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m6A-independent genome-wide METTL3 and METTL14 redistribution drives the senescence-associated secretory phenotype

Nature Cell Biology volume 23, pages 355–365 (2021)Cite this article

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Abstract

Methyltransferase-like 3 (METTL3) and 14 (METTL14) are core subunits of the methyltransferase complex that catalyses messenger RNA N6-methyladenosine (m6A) modification. Despite the expanding list of m6A-dependent functions of the methyltransferase complex, the m6A-independent function of the METTL3 and METTL14 complex remains poorly understood. Here we show that genome-wide redistribution of METTL3 and METTL14 transcriptionally drives the senescence-associated secretory phenotype (SASP) in an m6A-independent manner. METTL14 is redistributed to the enhancers, whereas METTL3 is localized to the pre-existing NF-κB sites within the promoters of SASP genes during senescence. METTL3 and METTL14 are necessary for SASP. However, SASP is not regulated by m6A mRNA modification. METTL3 and METTL14 are required for both the tumour-promoting and immune-surveillance functions of senescent cells, which are mediated by SASP in vivo in mouse models. In summary, our results report an m6A-independent function of the METTL3 and METTL14 complex in transcriptionally promoting SASP during senescence.

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Fig. 1: METTL3 and METTL14 regulate SASP.
Fig. 2: SASP is not regulated by m6A.
Fig. 3: Genome-wide redistribution of METTL3 and METTL14.
Fig. 4: METTL3 is localized to the pre-existing NF-κB sites within the promoters of SASP genes.
Fig. 5: METTL14 regulates the SASP gene enhancers.
Fig. 6: METTL3 and METTL14 mediate the formation of the promoter-and-enhancer loop of the SASP genes.
Fig. 7: METTL3 and METTL14 are required for the pro-tumorigenic and immune surveillance function of the SASP.

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Data availability

Cut-and-run, ChIP–seq and RNA-seq data that support the findings of this study have been deposited in the GEO under the accession number GSE141944 (RNA-seq for METTL3 knockdown and rescue, GSE159551; RNA-seq for METTL14 knockdown and rescue, GSE141991; cut-and-run and ChIP–seq, GSE141992; m6A-seq, GSE141993; and carRNA m6A-seq, GSE159550). For the correlation analysis between METTL14 and the SASP genes in human laser capture and microdissected PanIN lesion samples, gene expression data were obtained from the GEO (under the accession code GSE43288). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

The software and algorithms for the data analyses used in this study are all well-established from previous work and are referenced throughout the manuscript.

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Acknowledgements

This work was supported by the US National Institutes of Health (grant nos R01CA160331, R01CA163377, R01CA202919, R01CA239128, R01CA243142, P01AG031862 and P50CA228991 to R.Z.; and R50CA211199 to A.V.K.); US Department of Defense (grant nos OC180109 and OC190181 to R.Z.); The Honorable Tina Brozman Foundation for Ovarian Cancer Research and The Tina Brozman Ovarian Cancer Research Consortium 2.0 (to R.Z.); and Ovarian Cancer Research Alliance (Collaborative Research Development Grant no. 596552 to R.Z., and Ann and Sol Schreiber Mentored Investigator Award grant no. 649658 to J.L.). Support of the Core Facilities was provided by Cancer Centre Support Grant (CCSG; grant no. CA010815 to The Wistar Institute.

Author information

Authors and Affiliations

  1. Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA, USA

    Pingyu Liu, Jianhuang Lin, Takeshi Fukumoto, Timothy Nacarelli, Xue Hao, Andrew V. Kossenkov & Rugang Zhang

  2. Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA

    Fuming Li & M. Celeste Simon

  3. Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    M. Celeste Simon

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Contributions

P.L., F.L., J.L., T.F., T.N. and X.H. performed the experiments and analysed data. A.V.K. performed the bioinformatic analyses. P.L. and R.Z. designed the experiments. F.L., J.L., T.F. and T.N. contributed to the study design. P.L., A.V.K. and R.Z. wrote the manuscript. M.C.S. and R.Z. supervised the study. R.Z. conceived the study.

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Correspondence to Rugang Zhang.

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Extended data

Extended Data Fig. 1 METTL3 and METTL14-dependent changes in transcriptome during senescence.

a, Schematic of experimental timeline using oncogenic-H-RASG12V to induce senescence in IMR90 cells. b-c, IMR90 cells were induced to senesce by oncogenic RAS expressing a non-targeting siRNA control (siControl) or METTL14-targeted siRNA (siMETTL14) with or without the rescue of ectopically expressed wild-type or the R298P mutant METTL14 were subjected to RNA-seq analysis. Ingenuity pathway enrichment analysis of genes altered by siMETTL14 (b) and rescued by both wild-type and the R298P mutant METTL14 (c) are shown. d-e, Heatmap of RNA-seq data with 2 replicates in each of the groups for the genes whose expression significantly changed by METTL3 knockdown and rescued by both wild-type and the D394A/W397A mutant METTL3 (d). Ingenuity pathway enrichment analysis of genes altered by siMETTL3 (e) is shown. p = p value, Z = activation z-score, N = number of genes. P values were calculated using a two-tailed Fisher Exact test.

Extended Data Fig. 2 MTC regulates SASP during both oncogene and chemotherapy-induced senescence.

a-b, IMR90 cells were induced to senesce by oncogenic RAS (a) or Etoposide (b) with or without the expression of the indicated shRNAs and analysed for expression of the indicated SASP genes by qRT-PCR. Data represent mean ± s.d. of three biologically independent experiments. P values were calculated using a two-tailed t-test. Numerical source data for 2a and 2b are provided.

Source data

Extended Data Fig. 3 MTC regulates SASP in an enzymatic activity independent manner.

a-c, Control and RAS-induced senescent cells with or without knockdown of endogenous METTL3 and METTL14 were rescued by the indicated wild-type or mutant METTL3 or METTL14. Expression of IL6, IL1α, and IL1β (a); IL8, CXCL3 and CXCL5 (b); and SAA1 and SAA2 (c) was determined by RT-qPCR analysis. Data represent mean ± s.d. of three biologically independent experiments. P values were calculated using a two-tailed t-test. Numerical source data for 3a, 3b and 3c are provided.

Source data

Extended Data Fig. 4 Inhibition of MTC does not affect senescence-associated growth arrest.

a-b, IMR90 cells were induced to senesce by RAS with or without the expression of the indicated shRNAs. The indicated cells were examined for senescence-associated growth arrest by colony formation and stained for SA-β-gal activity (a). SA-β-gal-positive cells were quantified in the indicated treatment groups (b). c-d, IMR90 cells were induced to undergo senescence by etoposide with or without expression of the indicated shRNAs. SA-β-gal-positive cells were quantified (c) and expression of p16, a marker of senescence, was determined by immunoblot (d). Data represent mean ± s.d. of three biologically independent experiments. P values were calculated using a two-tailed t-test. Uncropped blots for 4d and numerical source data for 4b and 4c are provided.

Source data

Extended Data Fig. 5 METTL3 and METTL14 promote SASP.

a-d, IMR90 cells ectopically expressing METTL3, wild-type or a R298P mutant METTL14 were subjected to analysis for expression of the indicated proteins by immunoblots (a), colony formation assay (b), SA-β-gal staining (c) or expression of the indicated SASP genes by qRT-PCR (d). The experiment in 5a was repeated twice independently with similar results. e, IMR90 cells expressing oncogenic RAS with or without ectopically expressed wild-type or the R298P mutant METTL14 were subjected to qRT-PCR analysis for expression of the indicated SASP genes. f, IMR90 cells with or without expressing the indicated wild-type or mutant METTL3 or METTL14 were harvested at day 6 post infection and analysed for expression of the indicated proteins by immunoblot. The experiment was repeated twice independently with similar results. g-h, Conditioned medium collected from senescent cells with the indicated inducers were used to culture proliferating cells for 5 days. Changes in SA-β-gal (g) and BrdU incorporation (h) were examined. Data represent mean ± s.d. of three biologically independent experiments. P values were calculated using a two-tailed t-test. Uncropped blots for 5a and 5f and numerical source data for 5b, 5c, 5d, 5e, 5g and 5h are provided.

Source data

Extended Data Fig. 6 Kinetics of SASP gene expression.

a-g, ER:RAS-expressing IMR90 cells were treated with 100 nM 4-OHT to induce RAS expression and analysed for RAS expression by immunoblot (a), quantified for m6A levels from total RNAs (b), CCF formation (c) and quantification (d), expression of the indicated SASP genes (e), association of METTL3 and METTL14 with CXCL5 promoter and enhancer (f), or LINE1 and its regulated IFNα and IFNβ (g) by qRT-PCR analysis at the indicated time points. h, Expression of LINE1 and its regulated IFN β was determined by qRT-PCR in control and senescent cells without or with knockdown of METTL3 or METTL14. IL6 mRNA expression was used as a positive control. Arrows point to examples of CCF formed in RAS-induced senescent cells. Scale bar = 5 μm. Data represent mean ± s.d. of three biologically independent experiments. P values were calculated using a two-tailed t-test. Uncropped blots for 6a and numerical source data for 6b, 6d, 6e, 6f, 6g, and 6h are provided.

Source data

Extended Data Fig. 7 SASP genes are not subjected to m6A modification.

a, Distribution of m6A peaks across the indicated gene structure in control and RAS-induced senescent cells. b, Metagene m6A signal profile illustrating no global changes in m6A modifications around 5’ and 3’ end UTRs between control and RAS-induced senescent cells. c, Heatmap of changes in m6A modification on carRNAs in control and oncogenic RAS-induced senescent cells with or without knockdown of METTL3 or METTL14. d, Examples of m6A tracks at the boxed carRNAs that belong to each of the three indicated clusters with both forward and reverse strands indicated. H3K27ac modification levels in control (in blue) and senescent (in red) cells were used to identify the regulatory chromatin region (enhancer/promoter-associated RNAs). e, m6A tracks at the indicated SASP genes for both forward and reverse strands. Boxes indicated H3K27ac modification levels in control (red) and senescent (blue) cells used to identify the regulatory chromatin region. Please note that the changes in the gene body reflect changes in gene expression and specifically upregulation of SASP genes in senescent cells (and the associated increase in m6A modification was a reflection of an increase in input mRNA expression of these genes).

Extended Data Fig. 8 METTL3 redistribution to SASP gene promoters.

a, Distribution of METTL3 and METTL14 in the indicated genomic regions in control and RAS-induced senescent cells. b, Average binding signal from ChIP-seq analysis of RNA polymerase II occupancy on all genes in control and senescent cells without or with knockdown of METTL3 or METTL14. c, Transcription factor binding site enrichment analysis of increased cut-and-run peaks of METTL3 in senescent cells. d, Correlation between changes in binding signal of METTL3 (senescent vs. control) and NF-κB p65 binding signal in senescent cells. e, Co-immunoprecipitation analysis between NF-κB p65 subunit and METTL3 or METTL14 in the indicated cells. The experiment was repeated three times independently with similar results. f-g, ChIP–qPCR analysis of association of METTL3 on the CXCL5 promoter (f) or negative control regions of CXCL3 or CXCL5 gene promoters in the indicated cells (g). h-i, ChIP–qPCR analysis of association of NF-κB p65 on the CXCL5 promoter (h) or negative control regions of CXCL3 or CXCL5 gene promoters in the indicated cells (i). j, NF-κB reporter activity was determined in the indicated cells. k-m, The indicated ER:RAS IMR90 cells were induced by 4-OHT. Cells were harvested and analysed for expression of the indicated proteins by immunoblot (k), nuclear chromatin fraction of p65 (l), or association of p65 with the promoters of the indicated SASP genes by ChIP-qPCR assay (m). Data represent mean ± s.d. except for 8j mean ± s.e.m. of three biologically independent experiments. P values were calculated using a two-tailed t-test and a two-tailed Spearman correlation analysis for 8d. Uncropped blots for 8e, 8k and 8l and numerical source data for 8f, 8g, 8h, 8i, 8j and 8m are provided.

Source data

Extended Data Fig. 9 METTL14 regulates SASP gene enhancers.

a, List of direct METTL14 target genes that are upregulated in senescent cells, downregulated by METTL14 knockdown and rescued by both wild-type and the R298P mutant METTL14 with increased binding of METTL14 (≥2 fold) in senescent cells. b, Enrichment of SASP genes among direct METTL14 target genes. c, Enrichment of SASP genes among genes with increased binding of co-localized METTL14 and H3K27ac in senescent cells compared with control cells (≥2 fold). d, Cut-and-run peaks of METTL3, NF-κB p65, METTL14 and H3K27ac on the SAA1 and SAA2 gene loci in control and RAS-induced senescent cells. e, ChIP-qPCR analysis of the association of H3K27ac with enhancers of the indicated SASP gene loci in control and senescent cells with or without METTL14 knockdown. f, Pearson correlation analysis of METTL14 with the indicated SASP genes in human laser captured and micro-dissected PanIN lesion samples based on the GSE43288 dataset. n = 13 biologically independent samples. P values were calculated using a Pearson correlation analysis. g, ChIP–qPCR analysis of the association of METTL14 with enhancers of the indicated SASP genes in control and senescent cells with or without IKK inhibitor Bay 11-7082 (5 μM) treatment for 48 hrs. Data represent mean ± s.d. in 9e and mean ± s.e.m. in 9g of three biologically independent experiments. P values were calculated using a two-tailed t-test except in 9b-c by a two-tailed Fisher exact test and a two-tailed Pearson correlation analysis in 9f. Numerical source data for 9e, 9f and 9g are provided.

Source data

Extended Data Fig. 10 MTC is required for immune surveillance function of the SASP.

a, Validation of METTL3 and METTL14 knockdown by qRT-PCR analysis in mouse NIH 3T3 cells. n = 3 biologically independent experiments. b, Validation of METTL3 and METTL14 knockdown by immunofluorescence analysis in mouse NIH 3T3 cells. Arrows point to dsRed-expressing shRen control, shMETTL3 or shMETTL14. Bar = 10 μm. The experiment was repeated two times independently with similar results. c, Immunohistochemical staining of NRas in liver tissues injected with a mutant NRasV12/D38A that is incapable of inducing senescence at day 6. The experiment was repeated in 3 biologically independent mice with similar results. Bar = 50 μm. d, Quantification of CD45+/NRas+ foci in the livers isolated from the indicated mice at day 6. n = 6 biologically independent mice per group. Data represent mean ± s.d. P values were calculated using a two-tailed t-test. Numerical source data for 10a and 10d are provided.

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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 23, 355–365 (2021). https://doi.org/10.1038/s41556-021-00656-3

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