TIN2-tethered TPP1 recruits human telomerase to telomeres in vivo

Abstract

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres--TPP1 and the protein that tethers TPP1 to the complex, TIN2--results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.

FIG. 1.

TPP1 depletion results in loss of localization of telomerase to telomeres (assessed by FISH). (A) Mammalian chromosome end structure is regulated by a complex of six core telomere-associated proteins (indicated) that make up the shelterin complex (16, 51). TPP1 is associated with the double-stranded and single-stranded portion of telomeres via direct interactions with TIN2 and POT1, respectively (16, 51). (B) Fluorescence in situ hybridization (FISH) was used to detect hTR (red), and immunofluorescence (IF) was used to detect TRF2 (telomere marker, green) and hTERT (blue) or coilin (Cajal body marker, blue) in parental (−) and TPP1-depleted (+) super-telomerase HeLa cells. Cells were imaged by fluorescence microscopy. Merge panels in all microscopy figures show superimposition of the individual panels. A subset of hTR (hTERT) colocalizations with telomeres is indicated with arrowheads. Scale bars in all microscopy panels represent 10 μm. (C) The average number of hTR-telomere associations per cell (one focal plane) detected by FISH/IF in parental and TPP1-depleted cells represented in panel B is shown. Error bars in plots of localization data in all figures indicate standard errors (see Materials and Methods). (D) Parental (−) and TPP1-depleted (+) HeLa cells (no exogenous telomerase expression) were synchronized to mid-S phase during drug selection. hTR (red) and telomeres (green) were detected by FISH. hTR colocalizations with telomeres are indicated with arrowheads. (E) The average number of hTR-telomere associations per cell (one focal plane) detected by FISH/IF in parental and TPP1-depleted cells represented in panel D is shown.

FIG. 2.

Depletion of TPP1, TIN2, and POT1. (A) Immunoblot analysis of TPP1-depleted cells. Super-telomerase HeLa cells were transfected with pSuper-Puro (vector) or pSuper-Puro-TPP1 shRNA vector (shTPP1). Four days after transfection, TPP1, hTERT, TIN2, and CENP-A expression was analyzed by immunoblotting. The arrowheads indicate the position of endogenous TPP1 protein. Asterisks indicate nonspecific bands. CENP-A was used as a loading control. (B) Immunoblot analysis of TIN2-depleted cells. Four days after transfection, protein expression was analyzed as described for panel A. (C) qRT-PCR analysis of TIN2-depleted cells. qRT-PCR detection of mRNA levels of TIN2 in super-telomerase HeLa cells, 4 days after transfection with TIN2-shRNA, relative to empty-vector control. Error bars correspond to standard deviations of results of three independent experiments. Statistical analyses were done using a two-tailed Student's t test (**, P < 0.01). (D) Immunoblot analysis of POT1-depleted cells. Six days after transfection, protein expression was analyzed as described for panel A. (E) qRT-PCR analysis of POT1-depleted cells. qRT-PCR showing mRNA levels of POT1 in super-telomerase HeLa cells, 6 days after transfection with two different POT1-shRNAs, relative to empty-vector control. Error bars correspond to standard deviations of results of two independent experiments. (F) IP/immunoblot analysis of POT1-depleted cells. Coimmunoprecipitation of endogenous POT1 with TPP1 in POT1-depleted cells. TPP1-immunoprecipitated complexes from super-telomerase HeLa cells transfected with the indicated plasmids were resolved by 8% SDS-PAGE. Immunoblot antibodies are indicated on the left. No detection of TPP1 was observed in the supernatant fraction after IP (data not shown). A nonspecific band recognized by the TPP1 antibody in the IP fraction (**) served as a loading control. (G) Direct IF analysis of POT1-depleted cells. POT1 (red) and TRF2 (green) were detected by IF in parental (−) and POT1-depleted cells.

FIG. 3.

TPP1 depletion results in loss of physical association of hTERT with telomeres (assessed by ChIP). (A) ChIP of telomeric and Alu DNA with TPP1-specific (A) and hTERT-specific (B) antibodies in super-telomerase HeLa cells. The percentage of telomeric and Alu DNA recovered in each ChIP is indicated. Error bars correspond to standard deviations of results of three (hTERT ChIP) and five (TPP1 ChIP) independent experiments. Statistical analyses were done using a two-tailed Student's t test (***, P < 0.001; **, P < 0.01).

FIG. 4.

Telomerase recruitment depends on TIN2 but not POT1. (A) ChIP of telomeric and Alu DNA with hTERT antibody. (B) Quantification of ChIP data shown in panel A. Error bars correspond to standard deviations of results of three (TIN2 data) and two (POT1 data) independent experiments. Statistical analyses were done using a one-tailed Student's t test (*, P < 0.05). (C) hTR (red) was detected by FISH and TRF2 (green) and coilin (blue) were detected by IF in parental (−) and POT1- or TIN2-depleted cells. hTR colocalizations with telomeres are indicated with arrowheads. (D and E) The average number of hTR-telomere associations per cell (one focal plane) detected by FISH/IF in parental and POT1- or TIN2-depleted cells represented in panel C is shown.

FIG. 5.

Human telomerase is recruited to telomeres via the OB-fold of TPP1. (A) Immunoblot analysis of ectopically expressed FLAG epitope-tagged full-length TPP1 (TPP1), FLAG-tagged, shRNA-resistant full-length TPP1 (TPP1*), and FLAG-tagged, shRNA-resistant TPP1 lacking the OB-fold (TPP1ΔOB*). Super-telomerase HeLa cells were cotransfected with the indicated plasmids, and protein expression was analyzed 4 days after transfection. Black arrowheads indicate the presence of the respective TPP1 proteins, and white arrowheads indicate the lack of expression. Immunoblots were probed with anti-TPP1, anti-FLAG and CENP-A antibodies as indicated. (B) ChIP of telomeric and Alu DNA with hTERT antibody from cells in panel A. (C) Quantification of data in panel B representing percentage of telomeric and Alu DNA recovered in hTERT ChIP. Error bars correspond to standard deviations of results of four independent experiments. Statistical analyses were done using a two-tailed Student's t test (*, P < 0.05).

FIG. 6.

The TPP1 OB-fold is required to rescue telomerase recruitment to telomeres. An shRNA-resistant form of TPP1 is able to restore hTR localization to telomeres in TPP1-depleted cells. However, an shRNA-resistant form of TPP1 lacking the OB-fold cannot restore localization. (A) Parental and TPP1-depleted super-telomerase HeLa cells were subjected to FISH and IF to detect hTR (red), coilin (blue), and TRF2 (green). Merge panels show superimposition of hTR, coilin, and TRF2. Next, parental cells were cotransfected with shTPP1 and either TPP1* or TPP1ΔOB*. Treated cells were subjected to FISH and IF to detect hTR (red), FLAG (blue), and RAP1 (telomere marker, green). Merge panels show superimposition of hTR, FLAG, and RAP1. (B) Plot of the average number of telomere-associated hTR foci per cell in the parental cells and each experimental group. Error bars indicate standard errors calculated with N equal to the number of samples quantitated.

FIG. 7.

DNA damage response at telomeres following TPP1 depletion. (A) ChIP of telomeric and Alu DNA with γ-H2AX antibodies. Transfected plasmids are indicated. (B) The graph represents the quantification of the dot blot indicating the percentages of telomeric and Alu DNA recovered with γ-H2AX antibodies. Error bars correspond to standard deviations of four independent experiments. Statistical analyses were done using a two-tailed Student's t test (**, P < 0.01). (C) Immunoblot was probed with anti-γ-H2AX and CENP-A antibodies following transfection of super-telomerase HeLa cells with empty vector or TPP1 shRNA construct as indicated. (D) TPP1-depleted cells were subjected to FISH and IF to label for telomeres (green) and 53BP1 (TIF marker, red). Merge panels show superimposition of telomeres and 53BP1 (colocalizations are indicated by yellow).

FIG. 8.

Increased DNA damage response at telomeres upon TIN2 or POT1 depletion. (A) ChIP of telomeric and Alu DNA with γ-H2AX antibodies. (B) The graph represents the quantification of the dot blot. Error bars correspond to standard deviations of results of two independent experiments.

FIG. 9.

The presence of TIFs (telomere dysfunction-induced foci) does not impact the ability of TPP1 to rescue telomerase associations with telomeres. (A) ChIP of telomeric and Alu DNA with γ-H2AX antibodies. Transfected plasmids are indicated. Expression of TPP1* and TPP1ΔOB* partially rescues TIF formation observed in TPP1-depleted cells. (B) The graph represents the quantification of the dot blot indicating the percentages of telomeric and Alu DNA recovered with γ-H2AX antibodies. Error bars correspond to standard deviations of results of four independent experiments. Statistical analyses were done using a two-tailed Student's t test (***, P < 0.001; **, P < 0.01). (C) Although TIFs were detected in TPP1-depleted cells coexpressing TPP1* or TPP1ΔOB*, TIFs did not inhibit rescue of hTR recruitment to telomeres by TPP1*. Super-telomerase HeLa cells were cotransfected with shTPP1 and either TPP1* or TPP1ΔOB*. Treated cells were subjected to FISH and IF to label for hTR (red), FLAG (blue), 53BP1 (green), and telomeres (green). Merge panels show superimposition of hTR, 53BP1, and FLAG or hTR, telomeres, and FLAG.