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METTL3 regulates heterochromatin in mouse embryonic stem cells

Abstract

METTL3 (methyltransferase-like 3) mediates the N6-methyladenosine (m6A) methylation of mRNA, which affects the stability of mRNA and its translation into protein1. METTL3 also binds chromatin2,3,4, but the role of METTL3 and m6A methylation in chromatin is not fully understood. Here we show that METTL3 regulates mouse embryonic stem-cell heterochromatin, the integrity of which is critical for silencing retroviral elements and for mammalian development5. METTL3 predominantly localizes to the intracisternal A particle (IAP)-type family of endogenous retroviruses. Knockout of Mettl3 impairs the deposition of multiple heterochromatin marks onto METTL3-targeted IAPs, and upregulates IAP transcription, suggesting that METTL3 is important for the integrity of IAP heterochromatin. We provide further evidence that RNA transcripts derived from METTL3-bound IAPs are associated with chromatin and are m6A-methylated. These m6A-marked transcripts are bound by the m6A reader YTHDC1, which interacts with METTL3 and in turn promotes the association of METTL3 with chromatin. METTL3 also interacts physically with the histone 3 lysine 9 (H3K9) tri-methyltransferase SETDB1 and its cofactor TRIM28, and is important for their localization to IAPs. Our findings demonstrate that METTL3-catalysed m6A modification of RNA is important for the integrity of IAP heterochromatin in mouse embryonic stem cells, revealing a mechanism of heterochromatin regulation in mammals.

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Fig. 1: METTL3 binds endogenous retroviral elements.
Fig. 2: METTL3 is required for heterochromatin formation over repetitive elements.
Fig. 3: METTL3 regulates SETDB1 and TRIM28 localization to IAPEz elements.
Fig. 4: YTHDC1 recruited by METTL3-dependent m6A contributes to METTL3 binding and heterochromatin formation on IAPEz-int.

Data availability

The next-generation sequencing data generated by this study have been deposited to the GEO database under accession number GSE126243Source data are provided with this paper.

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Acknowledgements

We thank D. Moazed and E. Sendinc for critical reading of the manuscript and suggestions; J. Chen for suggestions and for sharing unpublished results; and Y. Shinkai for sharing the Setdb1 cKO cell line. H.S. was supported by the Shanghai Rising-Star Program (19QA1401300), National Science Foundation of China (81874157, 32070649, 31601060) and innovative research team of high-level local university in Shanghai. W.X. was supported by the National Science Foundation of China (31900469). Y.S. is an American Cancer Society Research Professor.

Author information

Affiliations

Authors

Contributions

W.X. and H.S. carried out most of the experiments and bioinformatics analyses. J.L. carried out YTHDC1W429A identification and genotyping. C.H. carried out LC–MS/MS analysis of m6A. J. Wen and J.D. provided discussions and advice on co-immunoprecipitation. L.T. provided discussions and advice on mES cell culture. L.W., J. Wang and B.R. provided discussions and advice on ChIP. H.M. and F.W. provided discussions and advice on m6A RIP and bioinformatics analyses, respectively. W.X., H.S. and Y.S. conceived the project and co-wrote the manuscript. H.S and Y.S. directed all the experiments with input from Y.G.S.

Corresponding authors

Correspondence to Yang Shi or Hongjie Shen.

Ethics declarations

Competing interests

Y.S. is a co-founder of and holds equity in Constellation Pharmaceuticals, Inc. and Athelas Therapeutics, Inc. Y.S. also holds equity in Imago Biosciences and is a consultant for Active Motif, Inc. The other authors declare no competing interests.

Additional information

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

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 METTL3 binds endogenous retroviral elements.

a, Accumulative plots showing the relative distances between peaks of METTL3 and histone modifications. b, Bar graph showing the overlapping ratios (calculated as Jaccard statistics, see Methods) of METTL3 peaks with repetitive elements. c, Accumulative plots showing the relative distances between METTL3 peaks and repetitive elements. d, Heat maps showing enrichment of METTL3, H3K9me3 and H4K20me3 on repetitive elements. e, Scatter plots showing the correlation between the enrichment levels of METTL3 and H3K9me3 (left) or H4K20me3 (right) on different subtypes (n = 277) of ERVK retrotransposons. Two-sided Pearson’s correlation test. f, Schematic representation of stitching of adjacent IAPEz fragments. g, Aggregation plots showing METTL3, H3K9me3, H4K20me3 and input patterns on IAPEz-int. h, Validation of METTL3 binding on IAPEz using three independent METTL3 antibodies. The mean of three biological replicates ± s.d. is shown. i, Schematic representation of uniquely mapped reads and randomly mapped non-unique reads. j, Aggregation plots and heat maps showing enrichment of METTL3, H3K9me3 and H4K20me3 on IAPEz-int elements with only uniquely mapped reads or uniquely + randomly mapped reads. Uniquely mapped ChIP–seq reads were used in ac, e. Uniquely and randomly mapped ChIP–seq reads were used in d, g. Heat maps were ranked according to METTL3 enrichment in parental cells in descending order in j. MTA and MaSat used in ChIP–qPCR are examples of repetitive elements not bound by METTL3.

Extended Data Fig. 2 METTL3 is required for heterochromatin formation.

a, Western blots showing METTL3 protein levels in parental, Mettl3 KO and rescued cell lines with METTL3WT or METTL3APPA. b, Bar plots showing m6A/A ratio on polyA RNAs in parental, Mettl3 KO and rescued cell lines with METTL3WT or METTL3APPA. The mean of three biological replicates ± s.d. is shown. c, ChIP–qPCR showing binding patterns of H3K9me3 (left) and H4K20me3 (right) on IAPEz-int elements in parental, Mettl3 KO and rescued cell lines with METTL3WT or METTL3APPA. The mean of three biological replicates ± s.d. is shown for ChIP–qPCR. *P < 0.05, **P < 0.01; two-sided t-test. Exact P values are provided in the Source Data. d, Box plots showing enrichment levels of H3K9me3 (left) and H4K20me3 (right) on IAPEz-int elements (n = 2,542) in parental, Mettl3 KO and rescued cell lines with wild-type or catalytically mutated METTL3. ****P < 0.0001 (exact P values from left to right: 0, 0, 0, 1.1 × 10−118, 6.2 × 10−269, 0), two-sided paired t-test. e, Aggregation plots showing enrichment levels of H3K9me3 (left) and H4K20me3 (right) on IAPEz-int elements in parental, Mettl3 KO and rescued cell lines. fh, Heat maps (f), box plots (g) and aggregation plots (h) showing enrichment levels of H3K9me3 on IAPEz-int elements (n = 2,542) in parental and Alkbh5 KO cell lines. = 0.00014, two-sided paired t-test. i, Western blots showing ALKBH5 protein levels in parental and Alkbh5 KO cell lines. j, k, Box plot showing density fold changes (log2[Mettl3 KO/parental]) of H3K9me3 (j) and H4K20me3 (k) on different types of repetitive elements upon Mettl3 KO. Uniquely mapped ChIP–seq reads were used in d, f, g, j, k. Uniquely + randomly mapped ChIP–seq reads were used in e, h. Heat maps were ranked according to METTL3 density in parental cells in descending order in f. For the box plots in d, g, j, k, the middle line and lower and upper hinges of the box plot correspond to the median and the first and third quartiles, respectively. The whiskers extend from the hinges to no further than 1.5 × IQR from the hinge. Outlying points are plotted individually. Blots are representative of two independent experiments in a, i. For blot source data, see Supplementary Fig. 1. MTA and MaSat used in ChIP–qPCR are examples of repetitive elements not bound by METTL3. Source data

Extended Data Fig. 3 METTL3 is required for heterochromatin formation.

a, Box plots showing enrichment levels of H3.3 on IAPEz-int elements (n = 2,542) in parental, Mettl3 KO and rescued cell lines with METTL3WT or METTL3APPA. ****P < 0.0001 (exact P values from left to right: 4.2 × 10−218, 2.7 × 10−274, 3.9 × 10−294), two-sided paired t-test. b, Box plots showing CpG methylation ratios on IAPEz-int elements (n = 2,542) in parental, Mettl3 KO and rescued cell lines with with METTL3WT or METTL3APPA. ****P < 0.0001 (exact P values from left to right: 4.87 × 10−111, 1.5 × 10−150, 0), two-sided paired t-test. c, Box plot showing density fold changes (log2[Mettl3 KO/parental]) of H3.3 on different types of repetitive elements upon Mettl3 KO. d, Box plot showing CpG methylation changes on different types of repetitive elements upon Mettl3 KO. Only elements with at least ten covered CpGs were used. e, RT–qPCR showing RNA levels of IAPEz-int in parental, Mettl3 KO and rescued cell lines with METTL3WT or METTL3APPA. The mean of three replicates ± s.d. is shown. *P < 0.05, **P < 0.01, two-sided t-test. Exact P values are provided in the Source Data. f, Box plot showing density fold changes (log2[Mettl3 KO/parental]) of RNAs of different types of repetitive elements upon Mettl3 KO. gj, Scatter plots showing correlation between METTL3 (g), H3K9me3 (h), H4K20me3 (i), DNA methylation (j) and RNA expression level on IAPEz-int (n = 2,542). Two-sided Pearson’s correlation test. k, Box plot showing RNA levels of IAPEz-int (n = 2,542) in parental, Mettl3 KO and Ythdf1/2/3 KO cell lines revealed by PolyA RNA-seq (GSE147849). ****P < 0.0001 (exact P values from left to right: 1.1 × 10−52, 7.3 × 10−37), two-sided paired t-test. Uniquely mapped ChIP–seq reads were used in ad, fk. For the box plots in ad, f and k, the middle line and lower and upper hinges of the box plot correspond to the median and the first and third quartiles, respectively. The whiskers extend from the hinges to no further than 1.5 × IQR from the hinge. Outlying points are plotted individually. Source data

Extended Data Fig. 4 METTL3 chromatin binding is dependent on its own catalytic activity.

a, ChIP–qPCR showing binding patterns of METTL3 on IAPEz-int elements in parental, Mettl3 KO and rescued cell lines with METTL3WT or METTL3APPA. The mean of three biological replicates ± s.d. is shown. *P < 0.05, **P < 0.01, two-sided t-test. Exact P values are provided in the Source Data. b, Aggregation plots showing METTL3 enrichment levels on IAPEz-int in parental, Mettl3 KO and rescued cell lines with METTL3WT or METTL3APPA. c, Western blot showing interactions of METTL14 with reintroduced METTL3 (METTL3WT or METTL3APPA) in Mettl3 KO cells. d, Aggregation plots showing METTL3 enrichment levels on IAPEz-int in Mettl3 KO rescued cells with METTL3WT, METTL3W475A or METTL3N477A. e, Western blot showing METTL3 protein levels in parental, Mettl3 KO and rescued cell lines with METTL3WT, METTL3APPA, METTL3W475A or METTL3N477A. f, Aggregation plots showing METTL3 enrichment levels on IAPEz-int in parental and Mettl14 KO cell lines. g, Western blots showing METTL14 protein levels in parental and Mettl14 KO cell lines. h, Aggregation plots showing METTL3 enrichment levels on IAPEz-int in parental and Rbm15/15b DKO cell lines. i, Western blots showing RBM15 and RBM15B protein levels in parental and Rbm15/15b DKO cell lines. j, Aggregation plots showing METTL3 enrichment levels on IAPEz-int in control and m6A methyltransferase complex components KD cell lines. k, Western blots showing protein levels of m6A methyltransferase complex components in control and KD cell lines. l, A cartoon illustrating the dCas9–METTL3 tethering assay in Mettl3 KO cell lines. m, Western blot showing Cas9 and METTL3 protein levels upon Dox treatment. n, ChIP–qPCR of Cas9 (left) and H3K9me3 (right) on IAPEz and control regions. The mean of three biological replicates ± s.d. is shown. *P < 0.05, **P < 0.01, two-sided t-test. Exact P values are provided in the Source Data. Uniquely + randomly mapped ChIP–seq reads were used in b, d, f, h, j. Blots are representative of two independent experiments in c, e, g, i, k, m. For blots source data, see Supplementary Fig. 1. MTA and MaSat used in ChIP–qPCR are examples of repetitive elements not bound by METTL3. Source data

Extended Data Fig. 5 METTL3 regulates SETDB1–TRIM28 recruitment.

a, Venn diagram showing overlaps of the IAPEz elements bound by METTL3, SETDB1 and TRIM28. b, Scatter plots showing correlation of METTL3 and SETDB1 (left) or TRIM28 (right) on IAPEz-int elements (n = 2,542). Two-sided Pearson’s correlation test. c, Box plots showing SETDB1 (left) and TRIM28 (right) enrichment levels on IAPEz-int elements in parental and Mettl3 KO cell lines. P = 0 (left), P = 0 (right), two-sided paired t-test. d, ChIP–qPCR showing binding patterns of SETDB1 (left) and TRIM28 (right) on IAPEz-int elements in parental and Mettl3 KO cells. The mean of three biological replicates ± s.d. is shown. *P < 0.05, **P < 0.01, two-sided t-test. Exact P values are provided in the Source Data. e, f, Box plots showing density fold changes (log2[Mettl3 KO/parental]) of SETDB1 (e) and TRIM28 (f) on different types of repetitive elements upon Mettl3 KO. g, Co-immunoprecipitation-coupled western blot showing interactions of SETDB1 (left) and TRIM28 (right) with reintroduced METTL3 (wild-type or catalytically mutated) in Mettl3 KO cells with or without triptolide treatment. Uniquely mapped ChIP–seq reads were used in b, c, e, f. For the box plots in c, e and f, the middle line and lower and upper hinges of the box plot correspond to the median and the first and third quartiles, respectively. The whiskers extend from the hinges to no further than 1.5 × IQR from the hinge. Outlying points are plotted individually. Blots are representative of two independent experiments in g. For blots source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 6 m6A exists on IAPEz-int transcripts.

a, RT–qPCR showing relative levels of IAPEz and control RNAs including Actb and Gapdh in different subcellular populations. The mean of three biological replicates ± s.d. is shown. b, Aggregation plot showing IAPEz-int ChIRP signals enriched on the IAPEz-int elements in the genome. c, Aggregation plot showing in situ ligated DNA of IAPEz-int transcripts revealed by GRID–seq (GSE82312) enriched on the IAPEz-int elements in the genome. d, Consensus motif of m6A enriched sites (chromatin ribominus RNA). e, Aggregation plot showing the average enrichment levels of m6A (log2[m6A/input]) over coding genes in parental and Mettl3 KO cell lines (chromatin ribominus RNA). f, UCSC snapshots showing m6A enrichment at the 3′ end of coding genes, which is depleted in Mettl3 KO cell lines. g, Western blots showing SETDB1 protein levels in parental and Setdb1 CKO cell lines. h, UCSC snapshots showing m6A enrichment at the 5′ end of IAPEz-int, which is depleted in Mettl3 KO cells. i, In vitro methyltransferase activity of METTL3–METTL14 with 20-nucleotide RNA substrates containing four repeats of the consensus sequence. The mean of three biological replicates ± s.d. is shown. Uniquely mapped MeRIP–seq reads were used in df, h. Uniquely + randomly mapped ChIRP–seq reads and GRID–seq reads were used in b, c. Blots are representative of two independent experiments in g. For blots source data, see Supplementary Fig. 1.

Extended Data Fig. 7 Recruitment of YTHDC1 to IAPEz chromatin depends on its m6A recognition ability.

a, ChIP–qPCR showing enrichment levels of nuclear-localized m6A reader proteins. The mean of three biological replicates ± s.d. is shown. b, Venn diagram showing overlaps between METTL3 and YTHDC1 binding events. c, Scatter plot showing correlation of METTL3 and YTHDC1 on IAPEz-int elements. d, e, UCSC snapshot (d) and ChIP–qPCR (e) showing YTHDC1 enrichment on IAPEz-int elements in parental, Mettl3 KO and rescued cell lines with wild-type or catalytically mutated METTL3. The mean of three biological replicates ± s.d. is shown. *P < 0.05, **P < 0.01, two-sided t-test. Exact P values are provided in the Source Data. f, Construction of YTHDC1WT, Ythdc1 KO, and YTHDC1W429A cell lines using an auxin-inducible degron (AID) system. g, Western blots showing IAA-induced rapid degradation of AID-YTHDC1. hk, Heat maps (h), UCSC snapshots (i), box plots (j) and aggregation plots (k) showing YTHDC1 levels on IAPEz-int in YTHDC1WT, Ythdc1 KO, and YTHDC1W429A cell lines. ****P < 0.0001. Exact P values from left to right: 0, 0; two-sided paired t-test. l, ChIP–qPCR showing YTHDC1 enrichment levels on IAPEz-int elements in control mES cells and mES cells treated with α-amanitin, flavopiridol and triptolide. The mean of three biological replicates ± s.d. is shown. * P < 0.05, ** P < 0.01; two-sided t-test. Exact P values are provided in the Source Data. Uniquely mapped ChIP–seq reads were used in bd, hj. Uniquely + randomly mapped ChIP–seq reads were used in k. Heat maps were ranked according to METTL3 density in parental cells in descending order in h. For the box plots in j, the middle line and lower and upper hinges of the box plot correspond to the median and the first and third quartiles, respectively. The whiskers extend from the hinges to no further than 1.5 × IQR from the hinge. Outlying points are plotted individually. Blots are representative of two independent experiments in g. For blots source data, see Supplementary Fig. 1. MTA and MaSat used in ChIP–qPCR are examples of repetitive elements not bound by METTL3. Source data

Extended Data Fig. 8 YTHDC1 stabilizes METTL3 on heterochromatin.

ac, Box plots (a), aggregation plots (b), and UCSC snapshot (c) showing METTL3 levels on IAPEz-int in YTHDC1WT, Ythdc1 KO and YTHDC1W429A cell lines. ****P < 0.0001 (exact P values from left to right: 2.3 × 10−176, 1.2 × 10−195), two-sided paired t-test. df, Box plots (d), aggregation plots (e), and UCSC snapshot (f) showing H3K9me3 levels on IAPEz-int in YTHDC1WT, Ythdc1 KO and YTHDC1W429A cell lines. ****P < 0.0001 (exact P values from left to right: 7.1 × 10−63, 4.8 × 10−279), two-sided paired t-test. gj, Heat maps (g), box plots (h), aggregation plots (i), and UCSC snapshots (j) showing H4K20me3 levels on IAPEz-int in YTHDC1WT, Ythdc1 KO and YTHDC1W429A cell lines. ****P < 0.0001 (exact P values from left to right: 8.8 × 10−30, 7.8 × 10−114), two-sided paired t-test. k, Western blots showing protein levels of METTL3 and YTHDC1 in control, Mettl3 KO, Ythdc1 KD and Mettl3 KO + Ythdc1 KD cell lines. l, ChIP–qPCR showing H3K9me3 enrichment level on IAPEz elements in control, Mettl3 KO, Ythdc1 KD and Mettl3 KO + Ythdc1 KD cell lines. *P < 0.05, **P < 0.01; two-sided t-test. Exact P values are provided in the Source Data. m, Western blots showing Cas9 protein levels upon Dox treatment in Mettl3 KO cell lines. n, ChIP–qPCR of H3K9me3 (left) and Cas9 (right) on IAP and control regions in Mettl3 KO cell lines expressing dCas9–YTHDC1. The mean of three biological replicates ± s.d. is shown. *P < 0.05, **P < 0.01; two-sided t-test. Exact P values are provided in the Source Data. o, Western blots showing Cas9 and METTL3 protein levels upon Dox treatment in Mettl3 KO + METTL3APPA cell lines. p, ChIP–qPCR of H3K9me3 (left) and Cas9 (right) on IAP and control in Mettl3 KO + METTL3APPA cell lines expressing dCas9–YTHDC1. The mean of three biological replicates ± s.d. is shown. Exact P values are provided in the Source Data. q, Co-immunoprecipitation coupled with western blots showing interactions of YTHDC1 with reintroduced METTL3 (wild-type or catalytically mutated) in Mettl3 KO cells with or without triptolide treatment. Uniquely mapped ChIP–seq reads were used in a, c, d, fh, j. Uniquely + randomly mapped ChIP–seq reads were used in b, e, i. Heat maps were ranked according to METTL3 density in parental cells in descending order in g. For the box plots in a, d, h, the middle line and lower and upper hinges of the box plot correspond to the median and the first and third quartiles, respectively. The whiskers extend from the hinges to no further than 1.5 × IQR from the hinge. Outlying points are plotted individually. Blots are representative of two independent experiments in k, mo, q. For blots source data, see Supplementary Fig. 1. MTA and MaSat used in ChIP–qPCR are examples of repetitive elements not bound by METTL3. Source data

Extended Data Fig. 9 SETDB1 regulates METTL3–YTHDC1 recruitment.

ad, Heat maps (a), UCSC snapshot (b), box plot (c) and aggregation plots (d) showing METTL3 enrichment levels on IAPEz-int in parental and Setdb1 CKO cells. P = 1.3 × 10−48, two-sided paired t-test. eh, Heat maps (e), UCSC snapshot (f), box plot (g) and aggregation plots (h) showing YTHDC1 enrichment levels on IAPEz-int in parental and Setdb1 CKO cells. P = 5.0 × 10−100, two-sided paired t-test. Uniquely mapped ChIP–seq reads were used in ac, eg. Uniquely + randomly mapped ChIP–seq reads were used in d, h. Heat maps were ranked according to METTL3 density in parental cells in descending order in a, e. For the box plots in c, g, the middle line and lower and upper hinges of the box plot correspond to the median and the first and third quartiles, respectively. The whiskers extend from the hinges to no further than 1.5 × IQR from the hinge. Outlying points are plotted individually.

Extended Data Fig. 10 RNA-dependent heterochromatin formation models.

a, RNA-dependent heterochromatin formation on IAPEz-int in mES cells. Specifically, METTL3 and other m6A methyltransferase components methylate IAPEz transcripts, which are recognized by the m6A reader protein YTHDC1. YTHDC1 in turn stabilizes METTL3 binding, possibly through protein–protein interactions. Chromatin-associated METTL3 enhances SETDB1–TRIM28 binding, which in turn stabilizes METTL3 recruitment. b, RNA-dependent heterochromatin formation on centromere regions in S. pombe. Specifically, heterochromatin generation over centromere regions is initiated by the base-paring recognition and binding of the RITS complex to RNAs transcribed from these regions, which in turn enhances sRNA generation through recruitment of RDRC. RITS then recruits CLRC to catalyse H3K9 methylation, which in turn promotes RITS binding. c, RNA-dependent heterochromatin formation on DSR genes in S. pombe. Specifically, Mmi1 protein recognizes the DSR consensus motif on RNAs transcribed from these genes and then recruits the H3K9 methyltransferase Clr4 through Red1.

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This file contains a Supplementary Discussion and Supplementary Fig. 1. Supplementary Fig. 1 contains all uncropped immunoblots shown in the main and Extended Data figs, dashed black boxes indicate cropped regions.

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Xu, W., Li, J., He, C. et al. METTL3 regulates heterochromatin in mouse embryonic stem cells. Nature 591, 317–321 (2021). https://doi.org/10.1038/s41586-021-03210-1

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