Article Figures & Data
Figures
- FIG 1
Generation of cell lines for extract-based in vitro telomerase assays. (A) Detailed schematic of the TIN2 protein. The TRF2/TPP1 interaction domain is indicated in green, with simplified TPP1 and TRF2 contacts illustrated at the top. A TRF1 FXLXP interaction motif is indicated in purple. The red gradient indicates the patient mutation cluster, where mutated residues cluster but differ in their frequency and disease severity. The blue hatched region indicates the variable C-terminal extension. At the bottom is a conservation track generated from the values from a multiple-sequence alignment performed with 35 known or predicted TIN2 proteins (see Materials and Methods and Table S1 in the supplemental material), with degrees of conservations indicated on a color scale ranging from white (score of 0 [not conserved]) to navy (score of 10 [highly conserved]). (B) Expression cassettes used in this study. All cassettes were expressed under the control of the cytomegalovirus (CMV) promoter in the pcDNA5/FRT backbone. Telomerase assay cell lines were generated as described in Materials and Methods. (C) Western blot of individually transfected TPP1, POT1, and TERT cDNAs next to telomerase assay cell lines with numbers corresponding to those in panel B. FLAG bands above POT1 are unidentified but may represent TERT degradation products. (D) Telomerase assays were stopped at 5, 10, 20, and 40 min for each cell line. Telomere repeats are indicated by +1, +2, etc. LC, loading and purification control.
- FIG 2
TINF2 expression stimulates telomerase processivity. (A) Western blot of duplicate transfections of mycTINF2 or GFP into TPP1/POT1/TERT cell lines. “TINF2” refers to the full-length gene, inclusive of introns. Samples were run on the same blot; the white line indicates cropping of lanes with samples not included in this study. (B) Telomerase assays of transfections performed as described for panel A were stopped after 5 or 10 min. LC, 18-mer loading and purification control; +1, +2, etc., repeat numbers; *, nonspecific band.
- FIG 3
TIN2 has three predominant isoforms in human cells. (A) Schematic of CRISPR and PacBio experiments. Scissors indicate the location of cut site and myc tag insertion. Blue line, oligo(dT20)-adapter used for reverse transcription; F1 and R1, primers used for PacBio sequencing (reported in panels D and E). (B) Western blot of edited myc-TIN2 CRISPR clones 1 to 7. +, positive-control transfection of myc-TINF2 plasmid; −, parental cell lines. Arrows indicate three distinct bands from TIN2 isoforms. Endogenous myc is apparent in the negative-control lane and runs at a size similar to that of TIN2L. (C) Myc Western blot of overexpressed cDNA for TIN2S and TIN2L and the full-length myc-TINF2 gene. Tubulin is shown as a loading control. (D) PacBio sequencing track showing the coverage of TIN2 exons followed by aligned sequence reads shown in blue and gray. Each line represents a single read. Blue; aligned sequence; gray, not covered (introns and indels). (E) StringTie-generated TIN2 transcripts from combined data from 293T, HeLa, RPE-1, K562, and LCL cell lines showing TIN2S, TIN2L, and the new isoform, TIN2M. Light blue, coding sequence; dark blue, unique TIN2M sequence; white, untranslated region.
- FIG 4
All TIN2 isoforms localized to telomeres. Images present immunofluorescence of cell lines expressing individual TIN2 isoforms. TRF2 marks telomeres (red), anti-myc antibody marks TIN2 (green), and nuclei were counterstained with DAPI. Merged images show telomeric foci with colocalized TRF2 and TIN2 staining for all three isoforms.
- FIG 5
All three TIN2 isoforms rescued TIF formation caused by TIN2 knockdown. (A) qRT-PCR was performed on HeLa GFP-expressing cell lines transduced with nontargeting (shNT) or TIN2-targeting (shTIN2) shRNA lentiviruses. Expression level was analyzed by 2−ΔΔCT relative quantification. shNT, n = 2; shTIN2, n = 3. (B) Western blot of HeLa cell lines expressing GFP or individual mycTIN2 isoforms, transduced with shNT or shTIN2 lentivirus. (C) Representative images of HeLa GFP cell lines transduced with shNT or shTIN2 lentivirus as indicated. Red, TRF2; cyan, 53BP1. TIFs are defined by colocalization of TRF2 and 53BP1. (D) Quantification of TRF2/53BP1 colocalizations per nucleus (TIFs), represented as a box plot with whiskers inclusive of all values (n > 250 nuclei per cell line). Data were analyzed with a Kruskal-Wallis one-way analysis of variance with Dunn’s correction for multiple comparisons. Significant P values are indicated on the graph.
- FIG 6
TPP1, POT1, and TERT coimmunoprecipitated with all TIN2 isoforms. myc-tagged TIN2 isoforms were transfected into TPP1/POT1/TERT (left) or TPP1TEL/POT1/TERT (right) cell lines. Anti-myc–agarose beads were used to pull down TIN2, and coimmunoprecipitation of FLAG-tagged TERT, TPP1, and POT1was assayed by Western blotting. *, IgG bands; IP, immunoprecipitation assay.
- FIG 7
TIN2 stimulates telomerase processivity beyond the TPP1/POT1 stimulation. (A) Western blots of GFP and myc-TIN2 isoform transfections into TPP1/POT1/TERT (left) or TPP1TEL/POT1/TERT (right) cell lines. FLAG bands above POT1 are unidentified but may represent TERT degradation products. Tubulin is shown as a loading control. (B) Telomerase assays were stopped at 10, 20, and 40 min (indicated by triangles above the gel). Quantification of the processivity is shown in panel C. LC, loading and purification control; +1, +2, and +3, repeat numbers. (C) Mean processivity values from 3 independent telomerase assays at the 40-min time point using the 15+ processivity method (see Materials and Methods). Orange bars represent a cell line overexpressing TERT/TR but not TPP1/POT1. Data were analyzed with a one-way analysis of variance (ANOVA) and Bonferroni’s multiple-comparison test against the GFP control. n = 3 independent transfections per cell line indicated. Error bars represent standard deviations (SD). **, P < 0.01; ***, P < 0.001.
- FIG 8
TIN2/TPP1/POT1 forms a stable shelterin subcomplex. (A) TIN2 completes the telomerase processivity complex. TIN2 enhances TPP1/POT1 stimulation of telomerase, forming a heterotrimeric processivity complex. (B) A cartoon proposing a dynamic, heterogeneous distribution of shelterin proteins across the length of human telomeres coordinating telomere length maintenance. TRF1 and TRF2 may direct TIN2/TPP1/POT1 to single-stranded DNA both at the telomere overhang and within the replication fork, aiding its roles in fork progression and telomerase stimulation.
Additional Files
Supplemental material
- Supplemental file 1 -
Table S1
PDF, 68K
- Supplemental file 1 -
