m6A RNA methylation orchestrates transcriptional dormancy during paused pluripotency

Abstract

Embryos across metazoan lineages can enter reversible states of developmental pausing, or diapause, in response to adverse environmental conditions. The molecular mechanisms that underlie this remarkable dormant state remain largely unknown. Here we show that N⁶-methyladenosine (m⁶A) RNA methylation by Mettl3 is required for developmental pausing in mouse blastocysts and embryonic stem (ES) cells. Mettl3 enforces transcriptional dormancy through two interconnected mechanisms: (1) it promotes global mRNA destabilization and (2) it suppresses global nascent transcription by destabilizing the mRNA of the transcriptional amplifier and oncogene N-Myc, which we identify as a crucial anti-pausing factor. Knockdown of N-Myc rescues pausing in Mettl3^-/- ES cells, and forced demethylation and stabilization of Mycn mRNA in paused wild-type ES cells largely recapitulates the transcriptional defects of Mettl3^-/- ES cells. These findings uncover Mettl3 as a key orchestrator of the crosstalk between transcriptomic and epitranscriptomic regulation during developmental pausing, with implications for dormancy in adult stem cells and cancer.

Extended Data Figure 1.. Dissection of paused pluripotency in Mettl3^−/− models.

a. m⁶A increase in paused ESCs was validated in an independent mass spectrometry experiment. Levels relative to control (Ctrl) for each replicate are shown (n=3 biological replicates). b. Validation of Rptor and Rictor knockdown by RT-qPCR in paused ESCs grown in FBS/LIF/2i (n=4 biological replicates). c. mTOR inhibition by Rptor and Rictor knockdown induces a paused phenotype with reduced cell proliferation and total RNA levels (n=4 biological replicates). d. Dot blot showing an increase in m⁶A levels in ESCs upon knockdown of Rptor and Rictor. Levels of m⁶A are normalized to RNA loading control (methylene blue staining, n=4 biological replicates). e. Design of Mettl3^−/− ESCs models used in this study. f. Validation of Mettl3^−/− ESCs, in control and pausing conditions, by western blot (representative of 3 biological replicates). g. Validation of Mettl3^{−/− #2–4} ESCs, in control and pausing conditions, by western blot (representative of 2 biological replicates). h. Mettl3^{−/− #2–4} ESCs also fail to suppress proliferation in paused conditions (n=3 biological replicates). i. Mettl3-knockout mutant model in mice (Mettl3^TCP−/−) and genotyping by PCR (left). Example of PCR genotyping of embryos resulting from Mettl3^TCP+/− crossing, representative of all genotyping performed in this study [n(Mettl3^TCP+/+) = 87, n(Mettl3^TCP+/+) = 132, n(Mettl3^TCP+/+) = 46]. +/+: wildtype, +/−: heterozygous, −/−: knockout. j. Validation of Mettl3TCP−/− in embryos by immunofluorescence. Representative staining images are shown. Number of embryos (n) as indicated. Scale bars = 30 μm. Data are mean ± SD (a-d) or mean ± SEM (h). P-values (as indicated on figure) by one-way ANOVA with Dunnett’s multiple comparison tests (a-c), two-tailed ratio paired Student’s t-tests (d), and linear regression test with interaction (h).

Extended Data Figure 2.. Paused Mettl3^−/− ESCs acquire a distinct gene expression profile.

a. Quantification of total RNA per cell in Mettl3^{+/+ #2} and Mettl3^{−/− #2–4} ESCs, in control and paused conditions. Data are mean ± SD, n=5 biological replicates. b. Decrease in nascent RNA per cell, as measured by EU incorporation with nuclear signal quantification, in wildtype ex vivo paused or hormonally diapaused blastocysts, compared to control E3.5 embryos. Number of embryos (n) as indicated. Scale bars = 30 μm. c. Strategy for RNA-seq with cell number-normalization using ERCC spike-in RNAs. d. Quantification of the number of expressed genes in Mettl3^+/+ and Mettl3^−/− ESCs, in control and paused conditions. Expressed genes are further defined as having high expression (log₂ normalized reads > 5, n=3 biological replicates). e. Number of differentially expressed genes (fold-change > 1.5 and adjusted P < 0.05) upon pausing, in Mettl3^+/+ and Mettl3^−/− ESCs. f. PCA plot for all expressed genes across all samples, showing across PC1 that Mettl3^+/+ ESCs acquire a more divergent expression profile upon pausing than Mettl3^−/− ESCs, relative to respective control condition. g-h. Gene expression changes (log₂ fold-changes) of gene sets selected from the “GO biological processes” (g) and “hallmarks” (h) collections, showing incomplete downregulation in paused Mettl3^−/− ESCs. i. Western blot of total and phosphorylated mTOR, and of the downstream targets of mTORC1 (Ulk1, 4Ebp1 and S6K1) (left, representative of 4 biological replicates). Quantification of phosphorylated levels, normalized to total levels, show no significant change in paused ESCs between Mettl3^+/+ and Mettl3^−/− (right). Data are mean ± SD (b, d, g-i). P-values (as indicated on figure) by two-way ANOVA with Dunnett’s multiple comparison tests (a, i), one-way ANOVA with Dunnett’s multiple comparison tests (b), two-tailed unpaired Student’s t-tests (d, g-h).

Extended Data Figure 3.. Mapping m⁶A distribution in the transcriptome of paused ESCs.

a. Strategy for MeRIP-seq in ESCs with cell number-normalization (CNN) using human cell spiking. b. Validation of the CNN strategy for the MeRIP-seq. By mixing different ratios of human cells to ESCs (1, 2 or 4%), we simulated global changes in methylation. Spiking normalization allows capture of these differences, as shown here by MeRIP-qPCR for 3 methylated mRNAs (NeuroD1, Nr5a2, Sox1). Data are mean ± SD, n=5 biological replicates with levels relative to 2% spike in each replicate. c. The specificity of the m⁶A capture was tested by spiking poly(A) RNA from ESCs with exogenous RNAs before performing MeRIP-qPCR. Data are mean ± SD, n=3 biological replicates. P-values (as indicated on figure) by two-tailed unpaired Student’s t-tests. d. Examples of gene track views of MeRIP-seq, for mRNAs of pluripotency factors previously shown to be methylated in ESCs. e. Motif analysis performed with DREME in a 100bp window surrounding MeRIP peak summits identifies several motifs corresponding to the consensus “DRACH” m⁶A motif (where D=A, G or U; H=A, C or U). f. Distribution of differential m⁶A peaks, according to the type of structural element within the transcript. g. Examples of gene track views of MeRIP-seq, for mRNAs with significant hypermethylation (Tial1, Ptrf) or hypomethylation (Cenpt) in pausing of ESCs. h. GSEA of m⁶A changes in paused ESCs relative to control ESCs, using the “hallmarks” collection. No single pathway is significantly enriched based on m⁶A changes (representative pathways are shown). P-values (as indicated on figure) by two-sided pre-ranked gene set enrichment analysis with Benjamini-Hochberg FDR correction.

Extended Data Figure 4.. Mapping the chromatin distribution of Mettl3 in paused ESCs.

a. m⁶A machinery (writers Mettl3, Mettl14 and Wtap; and erasers Fto and Alkbh5) in control (Ctrl) and paused ESCs by western blot in whole cell extracts (representative of 3 biological replicates). b. Increase of Mettl3 levels in chromatin extracts upon induction of paused pluripotency, measured by cell number-normalized (CNN) western blot (representative of 3 biological replicates). c. Strategy for Mettl3 ChIP-seq in ESCs with CNN approach using human cell spiking. d. Heatmap of the top 5000 most variable Mettl3 peaks by ChIP-seq across all samples, showing higher levels in paused ESCs (n=2 biological replicates per group). e. Density plot of the average levels of Mettl3 binding in the TSS of all genes by ChIP-seq, separated by expression and methylation status, in control and paused ESCs. Mettl3 binding is highest in the TSS of expressed genes with a methylated transcript, and in paused ESCs. Number of genes (n) as indicated. Data as mean normalized Mettl3 level (n=2 biological replicates per group). f. Examples of gene track views showing increased average levels (fold-change > 1.5) of m⁶A and Mettl3, by MeRIP-seq and Mettl3 ChIP-seq, respectively.

Extended Data Figure 5.. Capturing Mettl3-dependent changes in RNA stability in paused ESCs.

a. Strategy for RNA stability analysis based on intronic and exonic reads. b-c. Examples of genes with different (Slc16a1, Six4) and similar (Mtor, Gapdh) intronic and exonic mRNA fold-changes between Mettl3^−/− and Mettl3^+/+ ESCs based on RNA-seq data (b) and validation of stability changes by actinomycin D stability assay (c). N=3 biological replicates, t_1/2: half-life. d. Linear regression of log2 total conversion counts (relative to time 0h), as measured by SLAM-seq, showing an increase in transcriptome stability in paused Mettl3^−/− ESCs. e. Changes in RNA expression in paused Mettl3^−/− ESCs based on RNA-seq data (fold-change > 1.5) are associated with changes in RNA half-life in paused Mettl3^−/− ESCs. Data are mean ± SD (b) or mean ± SEM (c). P-values (as indicated on figure) by two-tailed paired Student’s t-tests (b), linear regression test with interaction (c) and one-way ANOVA (e). Boxes in the box plots define the interquartile range (IQR) split by the median, with whiskers extending to the most extreme values within 1.5 × IQR beyond the box.

Extended Data Figure 6.. Screening for candidate anti-pausing factors.

a. Quantification of the number of expressed genes in Mettl3^+/+ and Mettl3^−/− ESCs based on intronic RNA-seq, in control and paused conditions. Expressed genes are further defined as having high expression (log₂ normalized reads > 5, n=3 biological replicates). b. Heatmap of gene expression based on intronic reads for all genes expressed in Mettl3^+/+ or Mettl3^−/− ESCs (left) with average expression per sample (right, scored as median z-scores of all genes), showing defective hypotranscription in paused Mettl3^−/− ESCs. c. Identification of putative anti-pausing factors kept in check by m⁶A methylation and thereby destabilization of their transcript in paused pluripotency, based on RNA-seq, MeRIP-seq and SLAM-seq data in ESCs (see Methods for details). d. Expression levels (log₂ cpm) of the Myc factors in diapaused embryos (left, data from Boroviak et al.) and paused ESCs (right). Horizontal bars represent the mean, with 3 biological replicates per group, except for diapaused embryos which has 2 replicates. e. mTOR inhibition by dual knockdown of Rptor and Rictor reduces Mycn expression measured by RT-qPCR in ESCs in FBS/LIF/2i medium (n=4 biological replicates). Data are mean ± SD (a, b, e). P-values (as indicated on figure) by two-way ANOVA with Tukey’s multiple comparisons test (a), two-tailed Student’s t-tests (b), and one-way ANOVA with Dunnett’s multiple comparison tests (e).

Extended Data Figure 7.. Regulation of Myc family members and downstream targets by Mettl3 in paused pluripotency.

a. Quantification of N-Myc protein levels, showing increased expression in Mettl3^−/− ESCs, as shown in Fig. 5c. N=4 biological replicates. b. Representative western blot of N-Myc protein levels, showing increased expression in Mettl3^{−/− #2–4} ESCs. N=2 biological replicates. c. Increased expression of N-Myc in Mettl3^−/− ESCs grown in FBS/LIF/2i (compared to Mettl3^+/+ ESCs) measured by RT-qPCR (n=3 biological replicates). Levels are normalized to control Mettl3^+/+ ESCs grown in FBS/LIF, as shown in Fig. 5b. d. Mycn expression in Mettl3^−/− ESCs is restored to levels comparable to Mettl3^+/+ ESCs by transfecting a catalytically active form of Mettl3, and not its inactive mutant form (RT-qPCR, n=4 biological replicates). e. Validation of Mycn knockdown by RT-qPCR in paused ESCs grown in FBS/LIF (left) or FBS/LIF/2i (right). f-g. Blocking of Myc signaling by Mycn knockdown (e, in FBS/LIF/2i) or chemical inhibitor 10058-F4 (f, in FBS/LIF) partially restores the pausing phenotype in paused Mettl3^−/− ESCs in terms of cell proliferation (left, n=3 biological replicates) and total RNA levels per cell (right, n=4 and 5 biological replicates). h. Treatment with 10058-F4 partially restores the expression of Myc target genes in paused Mettl3^−/− ESCs (RT-qPCR, n=5 biological replicates). Data are mean ± SD (a, c-h), or mean ± SEM (g left). P-values (as indicated on figure) by two-tailed unpaired Student’s t-tests (a, d, f), by two-way ANOVA with Tukey’s multiple comparisons test (c) or Dunnett’s multiple comparison tests (d-e, h), one-way ANOVA with Dunnett’s multiple comparison tests (f-g).

Extended Data Figure 8.. m⁶A-dependent regulation of Mycn mRNA stability.

a. Knockout of Ythdf2 and triple knockout of Ythdf1–3 (TKO) phenocopy the knockout of Mettl3 in paused ESCs, with increased total RNA levels per cell (left, n=4 biological replicates) and proliferation (right, n=4 biological replicates) compared to wildtype (WT) ESCs. b. Increased expression of N-Myc in paused Ythdf2^−/− and TKO ESCs measured by RT-qPCR (n=4 biological replicates, relative to paused WT). c. Validation of m⁶A changes in Mettl3^+/+ and Mettl3^−/− ESCs by m⁶A-qPCR (n=6 biological replicates). d. Mettl3 and Ythdf2 binding of the Mycn transcript, measured by RIP-qPCR in 3 biological replicates. NeuroD1 and Sox2 were used as positive controls, and Actb and Gapdh were used as negative controls. e-f. Mycn is the only Myc family member regulated at the RNA stability level by Mettl3, as evidenced by analysis of exonic and intronic mRNA fold-changes (left, n=3 biological replicates per group) and SLAM-seq analysis of RNA half-life (right, with half-lives derived from 2 independent time courses). g. Nascent RNA capture by EU incorporation shows minimal changes in nascent transcription for Myc factors in Mettl3^−/− ESCs (n=5 biological replicates). h. Increased Mycn mRNA stability in paused Ythdf2^−/− ESCs compared to paused Mettl3^+/+ ESCs, as measured by an actinomycin D stability assay (n=3 biological replicates). t_1/2: half-life. Data are mean ± SD (a-e, g) or mean ± SEM (h). P-values (as indicated on figure) by one-way ANOVA with Dunnett’s multiple comparison tests (a-c), two-tailed Student’s t-tests (d-e), two-way ANOVA with Dunnett’s multiple comparison tests (g), and linear regression test with interaction (h).

Extended Data Figure 9.. Targeted m⁶A demethylation controls expression of Mycn in paused ESCs.

a. Model of lentiviral dCasRx epitranscriptomic editor with the m⁶A demethylase ALKBH5 and single guide RNA. b. Validation of the expression of the dCasRx-ALKBH5 fusion by western blot in parental (non-infected) ESCs, infected ESCs, and 2 infected clones. Clone 5 was used for all experiments (representative of 2 biological replicates). c. Guide RNAs (gRNAs) transfected for non-targeting control and Mycn-targeting conditions. d. Changes in m⁶A using the dCasRx-ALKBH5 editor in paused ESCs. Guides #2 and #3 significantly reduce m⁶A in Mycn transcripts, as measured by m⁶A-qPCR, and were selected for all subsequent experiments. N=7 biological replicates. NT: non-targeting. e. Dot blot showing that the global increase of m⁶A in paused ESCs is not affected by the dCasRx-ALKBH5 editor, with Mettl3^−/− ESCs as negative control. Representative of 3 biological replicates. MB: methylene blue. f. Quantification of N-Myc protein levels, showing increased expression with the dCasRx-ALKBH5 editor targeting Mycn in paused ESCs, with representative blot shown in Fig. 6e. N=3 biological replicates. g. Demethylation of Mycn increases the total RNA levels per cell in paused ESCs (n=6 biological replicates). Data are mean ± SD (d, f-g) and P-values (as indicated on figure) by one-way ANOVA with Dunnett’s multiple comparison tests (d, f-g).

Extended Data Figure 10.. Transcriptional changes by RNA-seq upon m⁶A demethylation of Mycn in paused ESCs.

a. Mycn expression is increased following targeting with the m⁶A demethylase ALKBH5 based on exonic reads, but not intronic reads, which is consistent with post-transcriptional regulation (n=4 biological replicates per condition). b. A global increase in transcripts levels is measured following Mycn mRNA demethylation using both exonic and intronic reads, which is consistent with globally elevated nascent transcription. c. Representative pathways of the GSEA of gene expression changes upon demethylation of Mycn mRNA in paused ESCs using the “hallmarks” collection. d-e. Genes upregulated upon demethylation of Mycn mRNA are enriched for N-Myc targets, as identified in ESCs by ChIP by Chen et al. GSEA using a random set of 100 N-Myc targets (c). Venn diagram showing a significant overlap between genes upregulated in paused Mettl3^−/− ESCs, genes upregulated following Mycn demethylation, and N-Myc ChIP targets. Data are mean ± SD (a). P-values (as indicated on figure) by two-way ANOVA with Dunnett’s multiple comparison tests (a), two-sided pre-ranked gene set enrichment analysis with Benjamini-Hochberg FDR correction (c, d), and one-sided simulation using hypergeometric distributions (e).

Figure 1:. The m⁶A methyltransferase Mettl3 is essential for paused pluripotency.

a. Screening of RNA modifications by mass spectrometry in poly(A) RNA. Levels in paused ESCs are normalized to control ESCs. Data are mean, n=2 biological replicates. b. Dot blot showing an increase in m⁶A levels in paused ESCs in FBS/LIF and FBS/LIF/2i media. Levels of m⁶A are normalized to RNA loading control (methylene blue staining). Data are mean ± SD, n=5 biological replicates. c-d. Growth curves showing that Mettl3^−/− ESCs fail to suppress proliferation in paused conditions in both FBS/LIF (c) and FBS/LIF/2i (d) media. Data are mean ± SEM, n=3 biological replicates. e. Mettl3 loss leads to the premature death of mouse blastocysts cultured ex vivo in paused conditions. Right: sample images of cultured embryos, with black arrow indicating a dead embryo and scale bar at 50μm. f. Quantification of recovered (live) embryos at E3.5 (control) or at Equivalent Days of Gestation (EDG) 6.5 and 8.5 following hormonal diapause, showing that Mettl3^TCP−/− embryos are impaired at undergoing hormonal diapause. Number of embryos (n) as indicated (e-f). P-values (as indicated on figure) by two-tailed Student’s t-tests (b), linear regression test with interaction (c-d), log-rank test (e), and χ2 test (f).

Figure 2:. Mettl3 regulates hypotranscription in paused pluripotency.

a. Quantification of total RNA per cell in Mettl3^+/+ and Mettl3^−/− ESCs, grown in control and paused conditions, in FBS/LIF or FBS/LIF/2i media. Data are mean ± SD, n=4 biological replicates. b. Representative histograms (left) of nascent transcription in Mettl3^+/+ and Mettl3^−/− ESCs grown in control or paused conditions and quantification (right) by median fluorescence intensity (MFI) relative to control Mettl3^+/+ in each experiment, showing increased transcription in paused Mettl3^−/− ESCs. Data are mean ± SD, n=4 biological replicates. Examples of FACS gating have been deposited on the Figshare repository (10.6084/m9.figshare.23551986). c-d. Immunofluorescence images and nuclear signal quantification of EU incorporation in ex vivo paused (c) and hormonally diapaused (d) blastocysts, showing increased nascent transcription in Mettl3^TCP−/−. Data are mean ± SD. Number of embryos (n) as indicated, with scale bar at 50μm. e. Quantification of poly(A) RNA per cell in Mettl3^+/+ and Mettl3^−/− ESCs, grown in control and paused conditions. Data are mean ± SD, n=4 biological replicates. f. Heatmap of gene expression (by RNA-seq) for all genes expressed in Mettl3^+/+ or Mettl3^−/− ESCs, showing defective hypotranscription in paused Mettl3^−/− ESCs. Data as z-score normalized per gene, with all samples displayed (n=3 biological replicates per group). g-h. Gene set enrichment analysis (GSEA) of gene expression changes in paused Mettl3^+/+ and Mettl3^−/− ESCs (as shown in Fig. 2f), using the “GO biological processes” (g) and “hallmarks” collections (h). Scatter plots (left) of the normalized enrichment scores (NES), with Spearman correlation coefficient (ρ). Representative pathways with defective hypotranscription in Mettl3^−/− (red dots) are highlighted (right). P-values (as indicated on figure) by two-tailed paired Student’s t-tests (a-b, e), one-way ANOVA with Dunnett’s multiple comparison test (c-d), two-sided pre-ranked gene set enrichment analysis with Benjamini-Hochberg FDR correction (f-g).

Figure 3:. The methyltransferase activity of Mettl3 sustains paused pluripotency.

a. Schematic of wildtype (WT) and catalytically inactive mutant (MUT) Mettl3 protein (top), and western blot of rescue by transfection in Mettl3^−/− ESCs (bottom, representative of 3 biological replicates). b-c. Transfection with wildtype Mettl3, but not its catalytic mutant, restores the in vitro pausing phenotype of hypotranscription (b) and suppressed proliferation (c) in paused Mettl3^−/− ESCs (n=4 biological replicates). d. PCA plot for all m⁶A peaks across all samples by MeRIP-seq, showing that paused ESCs have distinct m⁶A profiles (n = 3 biological replicates per condition). e. MeRIP-seq shows increased m⁶A in paused ESCs. Scatter plot (left) and number of peaks/genes with significant gain and loss of m⁶A (right, fold-change > 1.5 and adjusted P < 0.05). f. Heatmaps of Mettl3 ChIP-seq signal in control and paused ESCs, showing increased Mettl3 binding in paused ESCs. Signal was merged from 2 biological replicates per condition. g. Venn diagrams showing significant overlap between target genes of m⁶A (on related RNA) and Mettl3, identifying all target genes (top) or genes with increased levels of m⁶A and Mettl3 (fold-change > 1.5, no statistical threshold) in paused ESCs (bottom). h. Metagene profiles of peaks indicate that Mettl3 mainly targets the promoter and 5’UTR regions, while m⁶A mainly targets the stop codon and 3’UTR. All data are mean ± SD (b, c). P-values (as indicated on figure) by one-way ANOVA with Dunnett’s multiple comparison test (b), linear regression test with interaction (c), two-sided t-test adjusted by Benjamini-Hochberg FDR (e), and one-sided hypergeometric test (g).

Figure 4:. Mettl3 promotes RNA destabilization during pausing.

a. RNAs with increased m⁶A in pausing (as defined in Fig. 3e) are significantly more downregulated than RNAs with decreased m⁶A. This pattern is Mettl3-dependent, as analyzing the same RNAs in Mettl3^−/− ESCs shows no effect. RNA-seq data as shown in Fig. 2f (n=3 biological replicates per group). b. Differences in expression (log₂FC paused/Ctrl) between exonic and intronic RNA-seq data indicate a global decrease in RNA stability in Mettl3^+/+ ESCs upon pausing. This effect is absent in Mettl3^−/− ESCs. c. Schematic of the measurement of RNA degradation kinetics by SLAM-seq (left). In the paused state, Mettl3^−/− ESCs display an overall longer half-life of the transcriptome compared to Mettl3^+/+ ESCs (right). Half-lives were measured using 4 time points, with samples collected over 2 independent experiments (see Extended Data Fig. 5d). S4U: 4-thiouridine. d. Changes in RNA expression during pausing in wild-type ESCs (Paused/Ctrl, fold-change > 1.5) are anti-correlated with changes in RNA half-life in paused Mettl3^−/− ESCs (as measured in Fig. 4c). e. RNAs with increased m⁶A in pausing (as defined in Fig. 3e) are enriched among RNAs stabilized in Mettl3^−/− ESCs (half-life fold-change > 1.5). f. Increased RNA stability in control E3.5 blastocysts compared to diapaused blastocysts, as measured by treatment with actinomycin D for 2 hours followed by RT-qPCR (n = 4 biological replicates). Ribosomal 28S as negative control for RNA decay. All data are mean ± SD. P-values (as indicated on figure) by two-tailed Student’s t-tests (a-b, f), one-way ANOVA (d) and two-proportion z-tests (e). Boxes in the box plots define the interquartile range (IQR) split by the median, with whiskers extending to the most extreme values within 1.5 × IQR beyond the box.

Figure 5:. The transcription factor N-Myc is a key mediator of the pausing defects of Mettl3^−/− ESCs.

a. To identify m⁶A targets relevant in vivo, candidate genes were ranked by their Spearman correlation coefficient (ρ) with the m⁶A machinery in early mouse embryos (see Methods, n=11 independent samples). The top 10 ranked candidates are shown. b, c. Increased expression of N-Myc in Mettl3^−/− ESCs, shown by RT-qPCR (b, n=3 biological replicates) and western blot (c, representative of 4 biological replicates). d. Transfection with wildtype Mettl3, but not its catalytic mutant, restores N-Myc expression in Mettl3^−/− ESCs (representative of 3 biological replicates). e, f. Immunofluorescence images (left) and nuclear signal quantification (right) of N-Myc protein in ex vivo paused (c) and hormonally diapaused (d) blastocysts, showing increased levels in Mettl3^TCP−/−. Data are mean ± SD. Number of embryos (n) as indicated, with scale bar at 30μm. g. Scatter plots of the median log₂ fold-changes for each “hallmark” gene set, showing a positive correlation between in Myc/Mycn DKO ESCs and diapaused embryos or paused Mettl3^+/+ (but not Mettl3^−/−) ESCs, with spearman correlation coefficient (ρ). h. Knockdown of Mycn restores the in vitro pausing phenotype of suppressed proliferation (left, n=3 biological replicates) and total RNA levels per cell (right, n=4 biological replicates) in paused Mettl3^−/− ESCs. i. Nascent RNA capture by EU incorporation shows increased transcription for canonical Myc target genes in paused Mettl3^−/− ESCs (n=3 biological replicates). j. The majority of Myc module genes are not direct targets of m⁶A in paused pluripotency. The proportion in all expressed genes is shown for comparison. All data are mean ± SD (b, e-f, h-i). P-values (as indicated on figure) by two-way ANOVA with Tukey’s multiple comparisons test (b), one-way ANOVA with Dunnett’s multiple comparison tests (e-f, h right), two-sided Spearman correlation test (g), linear regression test with interaction (h left), and two-way Student’s t-tests (i).

Figure 6:. Mettl3 regulates pausing via m⁶A-mediated destabilization of Mycn mRNA.

a. Gene track view of MeRIP-seq and Mettl3 ChIP-seq for Mycn mRNA. b. Increased Mycn mRNA stability in paused Mettl3^−/− ESCs compared to paused Mettl3^+/+ ESCs, as measured by an actinomycin D stability assay (n=3 biological replicates). t_1/2: half-life. c. Insertion of the identified Mycn m⁶A site, but not its mutated version, reduces transcript stability in paused ESCs, as measured by a luciferase reporter assay (n=5 biological replicates). d. Site-specific demethylation of Mycn, achieved with a dCasRx conjugated to the m⁶A demethylaseALKBH5, directed by gRNAs (left), leads to increased Mycn mRNA stability (right, n=3 biological replicates). NT: non-targeting. e-f. Site-specific demethylation of Mycn phenocopies Mettl3^−/− in paused ESCs, with increased expression of N-Myc by RT-qPCR (e top, n=7 biological replicates) and western blot (e bottom, representative of 3 biological replicates), and higher proliferation (f, n=5 biological replicates). g. Differential gene expression, as measured by RNA-seq, induced by targeted demethylation of Mycn mRNA in paused ESCs (n=4 biological replicates per condition, fold-change > 1.5 and adjusted P < 0.05). h. Genes upregulated in paused Mettl3^−/− ESCs (compared to paused Mettl3^−/− ESCs) are also significantly upregulated following the demethylation of Mycn mRNA. Number of genes (n) as indicated. Data as z-score normalized per gene, with all samples displayed (n=4 biological replicates per group, left) and log₂ fold-change over NT gRNA control (right). (i) Scatter plots of the median log₂ fold-changes for each “hallmark” gene set, showing a significant negative correlation between paused ESCs with demethylated Mycn mRNA and paused Mettl3^+/+ (but not Mettl3^−/−) ESCs. Spearman correlation coefficient (ρ) is indicated. j. Model for the role of Mettl3-dependent m⁶A methylation in paused pluripotency. Elevated chromatin recruitment of Mettl3 increases m⁶A in the transcriptome. Hypermethylation destabilizes many transcripts, including the mRNA encoding the “anti-pausing” factor N-Myc. In absence of Mettl3, upregulated N-Myc enhances transcription and proliferation, disrupting pausing. All data are mean ± SD (c, e-f) or mean ± SEM (b, d). P-values (as indicated on figure) by linear regression test with interaction (b, d, f), two-tailed paired Student’s ratio t-tests (c), one-way ANOVA with Dunnett’s multiple comparison tests (e), two-way ANOVA (h), two-sided Spearman correlation test (i). Box plots present center lines as medians, with box limits as upper and lower quartiles and whiskers as 1.5×IQR.