropped data for gels are appended in Supplementary Fig. 1, and high-resolution images for whole-mount fluorescence imaging are available at Supplementary Fig. 2. qPCR primers, ChIRP probes and antibodies used in this study are listed in the supplementary tables.

References

  1. Fu, Y., Dominissini, D., Rechavi, G. & He, C. Gene expression regulation mediated through reversible m6A RNA methylation. Nat. Rev. Genet. 15, 293–306 (2014).

    Article  CAS  PubMed  Google Scholar 

  2. Zaccara, S., Ries, R. J. & Jaffrey, S. R. Reading, writing and erasing mRNA methylation. Nat. Rev. Mol. Cell Biol. 20, 608–624 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Yang, Y., Hsu, P. J., Chen, Y. S. & Yang, Y. G. Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism. Cell Res. 28, 616–624 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Liu, J. et al. YTHDF2/3 are required for somatic reprogramming through different RNA deadenylation pathways. Cell Rep. 32, 108120 (2020).

    Article  CAS  PubMed  Google Scholar 

  5. Luo, S. & Tong, L. Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain. Proc. Natl Acad. Sci. USA 111, 13834–13839 (2014).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  6. Xu, C. et al. Structural basis for selective binding of m6A RNA by the YTHDC1 YTH domain. Nat. Chem. Biol. 10, 927–929 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Kasowitz, S. D. et al. Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development. PLoS Genet. 14, e1007412 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Geula, S. et al. Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation. Science 347, 1002–1006 (2015).

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Shi, H. et al. m6A facilitates hippocampus-dependent learning and memory through YTHDF1. Nature 563, 249–253 (2018).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ivanova, I. et al. The RNA m6A reader YTHDF2 is essential for the post-transcriptional regulation of the maternal transcriptome and oocyte competence. Mol. Cell 67, 1059–1067 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hsu, P. J. et al. Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis. Cell Res. 27, 1115–1127 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhang, Y. et al. RNA-binding protein YTHDF3 suppresses interferon-dependent antiviral responses by promoting FOXO3 translation. Proc. Natl Acad. Sci. USA 116, 976–981 (2019).

    Article  CAS  PubMed  Google Scholar 

  13. Wu, Y. et al. Nuclear exosome targeting complex core factor Zcchc8 regulates the degradation of LINE1 RNA in early embryos and embryonic stem cells. Cell Rep. 29, 2461–2472 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. Liu, J. et al. N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science 367, 580–586 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  15. Macfarlan, T. S. et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature 487, 57–63 (2012).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hendrickson, P. G. et al. Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons. Nat. Genet. 49, 925–934 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Genet, M. & Torres-Padilla, M. E. The molecular and cellular features of 2-cell-like cells: a reference guide. Development 147, dev189688 (2020).

    Article  CAS  PubMed  Google Scholar 

  18. Wu, K. et al. SETDB1-mediated cell fate transition between 2C-like and pluripotent states. Cell Rep. 30, 25–36 (2020).

    Article  CAS  PubMed  Google Scholar 

  19. Batista, P. J. et al. m6A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15, 707–719 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chelmicki, T. et al. m6A RNA methylation regulates the fate of endogenous retroviruses. Nature https://doi.org/10.1038/s41586-020-03135-1 (2021).

  21. Patil, D. P. et al. m6A RNA methylation promotes XIST-mediated transcriptional repression. Nature 537, 369–373 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jachowicz, J. W. et al. LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo. Nat. Genet. 49, 1502–1510 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Percharde, M. et al. A LINE1-nucleolin partnership regulates early development and ESC identity. Cell 174, 391–405 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Matsui, T. et al. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature 464, 927–931 (2010).

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Li, X. et al. GRID-seq reveals the global RNA-chromatin interactome. Nat. Biotechnol. 35, 940–950 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Iturbide, A. & Torres-Padilla, M. E. A cell in hand is worth two in the embryo: recent advances in 2-cell like cell reprogramming. Curr. Opin. Genet. Dev. 64, 26–30 (2020).

    Article  CAS  PubMed  Google Scholar 

  27. Chen, J. et al. H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs. Nat. Genet. 45, 34–42 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Moazed, D. Small RNAs in transcriptional gene silencing and genome defence. Nature 457, 413–420 (2009).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zofall, M. et al. RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation. Science 335, 96–100 (2012).

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Wang, C. et al. A novel RNA-binding mode of the YTH domain reveals the mechanism for recognition of determinant of selective removal by Mmi1. Nucleic Acids Res. 44, 969–982 (2016).

    Article  CAS  PubMed  Google Scholar 

  31. Chen, Z. & Zhang, Y. Loss of DUX causes minor defects in zygotic genome activation and is compatible with mouse development. Nat. Genet. 51, 947–951 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Summers, M. C., McGinnis, L. K., Lawitts, J. A., Raffin, M. & Biggers, J. D. IVF of mouse ova in a simplex optimized medium supplemented with amino acids. Hum. Reprod. 15, 1791–1801 (2000).

    Article  CAS  PubMed  Google Scholar 

  33. Wu, G. et al. Establishment of totipotency does not depend on Oct4A. Nat. Cell Biol. 15, 1089–1097 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. He, J. et al. Transposable elements are regulated by context-specific patterns of chromatin marks in mouse embryonic stem cells. Nat. Commun. 10, 34 (2019).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  35. Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  38. Jin, Y., Tam, O. H., Paniagua, E. & Hammell, M. TEtranscripts: a package for including transposable elements in differential expression analysis of RNA-seq datasets. Bioinformatics 31, 3593–3599 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Hasson, D. et al. The octamer is the major form of CENP-A nucleosomes at human centromeres. Nat. Struct. Mol. Biol. 20, 687–695 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gu, J. et al. GoldCLIP: Gel-omitted Ligation-dependent CLIP. Genomics Proteomics Bioinformatics 16, 136–143 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Chu, C., Qu, K., Zhong, F. L., Artandi, S. E. & Chang, H. Y. Genomic maps of long noncoding RNA occupancy reveal principles of RNA–chromatin interactions. Mol. Cell 44, 667–678 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Fuchs, G. et al. 4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells. Genome Biol. 15, R69 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Feng, J., Liu, T., Qin, B., Zhang, Y. & Liu, X. S. Identifying ChIP-seq enrichment using MACS. Nat. Protocols 7, 1728–1740 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44 (W1), W160–W165 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Kim, D., Paggi, J. M., Park, C., Bennett, C. & Salzberg, S. L. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat. Biotechnol. 37, 907–915 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

    Article  ADS  CAS  PubMed  Google Scholar 

  51. Kumar, V. et al. Uniform, optimal signal processing of mapped deep-sequencing data. Nat. Biotechnol. 31, 615–622 (2013).

    Article  CAS  PubMed  Google Scholar 

  52. Ng, R. K. et al. Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5. Nat. Cell Biol. 10, 1280–1290 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Latos, P. A. & Hemberger, M. From the stem of the placental tree: trophoblast stem cells and their progeny. Development 143, 3650–3660 (2016).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank X. Quan, Z. Zhang, J.-Y. Ji, Y. Liu, S. Shu, W. Pang and S. Xu for experimental assistance; Y. Shi and H. Shen for valuable suggestions and for sharing unpublished results; H. Chen, C.-H. Hsu, M. Min, L. Shen, J. Wang and Y. Yu for discussion and constructive suggestions; and the Guangzhou Branch of the Supercomputing Center of Chinese Academy of Sciences, and the Cloud Computing Center of Chinese Academy of Sciences for their support. This work was supported by the National Key R&D Program of China (2019YFA0110200, 2017YFA0504100 and 2016YFA0100400), Key Research & Development Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory (2018GZR110104003), Frontier Science Research Program of the CAS (ZDBS-LY-SM007), the Science and Technology Program of Guangzhou (201804020052), National Natural Science Foundation of China (31771424, 32070794, 32000501, 32000503), and Science and Technology Planning Project of Guangdong Province (2020B1212060052).

Author information

Authors and Affiliations

Authors

Contributions

J.L. and M.G. performed the main experiments; J.H. performed the bioinformatics analysis. J.L. and J.C. initiated the study. K.W. performed the chimeric embryo assay, 2C reporter cell lines construction and Dux knockout. S.L., L.J. and Y.C. assisted in experiments including with cell culture, plasmid construction and imaging. G.W., M.Z. and K.W. performed chimeric embryo immunofluorescence staining assays and L.C. assisted embryo imaging. H.L. performed high-throughput sequencing. J.S. assisted with bioinformatics analysis. J.L. performed YTHDC1 RIP-seq, ChIRP-seq and 4sUDRB-seq with help from X.W., Y.L. and X.B. M.G. performed Mettl3 KO and Y.-L.Z. and G.-Z.L. performed the m6A RIP-seq. G.W. and X.Z. contributed to the work. J.C. wrote the manuscript and D.P., J.W., J.L. and J.H. helped to improve it. J.C. conceived and supervised the study.

Corresponding author

Correspondence to Jiekai Chen.

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Peer review information Nature thanks Miguel Branco and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 YTHDC1 depletion upregulates 2C-like genes and impairs proliferation of mouse ES cells.

a, PCR-based genotyping assay validates the generation of the Ythdc1-KO cell line. Genotyping assay was repeated at least twice with similar results. b, Upper: schematic diagram depicts the timeline of DMSO or 4OHT treatment. Lower: growth curves of mouse ES cells after Ythdc1-KO. Data are mean ± s.d. (n = 6 independent wells pooled from two independent experiments). c, Phase-contrast images of WT and Ythdc1 cKO mouse ES cells showing the viability of cell colonies. Micrographs was repeated at least three times with similar results. Scale bar, 200 μm. d, The ratio of annexin V positive cells after loss of Ythdc1 (n = 2 independent experiments). e, Expression changes of the Ythdc1-KO upregulated genes across indicated embryonic developmental stages (left), or between MERVL+/MERVL cells in WT mouse ES cells (right). P from one-sided Mann–Whitney U test. Boxplots denote the medians and the interquartile ranges (IQR). The whiskers of a boxplot are the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile. Data were from n  ≥ 2 independent experiments. f, Scatter plot for the differentiate expressed genes in Ythdc1 cKO and MERVL+/MERVLWT mouse ES cells. g, RNA-seq data showing the expression changes of indicated genes and MERVL-int. h, NANOG immunostaining in the Ythdc1-cKO mouse ES cells. Micrographs was repeated at least twice with similar results. Scale bar, 50 μm. i, Flow cytometry analysis showing the percentage of MERVL::tdTomato (x-axis) positive cells after loss of Ythdc1. j, Western blot validation of YTHDC1 and its mutants in the rescued assays. Immunoblotting was repeated at least twice with similar results. k, Fluorescence images display the cells by staining with EdU and DAPI. Micrographs were repeated at least twice with similar results. Scale bar, 50 μm. l, The percentage of Edu+/DAPI cells in MERVL+ and MERVL cells upon Ythdc1-KO. Number of counted cells are labelled on the top of the bar (n = 2 independent experiments). m, The percentage of phases/DAPI in WT and Ythdc1-KO cells. Numbers of counted cells are labelled on the top of the bar (n = 2 independent experiments). n, Phase-contrast images present the rescue effects of WT or mutant Ythdc1 overexpression (OE) upon Ythdc1-KO. Micrographs were repeated at least three times with similar results. Scale bar, 200 μm. o, Cell viability of mouse ES cells in n. Data are mean ± s.d. of three independent experiments. P values from two-sided Student’s t-test.

Extended Data Fig. 2 Ythdc1-KO cells incorporate into trophectoderm.

a, Fluorescence images of blastocysts that have been injected with the indicated mouse ES cells at the 8-cell state. Micrographs were repeated at least twice with similar results. b, A column graph showing the percentages of chimeric embryos with injected ES cells incorporated into ICM or trophectoderm. Two-sided Fisher’s exact test. c, Same as Fig. 1j, the E4.5 blastocysts developing from morula injected with Ythdc1-KO ES cells were stained for mCherry and CDX2. DAPI stains the nucleus. Micrographs were repeated at least twice with similar results. Scar bar, 20 μm. d, RT–qPCR detects the expression of Ythdc1 upon siRNA treatment, with siNC as the negative control (n = 2 independent experiments). e, A column graph quantifies the percentages of MERVL–tdTomato positive cells upon Ythdc1 knockout or knockdown. The data are presented as the mean ± s.d.; measurements from n = 3 independent experiments. P values determined by two-sided Student’s t-test. f, Whole-mount fluorescence imaging of representative 6.5 dpc ES cell chimeric embryos upon siYthdc1 or siNC. Fluorescence microscope (left) and confocal microscopy images (right)of siNC/siYthdc1 ES cells (mCherry+) in the ELF5-expressing ExE in the 6.5 dpc chimeras (ELF5 marks diploid trophoblast)52,53. DAPI stains the nucleus. Arrows point to mCherry+ cells in the ExE. Micrographs were repeated at least twice with similar results. g, A column graph quantifies the percentage of 6.5 dpc ES cell chimeric embryos upon siYthdc1 or siNC. P value was from two-sided Fisher’s exact test.

Extended Data Fig. 3 YTHDC1 protein binds to TE RNAs.

a, Western blot detection of the enrichment efficiency of Halo-tagged proteins in the RIP experiments. Immunoblotting was repeated at least twice with similar results. b, Normalized distribution of m6A peaks across 5′ UTR, coding sequence (CDS), and 3′UTR of mRNAs for peaks common from two biological replicates. c, Consensus sequence motif identified after analysis of common m6A peaks from two replicates. d, Pair-wise Pearson correlation for the m6A signal between different m6A RIP-seq datasets. e, Selected genomic views of m6A RIP-seq data for the indicated genes and TEs. f, Distribution of m6A signal density across intact L1Md_T elements. g, YTHDC1 RIP-seq motif was measured with one-tailed Fisher’s exact test. h, Genomic views of m6A and YTHDC1 RIP-seq data for Neat1. i, The genomic distribution of YTHDC1 RIP-seq peaks and input control. j, The m6A-marked TEs from 5 independent studies. k, The overlap between m6A-marked and YTHDC1-bound TE RNAs. l, Selected genomic views of m6A and YTHDC1 RIP-seq data for the indicated TEs with all mapped reads (All reads) or only unique mapped reads (Unique reads). m, H3K9me3-level changes for the Setdb1-dependent and -independent H3K9me3 regions after loss of Setdb1 (upper, n = 1 experiment) or Ythdc1 (lower, n = 2 independent experiments). Boxplots denote the medians and the interquartile ranges (IQR). The whiskers of a boxplot are the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile. Data were from n  ≥ 2 independent experiments. P from two-sided Student’s t-test. n, Same as panel m, but for different TEs. o, Analysis of newly transcribed IAPEz-int, L1Md_Gf and Zscan4b RNA at the indicated time points after DRB removal (n = 2 independent experiments). p, Western blot analysis of the expression of YTHDC1, METTL3 and SETDB1 in Ythdc1 cKO mouse ES cells treated with 4OHT. Immunoblotting was repeated at least twice with similar results. q, Boxplot shows the SETDB1 binding strength on IAPEz-int elements after loss of Ythdc1, n = 1 experiment. P value was from two-sided Student’s t-test.

Extended Data Fig. 4 YTHDC1 binds to H3K9me3-marked TEs chromatin.

a, Heat maps illustrating the density of YTHDC1 ChIP-seq reads upon Ythdc1 depletion. b, Read count tag density pileups of YTHDC1 ChIP-seq upon Ythdc1 depletion. c, Selected genomic views of YTHDC1 and H3K9me3 ChIP-seq data for the indicated TEs/genes with all mapped reads (All reads) or only unique mapped reads (Unique reads). d, A boxplot showing the expression of TEs with or without H3K9me3/YTHDC1 upon Ythdc1 depletion. Statistics were determined by one-sided Mann–Whitney U test. Data were from n = 2 independent experiments e, Left, RNA-seq showing the change in expression of TEs upon Ythdc1 depletion. TEs were ranked from upregulated to downregulated. Right, a moving-window average plot of the density of H3K9me3 and YTHDC1 binding. f, Pie charts showing the genomic distribution of the peaks identified by IAP and LINE1 ChIRP-seq. g, Read count tag density pileups of GRID-seq signal enriched on indicated TEs.

Extended Data Fig. 5 Mettl3-dependent m6A modification on H3K9me3-marked TEs.

a, Top, a schematic diagram showing the strategy of Mettl3 KO. Bottom, western blot validates the generation of the Mettl3-KO cell line. Immunoblotting was repeated at least three times with similar results. b, Metagene profiles of m6A signal along transcripts in two replicates for WT and Mettl3 KO mouse ES cells. c, Scatter plots showing the m6A level of m6A peak regions in WT and Mettl3 KO mouse ES cells. Hypermethylated peaks (green) and hypomethylated peaks (red) with m6A upon Mettl3 KO are shown. d, The overlap of m6A decreased TEs after Mettl3-KO from 4 independent studies. e, The m6A changes for TEs upon Mettl3 depletion from 4 independent studies. f, The m6A signal changes upon loss of Mettl3 in different studies for indicated TEs. Boxplots denote the medians and the interquartile ranges (IQR). The whiskers of a boxplot are the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile. P values were from two-sided Student’s t-test. Data were from n  ≥ 2 independent experiments. g, Read count tag density pileups of m6A RIP-seq reads for indicated TEs upon Mettl3 depletion from two independent studies. h, The overlap of Ythdc1 and Mettl3-dependent H3K9me3 marked TEs. P value is from one-tailed Fisher’s exact test. i, H3K9me3 level changes for Setdb1-dependent and -independent regions from Fig. 2e after Mettl3-KO (n = 2). P values are from two-sided Student’s t-test. Boxplots denote the medians and the interquartile ranges (IQR). The whiskers of a boxplot are the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile. j, Tag density pileups of H3K9me3 ChIP-seq reads for indicated TEs upon Mettl3 depletion. k, Same as panel i, but for different TEs. l, Same as Fig. 3i, but only the unique mapped reads were kept. m, Tag density pileups of IAP (left) and LINE1 (right) ChIRP for indicated TEs upon Ythdc1-KO. n, Same as panel m, but for Mettl3-KO.

Extended Data Fig. 6 Dux-KO does not impair YTHDC1-H3K9me3-mediated TE repression.

a, Heat map showing the expression of indicated genes upon Mettl3 depletion. b, Genome browser plot showing the GRID-seq signal in Dux loci. c, Top, a schematic diagram showing the strategy of Dux knockout. Bottom, PCR-based genotyping assay validates the generation of the Dux-KO cell line at the genome. Genotyping assay was repeated at least twice with similar results. d, RT–qPCR showing the expression of select genes/TEs upon Dux depletion in Ythdc1 cKO mouse ES cells treated with DMSO or 4OHT. Data are mean ± s.d. of three independent experiments. e, Read count tag density pileups of H3K9me3 ChIP-seq (from two replicates) reads for indicated TEs in Dux KO cells upon Ythdc1 depletion. f, Volcano plot showing the differential expressed genes after loss of Ythdc1 (left) or Mettel3 (right). g, GO analysis of the genes from panel f. h, Heat map showing the expression change of indicated genes after loss of Ythdc1 or Mettl3.

Supplementary information

Supplementary Figures

This file contains Supplementary Figure 1: Uncropped images of Western blot and PCR based genotyping gels; and Supplementary Figure 2: high-resolution images for whole-mount fluorescence imaging related to Extended Data Fig. 2f.

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Supplementary Table

Q-PCR primers, ChIRP probes and antibodies used in this study.

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Liu, J., Gao, M., He, J. et al. The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity. Nature 591, 322–326 (2021). https://doi.org/10.1038/s41586-021-03313-9

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