Expression of mouse telomerase catalytic subunit in embryos and adult tissues

The expressed sequence tag database dbEST was searched by using the tblast program for sequences homologous to the previously described telomerase reverse transcriptases of Euplotes aediculatus and Saccharomyces cerevisiae. Two putative human homologues were found, EST-A [GenBank accession no. AA281296 (also reported by Meyerson et al. in ref. 21)] and EST-B (AA311750).

A cDNA library from mouse embryonic stem cells was screened simultaneously with two different PCR fragments derived from EST-A and EST-B, respectively.

EST-A was obtained from Genome Systems (clone 712562A) and PCR-amplified by using the primers EST-A-5′ (5′-GGGGAATTCGCCAAGTTCCTGCACTGGCTGATG-3′) and ESTA-3′ (5′-CCCCCCCTGCAGCTACGCCCGCTCGTAGTTGAGCACGCT-3′) and was used to probe a mouse cDNA library. A second probe was obtained by amplifying a human cDNA library with primers EST-A-5′ (see above) and EST-B-3′ (5′- CCCCCCCTGCAGCTAAGGGAAGTTCACCACTGTCTTCCG-3′). These primers amplify a 1.1-kb fragment that extends from EST-A to EST-B. Both PCR fragments were labeled by random priming extension using labeled [α-³²P]dCTP and [α-³²P]-dGTP (3000 Ci/mmol; 1 Ci = 37 GBq).

The 5′ end of the mouse telomerase reverse transcriptase (mTERT) ORF was obtained by 5′RACE-PCR (GIBCO/BRL, version 2). Total RNA was obtained from mouse embryonic fibroblasts (MEFs) (31). The primers used were PRIMER 5 race1 (5′-AGGTGGAGGCTGTGAGA-3′) for cDNA synthesis, and two nested primers NESTED1 (5′-GGGGTCGACTTGGGCAACCAAAGTGCG-3′) and NESTED2 (5′-GGTCGACGCGGTAGATCTTCGGGTC-3′) for the PCR step.

Sequencing was done with a T7 Sequenase version 2.0 kit (Amersham) and an Applied Biosystems 377 DNA sequencer with the Prism dRhodamine Terminator Cycle Sequencing kit (Perkin-Elmer). Sequences were aligned with the MegaAlign DNA Star program.

The antigenic peptide FQKNRLFFYRKSVWC, also called PEPT-1, was synthesized on an automated multiple peptide synthesizer (AMS 422, Abimed) (32). The peptide was cleaved from the resin (33) and purified in reverse-phase HPLC. Peptide purity and composition were confirmed by reverse-phase HPLC and by amino acid analysis (Beckman 6300).

The peptide was used as immunogen to induce antibodies specific for human and mouse telomerase. Sera were collected and the IgG fraction was purified by protein G-Sepharose chromatography and used for the experiments described. The antibody was called K-370.

Mouse embryo fibroblasts (MEFs) were prepared from 13.5-day wild-type or mTR^−/− embryos (2). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

HeLa cells were synchronized by the double thymidine block method (34). MEFs were incubated for 48 hr in low-serum (0.1%) medium, then changed to rich medium (10% serum) plus aphidicolin (5 μg/ml) for 24 hr; cells were released from the block by adding rich medium without the drug. Progression into S phase was monitored by fluorescence-activated cell sorter (FACS) analysis at different times after the block release.

Adult mouse tissues were obtained from 2-month-old strain C57BL/6J or from wild-type and mTR^−/− littermates (2).

Telomerase activity was reconstituted in vivo in mTR^−/− cells by conventional calcium phosphate transfection with a plasmid containing an empty vector, the hTR gene under a cytomegalovirus (CMV) constitutive promoter, or a plasmid containing the mTR gene and upstream promoter sequences (2). Transfection was carried out as described (2). To measure telomerase activity, S-100 extracts were prepared from cell cultures as described (35). Telomerase activity was measured with a modified version (35) of the TRAP assay (TRAPeze kit; Oncor) (36). An internal control for PCR efficiency was included.

For RT-PCR analysis of mTERT mRNA, 1 μg of total RNA isolated from cells or tissues (31) was treated with DNase I (Boehringer) and hybridized to 100 ng of 6-nt random hexamers at 95°C for 10 min, followed by reverse transcription at 37°C for 1 h with SuperScript II reverse transcriptase (GIBCO/BRL). As a control, reactions were also carried out in the absence of reverse transcriptase (not shown). PCR amplification was performed using two sets of primers: clone2a (5′-CAGACATTTCCTTTACTC-3′) and clone2b (5′-ACCATATACCTGCCAGGG-3′) or Race I (5′-CTGCAATGTGACCTGAGG-3′) and 55/1 (5′-GAGCGCAACGAGAGAAACG-3′). After the PCR cycles (1 min at 94°C, 45 sec at 57°C, and 1 min and 30 sec at 72°C) in the presence of [α-³²P]dATP and [α-³²P]dGTP, the PCR products were separated in nondenaturing 4% polyacrylamide gels. The number of cycles used was in the linear range and varied from 20 to 25 cycles in different PCRs. To normalize the amounts of input RNA, amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was used as control.

A partial mTERT cDNA fragment (nucleotides 1629–3367), cloned in pBluescript SK (−), was digested at the 5′ or 3′ end of the insert and transcribed in the presence of digoxigenin (Riboprobe Kit; Promega) with T7 or T3 RNA polymerase, respectively. Mouse embryos were obtained at days 9–12 from wild-type or mTR^−/− pregnant females and fixed in 4% paraformaldehyde at 4°C for 12 hr. Embryos were treated with 6% H₂O₂ for clearing. Embryos were then prehybridized at 70°C for 1 hr (50% formamide, 5× SSC, 0.1 mg/ml yeast RNA, 1% SDS, and heparin), followed by the addition of the probe and incubation at 70°C for 16 hr in the same solution. Embryos were washed and transferred to blocking solution containing 1% Tween 20, 20% fetal bovine serum, and 1× blocking powder. They were then incubated with anti-digoxigenin antibody (anti-digoxigenin Fab fragments coupled to alkaline phosphatase) for 16 hr at 4°C, and the signal was developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Boehringer Mannheim).

As indicated, 20–100 μg of total protein from S-100 extracts (see below) was loaded per lane and subjected to electrophoresis in SDS/polyacrylamide gels (37). Equal amounts of protein were loaded. To determine protein concentration accurately in the extracts, the Micro BCA (bicinchoninic acid) protein assay kit (Pierce) was used. Gels were transferred to Problott membranes (Applied Biosystems) by using a Mini Trans Blot System (Bio-Rad). Membranes were blocked and incubated with rabbit serum samples for 90 min at room temperature, followed by horseradish peroxidase-labeled protein G and developed with enhanced chemiluminescence (ECL; Amersham). Antibody specificity was determined by immunoblotting using preimmune serum, anti-peptide antiserum, and anti-peptide antiserum preincubated with the immunizing peptide (PEPT-1) or with an unrelated peptide of the same length (PEPT-2)(TFLRTLVRGVPEYGC).

Human HeLa cells, MEFs, and human IMR-90 cells were seeded in chamber slides (Lab-Tek) and cultured overnight in DMEM supplemented with 10% fetal bovine serum. Cells were washed twice in PBS and fixed for 3 min in methanol. After two more washes in PBS, cells were blocked with 10% goat serum (Sigma) and incubated for 1 hr at 37°C with a 1/1000 dilution of the telomerase-specific K-370 antibody in 1% serum. As specificity control, the K-370 antibody was preincubated with the immunizing peptide (see above, not shown). Cells were washed in PBS, and a 1/400 dilution of rhodamine-conjugated anti-rabbit-immunoglobulin (Pierce) in 1% serum was added for 1 hr at 37°C. Nuclei were stained with Hoechst 33258. Images were captured with a Leitz DMRB fluorescence microscope (Leica) and the program q-fish (Leica).

Nuclear and cytoplasmic extracts were prepared from asynchronous and synchronous cultures (1–2 × 10⁶ cells) (38). Equal amounts of cytoplasmic and nuclear extracts were loaded per lane for Western blot analysis.

To isolate mTERT, we used two human expressed sequence tags that presented significant homology with the telomerase reverse transcriptases of ciliates and yeast to screen a mouse cDNA library (Materials and Methods). The mTERT gene contains an ORF that encodes a protein of 1,122 amino acids with a predicted molecular mass of 125 kDa.

The full sequence of the human telomerase reverse transcriptase, hTERT, has been reported by several groups (19–24). The nucleotide sequences of mTERT and hTERT are 74% identical, the protein identity is 64%, and the protein similarity, 69%. Mouse and human telomerase RNAs, mTR and hTR, respectively, are also relatively conserved (15). The high degree of identity between murine and human telomerase components suggested that they might share a similar structure. To test whether mouse and human telomerase complexes are functionally conserved, we asked whether the mTERT was able to reconstitute telomerase activity with the human telomerase RNA subunit in vivo. Murine cells that are genetically deficient for the mTR gene, mTR^−/−, are also deficient for telomerase activity (2). Telomerase activity in these cells can be rescued, however, by expressing an exogenous mTR gene (2) (Fig. 1). To test whether hTR was able to associate with mTERT and form a functionally active telomerase complex, we transfected the hTR gene into mTR^−/− cells. hTR was able to reconstitute telomerase activity in vivo in mouse cells lacking the mTR gene (Fig. 1), indicating that human and mouse telomerase proteins are functionally interchangeable and suggesting that mouse and human telomerase complexes are structurally conserved.

To analyze mTERT expression in mouse embryos, we performed whole-mount in situ hybridization using 9- to 12-day embryos and a digoxigenin-labeled mTERT RNA probe (see Materials and Methods). Fig. 2 a and b shows two 11-day wild-type embryos probed with sense or antisense mTERT RNA. The sense probe does not stain the embryos except for a low background signal (Fig. 2a). In contrast, the antisense probe shows that mTERT mRNA is abundant in the entire embryo and particularly in limbs, tip of the tail, and the nares (Fig. 2b; see also Fig. 2f). This expression pattern suggests that mTERT mRNA is expressed at higher levels in regions of intense proliferative activity. At day 12, the nares show a spotted pattern that coincides with the developing hair follicles of the whiskers (see Fig. 2 c and d, for embryos hybridized with sense or antisense probes, respectively).

To study whether the regulation of mTERT mRNA depends on the presence of other components of the telomerase complex, we analyzed mTERT expression in embryos lacking the essential mTR telomerase component. Embryos deficient in the mouse telomerase RNA gene, mTR^−/−, were hybridized with mTERT-derived sense and antisense riboprobes. Fig. 2e (sense probe) and f (antisense probe) show that mTR^−/− embryos have a pattern of mTERT mRNA expression similar to that in wild-type embryos (compare Fig. 2 b and f). These results suggest that mTERT mRNA expression is independent of the presence or absence of mTR, and consequently of the formation of active telomerase complexes.

We studied mTERT mRNA expression in adult murine tissues by RT-PCR using two sets of primers that amplify fragments of 363 and 988 bp (Materials and Methods). In a first set of experiments, we used cDNA derived from four adult tissues: testis and liver, which have telomerase activity, and brain and kidney, which are telomerase-negative or contain low levels of telomerase activity, respectively (5, 39). Using these two sets of mTERT-specific primers, we found that mTERT mRNA is detectable in all tissues, regardless of the presence or absence of telomerase activity, although its abundance appears more prominent in testis and liver, in agreement with the fact that these tissues also show high levels of telomerase activity (Fig. 3). As a control, GAPDH mRNA was detected by RT-PCR, showing equal amounts of GAPDH product in the four tissues tested (Fig. 3A).

The fact that mTERT mRNA is detectable in adult tissues lacking detectable telomerase activity suggests that the formation of active telomerase complexes could be subject to further regulation, such as the presence of the telomerase RNA (mTR) and/or posttranscriptional regulation of mTERT. To analyze whether mTERT mRNA expression in adult tissues depends on the presence of mTR, we studied mTERT mRNA expression in adult tissues derived from mTR^−/− mice. mTERT mRNA was detected by RT-PCR in three different adult tissues that normally show telomerase activity, spleen, thymus, and liver, independently of the presence (mTR^+/+ or wt) or absence (mTR^−/− or KO) of the mTR component (Fig. 3B). This indicates that mTERT mRNA expression is independent of the presence of the mTR component. Testis from wild-type mice had slightly higher amounts of mTERT mRNA than did the corresponding mTR^−/− tissue, but the significance of this is not clear, since the mTERT protein levels appear to be similar in the two tissues (see below).

We generated antibodies against hTERT and mTERT. Antiserum K-370 was obtained against a synthetic peptide (peptide K-370 or PEPT-1) encoded by EST1 from human telomerase reverse transcriptase and conserved in mTERT protein.

K-370 antibodies recognize a band in human and mouse immortal cell lines, 293 and FM3A, respectively (Fig. 4), in agreement with the fact that these cells have high telomerase activity levels (40, 41). The protein recognized in human extracts has a lower mobility than that present in mouse extracts, as expected from their predicted molecular masses, 127 kDa for hTERT and 125 kDa for mTERT. In both cases, the signal disappeared after preincubation of the antibody with the antigenic peptide (PEPT-1), but not when a nonspecific peptide (PEPT-2) was used (Fig. 4). Human primary cells IMR-90 do not present detectable amounts of the telomerase reverse transcriptase subunit (Fig. 4; see also Fig. 7A), in agreement with the fact that they lack telomerase activity (21). MEFs, with low telomerase activity levels (2), did not show detectable mTERT protein in this assay (Fig. 4); however, we detected mTERT protein in these cells by using a different assay (Fig. 7A).

To study mTERT protein expression, S-100 extracts from a number of tissues were prepared and analyzed by Western blotting using K-370 antibody. The mTERT-specific band is detected in telomerase-positive tissues such as thymus, testis, spleen, and liver (Fig. 5A) (5, 37) but is undetectable in ovary, muscle, brain, intestine, and heart, in agreement with the fact that all, except ovary, have been described as telomerase-negative or as low telomerase activity (Fig. 5A) (5, 40).

When mTERT protein was analyzed in thymus, spleen, and testis derived from wild-type (wt) or mTR^−/− (KO) littermate mice, we observed similar amounts of the mTERT-specific band in testis and spleen in the two genotypes (Fig. 5B; note that lane FM3A has 1/4 the protein loaded in Fig. 5A). This indicates that both mTERT mRNA (see above) and mTERT protein are expressed independently of the presence of active telomerase complexes in these tissues. In contrast, the mTR^−/− thymus lacked detectable mTERT protein, which was, however, present in wild-type thymus (Fig. 5B). This observation suggests that the presence of the mouse telomerase RNA component in the thymus may be necessary for the accumulation of mTERT protein and that this kind of regulation may be tissue specific. The fact that the mTR^−/− thymus and the wild-type thymus have similar mTERT mRNA levels indicates that the mTERT protein expression is regulated posttranscriptionally in this tissue.

To determine the subcellular localization of telomerase catalytic subunit in mammalian cells, such as human HeLa and IMR-90 cells, as well as in primary mouse cells (MEFs), we used K-370 telomerase antibody in Western blot analysis and in immunofluorescence.

Fig. 6 shows a double staining of two different asynchronous cell cultures by using Hoechst 33258 to stain the nucleus and K-370 antibody against the telomerase catalytic subunit. IMR-90 cells, previously shown to lack detectable levels of hTERT protein (Fig. 4), were not stained with K-370 antibody (Fig. 6 a and b, for Hoechst and K-370 staining, respectively). HeLa cells, which have high levels of telomerase activity (41), showed, in contrast, a clear staining of the nucleus (Fig. 6, c/e and d/f, for Hoechst and K-370 staining, respectively), and the fluorescent signal was blocked by preincubation of the antibody with the antigenic peptide (PEPT-1) (not shown). The nuclear staining of hTERT showed a punctuated pattern, supporting the previously described nuclear staining in HeLa cells by using an anti-peptide antibody to a different region of the hTERT (23). We are currently developing more potent antibodies to asses the nature of the speckles, which may correspond to the telomeres or to replication complexes (42). Mouse primary cells, MEFs, did not show a detectable specific signal in immunofluorescence (not shown), probably because mTERT protein levels in these cells are undetectable or very low (Fig. 4, also Fig. 7A). To confirm the nuclear localization of TERT, we prepared nuclear and cytoplasmic extracts from asynchronous cultures of both human (IMR-90 and HeLa) and mouse cells (MEFs) and analyzed the presence of the TERT protein by Western blotting. Equal protein amounts of cytoplasmic and nuclear extracts were analyzed. IMR-90 cells showed no detectable hTERT band, in agreement with the fact that these cells are negative for telomerase activity (Fig. 7A, see also Fig. 4). Both human immortal HeLa cells and primary mouse MEFs show expression of TERT protein in the nuclear fraction but not in the cytoplasmic fraction, confirming that TERT protein is located in the cell nucleus (see Fig. 7 A and C). As a control for the extract preparation, actin was found only in the cytoplasmic fraction of asynchronous HeLa extracts (see Fig. 7B).

To analyze whether telomerase activity is regulated by the subcellular localization of TERT protein during cell cycle progression, we studied TERT expression in synchronized HeLa cells and MEFs (Fig. 7 C and D). The percentage of cells in each cell cycle stage was monitored by FACS analysis. In both cell types, TERT is located in the cell nucleus throughout all the stages of the cell cycle, indicating that TERT availability is not regulated by nuclear transport (Fig. 7 C and D). The amounts of hTERT protein showed a moderate 3-fold increase as more cells entered the S phase (compare asynchronous cells to 0.0-hr synchronized cells in Fig. 7C).