METTL14 is a chromatin regulator independent of its RNA N⁶-methyladenosine methyltransferase activity

Abstract

METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m⁶A methyltransferase complex (MTC) that installs m⁶A. Surprisingly, depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell (mESC). While comparable global hypo-methylation in RNA m⁶A was observed in Mettl3 or Mettl14 knockout mESCs, respectively. Mettl14 knockout led to a globally decreased nascent RNA synthesis, whereas Mettl3 depletion resulted in transcription upregulation, suggesting that METTL14 might possess an m⁶A-independent role in gene regulation. We found that METTL14 colocalizes with the repressive H3K27me3 modification. Mechanistically, METTL14, but not METTL3, binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3. Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression. The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs. This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m⁶A, and critically impacts transcriptional regulation, stemness maintenance, and differentiation of mESCs.

Keywords

METTL14 / chromatin / H3K27me3 / m⁶A-independent / mESC differentiation

References

[1] Amemiya, HM, Kundaje A, Boyle AP. The ENCODE Blacklist: identification of problematic regions of the genome. Sci Rep 2019;9:9354.

[2] Anders, S, Pyl T, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 2015;31:166–169.

[3] Andersson-Rolf A, Mustata RC, Merenda A et al. One-step generation of conditional and reversible gene knockouts. Nat Methods 2017;14:287–289.

[4] Barbieri I, Tzelepis K, Pandolfini L et al. Promoter-bound METTL3 maintains myeloid leukaemia by m⁶A-dependent translation control. Nature 2017;552:126–131.

[5] Bolger, AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014;30:2114–2120.

[6] Cui Q, Shi H, Ye P et al. m⁶A RNA methylation regulates the self-renewal and tumorigenesis of glioblastoma stem cells. Cell Rep 2017;18:2622–2634.

[7] Dominissini D, Moshitch-Moshkovitz S, Schwartz S et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 2012;485(7397):201–206.

[8] Frye M, Harada BT, Behm M et al. RNA modifications modulate gene expression during development. Science 2018;361:1346–1349.

[9] He PC, He C. m⁶A RNA methylation: from mechanisms to therapeutic potential. EMBO J 2021;40:e105977.

[10] Heinz S, Benner C, Spann N et al. Simple combinations of line-age-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell 2010;38:576–589.

[11] Huang H, Weng H, Zhou K et al. Histone H3 trimethylation at lysine 36 guides m⁶A RNA modification co-transcriptionally. Nature 2019;567:414–419.

[12] Jia G, Fu Y, Zhao X et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol 2011;7:885–887.

[13] Kim, D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 2015;12:357–360.

[14] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012;9:357–359.

[15] Li H, Handsaker B, Wysoker A et al. The sequence alignment/map format and SAMtools. Bioinformatics 2009;25:2078–2079.

[16] Lin S, Choe J, Du P et al. The m⁶A Methyltransferase METTL3 promotes translation in human cancer cells. Mol Cell 2016;62:335–345.

[17] Lin Z, Hsu PJ, Xing X et al. Mettl3-/Mettl14-mediated mRNA N6-methyladenosine modulates murine spermatogenesis. Cell Res 2017;27:1216–1230.

[18] Liu J, Yue Y, Han D et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol 2014;10:93–95.

[19] Liu J, Eckert MA, Harada BT et al. m⁶A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer. Nat Cell Biol 2018;20:1074–1083.

[20] Liu J, Dou X, Chen C et al. N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science 2020;367:580–586.

[21] Liu J, Gao M, He J et al. The RNA m⁶A reader YTHDC1 silences retrotransposons and guards ES cell identity. Nature 2021a;591:322–326.

[22] Liu P, Li F, Lin J et al. m⁶A-independent genome-wide METTL3 and METTL14 redistribution drives the senescence-associated secretory phenotype. Nat Cell Biol 2021b;23:355–365.

[23] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014;15:550.

[24] Patil DP, Chen CK, Pickering BF et al. m⁶A RNA methylation promotes XIST-mediated transcriptional repression. Nature 2016;537:369–373.

[25] Ping XL, Sun BF, Wang L et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res 2014;24:177–189.

[26] Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 2010;26:841–842.

[27] Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 2010;11:R25.

[28] Schneider CA, Rasband WS, Eliceiri KW. NIH image to ImageJ: 25 years of image analysis. Nat Methods 2012;9:671–675.

[29] Sergiev PV, Aleksashin NA, Chugunova AA et al. Structural and evolutionary insights into ribosomal RNA methylation. Nat Chem Biol 2018;14:226–235.

[30] Skene PJ, Henikoff JG, Henikoff S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc 2018;13:1006–1019.

[31] Sledz P, Jinek M. Structural insights into the molecular mechanism of the m⁶A writer complex. eLife 2016;5:e18434.

[32] Wang X, Lu Z, Gomez A et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 2014;505:117–120.

[33] Wang X, Zhao BS, Roundtree IA et al. N6-methyladenosine modulates messenger RNA translation efficiency. Cell 2015;161: 1388–1399.

[34] Wang P, Doxtader KA, Nam Y. Structural basis for cooperative function of Mettl3 and Mettl14 Methyltransferases. Mol Cell 2016a;63:306–317.

[35] Wang X, Feng J, Xue Y et al. Structural basis of N6-adenosine methylation by the METTL3-METTL14 complex. Nature 2016b;534:575–578.

[36] Wang Y, Li Y, Yue M et al. N6-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications. Nat Neurosci 2018;21:195–206.

[37] Wen J, Lv R, Ma H et al. Zc3h13 regulates nuclear RNA m⁶A methylation and mouse embryonic stem cell self-renewal. Mol Cell 2018;69:1028–1038.e6.

[38] Whyte WA, Orlando DA, Hnisz D et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 2013;153:307–319.

[39] Wiles ET, Selker EU. H3K27 methylation: a promiscuous repressive chromatin mark. Curr Opin Genet Dev 2017;43:31–37.

[40] Wu C, Chen W, He J et al. Interplay of m⁶A and H3K27 trimethylation restrains inflammation during bacterial infection. Sci Adv 2020;6:eaba0647.

[41] Wuarin J, Schibler U. Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing. Mol Cell Biol 1994;14:7219–7225.

[42] Xu W, Li J, He C et al. METTL3 regulates heterochromatin in mouse embryonic stem cells. Nature 2021;591:317–321.

[43] Yue Y, Liu J, Cui X et al. VIRMA mediates preferential m⁶A mRNA methylation in 3’UTR and near stop codon and associates with alternative polyadenylation. Cell Discov 2018;4:10.

[44] Zhang Y, Liu T, Meyer CA et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol 2008;9:R137.

[45] Zheng G, Dahl JA, Niu Y et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell 2013;49:18–29.

[46] Zhong S, Li H, Bodi Z et al. MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor. Plant Cell 2008;20:1278–1288.

[47] Zhou KI, Shi H, Lyu R et al. Regulation of Co-transcriptional PremRNA splicing by m⁶A through the low-complexity protein hnRNPG. Mol Cell 2019;76:70–81.e9.

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2023 The Author(s) 2023. Published by Oxford University Press on behalf of Higher Education Press.