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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.

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.

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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Liu, S. Shu, W. Pang and S. Xu for experimental assistance; Y. Shi and H. Shen for valuable suggestions and for sharing unpublished results; H. Chen, C.-H. Hsu, M. Min, L. Shen, J. Wang and Y. Yu for discussion and constructive suggestions; and the Guangzhou Branch of the Supercomputing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).

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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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