TINF2 is a haploinsufficient tumor suppressor that limits telomere length
- Laboratory for Cell Biology and Genetics, Rockefeller University, United States
- Department of Human Genetics, Radboud University Medical Center, Netherlands
- Department of Molecular and Cellular Biology, University of California, Berkeley, United States
- Chan Zuckerberg Biohub, United States
Figures
Figure 1 with 2 supplements
Germline mutations in TINF2 identified in individuals with multiple malignancies.
(A) TINF2 mutations and clinical features of affected individuals in four different families. Telomere length percentile is based on Flow-FISH data (see below Figure 5—figure supplement 1A). (B, C) …
Figure 1—figure supplement 1
Pedigrees of two c.604G > C TINF2 families.
Pedigrees of two families with heterozygous c.604G > C mutations listed in Figure 1A. Probands are highlighted by arrows. Filled symbols indicate patients with confirmed TINF2 mutations and clinical …
Figure 1—figure supplement 2
Somatic mutations in the COSMIC cancer gene census identified in malignancies in TINF2 mutation carriers.
Table showing the somatic mutations identified in the tumors of families with TINF2 mutations.
Figure 2
Molecular analysis of transcripts resulting from TINF2 mutations.
(A) Transcript analysis in peripheral blood lymphocytes (with and without cycloheximide treatment, CHX) from patients with the c.604G > C TINF2 mutation (F1:III-3 and F1:II-4; see Figure 1A) and a …
Figure 3
Truncated TIN2 versions show altered binding to shelterin subunits and diminished telomeric localization.
(A) Co-immunoprecipitation of myc-tagged TRF1 (left panel), TRF2 (middle panel) and TPP1 (right panel) from 293T cells co-transfected with HA-tagged wt TIN2, S186fs, L170fs, E202fs, or the empty …
Figure 4 with 6 supplements
Heterozygous TINF2 mutations do not cause telomere damage or genome instability.
(A) Immunoblot for TIN2 and γtubulin in control cells and the indicated clones with targeted TINF2 alleles. (B) Quantification of the immunoblot shown in A. Unpaired t-test was used to determine …
Figure 4—figure supplement 1
Transcript analysis in 604G > C/+ cells reveals presence of two alternative TINF2 transcripts (604G > C I, 604G > C II).
Transcript analysis in control cells and cells with c.604G > C TINF2 mutations. Wild-type and alternative (604G > C I, 604G > C II, alt. splice exons 3–6) transcripts are indicated. Alt. splice …
Figure 4—figure supplement 2
Knock-in strategy for introduction of c.557del and c.604G > C mutations into RPE1 cells.
(A) Schematic of the TINF2 locus showing landmarks relevant to CRISPR/Cas9-mediated knock-in of TINF2 mutations. The single guide RNA (sgRNA) target regions and the primers used for genotyping (blue …
Figure 4—figure supplement 3
Strategy to generate TIN2+/- RPE1 clones.
(A) Schematic of the TINF2 locus showing landmarks relevant to CRISPR/Cas9-mediated targeting of exon1. The single guide RNA (sgRNA) target region and the primers used for genotyping (blue arrows) …
Figure 4—figure supplement 4
Sanger sequencing of CRISPR/Cas9-engineered clones with TINF2 mutations.
(A–C) Reference sequence with sgRNA sequence and PAM and the edited sequences (highlighted) of the obtained cell lines for c.557del (A), c.604G > C (B) and TIN2+/- cells (C) and control cell lines.
Figure 4—figure supplement 5
Characterization of cells with targeted TINF2 alleles.
(A) Growth curves of RPE1 control cells and cells with heterozygous c.557del or c.604G > C, homozygous c.604G > C mutations, or TIN2+/- cells. (B) Telomeric ChIP to determine the specificity of Abs …
Figure 4—figure supplement 6
Representation of TIFs, telomere fusions, and sister associations in the individual cell lines.
(A) Quantification of telomeres containing 53BP1 (≥50 nuclei per cell line, with individual cell lines from Figure 4D shown). (B) Quantification of telomere fusions (≥20 spreads per cell line, with …
Figure 5 with 3 supplements
Heterozygous TINF2 mutations induce telomere lengthening.
(A) Telomeric Southern blot of MboI/AluI-digested genomic DNA from immortalized and primary patient cells (lymphocytes), normal lung fibroblasts (MRC5), and HeLa1.3 cells. Median telomere length …
Figure 5—figure supplement 1
Long telomeres in TINF2 c.604G > C and c.557del patients and hESC CRISPR/Cas9 editing.
(A) Median telomere length (MTL) in individuals with TINF2 truncations as measured by Flow FISH. (B) Schematic of the TINF2 locus showing landmarks relevant to CRISPR/Cas9 mediated targeting of the …
Figure 5—figure supplement 2
No evidence for increased telomere recombination in c.604G > C mutant and Tin2+/- cells.
Quantification of telomeric sister chromatid exchanges (T-SCE) in RPE1 control cells, cells with heterozygous c.604G > C, or TIN2+/- cells (≥15 spreads per cell line, and two cell lines per …
Figure 5—figure supplement 3
Mutant and control RPE1 clones show similar telomerase activity.
(A–C) TRAP assay using extracts from the indicated c.604G > C (A), control and c.557del (B), or TIN2+/- (C) cells together with the respective control cell lines. Serially diluted extracts (500, …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (H. sapiens) | 293T | ATCC | ||
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- | Yang et al., 2017 | ||
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/604G > C.1 clone 1–3 m | This paper | Heterozygous forTINF2 c.604G > C | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/604G > C.2 clone 2–23 m | This paper | Heterozygous forTINF2 c.604G > C | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/604G > C.3 clone 1–21 m | This paper | Heterozygous forTINF2 c.604G > C | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- ctrl4 clone 1–4 c | This paper | Control cell line | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- ctrl5 clone 1–13 c | This paper | Control cell line | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- ctrl6 clone 5–8 c | This paper | Control cell line | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/557del.1 clone 1–9 m | This paper | Heterozygous forTINF2 c.557del | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/557del.2 clone 1–14 m | This paper | Heterozygous forTINF2 c.557del | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/557del.3 clone 2–17 m | This paper | Heterozygous forTINF2 c.557del | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- ctrl1 clone 2–7 c | This paper | Control cell line | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- ctrl2 clone 2–8 c | This paper | Control cell line | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- ctrl3 clone 1–2 c | This paper | Control cell line | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/-.1 clone 1-1het | This paper | Heterozygous for TINF2 | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/-.2 clone 1-3het | This paper | Heterozygous for TINF2 | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/-.3 clone 2-4het | This paper | Heterozygous for TINF2 | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/+ ctrl7 clone 2–9 c | This paper | Control cell line | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/+ ctrl8 clone 3–2 c | This paper | Control cell line | |
| Cell line (H. sapiens) | hTERT-RPE1 p53-/- Rb-/- +/+ ctrl9 clone 1–4 c | This paper | Control cell line | |
| Cell line (H. sapiens) | WIBR3 hESC | Lengner et al., 2010 | NIH stem cell registry number: 0079 | Wild-type |
| Cell line (H. sapiens) | WIBR3 hESC 1B1 | This paper | +/+ TINF2 Control cell line | |
| Cell line (H. sapiens) | WIBR3 hESC 3B4 | This paper | +/+ TINF2 Control cell line | |
| Cell line (H. sapiens) | WIBR3 hESC 3C2 | This paper | +/+ TINF2 Control cell line | |
| Cell line (H. sapiens) | WIBR3 hESC 5B1 | This paper | +/+ TINF2 Control cell line | |
| Cell line (H. sapiens) | WIBR3 hESC 3B3 | This paper | +/- TINF2 | |
| Cell line (H. sapiens) | WIBR3 hESC 4B4 | This paper | +/- TINF2 | |
| Cell line (H. sapiens) | WIBR3 hESC 5A4 | This paper | +/- TINF2 | |
| Cell line (H. sapiens) | WIBR3 hESC 5B3 | This paper | +/- TINF2 | |
| Anti-hTIN2 | Ye and de Lange, 2004 | #864 | ||
| Anti-γTubulin | Sigma | GTU88 | ||
| Anti-Myc | Cell signaling | 9B11 | ||
| Anti-HA | Abcam | Ab9110 | ||
| Anti-53BP1 | Abcam | ab175933 | ||
| Cy3-OO- (TTAGGG)3 | PNA bio | Telomere probe | ||
| FITC-OO-(CCCTAA)3 | PNA bio | Telomere probe | ||
| Alexa Fluor 647-OO-(TTAGGG)3 | PNA bio | Telomere probe | ||
| CENPB-AF488 | PNA bio | F3004 | Centromere probe |
Additional files
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Supplementary file 1
Uncropped images of immunoblots and telomere blots shown in the main figures.
- https://cdn.elifesciences.org/articles/61235/elife-61235-supp1-v1.jpg
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Supplementary file 2
Uncropped images of immunoblots and telomere blots shown in the main figures.
- https://cdn.elifesciences.org/articles/61235/elife-61235-supp2-v1.jpg
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Supplementary file 3
Uncropped images of immunoblots and telomere blots shown in the main figures.
- https://cdn.elifesciences.org/articles/61235/elife-61235-supp3-v1.jpg
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Supplementary file 4
p-Values and summary statistics for key data.
- https://cdn.elifesciences.org/articles/61235/elife-61235-supp4-v1.pdf
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Transparent reporting form
- https://cdn.elifesciences.org/articles/61235/elife-61235-transrepform-v1.docx
