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, 534 (7607), 335-40

Stem Cell Function and Stress Response Are Controlled by Protein Synthesis

Affiliations

Stem Cell Function and Stress Response Are Controlled by Protein Synthesis

Sandra Blanco et al. Nature.

Abstract

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.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Extended data Figure 1
Extended data Figure 1. Protein synthesis in epidermal populations.
a, Hair cycle stages and genetic lineage marking using K19- and Lgr5 tdTomato (tdTom) mice. Cell surface markers to isolated bulge stem cells are CD34 and Itgα6. Telogen: stem cells (CD34+/Itgα6+) 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: hair follicle and epidermal basal layer. Arrows: OP-purohigh cells in the hair follicle. Arrowheads: OP-purolow cells in the interfollicular epidermis. Nuclei (DAPI). c, tdTomato and OP-puro detection in back skin of K19tdTom and Lgr5tdTom mice in telogen and late anagen. Arrows: Tomato+ cells. Arrowheads: Tomato+/OP-purohigh cells. Dotted line: lower bulge. Merged panels in Figure 1c-f. e, Hair follicle lineages and differentiation markers used in Figure 1g-j. ORS: outer root sheet; Cp: Companion layer; IRS: inner root sheet; He: Henle’s layer; Hu: Huxley layer; Ci: cuticle of inner root sheet; Ch: cuticle; Co: cortex; Me: Medulla. f, P-cadherin and OP-puro detection in a late anagen. Scale bars: 50 μm.
Extended data Figure 2
Extended data Figure 2. Quantification of protein synthesis in epidermal populations.
a, b, Top 2.5%, 10%, 25%, and 50% translating epidermal cells (OP-purohigh) (a) were sorted for CD34 and Itgα6 (b). c, Protein synthesis in CD34+/α6+, CD34-/α6+ and CD34-/α6- epidermal populations in the top 2.5%, 10%, 25%, 50% or 100% (all) translating cells at indicated hair follicle stages. d, Percentage of CD34+/α6+, CD34-/α6+ and CD34-/α6- cells in the top 2.5%, 10%, 25%, 50% or 100% (all) of translating epidermal cells at indicated stages of the hair cycle. Bars: mean ± s.d. e, f, Violin plots of protein synthesis in top 2.5% OP-purohigh cells in tdTomato-negative (tdTom-) epidermal cells sorted for CD34 and Itgα6 from K19- (e) or Lgr5-tdTom mice (f) at all stages of the hair cycle. (n=mice). Source data: SI_EDF2.
Extended data Figure 3
Extended data Figure 3. Protein synthesis and cell cycle analyses in epidermal cells.
a-c, Violin plots of protein synthesis in indicated epidermal populations sorted for K19- (a) and Lgr5-tdTomato-positive (tdTom+) (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 (upper panel) and G1/G0 (lower panel) using all samples independent of hair cycle stage. Linear regression, correlation coefficient (r2) and P-value are shown. g, Box plots of protein synthesis (upper panel) and number of cycling cells (lower panel) 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 Lgr5tdTom mice. Shown are all cells (upper panel), tdTomato-negative (Tom-) (middle panel) and tdTomato-positive (Tom+) (lower panel) cells at the indicated hair cycle stages. Two-tailed Student’s t-test. **p < 0.01, ***p < 0.001, ****p<0.0001. (n=mice). Source data: SI_EDF3.
Extended data Figure 4
Extended data Figure 4. Protein synthesis in squamous tumours.
a-c, Co-labelling of OP-puro with markers for undifferentiated basal cells: Itgα6 (a), CD44 (b) and PDPN (c) in mouse tumours. Nuclei (DAPI). Arrows: low translating marker+ cells. Dotted line: invasive front of the tumour. Insets: magnified images (a’-c”). d, Gating of low, medium and high OP-puro cells in Nsun2+/+ (wt) and NSun2-/- K5-SOS skin tumours analysed in (e-g). e, Percentage of OP-purolow cells in tumours from Nsun2+/+ (wt) and NSun2-/- K5-SOS mice. f, g, Flow cytometry for Itgα6 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 Itgα6 and CD44 in unfractionated epithelial cells from mouse tumours. i, j, Histogram (i) showing OP-puro incorporation of cells as gated in (h) and quantification (h) (mean ± s.d.; n=4 mice). k, l, Endogenous expression of NSun2 (LacZ) in early (P23) (k) and late (30) 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 & Eosin staining (m) and immunostaining for LEF1 (n), K72 and DLX3 (o) in wild-type (WT) and NSun2-/- skin at P1. Nuclei (DAPI). Insets: magnified boxed area (i, ii). 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: SI_EDF4.
Extended data Figure 5
Extended data Figure 5. NSun2 in mouse skin squamous cell carcinomas.
a, Immunostaining for NSun2, Itgα6, K10 (differentiation marker), Laminin 5 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 deceased (†). Data represent mean, n ≥ 5 mice of each genotype. e, f, Haematoxylin & Eosin staining (e) and immunostaining for Itgβ1 (f) in sections from K5-SOS (K5-SOS/NSun2+/+) and K5-SOS/NSun2-/- skin tumours. b: Basal undifferentiated cells; sb: suprabasal layers. Arrows: Itgβ1+ cells. g, Relative mRNA expression levels of the indicated transcripts in skin tumours (mean ± s.d.; n=mice). h, Flow cytometry using Itgα6 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, TUNEL assay on sections of K5-SOS tumours expressing (K5-SOS NSun2+/+) or lacking NSun2 (K5-SOS NSun2-/-). Arrows: TUNEL+ (apoptotic) cells. Nuclei (DAPI) and dotted line: boundary of epithelia and stroma (f, j). Scale bars: 25 μm (a), 100 μm (e,f,j). Source data: SI_EDF5.
Extended data Figure 6
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 (H&E staining) (b), staining for GFP (c), Ki67 (d), Itgβ1 (e), and PDPN (f) in grafted tumour sections. Dotted line: boundary between epithelia and stroma. Arrows: basal and suprabasal expression. Nuclei (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 (Tumour, Node, Metastases) system). Arrows: NSun2high cells. Arrowheads: NSun2low cells. (m) Distribution of cells shown in (g-l) according to NSun2 protein levels. (n ≥ 3 samples). Scale bars: 100 μm. Source data: SI_EDF6.
Extended data Figure 7
Extended data Figure 7. NSun2-dependent RNA methylation of coding and non-coding RNA in tumours.
a, Percentage of NSun2-methylated sites (>0.15 m5C in NSun2+/+; <0.05 m5C in NSun2-/-) out of all covered sites (left hand panel) and in non-coding RNA (ncRNA) or introns and exons (right hand panel). 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+/+ (upper panels) and NSun2-/- (lower panels) tumours. f, Number of NSun2-methylated sites in exons 1 to 60 (upper panel) or distance from the transcriptional start site (TSS) in introns (lower panel). 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 NSun2-/- K5-SOS tumours. X-axis: nucleotide position in tRNA (j). Examples of NSun2-targeted tRNAs in NSun2+/+ (upper panels) and NSun2-/- (lower panels) K5-SOS tumours (k,l). Heat maps show methylated (red) and un-methylated (grey) cytosines. X-axis: cytosines. Y-axis: sequence reads. Numbers indicate the m5C position in the RNA (c-e;k,l). Bisulphite-seq and RNA-seq data represent average of 4 replicates per condition.
Extended data Figure 8
Extended data Figure 8. NSun2- deletion drives translational changes independent of mRNA expression.
a, Ribosome profiling and RNA sequencing experiments (see Figure 5) using NSun2-expressing (NSun2+/+) and NSun2-deficient (NSun2-/-) K5-SOS skin tumours, or cultured human skin fibroblasts (NSun2-/-Line1 and Line2 and healthy donors: NSun2+/-, NSun2+/+). HTS (High Throughput Sequencing). b, Correlation between protein synthesis (ribosome footprint density) in NSun2+/+ and -/- 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 translation start site for all open reading frames. d, Heat maps showing ribosome footprint reads around the translational start site (0) in NSun2+/+ and NSun2-/- tumours (3 replicates per condition; ribosome density > 0; colour: RPKM values of footprints). e, Log2 fold-change (FC) per transcript in normal skin (left hand panel) and tumour samples (right hand panel) 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 -/- (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 hand panel) and corresponding CDS (right hand panel) of the 192 transcripts in (k). ****p<0.0001 (two-tailed Student’s t-test).
Extended data Figure 9
Extended data Figure 9. RNA methylation-dependent changes of protein synthesis.
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 genes 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 (wt) 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 down-regulation 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, Itgα6 or Involucrin (IVL) and nuclei (DAPI). Control: empty vector (left hand panels); NSun2: wild-type NSun2 (middle panels), K190: enzymatic-dead NSun2 (right hand panels). Arrows: NSun2-expressing Itgα6-/IVL+ cells. Arrowheads: K190M-expressing Itgα6+/IVL- cells. l, Flow cytometry for Itgα6 of keratinocytes transduced with NSun2 (blue line, upper panel), K190M (blue line, lower 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 hours to stimulate differentiation. *p<0.05; **p<0.01 (two-tailed Student’s t-test) (h-m). Scale bar: 100 μm. Source data: SI_EDF9.
Extended data Figure 10
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 5’FU-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), Itgα6 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 characterised by the absence of NSun2 and low global protein synthesis. 2. Up-regulation 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 up-regulate NSun2 in response to the cytotoxic insult leads to cell death. Source data: SI_EDF10.
Figure 1
Figure 1. Hair follicle stem cells synthesize less protein than their progeny.
a, Epidermal populations analyzed. IFE: interfollicular epidermis, SG: sebaceous gland, BG: bulge, HG: hair germ, DP: dermal papilla. b, Treatment regimes. c-f, Detection of tdTomato (tdTom) and OP-puro in back skin of tdTom mice in telogen (c,d) and late anagen (e,f). Arrows: tdTom+ cells (magnification lower panels). Arrowheads: tdTom+/OP-purohigh cells. Dotted line: lower bulge. g-j, OP-puro and hair follicle lineage markers (late anagen). Dotted lines: cross section (i, ii). k, Schematic summary of (g-j). OP-puro+ layers (green). Scale bars: 50 μm.
Figure 2
Figure 2. Protein synthesis correlates with differentiation.
a-c, Experimental set up. d-f, Violin plots of normalized protein synthesis in OP-purohigh cells sorted for indicated epidermal populations (c). Itgα6: α6. g, Ki67 and OP-puro detection (late anagen). Arrowheads: Ki67-/OP-puro+ cells. Scale bar: 50 μm. h, Box plots of protein synthesis in cycling (S/G2/M) and non-dividing (G1/G0) OP-purohigh cells. n=mice. *p<0.05, **p < 0.01, ***p < 0.001, ****p<0.0001 (Two-tailed Student’s t-test). Source data: SI_Fig2.
Figure 3
Figure 3. Tumour-initiating cells synthesize less protein than their progeny.
a-c, Co-labeling OP-puro with indicated markers. Arrows: marker+ cells. d, Flow cytometry for OP-puro incorporation. e, Percentage of dividing cells (S/G2/M) and normalized protein synthesis (mean± s.d.; n=mice). f, Co-staining OP-puro, NSun2, Itgα6. Arrows: OP-puro+/NSun2+ cells. g-i, OP-puro-detection in sections (g, h) or by flow cytometry (i). Arrowheads: OP-purolow cells. Nuclei: DAPI. Scale bars: 50 μm. Dotted line: basal membrane. All analyses are in K5-SOS tumours. Source data: SI_Fig3.
Figure 4
Figure 4. NSun2-deletion promotes stem cell identity and tumourigenesis.
a, Tumour incidence. †: Deceased. b, c, Detection (b) and quantification (c) of pulsed-chased BrdU+ and EdU+ cells in tumours (see Methods). n = 5 slides x 3 mice. d, e, Immunostaining for Itgα6, K10 (d) and PDPN (e). Arrows: marker+ cells. Nuclei (DAPI); dotted line: basal membrane; scale bars: 100 μm. f-i, Flow cytometry (f, g) and quantification (h, i) of marker+ tumour cells. n=mice. j, NSun2 protein expression in human normal skin or tumours (mean ± s.d.). *p < 0.05; ***p< 0.001 (two-tailed Student’s t-test). Source data: SI_Fig4.
Figure 5
Figure 5. NSun2-deletion imposes distinct translational programmes.
a, b, Percentage of NSun2-methylated cytosines (dark red). c, Un-methylated tRNAs are cleaved and 5’tRNA fragments accumulate. d, Relative frequencies of tRNA fragments in tumours. e, No correlation between changes in protein synthesis and RNA expression in tumours. f, g, Significantly (p<0.05) changed 5’UTR ribosome densities in tumours (f) and human NSun2-/- lines (excluding significant (p<0.05) changes in NSun2+/-) (g) and corresponding CDS. ****p<0.0001 (two-tailed Student’s t-test). h, i, Frequency of ribosomal density values (p-value<0.05 and abs(log2FC)>0) (h) and Gene Ontology (GO) categories in tumours. j, GO categories of significant (p<0.05) changed 5’UTRs in NSun2-/- lines versus +/-. Data: average of 4 replicates per condition in human (g, j) and mouse (a-d) or 3 in (e,f,h,i).
Figure 6
Figure 6. NSun2-deletion sensitizes tumour-initiating cells to cytotoxic stress.
a-c, Tumour size (mean ± s.e.m) (a) and Ki67 detection (b, c) in control (Ctr) or 5’FU-treated mice. d, K10 and Itgα6 detection in treated NSun2-/- tumours. Arrows: K10+/Itgα6+-positive cells. e, Tumour size in mice treated with 5’FU and/or angiogenin inhibitor (AI). f, Quantification of tumour-initiating cells in tumours shown in (e) (mean ± s.d.). *p < 0.05; **p< 0.01 (two-tailed Student’s t-test). n=mice. Scale bars: 100 μm. Source data: SI_Fig6.

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