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. 2015 Jul 31;11(7):e1005410.
doi: 10.1371/journal.pgen.1005410. eCollection 2015 Jul.

The Shelterin TIN2 Subunit Mediates Recruitment of Telomerase to Telomeres

Amanda K Frank  1 , Duy C Tran  1 , Roy W Qu  1 , Bradley A Stohr  2 , David J Segal  3 , Lifeng Xu  1
Affiliations

The Shelterin TIN2 Subunit Mediates Recruitment of Telomerase to Telomeres

Amanda K Frank et al. PLoS Genet. .

Abstract

Dyskeratosis Congenita (DC) is a heritable multi-system disorder caused by abnormally short telomeres. Clinically diagnosed by the mucocutaneous symptoms, DC patients are at high risk for bone marrow failure, pulmonary fibrosis, and multiple types of cancers. We have recapitulated the most common DC-causing mutation in the shelterin component TIN2 by introducing a TIN2-R282H mutation into cultured telomerase-positive human cells via a knock-in approach. The resulting heterozygous TIN2-R282H mutation does not perturb occupancy of other shelterin components on telomeres, result in activation of telomeric DNA damage signaling or exhibit other characteristics indicative of a telomere deprotection defect. Using a novel assay that monitors the frequency and extension rate of telomerase activity at individual telomeres, we show instead that telomerase elongates telomeres at a reduced frequency in TIN2-R282H heterozygous cells; this recruitment defect is further corroborated by examining the effect of this mutation on telomerase-telomere co-localization. These observations suggest a direct role for TIN2 in mediating telomere length through telomerase, separable from its role in telomere protection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Heterozygous TIN2-R282H mutation induces progressive telomere shortening, but not gross telomere deprotection, in HCT116 cells.
(A) Telomeres were maintained in cells with wild-type TIN2, but progressively shortened in cells with heterozygous TIN2-R282H mutation. Cells were continuously passaged and collected at the indicated population doublings (PD). Genomic DNAs were extracted and bulk telomere lengths were examined by Telomere Restriction Fragment analysis using a telomeric repeat probe. (B) Growth curve of HCT116 knock-in cells. (C) Telomeric localization of TIN2 examined by immunofluorescence-FISH analysis in cells carrying heterozygous TIN2 mutation (clone R282H.1, PD8 cells) and wild-type TIN2 (clone WT.1, PD8 cells). For immunofluorescence staining, we used an anti-TIN2 antibody (Imgenex) (produced against an N-terminal epitope of TIN2 (a.a. 44–58)) which recognizes both the wild-type and mutant TIN2. Telomeres were detected by FISH using a PNA telomeric probe. (D) Telomeric localization of TRF1, TRF2 and TPP1 examined by immunofluorescence-FISH analysis in cells carrying heterozygous TIN2 mutation (clone R282H.1, PD8 cells). (E) Left panel: quantification of fraction of telomeres co-localizing with TPP1. Each circle on the graph represents a single nucleus. Telomeres in 10 nuclei were examined for each knock-in clone. Mean ± SD indicated by red lines. Right panel: TPP1 fluorescence intensity, normalized against telomere length (based on telomere FISH signal intensity). Each circle on the graph represents a single telomere. Mean ± SD indicated by red lines. Telomeres in 10 nuclei were analyzed for each clone. (F) Quantification of telomere dysfunction-induced DNA damage foci (TIFs) in early (PD3) and late PD (PD51 and PD76) HCT116 knock-in clones. For positive control, R282H.1 cells were treated with shRNA against TRF2 and TIFs quantified. DNA damage was assessed by immunostaining with an antibody against 53BP1 and telomeres were detected by PNA FISH using a telomeric probe. All quantifications were carried out blindly. Each circle on the graph represents a single cell. Mean ± SD indicated by red lines. TIFs in 100 cells were analyzed for each sample. (G) Quantification of telomeric abnormalities from metaphase spreads of HCT116 knock-in clones. Cells were collected at indicated PDs for metaphase spreading followed by FISH analysis using a PNA telomeric probe and a centromeric probe. ~4000 telomeres were analyzed for each sample. All quantifications were carried out blindly.
Fig 2
Fig 2. Telomerase inhibition in TIN2-R282H heterozygotes and TIN2-WT cells leads to similar rates of telomere erosion.
(A) Representative TRAP assay results from HCT116 knock-in clones infected with lentivirus expressing dominant-negative telomerase catalytic subunit (DN-hTERT) or luciferase control. Whole cell extracts from 500 and 100 cells at PD21 were analyzed for each line. (B) Telomere Restriction Fragment analysis of HCT116 knock-in clones. Cells from each infection were pooled, continuously passaged and collected at indicated population doublings (PD). (C) Left panel: Mean telomere lengths in (B) were determined by the ImageQuant software and plotted against PDs. Right panel: Quantification of changes in mean telomere length between PD8–28.
Fig 3
Fig 3. Heterozygous TIN2-R282H mutation decreases the frequency of telomere extension by telomerase.
HCT116 knock-in clones were infected with lentivirus expressing 47A-hTER to achieve a 1:1 steady state expression level of 47A-hTER: endogenous-hTER in each clone. 8 days after infection, parallel cultures of cells were collected for metaphase spreads followed by telomeric FISH, for quantitative PCR, for TRAP assay, and for counting of cell numbers. (A) Representative telomeric FISH images showing 47A-hTER-directed incorporation of TTTGGG variant repeats at telomeres in WT.1 cells. Cells were infected with an empty lentiviral vector control or lentivirus expressing 47A-hTER. Telomeric FISH was carried out using PNA probes for the canonical TTAGGG repeats (green) and the variant TTTGGG repeats (red). Telomeres incorporating TTTGGG repeats were marked with yellow arrows. Regions encircled in white boxes are enlarged at the bottom of the corresponding image for better visualization. (B) Relative steady state expression levels of 47A-hTER determined using QPCR. Expression levels were normalized to GAPDH and relative to respective endogenous telomerase RNA levels in each clone. Bars represent mean values of three experiments and SDs. (C) 47A-hTER assembles into equivalent levels of active telomerase in all knock-in clones. Top panel: whole cell extracts were examined for 47A-hTER-containing telomerase activity by 47A-hTER-specific TRAP assay using return primer 5’-GCGCGGTACCCATACCCATACCCAAACCCA-3’. Extracts from 400 and 100 cells were analyzed for each sample. WT.1 cells infected with the empty lentiviral vector was used as control to show the specificity of the TRAP assay conditions. Bottom panel: Quantification of relative TRAP activity of 47A-hTER-containing telomerase in each indicated clone, relative to that in R282H.1–47A cells. Bars represent mean values of three experiments and SDs. (D) Endogenous wild-type telomerase activity in knock-in clones infected with an empty lentiviral vector or lentivirus expressing 47A-hTER. Top panel: the same whole cell extracts as described in (C) were examined for endogenous wild-type telomerase activity using return primer 5’- GCGCGGTACCCTTACCCTTACCCTAACCCT-3’. Extracts from 100 and 25 cells were analyzed for each sample. Bottom panel: Quantification of relative endogenous wild-type telomerase activity in each indicated clone, relative to respective clones infected with a lentiviral vector control. Bars represent mean values of three experiments and SDs. (E) Quantification of fraction of telomeres incorporating TTTGGG repeats in knock-in clones expressing 47A-hTER. Data were obtained analyzing > 2500 chromosomes in ~60 metaphase spreads from each clone. All quantifications were carried out blindly. Each point on the graph represents a single metaphase spread. Mean values are indicated in red. *** (p≤0.001) ** (p≤0.01) calculated by two-tailed Student’s t-tests. (F) Quantification of telomeric TTTGGG fluorescence intensity. The distributions of fluorescence intensities, in arbitrary fluorescence unit, of more than 600 telomeric TTTGGG spots from metaphase spreads of each indicated clone are displayed. All quantifications were carried out blindly.
Fig 4
Fig 4. TIN2-R282H mutation reduces colocalization between endogenous telomerase RNA and telomeres.
(A) Immunostaining-FISH images of WT.1 cells showing co-localization between telomerase RNA and telomeres. Telomeres were identified by immunostaining using a mix of anti-TRF1 and anti-TRF2 antibodies. Telomerase RNA was labeled by RNA FISH. DNA was stained by DAPI. Telomerase RNA spots (red) co-localizing with telomeres (green) are marked by yellow arrows. (B) Quantification of telomerase RNA and telomere co-localization in HCT116 knock-in clones. Plot shows the average number of hTER-telomere co-localization per cell. 25 images (~8–15 cells per image) were taken randomly for each cell line at PD8. All quantifications were carried out blindly. Each point on the plot represents value obtained from one image. Mean values are indicated in red. *** (p≤0.001) ** (p≤0.01) calculated by two-tailed Student’s t-tests.
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