Whether protein synthesis and cellular stress response pathways interact to control stem cell function is currently unknown. Here we show that mouse skin stem cells synthesize less protein than their immediate progenitors in vivo, even when forced to proliferate. Our analyses reveal that activation of stress response pathways drives both a global reduction of protein synthesis and altered translational programmes that together promote stem cell functions and tumorigenesis. Mechanistically, we show that inhibition of post-transcriptional cytosine-5 methylation locks tumour-initiating cells in this distinct translational inhibition programme. Paradoxically, this inhibition renders stem cells hypersensitive to cytotoxic stress, as tumour regeneration after treatment with 5-fluorouracil is blocked. Thus, stem cells must revoke translation inhibition pathways to regenerate a tissue or tumour.
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We thank J. Marioni and D. Odom for their advice on analysing the sequencing data. This work was funded by Cancer Research UK, Worldwide Cancer Research, the Medical Research Council (MRC), the European Research Council, and EMBO. Research in M.F.’s laboratory is supported by a core support grant from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Cambridge Stem Cell Institute.
The authors declare no competing financial interests.
Extended data figures and tables
a, Hair cycle stages and genetic lineage marking using K19- and LGR5 tdTom mice. Cell surface markers to isolated bulge stem cells are CD34 and ITGA6. Telogen: stem cells (CD34+/ITGA6+) are quiescent and resting in the bulge (BG). Early anagen: stem cells divide and give rise to committed progenitors in the hair germ (HG), which then grow downwards into the bulb (BU) surrounding the dermal papilla (DP). Late anagen: cells differentiate upwards to form the hair. Catagen: intermediate phase, when the hair bulb degenerates into a new resting bulge. IFE, interfollicular epidermis; SG, sebaceous glands. Mouse transgenes label K19- (red) and LGR5- (orange) positive stem cells and their progeny. b, OP-puro detection in mouse epidermis at all hair cycle stages. Dotted lines indicate hair follicle and epidermal basal layer. Arrows indicate OP-purohigh cells in the hair follicle. Arrowheads indicate OP-purolow cells in the interfollicular epidermis. Nuclei are stained with DAPI. c, d, tdTom and OP-puro detection in back skin of K19tdTom and Lgr5tdTom mice in telogen and late anagen. Arrows indicate tdTom+ cells. Arrowheads indicate Tomato+/OP-purohigh cells. Dotted line indicates lower bulge. Merged panels from c, d are shown in Fig. 1c–f. e, Hair follicle lineages and differentiation markers used in Fig. 1g–j. Ci, cuticle of inner root sheet; Ch, cuticle; Co, cortex; Cp, companion layer; He, Henle’s layer; Hu, Huxley layer; IRS, inner root sheet; Me, medulla; ORS, outer root sheet. f, P-cadherin and OP-puro detection in a late anagen. Scale bars, 50 μm.
a, b, Top 2.5%, 10%, 25% and 50% translating epidermal cells (OP-purohigh) (a) were sorted for CD34 and ITGA6 (b). c, Protein synthesis in CD34+/ITGA6+, CD34−/ITGA6+ and CD34−/ITGA6− epidermal populations in the top 2.5%, 10%, 25%, 50% or 100% (all) translating cells at indicated hair follicle stages. d, Percentage of CD34+/ITGA6+, CD34−/ITGA6+ and CD34−/ITGA6− cells in the top 2.5%, 10%, 25%, 50% or 100% (all) of translating epidermal cells at indicated stages of the hair cycle. Error bars show mean ± s.d. e, f, Violin plots of protein synthesis in top 2.5% OP-purohigh cells in tdTom− epidermal cells sorted for CD34 and ITGA6 from K19-tdTom (e) or Lgr5-tdTom mice (f) at all stages of the hair cycle. (n = mice). Source Data for this figure is available in the online version of the paper.
a–c, Violin plots of protein synthesis in indicated epidermal populations sorted for K19-tdTom-positive (a) and LGR5-tdTom-positive (b) and -negative (c) populations. Protein synthesis is shown for top 10%, 25% or 50% OP-purohigh cells. d, e, Cell cycle analysis (d) and percentage of cells in G1/G0 or S/G2/M in the top 2.5%, 10%, 25% or 50% OP-purohigh cells in late anagen (e). Data represent mean ± s.d. f, Scatter plots correlating protein synthesis in the 2.5% OP-purohigh population with percentage of cells in S/G2/M (top) and G1/G0 (bottom) using all samples independent of hair cycle stage. Linear regression, correlation coefficient (r2) and P value are shown. g, Box plots of protein synthesis (top) and number of cycling cells (bottom) in the top 2.5% translating cell populations (OP-purohigh). h, Box plots of protein synthesis in cycling (S/G2/M) and non-dividing (G1/G0) cells in the 2.5% OP-purohigh population isolated from Lgr5-tdTom mice. Shown are all cells (top), tdTom− (Tom−) (middle) and tdTom+ (Tom+) (bottom) cells at the indicated hair cycle stages. **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed Student’s t-test. n = mice. Source Data for this figure is available in the online version of the paper.
a–c, Co-labelling of OP-puro with markers for undifferentiated basal cells: ITGA6 (a), CD44 (b) and PDPN (c) in mouse tumours. Nuclei are stained with DAPI. Arrows indicate low translating and marker-positive cells. Dotted line indicates invasive front of the tumour. Boxed areas are magnified on the right. d, Gating of low, medium and high OP-puro cells in Nsun2+/+ (wild type) and Nsun2−/−; K5-Sos skin tumours analysed in e–g. e, Percentage of OP-purolow cells in tumours from Nsun2+/+ (wild type) and Nsun2−/−; K5-Sos mice. f, g, Flow cytometry for ITGA6 and CD34 in unfractionated epithelial cells from mouse tumours (all cells) or epithelial cells with high, medium and low OP-puro incorporation (f) and quantification (g) (mean ± s.d.; n = 3 mice). h, Flow cytometry for ITGA6 and CD44 in unfractionated epithelial cells from mouse tumours. i, j, Histogram (i) and quantification (j) showing OP-puro incorporation of cells as gated and quantified in h (mean ± s.d.; n = 4 mice). k, l, Detection of endogenous expression of NSUN2 (LacZ) in early (P23) (k) and late (P30) anagen (l) hair follicles. Sections were co-stained with eosin or markers for bulge stem cells K15 and the hair lineages Huxley’s (Hu), cuticle (Ci) (GATA3), and cortex (Co) (LEF1). m–o, Haematoxylin and eosin staining (m) and immunostaining for LEF1 (n), K72 and DLX3 (o) in wild-type (WT) and Nsun2−/− skin at P1. Nuclei are stained with DAPI. Insets: magnified boxed area (1, 2). Scale bars, 50 μm. p, Correlation between proliferation and protein synthesis with differentiation of quiescent (QSC) or committed stem cells (CSC), committed progenitors (CP), differentiating progenitors (DP), and terminally differentiated (TD) cells. Source Data for this figure is available in the online version of the paper.
a, Immunostaining for NSUN2, ITGA6, K10 (differentiation marker), laminin 5α and K8 (tumour progression markers), and Slug (epithelial to mesenchymal transition-related gene) at different stages of DMBA-TPA-induced malignant progression to squamous cell carcinoma (SCC). b–d, Quantification of tumour diameter normalized to body weight (BW) (b), tumours per mouse (c), and mouse life span (d) in K5-Sos/Nsun2+/+ (K5-Sos), K5-Sos/Nsun2+/− and K5-Sos/Nsun2−/− littermates. Measurements start at P16. Data collection discontinued when mice died (indicated by a dagger). Data represent mean, n ≥ 5 mice of each genotype. e, f, Haematoxylin and eosin staining (e) and immunostaining for ITGB1 (f) in sections from K5-Sos (K5-Sos/Nsun2+/+) and K5-Sos/Nsun2−/− skin tumours. b, basal undifferentiated cells; sb, suprabasal layers. Arrows indicate ITGB1+ cells. g, Relative mRNA expression levels of the indicated transcripts in skin tumours (mean ± s.d.; n = mice). h, Flow cytometry using ITGA6 and CD44 in K5-Sos/Nsun2−/− and control K5-Sos (K5-Sos/Nsun2+/+) tumours. i, Percentage of cells in cell populations as gated in h (mean ± s.d.; n = mice). *P < 0.05; ***P < 0.001 (two-tailed Student’s t-test) (i). j, TdT-mediated dUTP nick end labelling (assay) (TUNEL) assay on sections of K5-Sos tumours expressing (K5-Sos/Nsun2+/+) or lacking Nsun2 (K5-Sos/Nsun2−/−). Arrows indicate TUNEL+ (apoptotic) cells. Nuclei are stained with DAPI. Dotted line indciates boundary of epithelia and stroma (f, j). Scale bars: 25 μm (a), 100 μm (e, f, j). Source Data for this figure is available in the online version of the paper.
Extended Data Figure 6 Deletion of Nsun2 enhances self-renewal of tumour-initiating cells in a cell-autonomous manner and NSUN2 expression in human skin tumours.
a, Tumour size after grafting of K5-Sos/Nsun2+/+ (K5-Sos) and K5-Sos/Nsun2−/− tumour cells subcutaneously into nude mice (mean ± s.d.; n = 3 mice). b–f, Histology (haematoxylin and eosin staining) (b), staining for GFP (c), Ki67 (d), ITGB1 (e) and PDPN (f) in grafted tumour sections. Dotted line indicates boundary between epithelia and stroma. Arrows indicate basal and suprabasal expression. Nuclei are stained with DAPI. g–l, Immunohistochemistry for NSUN2 in human normal skin, benign tumours, malignant basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) with increased malignancy (stages classified using the TNM system). Arrows indicate NSUN2high cells. Arrowheads indicate NSUN2low cells. m, Distribution of cells shown in g–l according to NSUN2 protein levels. (n ≥ 3 samples). Scale bars, 100 μm. Source Data for this figure is available in the online version of the paper.
Extended Data Figure 7 NSUN2-dependent RNA methylation of coding and non-coding RNA in mouse tumours.
a, Percentage of NSUN2-methylated sites (>0.15 m5C in Nsun2+/+; <0.05 m5C in Nsun2−/−) out of all covered sites (left) and in non-coding RNA (ncRNA) or introns and exons (right). b, Methylation level in coding and non-coding RNAs (>0.15 m5C in Nsun2+/+; <0.05 m5C in Nsun2−/−; coverage >10 reads). c–e, Examples of NSUN2-targeted non-coding RNA (Rpph1) and mRNA (Elf1 and Dscaml1) in Nsun2+/+ (top) and Nsun2−/− (bottom) tumours. f, Number of NSun2-methylated sites in exons 1 to 60 (top) or distance from the transcriptional start site (TSS) in introns (bottom). Plotted sites: >0.1 m5C in Nsun2+/+; <0.05 m5C in Nsun2−/−; coverage >10 reads. g, No correlation between NSUN2 methylation shown in b and RNA abundance in normal skin or K5-Sos skin tumours. NSun2 is highlighted as a control. h, Venn diagram with no significant overlap between NSUN2-methylation targets shown in b and differentially translated mRNAs (P < 0.05; measured as ribosome density of Nsun2+/+ versus Nsun2−/− tumours). i–l, NSUN2 methylation in tRNAs (>0.15 m5C in Nsun2+/+; <0.05 m5C in Nsun2−/−; coverage >10 reads) (i). Number and location of lost (red) or unchanged (grey) m5C sites in K5-Sos/Nsun2−/− tumours. Nucleotide position in tRNA is shown on the x-axis (j). Examples of NSUN2-targeted tRNAs in Nsun2+/+ (top) and Nsun2−/− (bottom) K5-Sos tumours (k, l). Heatmaps show methylated (red) and unmethylated (grey) cytosines. Cytosines are shown on the x-axis, and sequence reads on the y-axis. Numbers indicate the m5C position in the RNA (c–e; k, l). Bisulfite-seq and RNA-seq data represent average of 4 replicates per condition.
a, Ribosome profiling and RNA-sequencing experiments (see Fig. 5) using Nsun2-expressing (Nsun2+/+) and Nsun2-deficient (Nsun2−/−) K5-Sos skin tumours, or cultured human skin fibroblasts (NSUN2−/−line1 and NSUN2−/−line2 and healthy donors: NSUN2+/−, NSUN2+/+). HTS, high-throughput sequencing. b, Correlation between protein synthesis (ribosome footprint density) in Nsun2+/+ and Nsun2−/− tumours. c, Example of triplet periodicity in ribosome footprints (K5-Sos/Nsun2+/+, replicate 1) shown as number of reads against nucleotide position relative to the translational start site for all ORFs. d, Heatmaps showing ribosome footprint reads around the translational start site (0) in Nsun2+/+ and Nsun2−/− tumours (3 replicates per condition; ribosome density >0; colour indicates RPKM values of footprints). e, Log2 fold change (FC) per transcript in normal skin (left) and tumour samples (right) of significant (P < 0.05) expression differences. Nsun2 RNA levels (red). f–j, Scatter plots, linear regression lines and coefficient of correlation (r2) of mRNA expression and protein synthesis (density of ribosome footprints per kb) in Nsun2+/+ (grey) and Nsun2−/− (red) mouse tumours (f) and human fibroblasts (g–j). k, Venn diagram of transcripts with significant (P < 0.05) different ribosome footprint density in the 5′ UTR in NSUN2+/−, NSUN2−/−line1 and NSUN2−/−line2 human fibroblasts relative to NSUN2+/+ cells. l, Box plots of ribosome footprint read counts in the 5′ UTR (left) and corresponding CDS (right) of the 192 transcripts in k. ****P < 0.0001 (two-tailed Student’s t-test).
a, Venn diagram of transcripts with differential protein synthesis in NSUN2+/− and NSUN2−/− human fibroblasts relative to NSUN2+/+ cells. b, GO terms enriched in 424 commonly differentially translated transcripts in NSUN2−/− lines (a). c, Western blot for NSUN2 and tubulin in NSUN2−/− human fibroblasts rescued with viral constructs expressing wild-type NSUN2 (NSUN2-wt), two catalytically dead mutants (C271A and C321A) or the empty vector. d, Venn diagram of differentially translated transcripts in the indicated rescued cells relative to empty vector-infected control cells. Translation of 173 out of 746 of transcripts (23%) depended on the enzymatic activity of NSUN2. e–g, Differential translation of transcripts relative to NSUN2−/− cells (infected with empty vector) showing reduced translation in the presence of wild-type NSUN2 but not the enzymatic-dead versions of NSUN2 (C271A, C321A), corresponding GO categories (f) and examples (g). h, Boyden chamber migration assay towards epidermal growth factor (EGF) or control medium (ctr) using primary human keratinocytes transduced with a siRNA for NSUN2 (si_NSUN2) or a scrambled construct (si_ctr). Data represent mean ± s.d. (n = 3 assays). Western blot confirms downregulation of NSUN2 in the presence of the siRNA construct. i, j Reduced motility in keratinocytes expressing the enzymatic-dead NSUN2 construct (K190M) (K190M: n = 13; NSUN2: n = 19 cells) (i). Western blot confirms equal protein expression levels of K190M and NSUN2 (j). k, Reduced differentiation in primary human keratinocytes expressing the enzymatic-dead NSUN2 (K190M). Staining for NSUN2, ITGA6 or involucrin (IVL) and nuclei (DAPI). Control: empty vector (left); NSUN2: wild-type NSUN2 (middle); K190: enzymatic-dead NSUN2 (right). Arrows indicate NSUN2-expressing ITGA6−/IVL+ cells. Arrowheads indicate K190M-expressing ITGA6+/IVL− cells. l, Flow cytometry for ITGA6 of keratinocytes transduced with NSUN2 (blue line, top panel), K190M (blue line, bottom panel) or the empty vector (eVector) (red line). Negative control (grey line) represents unstained cells. m, Quantification of IVL+ infected keratinocytes grown in suspension for 24 h to stimulate differentiation. *P < 0.05, **P < 0.01 (two-tailed Student’s t-test) (h–m). Scale bar, 100 μm. Source Data for this figure is available in the online version of the paper.
Extended Data Figure 10 Protein expression differences, drug treatment of Nsun2−/− tumours and graphical summary.
a, b, Western blot analysis of translationally repressed (a) or induced (b) mRNAs in Nsun2−/− (−/−) compared to Nsun2+/+ (WT) skin tumours with quantification of band densitometry on the right (mean ± s.d.; n = 3 mice). *P < 0.05, ***P < 0.001 (two-tailed Student’s t-test). c, d, Control and 5FU-treated tumours, before and after treatment. e, f, Immunohistochemistry for p53 in tumours shown in c, d. g–i, Immunostaining for cleaved caspase 3 (Cl−CASP3) (g), Ki67 (h), ITGA6 and K10 (i) in K5-Sos tumours expressing (+/+) or lacking (−/−) Nsun2 and treated with CDDP (see Methods). Scale bars, 100 μm. j, Graphical summary: (1) quiescent undifferentiated stem and progenitor cells are characterized by the absence of NSUN2 and low global protein synthesis; (2) upregulation of NSUN2 counteracts angiogenin-mediated cleavage of tRNAs through site-specific methylation of tRNAs, allowing increased translation of lineage-specific transcripts driving terminal differentiation; (3) cytotoxic stress inhibits NSUN2 and global protein synthesis in particular of lineage-specific transcripts and promotes an undifferentiated quiescent cell state. Yet cell survival after the insult requires re-methylation of tRNAs by NSUN2 (see (2)); (4) the inability to upregulate NSUN2 in response to the cytotoxic insult leads to cell death. Source Data for this figure is available in the online version of the paper.
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Blanco, S., Bandiera, R., Popis, M. et al. Stem cell function and stress response are controlled by protein synthesis. Nature 534, 335–340 (2016). https://doi.org/10.1038/nature18282
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