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Molecular and Cellular Biology, April 2004, p. 3552-3561, Vol. 24, No. 8
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.8.3552-3561.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rescue of an hTERT Mutant Defective in Telomere Elongation by Fusion with hPot1

Blaine N. Armbruster,1 Corinne M. Linardic,1,2 Tim Veldman,1 Niharika P. Bansal,1 Diane L. Downie,1 and Christopher M. Counter1*

Department of Pharmacology and Cancer Biology and Department of Radiation Oncology,1 Department of Pediatrics, Division of Hematology and Oncology, Duke University Medical Center, Durham, North Carolina 277102

Received 23 May 2003/ Returned for modification 23 July 2003/ Accepted 21 January 2004


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ABSTRACT
 
The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations.


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INTRODUCTION
 
Telomeres are essential DNA-protein structures that cap and protect the ends of eukaryotic chromosome from illegitimate recombination, degradation, and detection as DNA damage (10). In humans, the telomere is composed of hundreds to thousands of tandem repeats of the G-rich sequence 5'-TTAGGG-3' (whereby the G strand overhangs the complementary C strand) and has been proposed to loop around, forming a T loop, and invade the duplex DNA to form a higher-order structure termed the D loop (25). The replication of telomeres poses a unique problem to eukaryotes, as removal of the terminal RNA primer during semiconservative replication of the leading strand leaves a gap that cannot be replicated by known DNA polymerases. In most eukaryotes, this is overcome by the de novo addition of telomeric DNA via the enzyme telomerase (9). Human telomerase is minimally composed of a reverse-transcriptase subunit (hTERT) that copies a template region of the accompanying RNA subunit (hTR) onto telomeres as DNA (34). Little is known regarding the mechanism by which the enzyme recognizes telomeres in vivo in higher eukaryotes. However, mutations to either the large N-terminal or smaller C-terminal DAT domains of hTERT render the enzyme catalytically active in vitro but unable to elongate telomeres or extend the life span of telomerase-negative cells in vivo (1, 3). Targeting N-terminal DAT mutants to telomeres by fusion to the double-stranded telomeric DNA-binding protein hTRF2 can extend the life span of telomerase-negative cells, although the growth of these cells was noticeably retarded (2). These results suggest that the DAT domain is involved in telomere-telomerase associations, although this model would be significantly strengthened if it could be demonstrated that a DAT mutant of hTERT could be completely rescued by targeting it to its substrate, the single-stranded G-rich telomeric DNA.

In the budding yeast Saccharomyces cerevisiae, the protein Cdc13p binds the telomere G-strand overhang, where it helps form a protective cap with other proteins (22, 23, 35) as well as recruit telomerase to telomeres, or at least activate the enzyme once bound to telomeres, through interactions with other proteins (17, 37). A putative human ortholog of Cdc13p (termed hPot1) was recently cloned (4). This protein shares sequence similarity with the DNA-binding domain of the {alpha} subunit of the telomere end-binding proteins from ciliates, which are functional and structural orthologs of Cdc13p (21, 24, 26, 31). hPot1 binds specifically to the G-rich single-stranded DNA of human telomeres in vitro (4) and (when overexpressed) colocalizes with the telomere-binding protein hTRF2 in vivo (5). Lastly, the presence of a null POT1 allele in the fission yeast S. pombe leads to dramatic telomere shortening and destabilized chromosome ends (4) whereas a similar loss of CDC13 in budding yeast leads to a catastrophic decrease of C-strand telomeric DNA and a RAD9-dependent growth arrest (17, 20, 35, 39). Collectively, these data suggest that hPot1 is a telomere-binding protein and that if the function of this protein is evolutionarily conserved, hPot1 should protect telomeres and positively regulate telomere length.

We now show that human Pot1 binds to telomeres in vivo and promotes elongation of telomeres in telomerase-positive human cells (in contrast to other human telomere DNA-binding proteins, which promote telomere shortening when overexpressed) (36, 38). Given that hPot1 binds single-stranded telomeric DNA, the substrate of telomerase, and promotes telomere elongation, we queried whether an N-terminal DAT mutant of hTERT could be rescued upon fusion to hPot1. We now show that this is the case but also find that this rescue does not depend entirely upon the DNA-binding activity of hPot1 but instead upon association with telomere chromatin. These results suggest the interesting possibility that the DAT domain might not be required for precise telomere binding but rather for general targeting to the telomere.


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MATERIALS AND METHODS
 
Plasmids. 5' BamHI, SnaBI, and NheI restriction sites followed by a Flag epitope tag and 3' SnaBI (eliminating the native stop codon to generate fusions) and SalI (to reintroduce a stop codon for experiments using unfused hPot1) sites were engineered into hPot1 cDNA by standard PCR; the resultant construct was sequenced to verify that the new open reading frame was correct. The BamHI-SalI fragment was subcloned into the same sites of pBabepuro (32) to create pBabepuro-Flag-hPot1. A C-terminal hemagglutinin (HA) epitope tag followed by a stop codon was introduced by PCR (and verified by direct sequencing) into a pBabepuro-Flag-hPot1 construct between the SnaBI and SalI sites to generate pBabepuro-Flag-hPot1-HA. pBabepuro-Flag-hTERT, pBabehygro-Flag-hTERT, pBabepuro-Flag-hTERT+128, and pBabehygro-Flag-hTERT+128 were previously described (2). The SnaBI fragment of Flag-hPot1 was introduced into the same site of pBabepuro-Flag-hTERT and pBabepuro-Flag-hTERT+128 to generate pBabepuro-Flag-hPot1-Flag-hTERT and pBabepuro-Flag-hPot1-Flag-hTERT+128. The same strategy was employed to produce these fusions in the background of pBabehygro. Mutations G76A, R80A, and R83A were introduced into Flag-hPot1 through the use of QuikChange (Stratagene) site-directed mutagenesis to generate Flag-hPot13A, which was sequenced to verify the mutations. hPot1{Delta}OB (29) was generated by subcloning into full-length hPot1 a PCR fragment containing BamHI, SnaBI, and NheI sites upstream of a Flag epitope tag adjacent to amino acid 127, thus creating pBabepuro-Flag-hPot1{Delta}OB. The SnaBI fragment of Flag-hPot13A or Flag-Pot1{Delta}OB was then introduced into the same site in pBabepuro-Flag-hTERT and pBabepuro-Flag-hTERT+128 to generate pBabepuro-Flag-hPot13A-Flag-hTERT, pBabepuro-Flag-hPot13A-Flag-hTERT+128, pBabepuro-Flag-hPot1{Delta}OB-Flag-hTERT, and pBabepuro-Flag-hPot1{Delta}OB-Flag-hTERT+128. Flag-hPot1, Flag-hPot13A, and Flag-hPot1{Delta}OB were digested with NheI and SalI and subcloned into the same sites of pCIneo (Promega) for use in the G-strand binding assays. pBabehygro-myc-hTRF2-YFP (used for colocalization experiments) was previously described (2).

Cell lines. HA5 cells, telomerase-negative human embryonic kidney (HEK) cells expressing the early region of simian virus 40 (15), or 293 cells (telomerase-positive HEK cells transfected with the E1 region of Ad5) (15) were stably infected with retroviruses derived from the pBabe plasmids described above, after which polyclonal populations were selected by treatment with 1.0 µg of puromycin/ml or 100 µg of hygromycin/ml as previously described (2). A population-doubling (pd) value of 0 was arbitrarily assigned to the first confluent plate under selection. HA5 or 293 cell lines were continually grown in dilutions of 1:8 or 1:16, respectively. Crisis was defined as the period when HA5 cell lines failed to become confluent within 3 weeks and displayed massive cell death. HA5 cell lines were considered to have an extended life span when they proliferated more than the number of population doublings required for two to three times vector control cells to reach crisis.

Chromatin immunoprecipitation assay. Chromatin immunoprecipitations were performed as previously described (reference 13 and reference therein) with the following modifications. A Branson sonifier microtip (Branson Ultrasonics) was used for sonication (output 3; duty cycle, 30% for five 10-s bursts), after which insoluble material was pelleted by microcentrifugation (13,000 x g for 5 min at 4°C), the remaining lysate was diluted in lysis buffer (1:2), and 40 µl of 50% slurry-precoupled anti-Flag M2 agarose affinity gel (Sigma) was added. Proteinase K digestion was also reduced to 2 h at 45°C. Lastly, duplicate dot blots were hybridized either with a 32P-labeled oligonucleotide telomeric probe [(T2AG3)4] in Church's buffer overnight at 50°C followed by two washes with 4x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 0.1% sodium dodecyl sulfate (SDS) or with an {alpha} satellite probe (derived from plasmid p82H (30) in Church's buffer overnight at 60°C followed by two washes with 0.1x SSC containing 0.1% SDS. Hybridization of the probes was confirmed through the use of 10 µg of total genomic DNA blotted on each membrane.

hPot1 G-strand in vitro binding assay. 35S-labeled hPot1 proteins were synthesized in vitro using a T7 quick-coupled TNT system by incubating 1 µg of pCIneo-Flag-hPot1, pCIneo-Flag-hPot1-HA, pCIneo-Flag-hPot13A, or pCIneo-Flag-hPot1{Delta}OB in rabbit reticulocyte lysate following the manufacturer's instructions (Ambion). Flag-hPot1 proteins were then immunoprecipitated using 50 µl of anti-Flag M2 agarose affinity gel per construct in 1x phosphate-buffered saline (PBS) containing 0.1 mM phenylmethylsulfonyl fluoride for 1 h at room temperature. Resin was then washed three times in 1x PBS for 5 min at room temperature, after which one-fifth of the immunoprecipitate was incubated for 30 min at room temperature in 20 µl of binding buffer (4) containing 10 nM of a G-strand oligonucleotide [(T2AG3)5], which was 32P labeled by T4 polynucleotide kinase (Invitrogen) and purified from unincorporated 32P with G-25 gel filtration Mini spin columns (Promega). Unbound G strand was removed by washing resin three times in 1 ml of 1x PBS for 5 min at room temperature. SDS loading buffer (1x) was added to samples and boiled; constituents were separated by electrophoresis on an SDS-6 to 20% polyacrylamide gel electrophoresis gradient gel, after which products were visualized by exposure to a Phosphorimager.

Telomere length and telomerase activity analysis. Lysates isolated from the described cells were diluted and assayed for telomerase activity as previously described (27). Telomere-containing terminal restriction fragments were visualized by resolving 5 µg of genomic DNA digested with HinfI and RsaI on 0.5% agarose gels, which were hybridized with a 32P-labeled (CCCTAA)3 probe followed by three washes with 15x SSC and then exposed to a Phosphorimager screen to visualize telomere-containing fragments, as previously described (15). Telomere lengths were recorded as the modal (peak) signals of the telomere-containing fragments.

Immunoblot analysis. A total of 150 µg of soluble lysate from HA5 stable cell lines was separated by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting as previously described (2) with the primary mouse monoclonal antibodies anti-Flag M2 (Sigma), anti-HA 12CA5 (Roche), anti-ß-tubulin (Sigma), and anti-ß-actin AC15 (Sigma) to detect Flag-hTERT, Flag-hPot1-Flag-hTERT (both wild-type and mutant configurations), Flag-hPot1-HA, tubulin, and actin, respectively, followed by incubation with goat anti-mouse immunoglobulin G-horseradish peroxidase (catalog no. 81-6520; Zymed Laboratories). Proteins were detected with ECL reagent (Amersham Pharmacia Biotech), following the manufacturer's protocol. Lysates were similarly prepared to detect Flag-hPot1 in 293 cells except that standard radioimmunoprecipitation buffer supplemented with 5 mM EDTA-2 µg of aprotinin/ml-1 µg of leupeptin/ml-1 µg of pepstatin A/ml-0.1 mM phenylmethylsulfonyl fluoride-1 mM Na3VO4-1 mM dithiothreitol was used. To immunoprecipitate Flag-tagged hPot1, 2 mg of soluble lysate was incubated with 10 µl of anti-Flag M2 agarose affinity gel at room temperature for 1 h in 1x PBS containing the aforementioned protease inhibitors. Similarly, HA-tagged hPot1 proteins were incubated with 10 µl of anti-HA affinity matrix (Roche). Resins were washed three times at room temperature for 5 min each time in 1x PBS buffer, after which the total resin was boiled in 1x SDS loading dye, resolved, and subjected to immunoblotting with the anti-Flag M2 antibody as described above. In the coexpression experiments, Flag-hPot1 proteins were similarly detected from HA5 cells through the use of 0.5 mg of total cell lysate.

Immunofluorescence. U2OS cells, a human cancer cell line with long telomeres (12), were stably infected with pBabepuro-Flag-hPot1 or pBabepuro-Flag-hPot1-Flag-hTERT and transiently transfected with pBabehygro-myc-hTRF2-YFP through the use of FuGENE 6 reagent (Roche). At 48 h posttransfection, cells were fixed, permeabilized, and blocked as previously described (2) except that 0.1% Triton X-100 was used to permeabilize the cells. Flag-tagged hPot1 and hPot1-hTERT proteins were detected with the anti-Flag M2 antibody recognized by a donkey anti-mouse antibody conjugated with Rhodamine RedX (Jackson ImmunoResearch). hTRF2-YFP was visualized by virtue of its fluorescence. Goat anti-PML (Santa Cruz Biotechnologies) was used in conjunction with donkey anti-goat conjugated with fluorescein isothiocyanate (Jackson ImmunoResearch) to detect endogenous PML bodies. Cells were examined at x630 magnification on an Olympus IX70 confocal fluorescent microscope.


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RESULTS
 
hPot1 binds telomeric DNA in vivo. Mutations in the N-terminal DAT domain of hTERT render the protein catalytically active but unable to elongate telomeres (1), a phenotype that could be partially alleviated by targeting the defective enzyme to telomeres via fusion with the telomere-binding protein hTRF2 (2). However, it is formally possible that such a fusion protein might create a dominant-negative hTRF2 molecule, which could rescue a DAT mutant of hTERT by virtue of disrupting chromatin and forming a more open confirmation that would facilitate access of hTERT to telomeres. To discount this possibility, we sought to fuse hTERT harboring a mutation in the N-terminal DAT domain to a completely different telomere-binding protein, namely, hPot1. Pot1 has been shown by immunofluorescence analysis to colocalize with the telomere-binding protein hTRF2 in vivo and to associate with oligonucleotides encoding the G-rich strand of telomeric DNA in vitro (4, 5). We now show biochemically that hPot1 associates with telomeres in vivo. Cells expressing an epitope-tagged hPot1 were treated with formaldehyde to cross-link hPot1 with telomeric DNA and proteins. hPot1-chromatin complexes were then immunoprecipitated with an antibody directed against the epitope tag and shown to associate with telomeric DNA, as detected by Southern hybridization with a telomeric probe (Fig. 1A). This association was specific, as little telomeric DNA was coimmunoprecipitated with hPot1 when the cells were not treated with formaldehyde or when an irrelevant antibody was used for immunoprecipitation and because other repetitive DNAs were not coimmunoprecipitated with hPot1 (Fig. 1A). As recently reported (29), this association is robust, as both hPot1 and hTRF1 coimmunoprecipitated telomeric DNA (Fig. 1A).



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  		FIG. 1. hPot1 binds telomeres and promotes telomere elongation. (A) 293 cells transiently transfected with Flag-tagged hPot1 or hTRF1 were treated with or without formaldehyde (X-link), lysed, and subjected to immunoprecipitation with the indicated antibodies. After purification, coimmunoprecipitated DNA was analyzed by Southern dot blot analysis with the indicated probes. Genomic DNA was used as a control for probe integrity. (B and C) 293 cells were stably infected with a control virus (vector), Flag-tagged hPot1{Delta}OB (f-hPot1{Delta}OB), or Flag- and HA-tagged hPot1 (f-hPot1-HA). The resulting cells were assayed for ectopic hPot1 expression by immunoprecipitation (IP) followed by immunoblotting (IB) with the indicated antibodies (B) and for changes in telomere length by Southern blot analysis with a telomeric probe and with genomic DNA isolated at the indicated pd (right panel) (C). 293 cells infected with vector or Flag-tagged hPot1 (f-hPot1) alone were similarly assayed (left panel).

Ectopic expression of hPot1 results in telomere elongation. Given that hPot1 bound telomeres in vivo, we next addressed the biological significance of this association to understand the effects of fusing hPot1 to hTERT. Previously, hTRF1 and hTRF2 were identified as the primary proteins that bind directly and specifically to telomere DNA in human cells (16). Both these proteins are structural components of telomeres and when overexpressed cause telomere shortening, presumably by altering chromatin structures such that access of telomerase to telomeres is inhibited (36, 38). We therefore tested whether ectopic expression of hPot1 in telomerase-positive cells would also induce telomere shortening. Telomere length in 293 cells stably expressing N-terminally Flag-tagged hPot1 was assayed by Southern blotting with a telomeric probe. Surprisingly, we found that cells overexpressing hPot1 exhibited a progressive increase in telomere length compared to vector control cells (Fig. 1C). Similar results were recently reported when hPot1 was overexpressed in HT1080 cells (14). hPot1 can therefore act as a positive regulator of telomere length.

It had recently been shown that expression of hPot1 with an N-terminal deletion to remove the DNA-binding domain results in telomere elongation in telomerase-positive cells (29). To rule out the formal possibility that the elongation observed to result from expressing full-length hPot1 in 293 cells was due to the generation of a truncated product defective in DNA binding (which would not be detected by an antibody against the N-terminal epitope tag), an HA epitope tag was engineered into the C terminus of the Flag-tagged hPot1 and the resultant protein was stably expressed in 293 cells. Immunoblot analysis following immunoprecipitation using the anti-HA antibody confirmed that only the full-length N- and C-terminal epitope-tagged hPot1 was stably expressed (Fig. 1B). Southern blot analysis confirmed that the 293 cells expressing this protein exhibited an increase in telomere length over time (Fig. 1C). Thus, ectopic expression of full-length hPot1 results in telomere elongation, at least in some telomerase-positive cell types.

Fusion of hPot1 to hTERT targets telomerase to telomeres. Having demonstrated that hPot1 binds telomeric DNA in vivo and having characterized the effect of overexpressing this protein, we could next address whether fusion of hPot1 could rescue the inability of hTERT harboring a mutation in the DAT domain (mutant +128) to elongate telomeres in vivo and extend cell life span. Flag-tagged hPot1 cDNA was therefore fused in frame with the cDNA encoding Flag-tagged wild-type hTERT or the DAT mutant hTERT+128. To determine whether this association altered hPot1 function, we monitored the telomere localization of hPot1 alone or fused to hTERT or hTERT+128 in human U2OS cells, which have long telomeres (12), facilitating the detection of proteins bound to telomeres by immunofluorescence. Expression of these proteins was confirmed by immunoprecipitation followed by immunoblotting with an anti-Flag antibody (Fig. 2A). The telomere localization of hPot1, hPot1-hTERT, and hPot1-hTERT+128 was then assayed by comparing the subcellular distribution of these proteins with that of transfected YFP-tagged hTRF2 (8, 11), a known telomere-binding protein. We found in all cases that hPot1 or derived fusion proteins colocalized with hTRF2-YFP (Fig. 2B). hTRF2 is presumably also associated with PML bodies in U2OS cells, which employ an alternative mechanism of telomere elongation (12, 42). We ruled out the possibility that hPot1-hTERT was at PML bodies instead of telomeres, because hPot1, hPot1-hTERT, and hPot1-hTERT+128 colocalize with hTRF2 nearly 100% of the time but with PML bodies only ~10 to 20% of the time (data not shown). Fusion of hTERT to hPot1 therefore does not disrupt the association of hPot1 to telomeres but instead redirects hTERT to chromosome ends.



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  		FIG. 2. Fusion of hPot1 to hTERT relocalizes catalytically active hTERT to telomeres. (A and B) Flag-tagged hPot1, hPot1-hTERT, and hPot1-hTERT+128 were stably introduced into U2OS cells. (A) Flag-hPot1 proteins were immunoprecipitated with an anti-Flag antibody and detected by immunoblotting with the same antibody. (B) hPot1-containing proteins stably expressed in U2OS cells were detected by immunofluorescence with an anti-Flag antibody (red) and compared with localization of transfected hTRF2-YFP (green), which was detected by virtue of its fluorescence. (C and D) HA5 cells stably infected with a control retrovirus (vector) or one encoding Flag-tagged hTERT, hPot1-hTERT, or hPot1-hTERT+128 in either pBabepuro or (for lower expression) pBabehygro (indicated by asterisks) were confirmed to express the appropriate protein by immunoblotting with an anti-Flag antibody (tubulin served as a loading control) (C) and were assayed for telomerase activity with threefold serial dilutions of cellular lysates, starting with 0.2 µg (internal control [IC] results and average TRAP activity levels [normalized to those of wild-type hTERT] are indicated at the bottom of the panel) (D). HT, heat treatment to inactivate telomerase.

The hPot1-hTERT and hPot1-hTERT+128 chimeras also retained the catalytic function of hTERT. Vectors encoding hPot1-hTERT or hPot1-hTERT+128 (or, as controls, vector alone or encoding hTERT or hTERT+128) were stably expressed in telomerase-negative human HA5 cells, as detected by immunoblot analysis (Fig. 2C). Since the fusion proteins were expressed at higher levels compared to the results seen with hTERT, additional cell lines were generated using a construct that reduced the expression of the fusion proteins (Fig. 2C). Ectopic expression of hTERT is known to restore telomerase activity in HA5 cells, which normally lack hTERT but express the hTR RNA subunit (1). Indeed, serial dilutions of extracts isolated from both hTERT- and hPot1-hTERT-expressing cells, but not from vector control cells, supported similar levels of elongation of a telomeric primer in vitro by the addition of telomeric repeats, which (when PCR amplified and resolved) formed a ladder indicative of telomerase catalytic activity. Similarly, fusion of hPot1 to hTERT+128, which itself is somewhat reduced in its ability to restore telomerase activity (see Fig. 5), had little effect on the catalytic activity of this mutant (Fig. 2D). The expression level of fusion proteins had no bearing on telomerase activity, likely because the presence of the hTR subunit is limiting (40). Thus, fusion of hPot1 to hTERT or hTERT+128 had no obvious effect on either the telomerase catalytic activity (as assessed by this assay) or the telomere-binding activity (as assessed by immunofluorescence) of hPot1.



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  		FIG. 5. Rescue of hTERT+128 by fusion with hPot1 mutants. HA5 cells stably infected with a vector control, hTERT, hPot1-hTERT, hPot13A-hTERT, hPot1{Delta}OB-hTERT, hTERT+128, hPot1-hTERT+128, hPot13A-hTERT+128, or hPot1{Delta}OB-hTERT+128 were confirmed by immunoblot analysis with an anti-Flag antibody to express the appropriate fusion proteins (actin serves as a loading control) (A) and assayed for telomerase activity with a ninefold dilution of cellular extracts, starting at a total of 0.2 µg (B). The internal control (IC) results and average values of TRAP activity (normalized to that of wild-type hTERT) are shown at the bottom of the panel. (C and D) Cell life span (mutants: hPot13A-hTERT [•], hPot13A-hTERT+128 [{circ}], hPot1{Delta}OB-hTERT [{blacktriangleup}], hPot1{Delta}OB-hTERT+128 [{triangleup}]; controls: hPot1-hTERT [{blacksquare}], hPot1-hTERT+128 [{square}], [hTERT+128 [{diamond}]) (C) and telomere length (as measured by Southern blotting analysis with a telomeric probe) (D) were monitored over time.

hTERT+128 function is restored by fusion to hPot1. We next determined whether fusion of hPot1 could overcome the inability of hTERT harboring a mutation in the DAT domain (hTERT+128) to arrest telomere shortening and extend the life span of telomerase-negative cells. Cell life span and telomere length were monitored in HA5 cells stably expressing hPot1-hTERT+128 or (as a control) hTERT, hTERT+128, hPot1-hTERT, or vector. Since HA5 cells lack telomerase activity, they lose telomeric DNA at each cell division until a critically short length is reached which leads to a period of genomic instability and cell death (termed crisis) (15). HA5 cells have therefore been used to monitor the ability of telomerase to function in vivo (1-3). As expected, vector control HA5 cells had short telomeres and were mortal (owing to the fact that these cells remain telomerase negative). On the other hand, cells expressing hTERT had a greatly reduced rate of telomere shortening and at late passage stably maintained telomere length as well as proliferating well beyond the point at which vector control cells perished at crisis (Fig. 3A and B). Interestingly, the telomeres of cells expressing hPot1-hTERT were 2 to 3 kb longer than those found in hTERT control cells (Fig. 3A). These telomeres were functional, as the cells continued to proliferate in a fashion identical to that seen with hTERT-expressing cells (Fig. 3B). Lastly, this effect was dependent upon direct fusion with hPot1 (as telomere elongation was not observed when the proteins were coexpressed), arguing that fostering the association of telomerase with telomeres underlies the ability of hPot1-hTERT to promote telomere elongation (see Fig. 6). Similar results were found when these cell lines were independently derived and again assayed for protein expression, telomerase activity, cellular life span, and telomere length or when cells expressing low levels of hPot1-hTERT were analyzed (data not shown). Thus, fusion of hPot1 to hTERT does not impede the ability of hTERT to elongate telomeres in vivo; rather, the forced targeting of telomerase to telomeres by hPot1 actually leads to telomere elongation.



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  		FIG. 3. Elongation of telomeres and life span extension by hPot1-hTERT and hPot1-hTERT+128 in mortal telomerase-negative cells. HA5 cells stably infected with a control retrovirus (vector) or one encoding Flag-tagged hTERT, hTERT+128, hPot1-hTERT, or hPot1-hTERT+128 were monitored for changes in telomere length by Southern blot analysis with a telomeric probe and with genomic DNA isolated at the indicated pd (A) and were grown in cultures to monitor cell life span (B). Average telomere length values [Avg length] are shown below panel A. hPot1-hTERT, {square}; hPot1-hTERT+128, {circ}; hTERT+128, •; vector, {blacktriangleup}; hTERT, {blacksquare}.



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  		FIG. 6. Rescue of in vivo functions of hTERT+128 requires direct fusion with hPot1. HA5 cells were stably coinfected with either hTERT or hTERT+128 in combination with control vector, hPot1, or hPot1{Delta}OB and were confirmed to express the appropriate proteins by immunoblotting with an anti-Flag antibody following immunoprecipitation (A), monitored for life span (hTERT with vector, {blacksquare}; hTERT with hPot1, •; hTERT with hPot1{Delta}OB, {blacktriangleup}; hTERT+128 with vector, {square}; hTERT+128 with hPot1, {circ}; hTERT+128 with hPot1{Delta}OB, {triangleup}) (B), and assayed for telomere length by Southern blot analysis with a telomeric probe and with genomic DNA isolated at the indicated pd (C).

Cells expressing hTERT+128, which cannot immortalize human cells owing to a defect in telomere elongation in vivo (1, 2), entered crisis like the vector control cell line with short telomeres. This mutant phenotype was completely rescued upon fusion to hPot1, as cells expressing hPot1-hTERT+128 proliferated indefinitely and maintained long telomeres (~6 kb), in similarity to control hPot1-hTERT cells (Fig. 3). Targeting of hTERT+128 to telomeres by hPot1 appears not only to rescue the inability of the crippled enzyme to replicate telomeres but also to enhance the ability of telomerase to elongate telomeric DNA. Similar results were found when these cell lines were independently derived and again assayed for protein expression, telomerase activity, cellular life span, and telomere length or when cells expressing low levels of hPot1-hTERT+128 were analyzed (data not shown).

The telomere-binding activity of hPot1 is required for rescue of hTERT+128. To test whether the telomere-binding activity of hPot1 is required for rescue of hTERT+128, we generated Flag epitope-tagged mutants hPot1{Delta}OB (lacking the OB-fold region of the protein) (29) and hPot13A (in which three conserved amino acids in the OB-fold region were mutated to alanines) (Fig. 4A). Both had a greatly reduced affinity for telomeric DNA in vitro, as there was a 10-fold decrease in the amount of hPot13A or hPot1{Delta}OB recombinant proteins bound to the G-strand telomeric oligonucleotide despite the fact that wild-type hPot1 readily associated with this DNA in manner specific for the G strand, but not the C strand, of telomeric DNA (Fig. 4B and data not shown). Flag-tagged hPot1{Delta}OB fused to Flag-tagged hTERT or hTERT+128 was still found to be telomeric by immunofluorescence analysis (data not shown), as expected on the basis of the association of this mutant with telomeric proteins (29). On the other hand, hPot13A fusion proteins failed to show punctate nuclear staining characteristic of telomere-binding proteins, suggesting a defect in telomere association, although this interpretation is tempered by the finding that hPot13A fusion proteins were more difficult to express in cells (Fig. 5A) and more cytoplasmic than hPot1-hTERT fusion proteins (data not shown).



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  		FIG. 4. Mutations in the hPot1 DNA-binding region diminish the ability of hPot1 to interact with a telomeric G-strand oligonucleotide in vitro. (A) Pictorial representation of hPot1{Delta}OB and hPot13A mutants (amino acid positions are indicated by numbers). Sequence alignment of putative telomere end-binding proteins from divergent eukaryotes (accession numbers: Macaca fascicularis [AB066545], Homo sapiens [NM_015450], Mus musculus [NM_133931], Euplotes crassus [M96818], and Caenorhabditis elegans [T22006]) through the use of a manually modified ClustalW alignment shows three identically conserved amino acids (asterisks) that were replaced with alanines in hPot13A. Conserved residues are show in gray-shaded boxes; identical residues are shown with white characters in black boxes. (B) Association of in vitro 35S-labeled hPot1 proteins with a 32P-labeled G-strand oligonucleotide. IP, immunoprecipitation.

hPot13A-hTERT+128 or hPot1{Delta}OB-hTERT+128 (or, as a control, hPot13A-hTERT, hPot1{Delta}OB-hTERT, hPot1-hTERT, or no transgene [vector]) was stably expressed in HA5 cells, as assessed by immunoblotting for the Flag epitope (Fig. 5A). These fusion proteins restored similar levels of telomerase activity to hTERT and hTERT+128, as assessed by dilution experiments (with the exception of hPot13A-hTERT+128, which exhibited a threefold decrease in enzyme activity compared to hTERT+128) (Fig. 5B). However, the presence of hPot13A does not appear to be detrimental to hTERT function in vivo, as expression of hPot13A-hTERT both extended the life span and stabilized the telomeres of HA5 cells. Fusion of hPot13A to hTERT+128 failed to prevent HA5 cells from losing telomeric DNA and entering crisis (Fig. 5C and D). These data suggest that telomere targeting of hPot1 is important for the rescue of the DAT mutant of hTERT, although the lower levels of telomerase activity and poorer expression might also contribute to the inability of hPot13A-hTERT+128 to arrest telomere shortening.

HA5 cells expressing hPot1{Delta}OB-hTERT proliferated extensively (Fig. 5C) and had telomeres as long as those detected in cells expressing hPot1-hTERT (Fig. 3A and 5D), reflecting either efficient targeting of the fusion protein to telomeres or the ability of hPot1{Delta}OB to independently promote telomere elongation or both. Consistent with these results, the same cells expressing hPot1{Delta}OB-hTERT+128 proliferated beyond the point of crisis at which vector control and hTERT+128- and hPot13A-hTERT+128-expressing cells died, although we note an increase in the doubling time of the culture (Fig. 5C). Telomeres of hPot1{Delta}OB-hTERT+128-expressing cells were initially long but then underwent a gradual decline over time (Fig. 5D) (unlike cells expressing hPot1-hTERT+128 or hPot1{Delta}OB-hTERT, in which telomeres were stably maintained at a long length) (Fig. 3A and 5D). It therefore appears that fusion with hPot1{Delta}OB rescues hTERT+128 by targeting the enzyme to telomere chromatin and not directly to telomeric DNA. However, this was not equivalent to the rescue by fusion with hPot1, which was more efficient at elongating telomeres and extending life span.

Rescue of hTERT+128 requires direct fusion to hPot1. Expression of hPot1 in telomerase-positive 293 cells caused telomere elongation (Fig. 1B). This raises the possibility that the rescue of the DAT mutant of hTERT by fusion with hPot1 might be due to overexpression of hPot1 (in the form of a fusion protein), which alters telomere structure to favor access of telomerase to telomeres. In contradiction to such a model, hPot1 needs to be physically tethered to hTERT+128 to rescue the inability of this mutant protein to extend cell life span. HA5 cells stably expressing hPot1 and hTERT+128 in trans entered crisis and died with short telomeres in a fashion identical to that seen with control cells expressing hTERT+128 (Fig. 6). Even when telomeric chromatin was converted to an open state by the expression of hPot1{Delta}OB, hTERT+128 could not extend cell life span. Specifically, telomeres were greatly elongated and cell life span was extended in HA5 cells coexpressing hPot1{Delta}OB and wild-type hTERT (indicating altered telomere chromatin), whereas the same cells coexpressing hPot1{Delta}OB and hTERT+128 were mortal and had short telomeres (Fig. 6).


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DISCUSSION
 
Expression of hPot1 clearly caused telomere elongation in telomerase-positive 293 cells, and this was not a result of cryptic translation from an internal ATG leading to an N-terminally truncated product. This effect appears to depend upon other unknown factors, as ectopic hPot1 induced telomere elongation in 293 cells, but not in hTERT-expressing HA5 cells or in certain populations of HT1080 cells (as shown by others) (14, 29). The mutant hPot1{Delta}OB was, as expected (29), also found to elongate telomeres, although we note that this mutant was more robust at increasing telomere length than wild-type hPot1 when expressed in 293 and HA5 (plus hTERT) cells, leading us to speculate that perhaps these two constructs induce telomere elongation by different mechanisms. For example, hPot1{Delta}OB may disrupt the overall conformation of the telomere (i.e., the T loop) by inhibiting appropriate formation of telomere protein complexes. Conversely, hPot1 may displace G-strand DNA at the D loop, making the region more accessible to telomerase, or (since Cdc13p helps recruit telomerase to telomeres) (17) hPot1 may target telomerase to G-strand telomeric DNA. In the latter case, it is worth noting that fusion of both Cdc13p to S. cerevisiae TERT (Est2p) and hPot1 to hTERT led to telomere elongation (Fig. 3B) (17). Alternatively, it is also possible that overexpression of both proteins perturbs the same telomere protein complexes; because hPot1{Delta}OB is not sequestered to the G strand, however, more of this protein is available to disrupt such complexes.

We suggest that the DAT domain is involved in telomere-telomerase associations in vivo. First, the phenotype of cells expressing hTERT with mutations in the DAT domain is consistent with an inability of hTERT to properly associate with telomeres in vivo (1, 3). Second, overexpression of proteins that associate with telomeres can rescue DAT mutants in yeast (19). Third, hTERT+128 fusion to two completely different telomere-binding proteins that bind to very different regions of telomeres rescues the inability of this mutant hTERT to elongate telomeres (Fig. 3) (2). This rescue depended on the ability of both fusion proteins to associate with telomeres in vivo (Fig. 5) (2) and could not be ascribed to the fusion generating a mutant telomere-binding protein that altered telomeric chromatin to favor access of telomerase to telomeres. How the DAT domain performs this function is not known. We show that targeting the hTERT+128 mutant to the telomere G-strand extension is the most efficient approach for rescue of the defects of this mutant. However, since the same mutant can be rescued almost as well by simply increasing the association of hTERT+128 with telomere chromatin (via fusion with hPot1{Delta}OB), the DAT domain may be involved in a more general association with telomeres.

The DAT domain is nestled within a larger conserved N-terminal region termed domain I (18) or the GQ domain (41). This larger region can complement catalytic activity in trans of N-terminally deleted hTERT (6), and mutations to this region generally abolish catalytic activity or decrease enzyme processivity (1, 7, 28), possibly related the fact that domain I has a weak affinity for the hTR RNA component of telomerase (33). The N terminus of hTERT therefore appears to encode an essential activity—perhaps processivity (7). Although the DAT domain resides in domain I, the +128 mutant (or nearby mutants) has little or no effect on telomerase activity (1, 28) and, in the case of the +128 mutant, is rescued by targeting to telomere chromatin. We speculate that the DAT domain may be related to an essential function of domain I, possibly by promoting an association with telomere chromatin in vivo through interactions with telomeric proteins. In this regard, it is worth noting that DAT mutants in yeast are complemented by proteins that facilitate telomere-telomerase association (19).


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ADDENDUM
 
While this paper was under review, two papers describing the effects of hPot1 expression on telomere length were published and are duly noted herein (14, 29).


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ACKNOWLEDGMENTS
 
We thank Tom Cech and Dominique Broccoli for generously providing hPot1 cDNA and plasmid p82H, respectively. Members of the Counter laboratory are thanked for helpful advice.

This work was supported by NIH grant CA82481. B.N.A. is supported by a Department of Defense Breast Cancer Research Predoctoral Fellowship. C.M.C. is a Leukemia and Lymphoma Society Scholar.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pharmacology and Cancer Biology and Department of Radiation Oncology, DUMC-3813, Durham, NC 27710. Phone: (919) 684-9890. Fax: (919) 684-8958. E-mail: count004{at}mc.duke.edu. Back


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Molecular and Cellular Biology, April 2004, p. 3552-3561, Vol. 24, No. 8
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.8.3552-3561.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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