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Molecular and Cellular Biology, June 2000, p. 4115-4127, Vol. 20, No. 11
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Telomere Maintenance in Telomerase-Deficient Mouse Embryonic Stem
Cells: Characterization of an Amplified Telomeric DNA
Hiroyuki
Niida,1,
Yoichi
Shinkai,2,*
M.
Prakash
Hande,3,
Takehisa
Matsumoto,1
Shoko
Takehara,4
Makoto
Tachibana,2
Mitsuo
Oshimura,4
Peter M.
Lansdorp,3,5 and
Yasuhiro
Furuichi1
Agene Research Institute, Kamakura
247-0063,1 Department of Cell Biology,
Institute for Virus Research, Kyoto University, Kyoto
606-8507,2 and Department of Molecular
and Cell Genetics, Faculty of Medicine, Tottori University, Yonago
683-8503,4 Japan; Terry Fox Laboratory,
British Columbia Cancer Research Center, Vancouver, British Columbia
V5Z 1L3,3 and Department of Medicine,
University of British Columbia, Vancouver, British Columbia V6T
2B5,5 Canada
Received 27 September 1999/Returned for modification 4 November
1999/Accepted 7 March 2000
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ABSTRACT |
Telomere dynamics, chromosomal instability, and cellular viability
were studied in serial passages of mouse embryonic stem (ES) cells in
which the telomerase RNA (mTER) gene was deleted. These
cells lack detectable telomerase activity, and their growth rate was
reduced after more than 300 divisions and almost zero after 450 cell
divisions. After this growth crisis, survivor cells with a rapid growth
rate did emerge. Such survivors were found to maintain functional
telomeres in a telomerase-independent fashion. Although
telomerase-independent telomere maintenance has been reported for some
immortalized mammalian cells, its molecular mechanism has not been
elucidated. Characterization of the telomeric structures in one of the
survivor mTER
/ cell lines showed
amplification of the same tandem arrays of telomeric and nontelomeric
sequences at most of the chromosome ends. This evidence implicates
cis/trans amplification as one mechanism for the
telomerase-independent maintenance of telomeres in mammalian cells.
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INTRODUCTION |
Telomeres are special structures at
the ends of eukaryotic chromosomes. Since the original work of Muller
and McClintock, the telomere has been thought to protect the chromosome
end from degradation and fusion to other chromosomes (38, 39,
43). In most eukaryotes, telomeric DNA consists of tandem repeats
of G-rich sequences, for example, TTAGGG in mammals and
other vertebrates (reviewed in reference 15). In
humans, all chromosome ends contain about 5 kb of telomeric DNA
(36), and telomeres shorten with each cell division in most
somatic cells (19, 22). However, cells that divide
indefinitely, such as germline cells and tumor cells, maintain the
length of their telomeres, suggesting that a telomere maintenance
pathway is activated in immortalized cells, as telomere maintenance is
essential for immortal cell growth (reviewed in references
11 and 20).
Telomerase is a ribonucleoprotein enzyme which elongates telomeres by
synthesizing telomeric DNA sequence onto the 3' ends of chromosomes.
Telomerase can compensate for the loss of telomeric DNA resulting from
incomplete replication of the 3' end of telomeres by normal cellular
DNA polymerases (reviewed in reference 2). In
humans, germline cells and more than 80% of primary tumor cells have
been shown to express telomerase activity, but most normal somatic
cells express low or undetectable levels of telomerase (reviewed in
reference 49). These results suggest that telomerase is the dominant pathway to maintain telomere length in human cells.
Cloning and expression of the mammalian telomerase RNA gene encoding
the template for telomeric DNA synthesis and the gene encoding the
catalytic reverse transcriptase subunit of the telomerase complex have
shown that these two gene products are essential and sufficient to
reconstitute telomerase activity (3, 13, 21, 28, 41, 44,
57). Results from two different recent studies have further
strengthened the notion that telomerase is the dominant mechanism for
telomere maintenance and that telomere maintenance is crucial for
cellular viability in mammals. It was shown that the expression of
telomerase in normal human somatic cells resulted in the extension of
their life span (5, 30, 55, 56). Studies with the telomerase
RNA component TER-deficient (mTER/) mice have revealed progressive
telomere shortening with each successive generation of the mutant mice
(4) and severe chromosomal instability (18).
Further studies of the late-generation mTER/
mice demonstrated that telomere dysfunction and associated chromosomal instability resulted in proliferative defects exemplified by germ cell
depletion in the testis, reduced growth capacity in highly proliferative organs, and a shortened life span (33, 48). Defective proliferation and a similar chromosomal instability were also
observed in mTER/ ES cells (46).
While these previous studies have clearly established the importance of
telomerase in the maintenance of functional telomeres, the telomerase
pathway does not appear to be the only way in which telomeres can be
maintained. For example, some human tumor cells and tumor-derived cell
lines do not express detectable telomerase activity and appear to
maintain their telomeres by a telomerase-independent, alternative
lengthening of telomeres (ALT) pathway (7, 8). Recent
analysis of the cells derived from the mTER/
mice has also clearly demonstrated that telomerase-independent mechanisms are capable of maintaining and elongating telomere length
under some circumstances (18). While it has been shown that
telomeres in ALT cells are very long and heterogeneous in size, the
details of the molecular mechanism of the ALT pathway(s) need to be elucidated.
Telomerase-independent telomere maintenance has been described for
cells from other species. Drosophila spp. maintain their telomere length by transposition of a set of retroposons (reviewed in
reference 37). The yeasts Saccharomyces
cerevisiae, Kluyveromyces lactis, and
Schizosaccharomyces pombe utilize recombination as a backup
mechanism for telomere maintenance (32, 35, 40, 45). Indeed,
telomere or subtelomeric sequence-mediated recombination has been
described (12, 24, 47). In this report, we describe further
studies with mTER/ ES cells. We observed
telomerase-independent telomere maintenance in
mTER/ cells that survived and proliferated
after prolonged culture. Analysis of the telomeric structure in such
survivors provided new insights into the nature of
telomerase-independent telomere maintenance.
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MATERIALS AND METHODS |
Cells.
mTER/ ES cells were
generated and cultured as described previously (46).
Pulsed-field gel electrophoresis and fragment analysis.
Genomic DNA from ES cells was prepared as described before
(42). DNA (15 µg) was digested with restriction
endonucleases and separated on a 1% agarose gel in 0.5×
Tris-borate-EDTA at 14°C with a CHEF DR-II pulsed-field apparatus
(Bio-Rad). Pulsed-field electrophoresis was performed at 6 V/cm for 12 to 18 h at a ramped pulse of from 1 to 10 s. The gel was
dried at 60°C for 1.5 h, denatured in 1.5 M NaCl-0.5 M NaOH
solution for 30 min, neutralized in 1.5 M NaCl-0.5 M Tris-HCl (pH 8.0)
buffer for 30 min, and hybridized to
5'-[
-32P]T2AG3 telomeric DNA
oligonucleotides in 5× SSC-5× Denhardt's solution-0.1× P wash
(1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate; P wash is 0.5 mM
pyrophosphate plus 10 mM Na2HPO4) at 37°C for
12 h. Following high-stringency washes in 0.1× SSC at 20°C for
1 h, the gel was autoradiographed. The 555-bp nontelomeric DNA
probe was amplified by PCR with oligonucleotides F01 (5'-TCT CTA TTA
TGG GGA TTA AAG G-3') and F02 (5'-TCC ATT TAT TCT CCA CAA CCG C-3').
Quantitative slot blot analysis for telomeric DNA contents was
performed as described before (46). The mouse major satellite DNA26 oligonucleotide probes were used as a reference for
signal intensity.
Telomere FISH analysis.
Metaphase chromosomes from ES cells
at different population doublings (PDL) were prepared, and fluorescence
in situ hybridization (FISH) with Cy-3-labeled
C3TA2 peptide-nucleic acid (PNA) probe and
subsequent quantitative analysis of digital images were performed as
described before (18, 59). FISH analysis of centromere (mouse major satellite DNA) and telomere was carried out by standard methods as described before (53). Centromere and telomere
probes were produced by using primers MM18a
(5'-TACACACTTTAGGACGTG-3') and MM18b
(5'-CACGTCCTAAAGTGTGTA-3') and telo-1
[5'-(T2AG3)5-3'] and telo-2
[5'-(C3TA2)5-3'] according to the
protocols reported previously (25, 26).
Cloning of telomeric DNA.
Genomic DNA from DKO741 at 860 PDL
was digested with HinfI, and the DNA fragments were
separated on a 1% low-melting-point agarose gel. DNA fragments of
about 1.6 kb were isolated from the gel, subcloned into the pZErO-2
vector (Invitrogen), and transformed into a Max Efficiency STBL2
competent cell (Gibco-BRL). Each single colony was formed on a
Luria-Bertani-agarose plate containing 25 µg of kanamycin per ml at
30°C for 16 h. Clones containing the TTAGGG repeat
sequences were screened by colony hybridization with the
5'-[-32P]T2AG3 telomeric DNA
oligonucleotide probe. Positive clones were isolated and sequenced by a
dye terminator method with an ABI Prism 377 sequencer (Applied
Biosystems). As described in the legend to Fig. 6, sequencing was
initiated from both ends of the insert, and the sequence at the
junction of the nontelomeric and telomeric regions was determined.
FISH with cloned nontelomeric DNA.
Approximately 3 to 5 µg
of nontelomeric DNA in the vector was labeled with Biotin-16-dUTP by
nick translation and then precipitated with ethanol, using salmon sperm
DNA as a carrier. The probe was dissolved in hybridization buffer
containing 50% deionized formamide, 2× SSC, 10% dextran sulfate, and
50 mM phosphate buffer (pH 7.0) to a concentration of 20 ng/µl.
Metaphase spreads at selected PDL from wild-type (WT) and
mTER/ ES cells were hybridized as described
earlier (17, 18). Slides were incubated with pepsin
(0.005%) in 10 mM HCl for 10 min at 37°C, washed with
phosphate-buffered saline (PBS) containing 50 mM MgCl2, and
then treated with 1% formaldehyde in PBS-MgCl2 for 10 min
at room temperature. After one more wash in PBS, slides were dehydrated
in a 70, 90, and 100% ethanol series. The labeled probe was diluted
with hybridization buffer to a final concentration of 4 to 8 ng/µl,
and 20 µl was added to each slide. The probe and the target DNA were
denatured simultaneously at 80°C for 3 to 4 min. Hybridization was
carried out overnight at 37°C in a moist chamber. After
hybridization, the slides were washed three times for 5 min each in
50% formamide-2× SSC buffer (pH 7.0) at 37°C, three times in 0.1×
SSC at 60°C, and twice for 5 min each in 2× SSC at room temperature.
For immunofluorescence detection, the slides were incubated with
avidin-fluorescein isothiocyanate (Vector Labs) for 30 min at 37°C in
a humid chamber. The signal was amplified using biotinylated goat
antiavidin antibody (Vector Labs). After dehydration in an ethanol
series, the slides were embedded with Vectashield mounting medium
(Vector Labs) containing 1 µg of propidium iodide (Sigma) per ml.
Slides were observed under a Zeiss Axioplan2 microscope (Carl Zeiss)
equipped with suitable filters and with a charge-coupled device camera
(SensiCam PCO).
Telomerase activity measurement.
Telomerase activity of the
mTER/ ES cells was measured with a
Telochaser detection kit (Toyobo) as described before (52).
BAL-31 nuclease digestion.
Genomic DNA (15 µg) was
digested with 2.5 U of BAL-31 nuclease (Takara) in 80 µl of 1×
BAL-31 buffer at 30°C for 10 and 20 min. The reaction was stopped by
adding 20 µl of 0.5 M EDTA and phenol-chloroform extraction. Digested
DNA was precipitated with 100 µl of isopropanol, washed with 70 and
100% ethanol, and then resolubilized in Tris-EDTA. One third of the
digested DNA (5 µg) was further digested with BamHI,
HapII, or HinfI and used for terminal restriction
fragment (TRF) analysis as described above.
Nucleotide sequence accession numbers.
The GenBank accession
numbers of the nontelomeric sequences are AB040048 and AB040049.
| |
RESULTS |
Isolation of survivors from late-passage cultures of
mTER-deficient ES cells.
Two independent
mTER/ ES cell lines (DKO301 and DKO741) were
established as previously described (46). These mutant ES
cells had no telomerase activity and showed progressive telomere
shortening under long-term culture conditions. Once telomere length
reached a critical size, chromosomal instability was induced, and
growth of the mutant cells was strongly suppressed. The growth-retarded mTER/ ES cells were mostly large cells that
apparently stopped proliferation at about 450 PDL. However, cultures
were continued, and from 2 × 104 to 10 × 104 cells were replated every 3 to 4 days. Rare
subpopulations of both mTER/ ES cell lines
survived the growth arrest and resumed proliferation, as shown in Fig.
1. The doubling time of the DKO301 and
DKO741 survivors was about 24 h, and they continued to grow up to
850 PDL, the endpoint of the experiment. The size of survivor
mTER/ ES cells was similar to that of WT
cells, and they were still telomerase negative (Fig.
2). The growth of control WT and
mTER+/ (KO6) ES cells was constant over 2 years, with a rate of 12 to 16 h per cell division. Due to the
limitations of the culture conditions employed in the present study, it
was not possible to determine the exact frequency of survivor cells in
the mTER/ ES cells. Also, it is uncertain if
the survivors were selected by mutation with genetic changes or induced
by a physiologically programmed pathway.
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FIG. 1.
Growth of mTER/ ES cells in
long-term culture. The growth characteristics of
mTER+/+ (WT), mTER/+
(KO6), and mTER/ (DKO301 and DKO741) ES
cells were monitored during long-term culture.
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FIG. 2.
Telomerase activity of mTER/
survivor ES cells. Total-cell lysates were prepared from WT, DKO301 at
663 PDL, and DKO741 at 638 PDL. The assay was performed multiple times,
and both DKO301 and DKO741 survivors still exhibited undetectable
levels of telomerase activity. IC, internal control.
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Telomere dynamics and chromosomal instability in survivor
mTER-deficient ES cells.
To analyze telomere dynamics
in the survivor mTER/ ES cells, we performed
Southern blot analysis of the TRFs containing telomeric DNA sequences.
As described previously (46) and shown in Fig. 3A,
Southern blot analysis of DNA digested
with restriction enzyme HinfI demonstrated that both the
size and the relative signal intensity of the TRFs hybridized with the
T2AG3 oligonucleotide probe were progressively
reduced in both mTER/ ES cell lines up to
the growth crisis stage (444 PDL for the DKO301 cells and 428 PDL for
the DKO741 cells). After growth crisis, the reduction in TRFs continued
in DKO741 survivor cells (from 655 to 834 PDL) but was reversed in
DKO301 survivor cells (from 569 to 824 PDL). Based on the reduction in
signal intensity during the first 200 PDL (46), no
T2AG3-hybridizing signals were expected for
either mTER/ ES cell line after 600 PDL.
However, analysis of short restriction fragments revealed a fragment of
about 1.6 kb which hybridized strongly to the
T2AG3 probe in the DKO741 survivor cells at 614 PDL, which was not observed in WT, KO6, DKO301, or precrisis DKO741 (187 PDL) cells (Fig. 3B). Using other 4- or 5-base restriction enzymes
(which were also not able to digest the TTAGGG sequence), we
further analyzed the DNA that hybridized with the telomere probe in the
DKO741 survivor cells. As shown in Fig. 3C and D, two types of
fragments were generated with such restriction enzymes. Like
HinfI digestion, AluI, HaeIII,
MboI, and RsaI digestion generated a dominant
single fragment of about 1 kb. HhaI and HapII,
however, produced more than one discrete size (about 6, 8, and >12 kb) and larger smears (>12 kb) of restriction fragments.
HapII-digested DNA from the mTER/
DKO741 survivor cells at different PDL showed ladders of fragments of
different sizes (Fig. 3E). Such short distinct fragments and ladders
were not observed with DNA from DKO301 survivor cells digested with
HapII and other restriction enzymes (Fig. 3E and data not
shown). Quantitative slot blot analysis also confirmed recovery of
T2AG3-hybridizing signals in both
mTER/ survivor ES cells between 448 and 764 PDL in DKO301 and 432 and 764 PDL in DKO741 cells (data not shown).
Therefore, we conclude that telomeric DNA in the
mTER/ survivor ES cells was maintained or
accumulated in a telomerase-independent manner.
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FIG. 3.
Telomere dynamics in mTER/ ES
cells. Genomic DNAs from mTER+/+ (WT) and
mTER/ (DKO301 and DKO741) ES cells at
selected PDL were digested with HinfI and separated on a
pulsed-field gel (A) or a 0.8% agarose gel (B) or digested with
HapII and separated on a pulsed-field gel (E). Genomic DNAs
from WT cells at 4 PDL, DKO301 cells at 824 PDL, and DKO741 cells at
764 PDL were digested with each 4- or 5-base restriction endonuclease
and separated on a pulsed-field gel (C) or a 0.8% agarose gel (D and
F). The
5'-[32P](T2AG3)3
telomeric DNA oligonucleotides (A to E) and 555-bp nontelomeric DNA
fragment (F) were used as probes.
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To further analyze telomere dynamics and chromosomal stability in the
mTER/ ES survivor cells, we performed
cytogenetic analysis of metaphase
spread chromosomes by quantitative
FISH (Q-FISH) with the (C
3TA
2)
3 PNA
probe (
31,
59). Representative FISH images of metaphase
spreads from the WT and
mTER/ ES cells at
different passages are shown in Fig.
4.
As summarized
in Fig.
5, the mean telomere fluorescence units
(TFU) of p-arm
and q-arm telomeres was 3.3 ± 0.2 and 9.3 ± 0.3 TFU, respectively,
in DKO301 cells and 2.9 ± 0.2 and
10.0 ± 0.3 TFU, respectively,
in DKO741 cells at the growth
crisis stage (about 450 PDL) (values
are means ± SE). These
values were between 10 and 21% of those
for WT ES cells at 10 PDL.
Different results were obtained with
DKO301 cells at 692 PDL and the
DKO741 cells at 683 PDL. The values
in these survivor cells were
4.7 ± 0.3 and 24.7 ± 0.7 TFU, respectively,
and 0.3 ± 0.1 and 23.5 ± 0.5 TFU, respectively, or 16, 58, 1,
and 55%
respectively, of those in WT cells at 10 PDL. These results
are
compatible with recovery of telomeric DNA sequences on the
q-arm but
not the p-arm telomeres.
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FIG. 4.
Metaphase spreads from WT and
mTER/ ES cells. FISH with a PNA-telomere
probe for WT (A and B), DKO301 (C to E), and DKO741 (F to H) at
selected PDL. Two-color FISH with the telomere (green) and mouse major
satellite (pink) probes for WT (I) at 1,134 PDL and DKO741 (J) at 683 PDL. Chromosomal stability and telomeric DNA content have been highly
sustained in the WT over 1,000 PDL. On the other hand, telomeric DNA
content in the mTER/ ES cells has decreased
progressively until the growth crisis stage (C and D; F and G) but
regained after the crisis by telomerase-independent mechanisms (E and
H). Furthermore, many fusions (mostly p-arm-to-p-arm fusions) were
induced during the growth crisis stage (D and G).
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FIG. 5.
Q-FISH analysis. Mean telomere fluorescence of
mTER+/+ (WT) and
mTER/ (DKO301 and DKO741) cells at selected
PDL. Fluorescence is expressed in TFU, where 1 TFU corresponds to 1 kb
of T2AG3 repeat in plasmid DNA (36).
For the survivors (DKO301 at 692 PDL and DKO741 at 683 PDL), the q-arm
TFU show a more appropriate telomere length at the ends of chromosomes.
Error bars indicate the SE.
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Cytogenetic analysis revealed that the progressive telomere shortening
in the
mTER/ ES cells with prolonged culture
resulted in increasing chromosomal
instability. The number and
frequency of fused chromosomes in
the WT and
mTER/ ES cells at different passages are
summarized in Table
1. The
chromosomes of
the WT ES cells were quite stable, and only three
fusion events in 18 metaphases were observed at 1,134 PDL (0.17
chromosome per metaphase).
The number of fused chromosomes was
significantly increased in both
mTER/ ES cell lines. Even before the growth
crisis stage, the DKO301
cells at 142 PDL and the DKO741 cells at 154 PDL contained 1.5
and 1.2 fusions per metaphase, respectively.
Furthermore, the
number of fused chromosomes was increased 10 times at
the growth
crisis stage in both mutant cells (11.1 to 16.3 and 13.6 to
14.9
fusions per metaphase in DKO301 and DKO741 cells, respectively).
These fusions were predominantly of a Robertsonian type (p- to
p-arm),
but other types of fusions, including a dicentric, p-
to q-arm,
tricentric, ring, and more complicated types, were also
observed.
Once survivors emerged from the cultures, the percentage of fused
chromosomes per metaphase was further increased; the frequency
of fused
chromosomes observed was increased, with 19.1 and 26.5
fusions per
metaphase at 692 PDL in DKO301 cells and at 683 PDL
in DKO741 cells,
respectively; therefore, 19.1 of 27.9 (68%) and
26.5 of 25.5 (>100%)
chromosomes were fused, respectively. Interestingly,
essentially all
chromosomes in metaphase spreads from these survivors
were end-to-end
Robertsonian type fusions, as shown in Fig.
4.
Hybridization with a
mouse major satellite probe specific for
a majority of centromeres in
WT cells (Fig.
4I) showed variable
staining of centromeres in the
mTER/ survivor ES cells (Fig.
4J),
confirming the metacentric nature
of the chromosomes in these cells. We
conclude that the vast majority
of the original p-arm telomeres in the
mTER/ survivor ES cells are no longer
present at the ends of the chromosomes,
which may explain why recovery
of TFU on the p-arm telomeres was
not induced in these cells (Fig.
5).
Cloning and sequencing of elongated telomere DNA.
To further
investigate the nature of the telomerase-independent acquisition of
telomeric DNA in the mTER/ survivor ES
cells, we cloned and sequenced the 1.6-kb HinfI TRF from the
DKO741 survivor cells (Fig. 3B). Five independent clones that
hybridized with the T2AG3 oligonucleotide probe
were obtained. Sequencing 200 to 300 bp of both ends on each insert
demonstrated that they were all identical. As shown in Fig.
6, further sequencing of the 1.6-kb
insert demonstrated that it consisted of the TTAGGG repeats
(about 700 bp) flanked with nontelomeric sequences. No sequence with
significant homology to the nontelomeric sequences was identified in
the GenBank database. Mapping with the 4- and 5-base restriction
enzymes (Fig. 6B) showed that the sizes of each fragment containing the
telomeric DNA region were consistent with those seen in the TRF
Southern blot analysis of the DKO741 survivor cells. Further
hybridization analysis demonstrated that the 1.6-kb HinfI
fragment hybridized to the nontelomeric DNA probe (555-bp PCR fragment
amplified from the nontelomeric region 5' to the telomeric DNA region,
as shown in Fig. 6B) was specifically and highly amplified in DKO741
survivors (Fig. 3F). Some additional bands (<500 bp) were detected in
DKO741 survivor DNA digested with AluI and MboI,
and their sizes were also consistent with the mapping of the 1.6-kb
fragment (Fig. 6B). In WT and DKO301 cell DNA, very faint bands were
detected (~0.5 kb for HinfI, ~0.3 kb for
AluI, and ~1.0 kb for RsaI). Slot blot analysis
demonstrated that the nontelomeric DNA signal was amplified by
>100-fold in the DKO741 survivor cells (not shown). Furthermore,
rehybridization confirmed that the fragments hybridizing to the
T2AG3 probe shown in Fig. 3D also hybridized to
the nontelomeric DNA probe (not shown).
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FIG. 6.
Sequencing and structure of the 1.6-kb HinfI
TRF from the DKO741 survivor ES cells. Genomic DNA from DKO741 at PDL
860 was digested with HinfI. Fragments of about 1.6 kb were
isolated and subcloned. (A) Nucleotide sequence of the cloned gene. (B)
Structure of the cloned 1.6-kb HinfI fragment. Open and
shaded boxes indicate telomeric and nontelomeric DNA regions,
respectively. The recognition sites of the 4- and 5-base restriction
endonucleases used in Fig. 2 are indicated: A, AluI; HIII,
HaeIII; M, MboI; Hi, HinfI; R,
RsaI. The sizes of the restriction fragments are shown. The
numbering of restriction sites 3' to the telomeric region is based on
the 3'-most HinfI site as base 1600. With the
T2AG3 probe, we expected fragment sizes of 868, 1,315, >1,070, 1,600, and >1,432 bp with AluI,
HaeIII, MboI, HinfI, and
RsaI, respectively. With the 555-bp nontelomeric DNA probe,
we expected fragment sizes of 320 and >232, 1,315, >302 and 231, 1,600, and >1,432, respectively, for the same enzymes.
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To confirm the localization of the cloned 1.6-kb sequence at the
chromosome ends, we performed FISH analysis on WT and both
mTER/ ES cell lines with the nontelomeric
sequence probe which was
used for the previous TRF Southern blot
analysis. As shown in
Fig.
7C, most of
the chromosome ends (85 ± 6%) from DKO741 survivors
at 683 PDL
specifically hybridized to the nontelomeric DNA probe
(green signals).
However, no specific signals on the metaphase
spread chromosomes in WT,
DKO301, and precrisis DKO741 (at 154
PDL) cells were observed (Fig.
7A
and B and data not shown). The
endogenous locus most likely escaped
detection by FISH due to
the small size of the oligonucleotide probe.
This FISH analysis
clearly showed accumulation of the cloned
nontelomeric/telomeric
DNA sequence at the ends of chromosomes
specifically in DKO741
survivor cells.
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FIG. 7.
FISH analysis of nontelomeric DNA sequence. FISH with
the 555-bp nontelomeric DNA probe used for Fig. 3F (lower panel). (A)
WT cells at 10 PDL; (B) DKO741 at 154 PDL; (C) DKO741 at 683 PDL.
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To further investigate the terminal nature of the 1.6-kb DNA unit, DNA
from DKO741 survivor cells at 638 PDL and control cells
(WT and DKO301
survivors at 663 PDL) was digested with BAL-31
nuclease for increasing
lengths of time. Because this enzyme progressively
shortens DNA from
the ends, sequences at chromosome ends such
as telomeric sequences are
sensitive to BAL-31 digestion (
29).
As shown in Fig.
8, TRFs of WT DNA generated by
HapII digestion
were progressively shortened by BAL-31
predigestion. The average
TRF length of DKO301 survivor DNA was also
decreased in a time-dependent
fashion. However, DKO741 survivor DNA
behaved somewhat differently.
The amounts of higher-molecular-weight
HapII fragments (>23 kb)
were diminished after 10 min of
BAL-31 predigestion, but no further
TRF shortening was observed after
further incubation with BAL-31
(20 min [Fig.
8] and up to 60 min
[not shown]). This result indicates
two possibilities. (i) The tandem
arrays of the telomeric/nontelomeric
DNA which were observed at the end
of most of the chromosomes
in the DKO741 survivor cells by FISH
analysis are not the very
end of the chromosomes, and (ii) the
telomeric/nontelomeric DNA
is at the very end of the chromosomes but
resistant to BAL-31
digestion due to the unusual structure of these
arrays or the
poor degradation rate of the nontelomeric DNA. In the
absence
of any conclusive evidence, we prefer the second possibility.
If the first possibility is true, it is difficult to envisage
the role
of such ends as "functional telomeres" (telomeric sequence
arrays
existed only in the characterized ~1-kb telomeric/nontelomeric
TRF
fragments, such as shown in Fig.
3D).
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FIG. 8.
BAL-31 nuclease treatment assay. Genomic DNA (5 µg)
from WT at 1,008 PDL, DKO301 at 663 PDL, or DKO741 at 638 PDL was
digested with BAL-31 nuclease for 0, 10, or 20 min. Then, DNA was
further digested with HapII and used for TRF analysis.
(T2AG3)3 was used as the probe.
|
|
| |
DISCUSSION |
In the present study, we demonstrate that telomeres in the
mTER/ survivor ES cells were maintained
independently of telomerase activity. Furthermore, cloning and
sequencing analysis of the TRF from the DKO741 survivor cells provides
the first evidence at the nucleotide level that telomere length
maintenance and extension in telomerase-negative mammalian cells may
involve amplification of (sub)telomeric repeat sequences that is
independent of the "telomerase" system.
Growth and telomere dynamics in mTER-deficient ES
cells.
As described previously (46), growth retardation
and chromosomal instability were coincidentally induced in
mTER/ ES cells under long-term culture
conditions. Complementation of mTER gene expression in the
mTER/ ES cells before the growth crisis
stage prevented growth retardation. However, as telomere length
continued to shorten, chromosomal stability was significantly
maintained (H. Niida and Y. Shinkai, unpublished data). Since
mTER/ ES cells complemented with the
mTER gene contained less than 5% of the telomerase activity
measured in WT ES cells, we speculate that the low level of telomerase
activity may prevent end-end fusions but not telomere shortening.
Similar results were recently reported by Zhu et al. (58),
who showed that ectopic expression of telomerase in normal human
fibroblast cells may result in the maintenance of chromosome stability
and an extended life span despite an initial overall shortening of
telomere length. Most likely, the growth defect of the
mTER/ ES cells was similarly associated with
chromosomal instability of specific chromosomes with a
shorter-than-average telomere length.
Growth or proliferative defects were previously observed in
mTER-deficient mice with shortened telomeres (
33,
48). Typically,
in the sixth generation (G6) of
mTER/ mice, the development of male germ
cells was impaired and the
proliferative capacity of the skin and
hematopoietic cells was
decreased. Furthermore, aneuploidy and
chromosomal fusions were
significantly increased in splenocytes from G6
mice. Mouse embryonic
fibroblasts (MEFs) derived from the
late-generation animals showed
biphasic cell cycle arrest at an early
passage (
9). However,
the MEFs have never shown growth
defects even after many chromosomal
fusions were induced due to severe
telomere shortening (
4,
18). On the other hand, in the
INK4a/ background, loss of telomere function
was associated with a decreased
rate of Myc/RAS focus formation (growth
defect) in late-generation
mTER/ MEFs
(
14). Although we do not have a clear explanation for
this
discrepancy, the response to telomere shortening and the
capacity to
grow in the presence of chromosomal instability may
vary among cell
types or genetic backgrounds. A possible mechanism
is differential
checkpoint thresholds. A good example is the well-known
difference
between cells upon activation of p53; some cells will
die by apoptosis,
and others will respond with cell cycle arrest.
Indeed, the growth
defect of the
INK4a/
mTER/ MEFs and apoptosis of specific cell
types in G6 mice were p53
dependent (
9,
14). This evidence
may suggest that the growth
defect of our
mTER/ ES cells was also p53 mediated. In
addition, the much more severe
phenotype of
mTER/ C57BL/6 mice (
23) compared
to
mTER/ mice on a mixed C57BL/6/129
background (
33,
48) supports
the idea of the genetic
background
difference.
Once survivor cells predominated in the cultures of
mTER/ ES cells, essentially all chromosomes
were fused and the total number
of chromosomes per metaphase spread was
reduced to about half
(Table
1). Most of the fusions can be
characterized as (pseudo-)Robertsonian
fusions. In early passages of WT
ES cells, the mean TFU value
on q-arm telomeres was about 1.5 times
greater than that on p-arm
telomeres (Fig.
3). The appearance of
Robertsonian-type fusion
as the dominant type in the
mTER/ ES cells would be expected if telomere
size were most critical
for induction of end-to-end fusion. Other types
of fusions were
also observed in both lines of
mTER/ ES cells at the growth crisis stage.
However, fewer such abnormalities
were observed in later passages of
the survivor cells, suggesting
that cells with other types of fusions
than (pseudo-)Robertsonian
fusions had a growth disadvantage.
Nevertheless, 8 to 13% of chromosomes
in the
mTER/ survivor ES cells at >680 PDL were
still contained in dicentrics,
tricentrics, and other chromosomal
abnormalities. One possibility
is that only one centromere was active
not only in the (pseudo-)Robertsonian-type
fusions, but also in the
dicentric or tricentric chromosomes,
as described elsewhere
(
51). In this case, breakage-fusion-bridge
cycles could not
be triggered. Therefore, once further end-to-end
fusions were
suppressed by telomerase-independent telomere lengthening,
the growth
defect resulting from the chromosomal instability in
the surviving
mTER/ ES cells may have been
attenuated.
In the DKO741 survivor cells at 683 PDL, 92% of the chromosomes were
(pseudo-)Robertsonian-type fusions (Fig.
3) with a mean
p-arm telomere
value of 0.3 ± 0.1 TFU. The critical size of telomeric
DNA
sequences required for chromosomal stability in mammals is
most likely
higher than this estimate, as many of the observed
fusions are likely
to have evolved from more than one breakage-fusion-bridge
cycle. In
general, our results support the idea that a minimum
telomere length is
required at chromosome ends to maintain chromosome
stability and
prevent chromosome
fusions.
Telomerase-independent telomere maintenance.
Although
telomerase has been shown to be the dominant pathway to maintain
telomere length in mammalian cells, it was reported that some human
tumor cells and tumor-derived cell lines do not express detectable
levels of telomerase activity and appear to maintain their telomeres by
the ALT pathways (7, 8). Recent studies of cells derived
from mTER/ mice also clearly demonstrated
that the telomerase-independent ALT mechanisms can maintain and
elongate telomeres in murine cells under some circumstances
(18). While it has been shown that telomeres in ALT cells
are typically very long and heterogeneous in size, the details of the
molecular mechanism(s) involved in ALT remain to be elucidated.
As described for the
mTER/ ES cells,
telomerase-defective cells from certain yeast strains have shown a very
similar phenotype
of telomere dynamics and growth abilities (
34,
40,
45,
50).
Accompanying the progressive telomere shortening, a
severe growth
defect was induced once the telomere length reached a
critical
size. Then, a minor subpopulation of mutant cells without an
apparent
growth defect emerged. Genetic studies further demonstrated
that
telomerase-independent telomere maintenance in the surviving
mutant
yeast strains was dependent on the
RAD52 gene,
suggesting the
involvement of recombination (
32,
35,
40). In
S. cerevisiae,
an unusual telomeric DNA structure consisting
of tandem arrays
of telomeric and subtelomeric DNA sequences was
observed in the
telomerase-defective survivor cells (
35).
Such arrays can be
amplified by homologous recombination between two
distant telomeric
or subtelomeric DNA sequences. However, the surviving
cells from
K. lactis and
S. pombe seemed to
elongate and maintain the telomeric
DNA sequence only (
40,
45). Therefore, the specific telomeric
structure observed in the
S. cerevisiae survivor strains was considered
nonessential
for recombination-mediated telomere maintenance and
simply the
by-product of recombination
reaction.
One of the
mTER/ survivor ES cell lines,
DKO741, showed a telomeric structure similar to that of
telomerase-negative
S. cerevisiae survivors in that most of
the (fused) chromosome ends contained
repeats of a DNA sequence unit
containing both telomeric and nontelomeric
(subtelomeric?) sequences.
Based on this similarity, it could
be hypothesized that the
recombination-based reaction is also
involved in telomerase-independent
telomere maintenance in the
DKO741 survivor cells. That is, the DNA
sequence unit consisting
of the telomeric/nontelomeric/telomeric (or
vice versa) sequences
may exist on a border between the subtelomeric
and telomeric regions.
Alternatively, the nontelomeric DNA may contain
a fragile site,
and this site may have been preferentially broken
following the
breakage-fusion-bridge cycles during the growth crisis
stage.
Upon fusion with a very short telomere on another chromosome,
the telomeric/nontelomeric sequences at the end of a chromosome
could
be generated relatively easily. Then, once a critical but
unidentified
change(s) is induced, all chromosome ends in the
DKO741 cells may
eventually have acquired and amplified this specific
DNA unit as a
consequence of serial recombination-based reactions.
The
recombination-based reaction can be initiated by interchromosomal
pairing, intrachromosomal pairing, or recently described telomere
t-looping (
16). On the other hand, DKO301 survivor cells and
other telomerase-negative human cell lines (
7) have not
shown
such a specific telomeric structure and appear to have only
elongated
telomeric DNA sequences on each chromosome end. As described
for
other telomerase-defective yeast strains (
40,
45), they
may
also have utilized a recombination-based reaction with only
telomere
repeat sequences as the telomere maintenance pathway. Of
course,
this is still one of several possible mechanisms. These cells
or all of the survivor cells may have used a non-recombination-based
mechanism. Further investigation will be needed to elucidate the
entire
spectrum of the telomerase-independent telomere maintenance
mechanisms.
Because telomere maintenance in normal cells appears to be highly
dependent on the telomerase pathway, the ALT pathway in
normal cells
may be negatively regulated at different levels.
Therefore,
accessibility of the telomere region to the ALT pathway
may be
different before and after the growth crisis stage. One
possible level
of regulation could be telomere binding proteins
(
1,
6,
10).
It was reported that inactivation of one such
protein, human TRF2,
induced severe end-end fusions even though
these chromosomes contained
long stretches of telomere repeats
(
54). Another telomere
binding protein, Taz1p in
S. pombe, was
also shown to be
involved in the regulation of telomeric recombination
(
45).
Therefore, TRF2 may regulate the accessibility of telomeres
to the
telomerase-independent pathway in mammals. Inhibition of
TRF2 was found
to result in immediate deprotection of chromosome
ends, and TRF2 may be
required to form a large duplex loop that
protects telomeres from DNA
repair machinery, including recombination
events (
16). The
unusual telomeric DNA structure observed in
the DKO741 survivor cells
may potentially disrupt the regulation
mediated by telomere binding
proteins such as TRF2 and facilitate
access to the ALT pathway(s).
Alternatively, the unusual telomeric
DNA structure may facilitate
t-loop formation, and this telomeric
t-loop could indeed self-prime for
telomeric DNA extension by
an ALT pathway(s). Furthermore, inactivation
of TRF2 also induced
cell death in an ATM/p53-dependent manner,
suggesting that telomeres
lacking TRF2 were recognized as broken DNA
ends and that cells
containing such deprotected telomeres are
eliminated by the ATM/p53-mediated
apoptotic pathway (
27).
Recent studies with late-generation
mTER/
mice in the
p53/ background demonstrating
that growth arrest and apoptosis in
G6
mTER/
mice is mostly mediated through the p53 pathway are in agreement
with
this notion (
9). Short telomeres themselves (e.g., at
a
length that is unable to form a t-loop) and abnormal TRF2 may
both
trigger the DNA damage signal. In cells without telomerase,
the only
way to revert this block is to extend telomeres by ALT
pathways or to
bypass the signals involved in the ATM/p53 pathway.
Once we solve the
problems addressed above, we will have a clearer
idea of the essential
original proposed function of telomeres,
that telomeres protect
chromosomes from random degradation and
fusion.
| |
ACKNOWLEDGMENTS |
Y.S. is supported by the Japanese Foundation for
Multidisciplinary Treatment of Cancer and the Cell Science Research
Foundation. Research in the laboratory of P.M.L. is supported by NIH
grants ROIAI29524 and GM56162 and by a grant from the National Cancer Institute of Canada with funds from the Terry Fox Run.
We thank Terumi Kohwi-Shigematsu and Dag H. Yasui for critical reading
of the manuscript and Cheryl Helgason for experimental help.
H.N. and M.P.H. contributed equally to this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Cell Biology, Institute for Virus Research, Kyoto University, 53 Shogoin, Kawara-cho, Kyoto 606-8507, Japan. Phone: 81-75-751-3990. Fax: 81-75-751-3991. E-mail: yshinkai{at}virus.kyoto-u.ac.jp.
Present address: Life Sciences Division, Lawrence Berkeley National
Laboratory, University of California, Berkeley, CA 94720.
Present address: Center for Radiological Research, Columbia
University, New York, NY 10032.
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Molecular and Cellular Biology, June 2000, p. 4115-4127, Vol. 20, No. 11
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
