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Thymidine nucleotide metabolism controls human telomere length

Nature Genetics volume 55, pages 568–580 (2023)Cite this article

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A Publisher Correction to this article was published on 10 May 2023

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Abstract

Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed genome-wide CRISPR–Cas9 functional telomere length screening and identified thymidine (dT) nucleotide metabolism as a limiting factor in human telomere maintenance. Targeted genetic disruption using CRISPR–Cas9 revealed multiple telomere length control points across the thymidine nucleotide metabolism pathway: decreasing dT nucleotide salvage via deletion of the gene encoding nuclear thymidine kinase (TK1) or de novo production by knockout of the thymidylate synthase gene (TYMS) decreased telomere length, whereas inactivation of the deoxynucleoside triphosphohydrolase-encoding gene SAMHD1 lengthened telomeres. Remarkably, supplementation with dT alone drove robust telomere elongation by telomerase in cells, and thymidine triphosphate stimulated telomerase activity in a substrate-independent manner in vitro. In induced pluripotent stem cells derived from patients with genetic telomere biology disorders, dT supplementation or inhibition of SAMHD1 promoted telomere restoration. Our results demonstrate a critical role of thymidine metabolism in controlling human telomerase and telomere length, which may be therapeutically actionable in patients with fatal degenerative diseases.

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Fig. 1: Telomere length CRISPR–Cas9 screening reveals that dT nucleotide metabolism genes are required for telomere length control.
Fig. 2: dT treatment drives telomerase-dependent telomere lengthening in human cells.
Fig. 3: dT nucleotide metabolism genes control dT-mediated telomere lengthening.
Fig. 4: SAMHD1 restricts human telomere length and limits dT-mediated telomere elongation.
Fig. 5: Telomere lengthening from dT treatment occurs without inhibiting cell growth.
Fig. 6: Induction of replication stress is insufficient to explain telomere lengthening from dT treatment.
Fig. 7: dT nucleotides enhance human telomerase activity independent of dTTP’s role as a telomerase substrate.
Fig. 8: dT supplementation or SAMHD1 knockdown drives telomere lengthening in iPSCs from patients with TBDs.

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Data availability

The sgRNA library sequencing data and T-free TRAP sequencing data used for analysis have been deposited in the Sequence Read Archive and are available via BioProject accession code PRJNA851386. Source data are provided with this paper.

Code availability

The MATLAB script used to analyze the T-free TRAP sequencing data has been posted to a public repository76. Version 1.0 was used in this manuscript (https://doi.org/10.5281/zenodo.7607615).

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Acknowledgements

We thank the patients and families for research participation. We thank R. Mathieu and the Harvard Stem Cell Institute (HSCI)-Boston Children’s Hospital (BCH) Flow Cytometry Research Lab; the Molecular Biology Core Facilities at the Dana-Farber Cancer institute (for high-throughput sequencing support); J. Asara and the Beth Israel Deaconess Medical Center Mass Spectrometry Core Facility; and A. Shimamura, M. Fleming and the BCH Bone Marrow Failure/Myelodysplastic Syndrome Registry (National Institutes of Health (NIH) grant R21DK099808). We thank L. Zon and Y. Fong for critical input. We thank A. Gutierrez and K. Bodaar for guidance and support with the CRISPR–Cas9 screening. We thank D. Moon for generating the TERC-null 293T cell line. We acknowledge the following funding sources: NIH grants T32GM007226, T32GM007753 and T32GM144273 (to W.M.), NIH grants R01DK107716 and R33HL154133 (to S.A.), the BCH Translational Research Program, HSCI, Team Telomere, the Million Dollar Bike Ride/Penn Medicine Orphan Disease Center and philanthropic gifts (to S.A.). This content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Division of Hematology/Oncology and Stem Cell Program, Boston Children’s Hospital, Boston, MA, USA

    William Mannherz & Suneet Agarwal

  2. Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    William Mannherz & Suneet Agarwal

  3. Biological and Biomedical Sciences Program, Harvard/MIT MD-PhD Program, Harvard Stem Cell Institute, Harvard Initiative for RNA Medicine, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    William Mannherz & Suneet Agarwal

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S.A. and W.M. conceived of the study and designed the experiments. W.M. performed the experiments and analyzed the data. S.A. and W.M. wrote the manuscript.

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Correspondence to Suneet Agarwal.

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S.A. and W.M. are named as inventors on provisional patent application 63/394,588 relating to the data shown.

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Extended data

Extended Data Fig. 1 Telomere length CRISPR/Cas9 screening using flow-FISH.

a-c, Histogram of GFP fluorescence from K562 cells (a), K562 cells transduced with the pXPR-011 vector which expresses eGFP and an sgRNA targeting eGFP (b), and K562 cells expressing Cas9 and transduced with the pXPR-011 vector, 13 days post transduction (c). Presence of GFP-negative cells in c indicates functional Cas9 nuclease activity. d-g, Representative gating strategy for flow-FISH telomere length screening. Data from nucleotide metabolism library infected K562 cells, replicate 1. Cells are gated to enrich for single cells (e, f), and gated on low DAPI fluorescence to enrich for cells with 2 N genome copy number and aid in identifying sgRNAs which promote telomere elongation independent from changes in total DNA content (g) followed by gating on high and low TelC-Alexa 647 probe fluorescence populations. Gates adjusted to maintain approximately 5% of cells throughout the duration of the sort. h, i, sgRNA enrichment in high (h) and low (i) telomere fluorescence populations compared to unsorted populations from K562 cells expressing Cas9 that were infected with the Brunello sgRNA library and then cultured for 49 days followed by flow-FISH sorting of the 5% of cells with the highest and lowest telomere fluoresence in two replicates performed on consecutive days. Enrichment score calculated using the MAGeCK RRA software. Known telomere length regulating genes indicated with orange dots; other genes indicated are involved in nucleotide metabolism. j, KEGG pathway enrichment analysis performed on the genes with sgRNAs enriched in the sorted short telomere population (i), analysis performed using the MAGeCKFlute software package (see Methods). Plot includes top enriched KEGG terms, plotting -log10 adjusted P value, which includes q-value estimation for false discovery rate control; dot size indicates number of genes identified in that pathway out of the short telomere enriched genes.

Extended Data Fig. 2 Characterization of TERC-null 293T cells.

a, Schematic of TERC genotypes in TERC-null 293T cells generated by genome-editing, including a deletion of the essential box H domain on one allele, and an 821-bp TERC locus deletion that encompasses 74 bp from the 3’ end of TERC on the other allele. b, Ethidium bromide stained agarose gel of PCR of 293T or 293T TERC-null genomic DNA using primers flanking the deletions indicated in a. c, Sanger sequencing of gel-purified PCR products from the (1) higher molecular weight bands in b, indicating that the non-deleted allele lacks the box H domain, and (2) the ∆821 bp deleted band from b, with trace file showing the deletion junction in a genomic context. d, RT-qPCR of TERC expression relative to GAPDH in wild-type 293T and TERC-null 293T cells, performed in technical triplicate. P value calculated by unpaired t test. Data are shown are means and error bars indicate standard deviation. e, Telomerase activity measured via the TRAP assay, performed on 5-fold serial dilutions of lysates. HI indicates heat-inactivated lysate. IC indicates the internal control product. f, TRF of wild-type and TERC-null 293T cells. Days of culture were recorded beginning approximately two months after gene editing. Telomere length gradually declines with passage until cells universally senesce. g, Quantification of f, line fit using simple linear regression. Data presented in this figure are the results of single experiments unless otherwise indicated.

Extended Data Fig. 3 dT nucleotide metabolism perturbations and their effects on telomere length and polar metabolite homeostasis.

a, TRF of 293T cells treated with the indicated dose of dT for 10 days. The 0 µM dT lane is the same image as the rightmost lane in Fig. 2f. Manufacturer 2, Santa Cruz Biotechnology; Manufacturer 3, MP Biomedicals. b,c, Genomic DNA from 293T or K562 cells manipulated with the indicated sgRNA(s) followed by dT treatment was PCR amplified using primers specific to the TK1 (b) or TK2 (c) genomic loci. Amplicons were separated by agarose gel electrophoresis, demonstrating the three pooled sgRNAs targeting either TK1 or TK2 generated on-target genomic deletions. First lane is a molecular weight marker. PCR products were Sanger sequenced and editing efficiency was quantified using the Synthego ICE algorithm (shown as ‘Knockout Score’). The model fit of the ICE quantification is also displayed. Genomic DNA used in this figure was the same as the DNA used to generate Fig. 3b–e. d, Quantification of Fig. 3b. n = 3 biological replicates for each cell line, P value calculated using paired two-sided t test. e, Polar metabolite profiling by liquid chromatography mass spectrometry of 293 T cells treated with or without 100 µM dT for 24 hours, performed in biological triplicate. P value calculated by unpaired two-sided Student’s t test of average signal intensity in treatment vs. control samples; nucleotide and nucleoside species detected in all samples displayed. Note: dGTP not detected. f, TRF of 293T cells treated with the indicated compound for 10 days. dU, deoxyuridine. g, Detection of CRISPR/Cas9 editing of TYMS locus using TYMS-specific primers, performed as in b and c. Genomic DNA used was the same as the DNA used to generate data in Fig. 3g–j. Data in d and e are shown as means, and error bars indicate standard deviation.

Extended Data Fig. 4 Manipulation of SAMHD1 levels by CRISPR/Cas9, shRNA, and lentiviral expression.

a, Immunoblot of 293T and K562 cells electroporated with Cas9 and the indicated sgRNA(s) using primary antibodies against SAMHD1 or β-Actin, corresponding to cell lines evaluated in Fig. 4b. b, Immunoblot of K562 cells transduced with vectors expressing the indicated shRNA, corresponding to cell lines evaluated in Fig. 4c. c, qRT-PCR of SAMHD1 expression compared to β-Actin, performed in technical triplicate. Means of the replicates are shown. d, Immunoblot of 293T cells transduced with vectors expressing the indicated shRNA. e, TRF of indicated cell lines transduced with the indicated shRNA and cultured for 15 days, and quantification of the TRF using the WALTER webtool. The boxplot displays the 75th, 50th and 25th percentile molecular weight of the telomere signal distribution in the TRF blot. f, Immunoblot of 293T cells transduced with vectors to overexpress either eGFP or the indicated SAMHD1 variant and treated with the indicated dose of dT, corresponding to cell lines evaluated in Fig. 4i, j. Data presented in this figure are the results of single experiments unless otherwise stated. Full-length western blots are presented as source data.

Source data

Extended Data Fig. 5 Cell cycle assessment of cells treated with dT.

a–d, DAPI staining of 293T (a, b) and K562 (c, d) cells treated with the indicated dose of dT for 7 or 8 days, respectively, measured by flow cytometry, plotted as histograms of DAPI intensity, displaying representative samples from each treatment arm (a, c), and the percentage of cells in different stages of the cell cycle (b, d) gated based on the lines drawn on the histogram, gates determined based on untreated samples. n = 2 biological replicates for 293T cells treated with 200 µM dT and n = 3 biological replicates for all other conditions. Data presented are means; error bars indicate standard deviation.

Extended Data Fig. 6 Evaluation of telomere length and cell cycle progression changes from treatment with dT, aphidicolin, 5FU, or hydroxyurea.

a, TRF Southern blot of 293T cells treated with the indicated doses of aphidicolin and hydroxyurea for 10 days. b, TRF Southern blot of 293T TERC-null cells transfected with TERT in addition to the indicated vector, cultured for 18 hours, then treated with the indicated dose of dT for 30 hours. c, TRF Southern blot of 293T TERC-null cells transfected with TERT in addition to the indicated vector, cultured for 18 hours, then treated with the indicated dose of dT for two days. d, TRF Southern blot of 293T TERC-null cells transfected with the indicated expression vectors, cultured for 18 hours, then treated with the indicated dose of dT for five days. e–h, Cell cycle analysis by DAPI staining and flow cytometry of 293T TERC-null cells transfected with TERC and TERT expression vectors, cultured for 18 hours, then treated with the indicated of dose of dT (e), aphidicolin (f), 5FU (g), or hydroxyurea (h), displayed as histograms of DAPI intensity of representative samples from each treatment arm, corresponding to cells in Fig. 6b–l. Gating based on untreated cells. TRFs presented in this figure show the results of single experiments.

Extended Data Fig. 7 T-free telomerase is sensitive to dT nucleotide manipulations.

a, Representative modified TRAP assay performed on super-telomerase extracts using the indicated dose of dTTP and physiologic levels of dATP, dCTP and dGTP (see Methods). b, Quantification of a. n = 2 biological replicates. c, GGAAAG TRAP assay performed on lysates from 293T TERC-null cells overexpressing T-free super-telomerase demonstrates linearity between cell input amount and telomerase signal. Five-fold serial dilutions performed. HI, heat inactivated. d, Quantification of lanes 1–3 from c. e, Representative modified GGAAAG TRAP assay performed on super-telomerase extracts generated using the indicated TERC vector. Assay performed with the indicated dose of dTTP and physiologic levels of dATP, dCTP and dGTP (see Methods). HI, heat inactivated. f, Quantification of e using two-sided unpaired Student’s t test; n = 3 biological replicates. g, Representative modified GGAAAG TRAP assay performed on T-free super-telomerase extracts supplemented with the indicated dose of dTTP and physiologic levels of dATP and dGTP. h, Quantification of g as in f, n = 3 biological replicates. i, Diagram of GGAAAG TRAP product sequencing and analysis strategy. Note * indicates T’s encoded by the partially complementary reverse primer, preventing analysis of base composition in that portion of the read. j, Quantification of base pair composition of representative GGAAAG TRAP products from g with 0 μM or 25 μM dTTP by nanopore sequencing (see Methods). Bits of information calculated using Shannon entropy and plotted using ggseqlogo. k, Quantification of base pair composition of GGAAAG TRAP products from g using nanopore sequencing (see Methods). P value calculated using two-sided Student’s t test; n = 3 biological replicates. l, Quantification of Fig. 7d, plotting the signal in the indicated telomerase product repeat relative to the signal of the corresponding repeat in the lane without dTTP added, normalized for loading (see Methods). m, TRF Southern blot of 293T TERC-null cells transfected with TERT in addition to the indicated vector, cultured for 18 hours, then treated with the indicated dose of dT for 30 hours, and probed with a GGTTAG complementary probe. Lanes 1–4 are the same blot shown in Extended Data Fig. 6b. n, Blot from m was stripped and re-probed with a probe complementary to the GGAAAG repeat. o, Slot blot of DNA from 293T TERC-null cells overexpressing eGFP and TERT showing linear relationship between DNA input and signal; rows are technical triplicates. p, Quantification of o. q, Slot blot of DNA from 293T TERC-null cells overexpressing T-free super-telomerase; rows are technical triplicates. r, Quantification of q. s, Slot blot of DNA from 293T-TERC null cells transfected with TERT and TERC, cultured for 18 hours, then treated with dT as indicated for 30 hours. Denatured DNA for each sample was split and loaded onto parallel blots, which were probed for the indicated target. Performed in technical triplicate. t, Quantification of s. P values calculated with one way ANOVA using Dunnett’s multiple comparisons test for each probe. For b, d, f, h, k, p, r and t, the mean of the data is presented and error bars indicate s.d.; ns, P > 0.05.

Extended Data Fig. 8 Effects of dT on iPSC cell cycle progression and replication stress signaling.

a, Cell cycle analysis of wild-type iPSCs cultured in the indicated of dose of dT for 24 hours, measured by DAPI staining and flow cytometry, displayed as histograms of DAPI intensity. n = 2 biological replicates; the mean of the replicates is presented. b, Representative histograms of DAPI signal for cells in a. Gates defined based on untreated cells. c, Immunoblot of cells treated as in a; all images of the same membrane blotted with the indicated primary antibodies. UV- treated cells used as a positive control. Blot shows the results from a single experiment. Full-length blots are provided as source data.

Source data

Extended Data Fig. 9 Model of relationship between dT nucleotide metabolism and telomere synthesis.

a-e, Schematics illustrate conditions of homeostasis (a), dT supplementation (b), dU supplementation (c), loss of SAMHD1 (d), and treatment with hydroxyurea or 5-fluorouracil (e).

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Source Data Fig. 6

Unprocessed western blots.

Source Data Fig. 7

Unprocessed gels.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 8

Unprocessed western blots.

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Mannherz, W., Agarwal, S. Thymidine nucleotide metabolism controls human telomere length. Nat Genet 55, 568–580 (2023). https://doi.org/10.1038/s41588-023-01339-5

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