Naked mole rat TRF1 safeguards glycolytic capacity and telomere replication under low oxygen

Abstract

The naked mole rat (NMR), a long-lived and cancer-resistant rodent, is highly resistant to hypoxia. Here, using robust cellular models wherein the mouse telomeric protein TRF1 is substituted by NMR TRF1 or its mutant forms, we show that TRF1 supports maximal glycolytic capacity under low oxygen, shows increased nuclear localization and association with telomeres, and protects telomeres from replicative stress. We pinpoint this evolutionary gain of metabolic function to specific amino acid changes in the homodimerization domain of this protein. We further find that NMR TRF1 accelerates telomere shortening. These findings reveal an evolutionary strategy to adapt telomere biology for metabolic control under an extreme environment.

Fig. 1. NMR TRF1 improved glycolytic capacity at low oxygen.

(A) Unique amino acid changes of NMR TRF1 (in red) in conserved sequences. The first row shows amino acids involved in TRFH dimerization (in red) and TIN2 binding cavity (in brown). The last two rows show mutGLAD and mutTIN mutations in the mouse sequence. NMR, naked mole rat; DMR, Damaraland mole rat; BMR, blind mole rat. (B) Dimerization of human TRFH, with the TRF binding motif (TBM) of TIN2 shown in orange. Enlarged details of TRFH of TRF1 show the surface of TIN2 binding cavity, with Ala¹⁰⁵ in purple and TIN2 TBD in orange sticks. Models were prepared with I-TASSER (iterative threading assembly refinement) based on 3BQO. (C) Analysis of glycolysis: Kinetics of ECAR in MEFs with TRF1^F/F + recombinase (CRE) + ectopic TRF1 variants and of MEFs with p53^−/− CRISPR-Cas9 “WT” and “GLAD” in response to glucose and oligomycin and 2-DG at atmospheric oxygen and 1% oxygen. The ECAR value was not normalized, and each data point represents the mean ± SD (n = 3 to 5). *P ≤ 0.05 (Wilcoxon-Mann-Whitney test), calculated glycolytic capacity based on the difference between the maximum ECAR following oligomycin injection and the last rate measurement before glucose injection in the case of NMR TRF1 and mouse TRF1 and CRISPR-Cas9 GLAD and WT.

Fig. 2. NMR TRF1 displays enhanced localization at and binding to telomeres compared to mouse TRF1.

(A and B) Representative images (A) and quantification (B and C) of colocalization between flag-TRF1 foci (green) and telomeric repeats foci (purple) under atmospheric O₂ and 3% O₂ for MEFs with TRF1^F/F + CRE + ectopic TRF1 variants (B) and for MEFs with p53^−/− CRISPR-Cas9 WT and “GLAD” (C) . (D) Representative image and (E and F) quantification of ChIP analyses for TRF1 binding to telomeric repeats (E) or to SINE B elements (F) in MEFs with SV40-LT TRF1^F/F + CRE + TRF1 rescue based on NMR or mouse TRF1 orthologs or TRF1 variants. Immunoprecipitation with flag antibody; bead controls were spotted onto slot blots and hybridized with a telomeric probe. Quantification of four independent experiments (in duplicate for each) that were performed for NMR and mouse TRF1 samples. Two independent experiments (in duplicate) were performed for mutGLAD and for telomeric repeats, and one experiment (in duplicate) was performed for other samples. Statistical analyses: Student’s t test, n ≥ 30 samples, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; n.s. not significant.

Fig. 3. NMR TRF1 enhances telomere protection under low oxygen and during replication.

(A and D) Quantification of TIF of cells under 3% O₂ and after aphidicolin treatment revealed by γH2A.X staining (A) or without drug treatment (D). (B and C) Representative images (B) and quantification (C) of the MTS rate per chromosome spreads. Cells for (A) to (D) experiences: MEFs with SV40-LT TRF1^F/F + CRE + TRF1 rescue based on NMR or mouse TRF1 orthologs or TRF1 variants. (E) Quantification of TIFs, without the drug and with aphidicolin (+ APH) treatment revealed by γH2A.X staining at atmospheric oxygen and under 3% oxygen in MEFs with p53^−/− CRISPR-Cas9 WT and GLAD. Statistical analyses: Student, Wilcoxon-Mann-Whitney, and ANOVA tests, n ≥ 30 samples, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Fig. 4. NMR TRF1 supports improved TRF1 and TIN2 nuclear localization and limits telomere elongation.

(A to D) Representative images (A) and quantification (B and D) of colocalization between flag-TRF1 foci (green) and myc-TIN2 (purple) (B); quantification of total number of flag-TRF1 foci in nucleus without and with ectopic myc-TIN2 expression (C) and of total number of myc-TIN2 foci in nucleus (D) in MEFs with SV40-LT TRF1^F/F following KO of endogenous TRF1 by CRE and rescue by ectopic NMR or mouse TRF1 orthologs under atmospheric oxygen or at 3% oxygen. (E) Telomere length distribution averages are represented by the shape of each beanplot, with small vertical lines that represent the mean of telomere length for each metaphase spread. Statistics: Student and ANOVA tests, n ≥ 30 samples, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. AU, arbitrary units.