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

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.

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

a, Schematic of experimental timeline using oncogenic-H-RAS^G12V 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 wildtype 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 wildtype 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 wildtype 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 analyzed 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.

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 wildtype 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.

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.

Extended Data Fig. 5. METTL3 and METTL14 promote SASP

a-d, IMR90 cells ectopically expressing METTL3, wildtype 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 wildtype 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 wildtype or mutant METTL3 or METTL14 were harvested at day 6 post infection and analyzed 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.

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 analyzed 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.

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 NFκB p65 on the CXCL5 promoter (h) or negative control regions of CXCL3 or CXCL5 gene promoters in the indicated cells (i). j, NFκB 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 analyzed 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.

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 wildtype 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.

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 NIH3T3 cells. n=3 biologically independent experiments. b, Validation of METTL3 and METTL14 knockdown by immunofluorescence analysis in mouse NIH3T3 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 NRas^V12/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.

Figure 1:. METTL3 and METTL14 regulate SASP

a, 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 wildtype or the R298P mutant METTL14. Cells were harvested and analyzed for expression of the indicated proteins by immunoblot. The experiment was repeated three times independently with similar results. b-d, Numbers of genes significantly changed in the indicated cells determined by RNA-seq analysis (b), and heatmap of RNA-seq data with 3 biologically independent replicates in each of the groups for the genes whose expression significantly changed by METTL14 knockdown and rescued by both wildtype and the R298P mutant METTL14 (c), among which SASP genes were significantly enriched (d). e-g, IMR90 cells were induced to senesce by oncogenic RAS expressing a non-targeting siRNA control (siControl) or METTL3-targeted siRNA (siMETTL3) with or without the rescue of ectopically expressed wildtype or the D394A/W397A mutant METTL3. Cells were harvested and analyzed for expression of the indicated proteins by immunoblot (e). The experiment was repeated three times independently with similar results. Numbers of genes significantly changed in the indicated cells determined by RNA-seq analysis (f), and SASP genes were significantly enriched among the genes whose expression significantly changed by METTL3 knockdown and rescued by both wildtype and the D394A/W397A mutant METTL3 (g). h, IMR90 cells were induced to senesce by RAS with or without the expression of the indicated shRNAs. Expression of the indicated proteins was determined by immunoblot. The experiment was repeated three times independently with similar results. i, The secretion of soluble factors under the indicated conditions was detected by antibody arrays. The heatmap indicates the fold change (FC) in comparison to the control or RAS-induced senescent condition. Relative expression levels per replicate and average fold change differences are shown (n=4 biologically independent replicates). P values were calculated using a two-tailed Fisher Exact test in 1d and 1g. Uncropped blots for 1a, 1e, 1h, and numerical source data for 1i are provided.

Figure 2:. SASP is not regulated by m⁶A

a, Tracks of m⁶A distribution on the representative SASP genes based on RNA immunoprecipitation followed by sequencing using an anti-m⁶A antibody. The m⁶A signal was normalized with the corresponding input and the relative fold change was shown. m⁶A modified non-SASP gene PHLPP2, BCL2A1 and CENPA were used as positive controls. Red arrows point to statistical cut off in peak calling. b-d, m⁶A levels from total RNAs in control and RAS-induced senescent cells with or without knockdown of endogenous METTL3 or METTL14 and rescued by the indicated wildtype or mutant METTL3 or METTL14 (b). m⁶A modifications on the indicated SASP genes (c) or a positive control PHLPP2 gene (d) were quantified with RNA immunoprecipitation using an anti-m⁶A antibody followed by RT-qPCR. Data represent mean ± s.d. of three biologically independent experiments. P values were calculated using a two-tailed t-test. Numerical source data for 2b, 2c and 2d are provided.

Figure 3:. Genome-wide redistribution of METTL3 and METTL14

a, Heatmap clustering of cut-and-run seq profiles of METTL3 and METTL14 in control and senescent cells. b-c, Correlation of binding signal between METTL3 and METTL14 in control vs. senescent cells (b) and correlation of the specific binding signal of METTL3 or METTL14 between control and senescent cells (c). d, Distribution of relative normalized density of METTL3 and METTL14 cut-and-run seq peaks within gene body context in control (C) and senescent (S) cells. e, Average profiles of RNA polymerase II (Pol II) occupancy on SASP gene loci in control (C) and senescent (S) cells with or without METTL3 or METTL14 knockdown determined by ChIP-seq analysis.

Figure 4:. METTL3 is localized to the pre-existing NF-κB sites within the promoters of SASP genes

a, Enrichment of SASP genes among genes whose promoters showed an increased association with METTL3 in senescent cells. b, Heatmap of binding signal around METTL3 peaks found in control and RAS-induced senescent cells from cut-and-run seq for METTL3 and NF-κB p65. c, Co-immunoprecipitation analysis between METTL3 or METTL14 and NF-κB p65 subunit and RNA polymerase II in control and RAS-induced senescent cells. The experiment was repeated three times independently with similar results. d-e, ChIP–qPCR analysis of the association of METTL3 and NF-κB p65 with the promoters of the indicated SASP genes in control and RAS-induced senescent cells with or without METTL3 or METTL14 knockdown or treated with IKK inhibitor Bay 11–7082 (5 μM) for 48 hrs. Data represent mean ± s.e.m. of three biologically independent experiments. P values were calculated using a two-tailed t-test except in 4a by a two-tailed Fisher Exact test. Uncropped blots for 4c and numerical source data for 4d and 4e are provided.

Figure 5:. METTL14 regulates SASP gene enhancers

a, Overlap between genes with expression rescued by both wildtype (wt) and the R298P mutant (mt) METTL14 in senescent cells with METTL14 knockdown and genes with ≥2 fold increase in METTL14 binding in senescence (S) compared with controls (C). n=3 biologically independent experiments for RNA-seq analysis. b, Average profiles of the cut-and-run signal for METTL14 and the ChIP-seq signal for H3K27ac for genomic loci with increased association for both METTL14 and H3K27ac in senescent (S) compared with control (C) cells (≥2 fold). c, Representative cut-and-run peaks of METTL3, NF-κB p65, METTL14 and H3K27ac on the indicated SASP genes loci in control and RAS-induced senescent cells. d, ChIP-qPCR analysis of the association of H3K27ac with the enhancers of the indicated SASP gene loci in control and RAS-induced senescent cells with or without METTL14 knockdown. Data represent mean ± s.d. of three biologically independent experiments. P values were calculated using a two-tailed t-test except in 5a by a two-tailed Fisher Exact test. Numerical source data for 5d are provided.

Figure 6:. METTL3 and METTL14 mediate SASP gene enhancer and promoter loop formation

a, 3C-qPCR analysis of the promoter-enhancer interaction frequency on the indicated SASP gene loci in control and RAS-induced senescent cells with or without METTL14 knockdown. Schematic illustrates the 3C primers targeting the enhancer and promoter of the SASP gene loci according to the cut-and-run seq peaks for H3K27ac and METTL14 in control and RAS-induced senescent cells. b, A model for the mechanism by which the redistributed METTL3 and METTL14 promote SASP gene expression in senescent cells by mediating promoter and enhancer looping. c-d, Representative images of 3D DNA-FISH for IL1β locus in control and RAS-induced senescent cells with or without knockdown of METTL3 or METTL14. Dual labelled DNA-FISH was performed with a probe for promoter (in red) and another probe for enhancer (in green) (c). Scale bars = 5 μm. The distance between IL1β promoter and enhancer probes was determined using ImageJ software (d). At least 40 loci were quantified for each of the indicated groups. e-f, ChIP–qPCR analysis of association of HA-tagged wildtype and mutant METTL3 (e) and FLAG-tagged wildtype and mutant METTL14 (f) with the promoters and enhancers of the indicated SASP genes in control and senescent cells with or without endogenous METTL3 or METTL14 knockdown, and rescued with HA-tagged wildtype or mutant METTL3 or FLAG-tagged wildtype or mutant METTL14. Data represent mean ± s.d. of three biologically independent experiments unless otherwise stated. P values were calculated using a two-tailed t-test. Numerical source data for 6a, 6d, 6e and 6f are provided.

Figure 7:. METTL3 and METTL14 are required for pro-tumorigenic and immune surveillance function of the SASP.

a, TOV21G ovarian cancer cell growth in conditioned media collected from control and senescent IMR90 cells without or with knockdown of METTL3, METTL14 or WTAP. After 7 days of incubation, cell number was counted and normalized against cell number from cells cultured in conditioned media collected from control proliferating IMR90 cells. b-c, Tumor growth stimulated by co-injected senescent IMR90 fibroblasts in a xenograft mouse model was inhibited by knockdown of METTL3, METTl14 or WTAP. TOV21G cells were subcutaneously co-injected with the indicated senescent IMR90 cells into 6–8-week-old NSG female mice. Tumor volume (b) was measured at the indicated time points (n=6 biologically independent mice per group) and tumor weight (c) was measured at the end of the experiment (n=6 mice/group). d, Schematic of experimental design and transposon-based constructs. e-f, Representative images of SA-β-gal staining of liver tissues from the indicated groups at day 6 and 14 post injection (e), and SA-β-gal positive cells were quantified in the indicated groups (f) (n=6 biologically independent mice per group). g-i, Representative images of immunohistochemical staining for NRas and CD45 expression in each of the indicated groups at the indicated time points. The immune clearance was indicated by comparing NRas positive cells at day 6 and remaining NRas positive cells at day 14 (h). Clusters of immune cells at day 6 were quantified (i), n=6 biologically independent mice per group. n.s., not significant. Scale bars = 50 μm. Data represent mean ± s.d. except in 7c with mean ± s.e.m. P values were calculated using a two-tailed t-test. Numerical source data for 7a, 7b, 7c, 7f, 7h and 7i are provided.