Telomere Maintenance through Spatial Control of Telomeric Proteins

Abstract

The six human telomeric proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 can form a complex called the telosome/shelterin, which is required for telomere protection and length control. TPP1 has been shown to regulate both POT1 telomere localization and telosome assembly through its binding to TIN2. It remains to be determined where such interactions take place and whether cellular compartmentalization of telomeric proteins is important for telomere maintenance. We systematically investigated here the cellular localization and interactions of human telomeric proteins. Interestingly, we found TIN2, TPP1, and POT1 to localize and interact with each other in both the cytoplasm and the nucleus. Unexpectedly, TPP1 contains a functional nuclear export signal that directly controls the amount of TPP1 and POT1 in the nucleus. Furthermore, binding of TIN2 to TPP1 promotes the nuclear localization of TPP1 and POT1. We also found that disrupting TPP1 nuclear export could result in telomeric DNA damage response and telomere length disregulation. Our findings highlight how the coordinated interactions between TIN2, TPP1, and POT1 in the cytoplasm regulate the assembly and function of the telosome in the nucleus and indicate for the first time the importance of nuclear export and spatial control of telomeric proteins in telomere maintenance.

Telomere maintenance by telomerase and telomeric proteins is essential for normal cell growth, and its disregulation may have direct consequences in aging and cancer (2, 10, 11, 22, 28, 29, 44). Recent biochemical and genetic experiments have led to a much clearer understanding of how telomere ends are protected in mammalian cells. To date, six major telomeric proteins—namely, TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 (previously PTOP, PIP1, or TINT1)—have been identified in human cells and shown to participate in telomere regulation (2, 9, 38). These proteins perform related but distinct functions at the telomeres. Understanding the function and molecular interaction of these telomeric proteins is fundamental to telomere biology.

Telomeres in mammalian cells consist of long telomeric double-stranded DNA (dsDNA) repeats and short single-stranded DNA (ssDNA) overhangs (12, 13). TRF1 and TRF2 directly associate with the repeats through their myb domains (4). TRF1 is a negative regulator of telomere length since overexpression of TRF1 results in gradual shortening of telomeres (41). Overexpression of dominant-negative mutants or deletion of TRF2 results in DNA damage responses at the telomeres and chromosomal end-to-end fusions, underlining its essential role in telomere end protection (5, 42). TRF2 also works closely with its associated protein RAP1 (24, 31). In TRF2 KO mouse embryonic fibroblast cells, protein levels of RAP1, but not TRF1, were diminished, suggesting that TRF2 and RAP1 act together (5).

The human telomere ssDNA overhangs are protected by POT1 and TPP1. POT1 and TPP1 are OB-fold-containing proteins that are structurally homologous to ciliate telomere end binding proteins TEBP-α and -β (16, 43, 46). Originally identified in yeast, POT1 family proteins have been shown to regulate telomere length and protect telomeres from DNA damage, rapid degradation, and chromosomal fusion (6, 7, 14, 19, 27, 36, 45). Human POT1 harbors two OB folds in its N terminus and recognizes telomeric ssDNA with high affinity (23). Surprisingly, the telomeric targeting of POT1 is controlled by TPP1, a POT1- and TIN2-interacting protein (26, 46, 48). However, whether TPP1 controls POT1 telomere targeting by regulating POT1 nuclear localization remains to be determined. TPP1 heterodimerizes with POT1 and enhances its affinity toward telomere ssDNA (43, 46). In addition to binding and protecting the G-strand telomere overhangs, the POT1-TPP1 complex is also capable of recruiting and stimulating telomerase activity, thereby regulating telomere length through TPP1-telomerase interaction (43, 46). Elucidating the interaction and the function of these two proteins should continue to prove pivotal to our understanding of telomere maintenance.

Human telomeres are regulated by various telomeric protein complexes, including the high-molecular-weight telosome/shelterin that contains all six major telomeric proteins (9, 25). Within the telosome, TRF1 and TRF2 are connected to POT1 through TIN2 and TPP1, two key factors for telosome assembly (32). In the absence of TIN2, only smaller telomeric subcomplexes were formed (32). TIN2 has emerged as the key telosome element because of its ability to interact with TRF1, TRF2, and TPP1 (17, 21, 25, 26, 47). Meanwhile, TPP1 promotes the TRF1-TIN2-TRF2 subcomplex and telosome formation through TPP1-TIN2 interaction (32). Such interdependence during high-order complex formation argues that the spatial and/or temporal restriction of telomeric protein-protein interactions may determine the ultimate composition and function of telomeric protein complexes. Although there have been numerous studies on the function of telomeric proteins in the nucleus, it remains unclear, for example, whether the formation of various telomeric complexes can be directly regulated by the subcellular localization of telomeric proteins.

Apart from their known telomeric association, little is known regarding the total cellular distribution of the six mammalian telomeric proteins. To visualize telomere targeting of proteins by immunostaining, cells are often doubly permeabilized (24). This method disrupts proteins in both the cytoplasm and the nucleoplasm, providing a much more limited view of localization of telomeric proteins inside the cell. In order to understand the role of cellular localization of telomeric proteins in telomere maintenance, we have systematically investigated the localization and in vivo interactions of the six human telomeric proteins. Notably, we found TIN2, TPP1, and POT1 to localize and interact with each other not only in the nucleus but also in the cytoplasm. Unexpectedly, we found TPP1 to contain a nuclear export signal. We showed here that TPP1 shuttles between the cytoplasm and the nucleus, which in turn regulates the nuclear localization of POT1. In addition, TIN2 binding to TPP1 helps to promote the nuclear retention of POT1. Furthermore, disruption of TPP1 nuclear export results in telomere dysfunction. Our results suggest that the coordinated interactions between TIN2, TPP1, and POT1 in the cytoplasm regulate the assembly and function of the telosome in the nucleus, and underscore the importance of spatial control of telomeric proteins and their interactions in regulating POT1 localization and telomere maintenance.

MATERIALS AND METHODS

Expression constructs, cell cultures, and antibodies.

Rabbit anti-TPP1 and anti-TIN2 (26), anti-RAP1 (Bethyl), and goat anti-TRF1 (32), anti-POT1 (NB500-176; Novus Biologicals), anti-TRF2 (OP129; Calbiochem), anti-GRB2 (catalog no. 610112; BD Transduction Laboratories), anti-α-tubulin (T9026; Sigma), anti-lamin (sc-6215; Santa Cruz), anti-ORC2 (catalog no. 551179; BD Transduction Laboratories), and anti-Flag (M2; Sigma) antibodies were used for Western blot analysis. Anti-POT1N and anti-53BP1 (46), anti-TRF2 (OP129; Calbiochem), and anti-Flag (M2; Sigma) antibodies were used for immunostaining.

To generate cell lines stably expressing fluorescence protein-tagged or Flag-tagged telomeric proteins, Gateway cloning (Invitrogen) was performed to clone the cDNAs into pCL-based Gateway destination vectors containing green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), DsRed, or Flag. All of the fluorescence proteins were fused to the N terminus of the targeting proteins. V5-tagged POT1 was generated by TOPO cloning into pDNA3-based vector (Invitrogen). Mutations on TPP1 were generated by QuikChange mutagenesis (Stratagene). To generate cell lines stably expressing telomeric proteins fused to the N- or C-terminal fragments of YFP, the cDNAs of the telomeric proteins were Gateway cloned into pBabe-based retroviral vectors containing the sequences encoding either YFPn (amino acids 1 to 155 of Venus YFP) or YFPc (amino acids 156 to 239 of YFP). To generate cell lines stably expressing short hairpin RNAs (shRNAs) specifically targeting TPP1 or GFP, mouse U6 promoter-driven expression of shRNA constructs were generated on a pCL-based vector. The RNA interference sequences for sh-TPP1-A and shGFP are 5′-GTGGTACCAGCATCAGCCTT-3′ and 5′-CACAAGCTGGAGTACAACT-3′ (32), respectively.

Stable cells expressing various constructs were established by retroviral infection. The cells were selected in the presence of puromycin, G418, or both. The selected cells were maintained as a pool for experiments described here.

Coimmunoprecipitation.

To prepare cell lysates for coimmunoprecipitation, the cells were lysed in NETN (1 M Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl, 0.5% NP-40) buffer supplemented with protease inhibitor cocktails (Sigma) and subjected to centrifuge at 14,000 × g for 15 min. After centrifugation, the supernatant was used for immunoprecipitation with anti-Flag M2 affinity resins (Sigma) on ice for 1 h. After four washes with NETN buffer, the proteins were eluted with Flag peptide (200 μg/ml) and subjected to Western blot analysis.

Immunofluorescence and BiFC.

Cells for immunofluorescence were grown on glass coverslips. The cells were then fixed with 4% paraformaldehyde prepared in phosphate-buffered saline for 15 min and permeabilized with 0.2% Triton X-100. After blocking with 1% goat serum, the proteins were detected with appropriate antibodies, followed by secondary antibodies conjugated with fluorescein isothiocyanate or Texas red. To detect telomere dysfunction-induced foci, the cells were costained with anti-TRF2 monoclonal and anti-53BP1 polyclonal antibodies. To visualize the nuclei, DAPI (4′,6′-diamidino-2-phenylindole) was used to stain DNA. Fluorescence microscopy was performed on a Nikon TE200 microscope equipped with a Coolsnap-fx charge-coupled device camera. GFP fusion proteins are ready for analysis after fixation. For bimolecular fluorescence complementation (BiFC) experiments, the cells were grown on glass-bottom culture plates (MatTek Corp.). The cell nuclei were stained with Hoechst 33342, a cell-permeable DNA dye. The fluorescence emissions of the living cells were imaged as described above.

Subcellular fractionation.

A total of 10⁶ of HTC75 cells were treated with trypsin, washed with culture medium and PBS, and resuspended in 20 μl of hypotonic buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl₂, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol, and protease inhibitors). A limiting detergent lysis was performed bythe addition of 0.1% of Triton X-100 to the cells, followed by incubation at 4°C for 5 min. After incubation, the lysates were centrifuged at 1,300 × g for 4 min. After centrifugation, the supernatant and the pellet were saved as the cytosolic and the nuclear/heavy membrane fractions, respectively, for Western blot analysis.

In vitro pull-down assay.

The glutathione S-transferase (GST)-POT1 protein was produced in Sf9 insect cells and purified by glutathione agarose as previously described (46). The various TPP1 proteins labeled with [³⁵S]methionine were prepared by using TnT quick-coupled transcription/translation systems (Promega). The binding reaction of GST-POT1 with TPP1 proteins was performed in NETN buffer at 4°C for 1 h. The precipitates were washed with NETN buffer, eluted with sodium dodecyl sulfate (SDS) sample buffer, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE).

Telomere restriction fragment assay.

HTC75 cells stably expressing various TPP1 proteins were generated by retrovirus infections and subsequent puromycin selection. After drug selection (at which point designated as PD0), the surviving cells were passaged and collected at various time points. The genomic DNA was extracted with a DNeasy kit (QIAGEN) and used for telomere restriction fragment assay performed as previously described (26). The data were then analyzed by using ImageQuant and the Telorun analysis tool (33).

RESULTS

Intracellular localization of telomeric proteins.

Numerous studies have established the critical roles for the six telomeric proteins in regulating mammalian telomere chromatin (1, 2, 9, 34, 38). The majority of these studies have focused on the telomere functions of these proteins; the cellular localization of these proteins remains poorly characterized. We therefore set out to systematically investigate the localization of the six telomeric proteins in human cells. To study the localization of human telomeric proteins, we generated cell lines stably expressing GFP-tagged TRF1, TRF2, RAP1, TPP1, TIN2, and POT1 and examined their localization. Human TPP1 mRNA contains two potential Kozak sequences for translation initiation, which encodes, respectively, the long-form TPP1-L (544 amino acids) and the short-form TPP1-S (458 amino acids, also called TPP1Δ86). The additional N-terminal 86 amino acids in TPP1-L appear to be absent in other species, including the mouse (26). Mass spectrometry sequencing and Western blotting experiments suggest that TPP1-S is the predominant form of TPP1 in human cells (data not shown). We therefore focused on TPP1-S for the present study.

Interestingly, the GFP-tagged telomeric proteins exhibited different localization patterns. While GFP-TRF1, -TRF2, -RAP1, and -TIN2 localized primarily in the nucleus (Fig. 1A), GFP-TPP1 and GFP-POT1 were clearly present in both the nucleus and the cytoplasm (Fig. 1A). A small amount of GFP-TIN2 could also be detected in the cytoplasm. On close examination, all GFP fusion proteins showed telomere localization (data not shown).

FIG. 1.

Subcellular localization of human telomeric proteins. (A) Localization of GFP-tagged human telomeric proteins. HTC75 cells were infected with retroviral vectors for stable expression of different GFP-tagged telomeric proteins. The cells were fixed, and fluorescence signals were visualized by using a microscope. The cell nuclei were stained with DAPI. (B) Fractionation and Western blot analysis of endogenous telomeric proteins. HTC75 cells were fractionated into cytoplasmic (S1) and nuclear/heavy membrane (P1) fractions as described in Materials and Methods and resolved by SDS-PAGE. Whole-cell extracts (WCE) were also prepared. Western blotting was performed with antibodies for cytoplasmic marker proteins (GRB2 and tubulin), nuclear marker proteins (lamin A/C and ORC2), and telomeric proteins (TPP1, POT1, TIN2, TRF1, TRF2, and RAP1). An arrow indicates endogenous TPP1 or POT1. POT1 has a 70-kDa and a 55-kDa form (15).

To further confirm this observation, cytoplasmic and nuclear fractions of HTC75 cells were prepared by limiting detergent lysis for Western blotting (30). As shown in Fig. 1B, marker proteins for the S1 cytoplasmic fraction (GRB2 and tubulin) and the P1 nuclear/heavy membrane fraction (lamin A/C and ORC2) were largely consistent with their respective localizations. A trace amount of cytoplasmic marker proteins in the nuclear fraction was likely a result of incomplete cell lysis. In agreement with previous findings, all six telomere proteins mainly localized to the nucleus. In particular, endogenous TIN2, TRF2, TRF1, and RAP1 are primarily nuclear localized, a finding consistent with published studies (24, 37). Importantly, a significant amount of endogenous TPP1 and POT1 was also detected in the cytoplasmic faction (Fig. 1B). Taken together, these data are consistent with our GFP fusion protein analyses (Fig. 1A) and demonstrate that TPP1 and POT1 may also localize to the cytoplasm.

TIN2, TPP1, and POT1 interact with each other in both the cytoplasm and the nucleus.

Given their critical roles in regulating telomere length and protecting telomere ends, the nuclear assembly and telomere targeting of the six main telomere proteins must be essential for telomere maintenance. However, the cytoplasmic localization of TPP1 and POT1 suggests that these three proteins may also interact with each other in the cytoplasm. In order to verify where interactions of telomeric proteins occur in live cells, we utilized BiFC to analyze pairwise interactions among TRF2, TIN2, TPP1, and POT1 (18). Previously, we and other groups demonstrated that TIN2 binds directly to TRF2 and TPP1, whereas TPP1 interacts directly with POT1 (Fig. 2A) (17, 21, 25, 26, 48).

FIG. 2.

In vivo interactions among human telomeric proteins revealed by BiFC. (A) Diagram of known pairwise interactions within the telosome. (B) Schematic representation of the BiFC technique in detecting TIN2-TRF2 interaction. (C) Visualization of telomeric protein interactions in live cells through BiFC. HTC75 cells stably coexpressing YFP fragments alone or YFP fragment-tagged TRF2, TIN2, TPP1, or POT1 (as indicated) were established. YFPn, Venus YFP N-terminal fragment (amino acids 1 to 155); YFPc, YFP C-terminal fragment (amino acids 156 to 239). To detect telomeres in these cells, dsRed-TRF1 was also coexpressed. The cells were stained with Hoechst 33342 to visualize the nuclei and analyzed directly under a fluorescence microscope. (D) Positive BiFC fluorescence signal by YFPn-TPP1 and YFPc-TIN2 required direct interaction between TPP1 and TIN2. HTC75 cells coexpressing YFP fragments alone or YFP fragment-tagged TIN2, wild-type, or mutant TPP1 were analyzed by fluorescence-activated cell sorting. The percentages of GFP-positive cells were indicated in the histograms. Control, parental HTC75 cells.

In BiFC assays, two proteins are fused to separate fragments of YFP. When these proteins interact, the YFP fragments will be brought into close proximity to form a functional fluorescent complex (Fig. 2B). As expected, YFP signals could be detected in cells coexpressing TIN2-TRF2, TPP1-TIN2, and TPP1-POT1 pairs, but not the YFP fragments alone (Fig. 2C). The TIN2-TRF2 interaction appeared to occur largely (>95%) in the nucleus. Furthermore, the signals in these cells displayed the punctate pattern that was superimposable with the telomere marker dsRed-TRF1. In contrast, in all of the fluorescence-positive interphase cells examined, TIN2-TPP1 and POT1-TPP1 interactions took place in both the cytoplasm and the nucleus (Fig. 2C). In the nucleus, the punctate YFP fluorescence signals overlapped with dsRed-TRF1, indicating that TIN2-TPP1 and POT1-TPP1 interactions could occur at the telomeres (Fig. 2C). In the cytoplasm, the fluorescence signals appeared evenly distributed. As a negative control, cells that coexpressed TIN2 with the TPP1 mutant (TPP1ΔC22) that no longer binds TIN2 (32) failed to exhibit fluorescence in the BiFC assay (Fig. 2D), suggesting that fluorescence complementation required direct interactions between TPP1 and TIN2. These data are consistent with our observed cytoplasmic localization of TPP1 and POT1 (Fig. 1) and raise the question of whether the interactions of telomeric proteins and the likely formation of subcomplexes in different subcellular compartments may affect the function of these proteins at the telomeres.

Regulation of POT1 and TPP1 cellular localization by TIN2.

Previous work has demonstrated that TIN2, TPP1, and POT1 interact with each other in the order of TIN2-TPP1-POT1 to form the telosome (32). TPP1 controls POT1 telomere localization, which is in turn regulated by TIN2-TPP1 interaction. Given the data in Fig. 1 and 2, we next sought to determine whether TIN2 could modulate TPP1 and POT1 cellular localization. We reasoned that TIN2 might regulate TPP1 and POT1 telomere localization through the nuclear import and/or retention of TPP1 or POT1. The higher intensity of GFP-TPP1 and GFP-POT1 in the cytoplasm may be due to overexpression of these proteins and limited endogenous TIN2. If this is the case, overexpression of TIN2 in GFP-TPP1 or GFP-POT1 cells should lead to increased GFP-TPP1 and GFP-POT1 in the nucleus. As shown in Fig. 3A, in all of the interphase cells examined (∼100%), GFP-TPP1 was predominantly found in the cytoplasm. However, coexpression of dsRed-tagged TIN2 resulted in increased nuclear TPP1 signal in all of the interphase cells examined, suggesting that TIN2-TPP1 interaction promotes nuclear localization of TPP1. In further support of this notion, the TPP1 mutant (TPP1ΔC22) that no longer binds TIN2 remained cytoplasm localized when TIN2 was coexpressed (Fig. 3A).

FIG. 3.

TIN2 promotes TPP1 and POT1 nuclear localization. (A) TIN2 increases nuclear TPP1 through TIN2-TPP1 interaction. Cells stably expressing GFP-TPP1 or GFP-TPP1ΔC22 alone or with dsRed-TIN2 were fixed, and the localization of GFP-TPP1 proteins was determined by fluorescence microscopy. (B) TIN2 increases nuclear POT1. Cells stably expressing YFP-POT1 with or without CFP-TIN2 were fixed, and the localization of YFP-POT1 proteins was determined by fluorescence microscopy. (C) Knockdown of TPP1 in HTC75 cells. Stable TPP1 knockdown cells were established by transduction with retroviral vectors expressing a control shRNA sequence against GFP (shGFP) or a shRNA sequence (shTPP1-A) against human TPP1. Whole-cell extracts from these cells were Western blotted for endogenous TPP1 (marked by the arrow), TRF2, RAP1, and tubulin. Tubulin was used as a loading control. (D) TIN2 promotes POT1 nuclear retention in a TPP1-dependent manner. Cells stably expressing YFP-POT1 with or without Flag-TIN2 and cells expressing YFP-POT1 with Flag-TIN2 and TPP1 shRNA-A were generated. The cells were fixed, and the localization of YFP-POT1 proteins was determined by fluorescence microscopy. Nuclei were detected with DAPI staining. (E) The data in panel D were quantified based on the percentages of cells with cytoplasmic and nuclear YFP-POT1.

Similar to TPP1, POT1 nuclear localization appeared to be regulated by TIN2 as well. As shown in Fig. 3B and E, coexpression of CFP-TIN2 concentrated POT1 in the nucleus. Because of the TIN2-TPP1-POT1 link, we speculated that TIN2 might increase POT1 nuclear retention through TPP1. If this is the case, reducing endogenous TPP1 level by RNA interference should affect nuclear localization of POT1. Indeed, in TPP1 knockdown cells, coexpression of TIN2 failed to promote YFP-POT1 accumulation in the nucleus (Fig. 3C, D, and E). These results are not only consistent with our model that the TIN2-TPP1-POT1 link is essential for POT1 nuclear localization but also suggest that POT1 activity is controlled by TIN2 and TPP1 through both nuclear localization and telomere targeting.

TPP1 shuttles between the cytoplasm and the nucleus.

TPP1 is a key factor in telosome assembly and regulates POT1 localization (26, 32). The finding of TPP1 residing in both the cytoplasm and the nucleus prompted us to investigate the mechanism that controls TPP1 subcellular localization. We hypothesized that TPP1 localization might be regulated by nuclear import or export. To investigate this possibility, cells stably expressing GFP-TPP1 were treated with leptomycin B (LMB). LMB inhibits CRM-1/exportin 1, a protein necessary for nuclear export signal (NES)-mediated protein export. As shown in Fig. 4A, the accumulation of wild-type TPP1 in the nucleus was found in LMB-treated cells, suggesting that TPP1 can be exported to the cytoplasm in a CRM-1-dependent manner. When these LMB-treated cells were further incubated in the absence of LMB, translocation of TPP1 to the cytoplasm could be observed within 1 h. These results suggest that TPP1 is imported into the nucleus and then exported back to the cytoplasm.

FIG. 4.

TPP1 localization is controlled by CRM-1 mediated nuclear export and the RD domain. (A) Effect of LMB on TPP1 localization. GFP-TPP1 expressing cells were treated with LMB (5 nM) for 3 h, washed, and further incubated in the absence of LMB. Cells were collected at different time points and fixed for analysis by fluorescence microscopy. (B) Deletion of TPP1 RD domain results in nuclear retention of TPP1. The localization of GFP-tagged wild-type TPP1 and TPP1 deletion mutants was determined by fluorescence microscopy. (C) Alignment of putative NESs in TPP1 homologues, human p53, and HDM2.

To identify the domain that is responsible for TPP1 nuclear import or export, we examined the localization of TPP1 deletion mutants. Deletion of the POT1 recruitment domain (TPP1ΔRD) enabled TPP1 to accumulate in the nucleus in ∼100% of the interphase cells (Fig. 4B), suggesting that cytoplasmic localization of TPP1 may be maintained by either TPP1 interaction with POT1 or an active NES located within the RD domain. Interestingly, disruption of the TIN2-interacting domain (TPP1ΔC22) did not change TPP1 localization. In addition, further mutation of the TIN2 binding site on TPP1ΔRD did not prevent its nuclear retention (Fig. 4B), implying independent mechanisms for nuclear export mediated through the RD domain versus nuclear retention mediated through TIN2-TPP1 interaction. These data, along with our finding that TIN2-TPP1 interaction promotes wild-type TPP1 nuclear retention (Fig. 3A), suggest that TPP1 possesses intrinsic nuclear export property, and the binding of TIN2 to TPP1 may counteract this export activity to help retain TPP1 in the nucleus.

Nuclear export of TPP1 is mediated by an NES.

Proteins such as p53 and MDM2 are actively transported to the cytoplasm from the nucleus via an NES that contains a leucine-rich sequence of conserved spacing and hydrophobicity (3, 20, 35, 39) (Fig. 4C). Sequence analysis of human TPP1 revealed two putative NESs: one in the RD domain (NES1) and one in the serine-rich domain (NES2) (Fig. 4C). Both sequences show high degrees of conservation between mammals. In particular, the NES1 sequence is also conserved between humans, mice, and frogs. To examine whether these putative NESs mediate TPP1 nuclear export, we established cell lines stably expressing GFP-TPP1 mutants in which the NESs had been disrupted. As shown in Fig. 5A, the fluorescence signal was enriched in the nucleus in most cells (>95%) expressing the TPP1 NES1 mutant but not the TPP1 NES2 mutant. Disruption of both NES1 and NES2 did not lead to a further increase of GFP-TPP1 nuclear localization. These results indicate that NES1 mediates TPP1 nuclear export.

FIG. 5.

TPP1 contains a functional NES in the RD domain. (A) Mutation of NES1 but not NES2 abolishes TPP1 nuclear export. Cells stably expressing GFP-tagged wild-type TPP1 and various TPP1 NES mutants were analyzed by fluorescence microscopy. (B) Mutation of TPP1 NES1 does not affect TPP1-POT1 interaction in cells. 293T cells were transiently transfected with V5-POT1 alone or in combination with Flag-tagged wild-type or mutant TPP1. Whole-cell extracts were prepared for immunoprecipitation by using anti-Flag M2 antibodies. The whole-cell extracts and immunoprecipitates were Western blotted with anti-POT1 or anti-Flag antibodies. (C) Mutation of TPP1 NES1 does not affect TPP1-POT1 interaction in vitro. Wild-type and mutant TPP1 were in vitro translated in the presence of [³⁵S]methionine and used for in vitro pull-down assays with GST-POT1 proteins purified from Sf9 cells. The samples were resolved by SDS-PAGE, and the radioactive signals were detected by using a PhosphorImager.

The identification of NES1 within the RD domain is consistent with our observation that deletion of the RD domain led to TPP1 nuclear localization (Fig. 4B). However, this does not exclude the possibility that TPP1 nuclear export may be controlled by POT1 interaction with the TPP1 RD domain. For example, sequences surrounding NES1 may be required for POT1 binding, and mutation of NES1 might have negatively affected POT1 binding. To address this possibility, the wild type and various Flag-tagged TPP1 mutants were cotransfected with V5-POT1 into HTC75 cells. As shown in Fig. 5B, POT1 could coimmunoprecipitate with wild-type TPP1 and TPP1ΔC22 but not TPP1ΔRD. Importantly, mutation of NES1 (TPP1-NES1m1) did not affect POT1 binding. In addition, similar results were obtained with insect cell-expressed GST-POT1 and in vitro-translated TPP1 proteins (Fig. 5C). GST-POT1 could bring down both wild-type TPP1 and TPP1-NESm1. Taken together, these results indicate that NES1 is a bona fide export sequence that mediates TPP1 nuclear export, and it is separable from the sequence motifs that mediate TPP1-POT1 interaction.

Disruption of TPP1 nuclear export triggers telomere dysfunction.

TPP1 protects telomeres through heterodimerizing with POT1 and controlling telomerase access and activity, which occurs in the nucleus (43, 46). It was therefore surprising to find that TPP1 is regulated by nuclear export. We next sought to determine whether such regulation was required for TPP1-mediated telomere protection. Loss of telomere end protection results in activation of DNA damage responses (e.g., 53BP1 foci) at the telomeres, as evidenced by the presence of telomere-dysfunction-induced foci (TIFs) (8, 40). Consistent with our recent studies, relatively minor DNA damage responses were observed in cells overexpressing wild-type TPP1, whereas the expression of TPP1 mutants that failed to interact with either POT1 (TPP1ΔRD) or TIN2 (TPP1ΔC22) activated DNA damage response at the telomeres (Fig. 6A and B) (46). In this case, more than 80% of 53BP1 foci were colocalized with TRF2 (Fig. 6C). Notably, the TPP1 NES1 mutant, which was expressed at levels comparable to TPP1 and TPP1ΔC22, also induced robust TIFs (Fig. 6), suggesting that mislocalization of TPP1 to the nucleus led to telomere dysfunction.

FIG. 6.

Expression of TPP1 NES1 mutant induces telomere dysfunction. (A) Formation of TIFs in TPP1 mutant-expressing cells. Cells expressing Flag-tagged wild-type or mutant TPP1 proteins were fixed and immunostained with anti-TRF2 (in green) and anti-53BP1 (in red) antibodies. (B) Quantification of the results in panel A. Error bars indicate the standard error (n = 5). (C) Percentages of 53BP1 foci that colocalized with TRF2 were quantified in TIF-positive cells. (D) The expression levels of Flag-tagged TPP1 proteins in cells from panel A were analyzed by Western blotting with anti-Flag antibodies.

TPP1 is also a critical regulator of telomere length (17, 26, 48). To understand the role of TPP1 nuclear export in telomere length control, we compared the average telomere lengths of HTC75 cells expressing different TPP1 mutants. Similar to TPP1ΔRD and TPP1ΔC22, overexpression of the TPP1 NES1 mutant led to telomere length extension (Fig. 7). These data indicate that TPP1 nuclear export is necessary for proper telomere maintenance in human cells.

FIG. 7.

TPP1 NES1m1 expression results in telomere elongation. (A) Telomere restriction fragment analysis of TPP1 mutant-expressing HTC75 cells. Genomic DNA was purified from vector control and TPP1 wild-type and mutant-expressing cells. The DNA was digested, and Southern blotting was performed with ³²P-labeled (TTAGGG)3 probes. The radioactive signals were analyzed by using a PhosphorImager. (B) Quantification of the results in panel A. PD, population doubling.

Disruption of TPP1 nuclear export alters POT1 nuclear localization.

TPP1ΔRD and TPP1ΔC22, which failed to bind POT1 and TIN2, respectively, likely cause telomere defects through a dominant-negative effect. However, TPP1-NES1 still maintains the ability to bind POT1 (Fig. 5B and C) and TIN2 (data not shown). What is the mechanism that led to telomere dysfunction in TPP1-NES1m1 expressing cells? We reasoned that the forced retention of TPP1 (as seen with TPP1-NES1m1) might have altered POT1 nuclear localization that would result in telomere dysfunction.

To test this possibility, we investigated endogenous POT1 localization in HTC75 cells expressing a panel of TPP1 mutants. Through Western blotting (data not shown) and indirect immunofluorescence staining, we found that overexpression of TPP1 wild-type or mutant proteins led to increased endogenous POT1 levels (Fig. 8A). In TPP1 wild-type and TPP1ΔC22-expressing cells, the majority of POT1 was found in the cytoplasm, a finding consistent with the intact export activity of these TPP1 proteins. In contrast, nuclear localization of POT1 was upregulated in TPP1-NES1m1-expressing cells compared to TPP1 wild-type cells. Taken together with the data that expression of the TPP1 NES1 mutant resulted in telomere dysfunction (Fig. 6 and 7), these findings suggest that the nuclear concentration of POT1 may be an important factor in POT1-mediated telomere maintenance. Furthermore, TPP1 may also function as a gauge for maintaining proper POT1 concentration in the nucleus through its nuclear export activity.

FIG. 8.

TPP1 nuclear export regulates POT1 nuclear localization. (A) TPP1-NES1m1 expression alters endogenous POT1 localization. Parental and various Flag-TPP1-expressing cells were fixed and immunostained with anti-POT1 (in green) and anti-Flag (in red) antibodies. Subcellular localization of endogenous POT1 and Flag-TPP1 proteins was determined by fluorescence microscopy. The exposure time for the green channel is listed. The cell nuclei were stained with DAPI. (B) A proposed model indicates the role of TPP1 and TIN2 in regulating localization and assembly of telomeric complexes.

DISCUSSION

Differential subcellular localization of telomeric proteins.

Our study here explored for the first time how compartmentalization of mammalian telomeric proteins and their interactions might contribute to telomere regulation. Among the six main telomeric proteins, TRF1, RAP1, TIN2, and TRF2 mainly localize to the nucleus, whereas TPP1 and POT1 are found in both the cytoplasm and the nucleus. These findings suggest a more comprehensive picture of spatial control of telomere proteins, which can occur in the cytoplasm, in the nucleoplasm, or on the telomeric chromatin (Fig. 8B). It is also tantalizing to speculate that cytoplasmic telomeric proteins may carry out additional functions distinct from their nuclear counterparts. Moreover, differential localization of telomeric proteins may be required for their modification, and compartmentalization of their interactions may be important for the proper folding and stoichiometric assembly of various telomeric complexes.

Role of nuclear export in telomere maintenance.

We have identified a highly conserved NES in the TPP1 RD domain that is likely responsible for the cytoplasmic pool of TPP1. In addition, we showed that distinct sequence motifs appeared to mediate POT1 binding versus TPP1 nuclear export. Nonetheless, the close proximity of these motifs suggests possible cross talk between these two activities, which warrants future investigation.

The data presented here support the model that TPP1 controls POT1 subcellular localization (in addition to telomere targeting) by directly binding to POT1. First, TPP1 and POT1 are tightly connected functionally. As the mammalian homologue of ciliate TEBP heterodimer, TPP1-POT1 functions as a unit in telomere protection. Therefore, changes in TPP1 localization likely impact on POT1 as well. Second, it has been shown previously that POT1 telomere chromatin targeting was severely affected in TPP1 knockdown cells (46), implying that POT1 subcellular localization may be TPP1 dependent. Finally, in cells expressing Flag-tagged TPP1 mutants, the localization of endogenous POT1 closely mirrors TPP1 mutant proteins (Fig. 8A).

Interestingly, POT1-TPP1 interaction appears necessary but insufficient for retaining POT1 in the nucleus. For example, our data suggest a critical role of TIN2 in promoting the nuclear retention of both TPP1 and POT1. It is also possible that POT1 may harbor a nuclear localization signal that is regulated by TIN2-TPP1-POT1 interactions. In addition to TIN2 binding and the nuclear import-export apparatus, it remains possible that other proteins or signaling cues in the cytoplasm or nucleus may be required for POT1 nuclear retention. Alternatively, POT1-TPP1 interaction in the cytoplasm may be important for POT1 function in the nucleus. Forcing TPP1 into the nucleus as seen in TPP1NES1m1 may disrupt such regulation and therefore deprotect telomeres. An interesting future question is whether POT1 localization is modulated by posttranslational modifications of telomeric proteins.

Mechanism of targeting POT1 to the telomeres.

POT1 contains multiple OB folds that mediate its binding to telomere ssDNA overhangs (23). POT1 mutants that carry intact OB folds only do not interact with TPP1 and are largely defective for telomere chromatin localization (26, 46). This is somewhat puzzling considering that the POT1 OB folds can bind telomere ssDNA with very high affinity (∼10 nM) (23). One possibility is that TPP1 heterodimerization with POT1 is needed to enhance its affinity to telomere ssDNA (43, 46). The results presented here offer an additional explanation: in the absence of TPP1 interaction, POT1 may be unstable and/or sequestered in the cytoplasm and therefore fail to localize to the telomeres. The findings to date suggest an intricate and a multilevel process that controls POT1 targeting to the telomeres.

Subcellular compartmentalization and telosome assembly.

TIN2 is essential for telosome assembly through interacting with both TRF1 and TRF2, whereas TPP1 promotes the connectivity of the TRF1 and TRF2 subcomplexes (32). The TIN2-TPP1 interaction seems vital to this process. Our findings are not only consistent with the crucial roles of TPP1 and TIN2 in telosome formation but also suggest that the formation of telosome and its subcomplexes may be compartmentally regulated. Using BiFC, we have shown that TIN2 primarily interacts with TRF1 and TRF2 in the nucleus, whereas TPP1 binds POT1 and TIN2 in both the cytoplasm and the nucleus (Fig. 2). Our failure to detect cytosolic TIN2-TRF1 or TRF2 BiFC signals may be caused by unfavorable protein conformations for YFP fluorescence complementation to occur. However, the lack of cytoplasmic localization of TRF1 and TRF2 argues against this possibility. It is possible that POT1-TPP1 and TIN2-TPP1 interactions initially take place in the cytoplasm. Once the stoichiometric complex is formed among TIN2, TPP1, and POT1, the complex is transported into the nucleus, perhaps to facilitate telosome assembly. Therefore, TIN2-TPP1 binding could function as a switch to determine nuclear import and/or retention of the POT1-TPP1 complex (Fig. 8B). TIN2 may enhance POT1 and TPP1 nuclear localization through the formation of a stoichiometric TIN2-TPP1-POT1 complex that either “masks” the TPP1 nuclear export signal or provides additional nuclear localization signals, which ultimately leads to the formation of the telosome in the nucleus.

Our model also implies that TIN2 could be a limiting factor in telosome formation, in terms of both bridging different subcomplexes and regulating their localization. Consistent with this model, we found that TIN2 expression not only enhanced TPP1 and POT1 nuclear localization but also increased their targeting to the telomeres (Fig. 3). The punctate telomere fluorescence signal of YFP-POT1 was more apparent in cells coexpressing TIN2. In addition, among the six telomeric proteins, only TIN2 when expressed in HeLa cells stoichiometrically copurified with other telosome subunits (26). A possible benefit of such regulation is that concentrations of telosome subunits in the nucleus can be properly controlled. Excess TPP1 or POT1 (e.g., due to mis-expression) can be exported to the cytoplasm without affecting the stoichiometry of TPP1 and POT1 in the nucleus. In line with this notion, forcing TPP1 into the nucleus by mutating its NES sequence resulted in higher POT1 concentration in the nucleus (Fig. 8A) and telomere dysfunction (Fig. 6 and 7). Conversely, endogenous POT1 levels dropped in TPP1 knockdown cells (data not shown). Because TIN2 also associates with TRF1 and TRF2, TIN2-dependent nuclear retention of POT1-TPP1 may also influence the concentration and stability of telomeric complexes in the nucleus, which ultimately modulate telomere end protection and telomere length.

In summary, our results have revealed spatial control of protein-protein interactions of human telomeric proteins and highlighted the importance of nuclear export and retention of telomeric proteins in telomere maintenance.