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Molecular and Cellular Biology, March 2000, p. 1659-1668, Vol. 20, No. 5
The Rockefeller University, New York, New
York 10021,1 and Boehringer Ingelheim
Pharma KG, Biberach, Germany2
Received 10 August 1999/Returned for modification 5 October
1999/Accepted 18 November 1999
Telomere length in human cells is controlled by a homeostasis
mechanism that involves telomerase and the negative regulator of
telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report
that TRF2, a TRF1-related protein previously implicated in protection
of chromosome ends, is a second negative regulator of telomere length.
Overexpression of TRF2 results in the progressive shortening of
telomere length, similar to the phenotype observed with TRF1. However,
while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the
expression of exogenous TRF2 was extinguished and the telomeres
eventually regained their original length. Consistent with their role
in measuring telomere length, indirect immunofluorescence indicated
that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are
more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1
nor TRF2 affected the expression level of telomerase. Furthermore, the
presence of TRF1 or TRF2 on a short linear telomerase substrate did not
inhibit the enzymatic activity of telomerase in vitro. These findings
are consistent with the recently proposed t loop model of telomere
length homeostasis in which telomerase-dependent telomere elongation is
blocked by sequestration of the 3' telomere terminus in TRF1- and
TRF2-induced telomeric loops.
The length of mammalian telomeres is
governed by a homeostasis mechanism. Telomeres have a species-specific
length setting (26) which is constant over the generations,
despite high levels of telomerase activity in the germline
(47). For instance, the telomeres of Mus musculus
are maintained at 20 to 50 kb, while the closely related mouse species
Mus spretus has telomeric tracts closer in length to human
telomeres, usually ranging from 5 to 15 kb. Similarly, despite high
levels of telomerase, the telomeres of many human tumor cell lines do
not grow but are stably maintained at a setting characteristic for each
individual cell line (12, 13, 44). Telomere length
homeostasis is also evident when new telomeres are generated by
transfection of short stretches of telomeric DNA into cultured cells.
The transfected telomeric tracts are elongated, presumably by
telomerase, until their length matches the other telomeres in the
transfected cells (3, 23, 43). The new telomere presumably
recruits telomere binding proteins and so attains all functions of
telomeres, including the length regulation characteristic of a given
cell. Regulated growth of a single telomere, as observed in this
context, suggests that cells can measure and modulate the length of the
telomeric repeat array at individual chromosome ends, implying a
cis-acting regulatory mechanism.
Human telomeres contain two related TTAGGG repeat binding factors, TRF1
and TRF2 (7, 10, 11). Both TRF proteins have a Myb-like
helix-turn-helix domain in their carboxy terminus and a central
conserved domain that includes sequences responsible for the formation
of homodimers. The two proteins do not heterodimerize, and they differ
substantially at the N terminus, which is acidic in TRF1 but basic in
TRF2 (10). TRF1 and -2 are most closely related in their
Myb-type DNA binding domains (56% identity), and both proteins can
bind duplex telomeric DNA in vitro. The DNA binding site of TRF1, as
determined by systematic evolution of ligands by exponential enrichment
(SELEX), is composed of two identical YTAGGGTTR half sites which each
engage one Myb domain in the TRF1 homodimer (6). There is no
constraint on the distance between the two half sites, and the sites
can be bound in direct or inverted orientation. TRF1 has architectural
features, including the ability to loop the sequence between two half
sites (6) and the ability to pair two telomeric tracts
(20). Although TRF2 displays a similar preference for duplex
TTAGGG repeats in vitro (4, 10), its DNA binding features
have not been established in detail.
In human cells, both TRF1 and TRF2 are predominantly located at
chromosome ends where they contribute to the protection and maintenance
of telomeric DNA. TRF1 has been demonstrated to regulate telomere
length (44). Overexpression of TRF1 in the
tetracycline-responsive human fibrosarcoma cell line HTC75 results in a
gradual decline in telomere length at a rate of ~10 bp/population
doubling (PD). Conversely, the expression of a dominant negative allele
of TRF1, which removes endogenous TRF1 from telomeres, leads to
telomere elongation. In this system, TRF1 did not affect the activity
of telomerase detectable in cell extracts, suggesting that TRF1 does not affect telomerase activity globally in the cell. Instead, we have
proposed that TRF1 acts in cis as a negative length
regulator at each individual telomere. According to the current model,
an inappropriately long telomere would recruit a large amount of TRF1
protein, blocking telomerase-mediated elongation of that particular
chromosome end and thus leading to a resetting of the telomere length
in cis. A similar protein-counting model was proposed for
telomere length homeostasis in yeast (31).
An important question is how TRF1, which binds along the length of the
telomere, modulates telomerase, an enzyme that acts at telomere
termini. One mechanism that could be considered in this context is that
accessibility of a DNA end to telomerase is diminished by the presence
of TRF1 on or near the telomere terminus. An alternative proposal has
recently emerged from the finding that telomeres fold back, forming a
large duplex lariat called the t loop (21). In the t loop,
the 3' single-stranded telomeric overhand of TTAGGG repeats is tucked
into the duplex part of the telomeric repeat tract. The t loop is
proposed to sequester telomeres from activities that might act on
chromosome ends, including telomerase. In vitro studies suggest that
telomerase requires an accessible 3' overhang (28, 30, 46),
a structure predicted to be absent from t loops. Therefore, t loops
could control the action of telomerase at individual chromosome ends. Based on biochemical studies, the formation of t loops was proposed to
involve both TRF1 and TRF2. TRF1 has the ability to induce bending,
looping, and pairing of duplex telomeric DNA (5, 6, 20, 21),
activities that could facilitate the folding back of the telomere. TRF2
was found to induce the invasion of the 3' single-stranded TTAGGG
repeat tail into duplex telomeric DNA, forming t loops in vitro
(21; R. Stansel, T. de Lange, and J. D. Griffith, unpublished data). Thus, a t-loop-based mechanism for
telomere length regulation would predict that both TRF1 and TRF2 are
required for the length homeostasis of human telomeres. Specifically,
the model predicts that, like TRF1, TRF2 acts as a negative regulator
of telomere length.
The role of TRF2 in telomere length regulation cannot easily be
examined through the inhibition of its function. Interference with the
activity of TRF2 results in immediately deleterious phenotypes, possibly due to inappropriate exposure of unfolded telomere termini to
DNA damage checkpoints and repair activities. For instance, cells
forced to express a dominant negative allele of TRF2 rapidly initiate
an apoptotic pathway (24), and telomeres lacking TRF2 lose
their 3' overhangs and undergo covalent fusions (45;
A. Smogorzewska and T. de Lange, unpublished data). Therefore, we have
addressed the role of TRF2 in telomere length regulation by
overexpression of the full-length protein. The results demonstrate that
TRF2 is a second negative regulator of telomere length in mammalian
cells and are consistent with a t-loop-based mechanism for telomere
length homeostasis.
Cell lines.
Four clonal HTC75 cell lines (P4, P7, P12, and
P33) expressing full-length TRF2 from a tetracyclin-repressed promoter
were described previously (45). Two HTC75-derived clonal
cell lines (J3 and J24) expressing an N-terminally FLAG-tagged deletion
allele of TRF1 (TRF166-439) were generated by
cotransfection of the pTetFLAGhTRF166-439 plasmid with a
neomycin resistance marker. Inducible expression of
TRF166-439 was tested by indirect immunofluorescence and
immunoblotting with the FLAG-specific M2 monoclonal antibody (Kodak) on
24 independent clones isolated after selection in media containing 400 µg of G418 per ml. A control HTC75 cell line, B27, contained the
empty vector (44). Cells were grown in Dulbecco modified
Eagle medium (DMEM) supplemented with L-glutamine,
penicillin-streptomycin, nonessential amino acids, 10% bovine calf
serum (HyClone) and either hygromycin (90 µg/ml) or G418 (150 µg/ml). Drugs were alternated every other week. Cells were passaged
at 1:16 when they reached 80% confluence. Noninduced and induced cells
were grown in parallel with or without doxycycline (Sigma, 100 ng/ml).
The cells from hamster cell lines AHL-1 (ATCC CCL 195), BHK-21 (ATCC
CCL 8544), and CHO-K1 (ATCC CCL 61) were grown in DMEM supplemented as above.
Whole-cell extracts.
Cells grown in 10-cm-diameter dishes
were harvested by scraping in 5 ml of cold phosphate-buffered saline
(PBS) and were collected after centrifugation at 1,000 × g for 5 min. Cell pellets were resuspended in 4 to 6 volumes (50 to 200 µl) of ice-cold buffer C (20 mM HEPES-KOH [pH 7.9], 420 mM
KCl, 25% glycerol, 0.1 mM EDTA, 5 mM MgCl2, 1 mM
dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride [PMSF],
0.2% Nonidet P-40, 1 µg of leupeptin, pepstatin, and aprotinin per
ml) and were incubated for 30 min on ice and centrifuged for 10 min at
14,000 × g at 4°C. The supernatant was dialyzed for
2 h against buffer D (20 mM HEPES-KOH [pH 7.9], 100 mM KCl, 20%
glycerol, 0.2 mM EDTA, 0.2 mM EGTA, 0.5 mM DTT, and 0.5 mM PMSF) at
4°C, was snap-frozen in liquid nitrogen, and was stored at Western blotting.
Proteins (30 µg) were separated on a
sodium dodecyl sulfate (SDS)-9% polyacrylamide gel and were
transferred to nitrocellulose by electroblotting. Membranes were
stained with Ponceau S to verify that equal amounts of protein were
fractionated in each lane. After blocking for 30 min in PBS containing
10% nonfat milk powder and 0.5% Tween 20, blots were incubated for 12 to 16 h at 4°C with the anti-TRF2 antibody 508 or 647 or with
anti-TRF1 antibody 371 (44, 45; X.-D. Zhu and T. de
Lange, unpublished data) or anti-cyclin D antibody (Santa Cruz),
followed by three washes in PBS containing 0.1% nonfat dry milk powder
and 0.1% Tween 20. Blots were incubated for 45 min with horseradish
peroxidase-conjugated donkey anti-rabbit antibody (for 508, 647, and
371) or sheep anti-mouse antibody (for cyclin D) (Amersham) and were
washed three times for 10 min. The secondary antibody was detected by
using the ECL kit (Amersham).
Genomic blotting.
Cells from a subconfluent 15-cm-diameter
dish (approximately 107 cells) were trypsinized, washed in
cold PBS, and collected by centrifugation for 5 min at 1,000 × g, and cell pellets were frozen at Transient expression of TRF166-439.
Logarithmically growing HeLa 1.2.11 cells (107 cells in 0.8 ml of HBS [21 mM HEPES, pH 7.05, 137 mM NaCl, 0.7 mM
NaHPO4, 5 mM KCl, 6 mM glucose]) were electroporated (960 µF/320 mV) with 7.5 µg each of
pTetFLAGhTRF166-439 and the tTA-expression vector pUHD15-1
(19), and the cells were grown on autoclaved
coverslips in DMEM with 10% fetal bovine serum for 24 h before
being processed for indirect immunofluorescence.
FISH on metaphase spreads.
Hamster cells were incubated with
0.5 µg of colcemid per ml of growth media at 37°C for 30 min. Cells
were harvested by trypsinization and were incubated in 0.075 M KCl at
37°C for 7 min. Subsequently, cells were fixed in methanol-acetic
acid (3:1) and were dropped onto water-wetted slides. Fluorescence in
situ hybridization (FISH) was carried out as described before
(27) with 0.5 µg of fluorescein isothiocyanate-conjugated
(C3TA2)3 peptide nucleic acid (PNA) probe (Biotech BmpbH) per ml. The cells were embedded in a mixture of
90% glycerol and 10% PBS containing 1 mg of p-phenenylene
diamine (Sigma) per ml with 0.2 µg of 4',6-diamidino-2-phenylindole
(DAPI) per ml.
Immunofluorescence and microscopy.
FLAG-tagged
TRF166-439 was detected by using the anti-FLAG monoclonal
antibody M2 (Sigma), endogenous full-length TRF1 was detected by using
antibody 371C2 (44), and endogenous TRF2 was detected by
using antibody 508 (45). M2 was detected with fluorescein isothiocyanate-conjugated sheep anti-mouse antibody (Jackson), and
371C2 and 508 were detected with tetramethyl rhodamine
isocyanate-conjugated donkey anti-rabbit antibody (Jackson). Secondary
antibodies did not cross-react, as shown by control experiments
omitting either of the primary antibodies. DNA was counterstained with
DAPI. Endogenous TRF1 and TRF2 on telomeres in hamster, HeLal.2.11, and
HeLall cells (39, 45) were detected in parallel by using
affinity-purified rabbit polyclonal antibodies 371 and 647, respectively (44; X.-D. Zhu and T. de Lange,
unpublished data), and tetramethyl rhodamine isocyanate-conjugated
donkey anti-rabbit secondary antibodies (Jackson). For analysis of the
relative abundance of TRF1 and TRF2 on long and short HeLa telomeres,
exposure times were 1.0 s for all photographs, and the images are
presented without changes in settings. Photographs were taken with a
Photometrix SenSyn camera installed on a Zeiss Axioplan2 microscope
with IPLab software (Scanalytics, Inc.).
RNA isolation.
Total RNA was isolated as described
(2). Briefly, cells were washed in PBS, lysed in 3 M LiCl-6
M urea, sonicated, and incubated on ice for 6 h. RNA was collected
by centrifugation at 14,000 × g for 20 min at 4°C, was
treated with proteinase K (50 µg/ml) in 200 µl of a solution
containing 10 mM Tris-HCl [pH 7.5], 10 mM EDTA, 100 mM NaCl, and 1%
SDS for 10 min at 37°C, was extracted with
phenol-chloroform-isoamylalcohol (24:24:1), and was precipitated with
isopropanol in the presence of 0.2 M Na-acetate, pH 5.5.
RNase protection.
The hTERT sequence was subcloned from pCl
Neo-hEST2-HA (a gift from R. A. Weinberg) (32) into
pcDNA3 (Invitrogen). A Styl fragment containing part of the hTERT
sequence (nucleotide [nt] 3229 to 3452) and flanking vector sequences
was purified and used in the MAXIscript in vitro transcription reaction
(Ambion) with 5 µl of [ Partial purification of human telomerase.
Nuclear extracts
from HeLa cells (16) were dialyzed against buffer A-100 (20 mM HEPES [pH 7.9], 1 mM EDTA, 1 mM EGTA, 10% glycerol, 100 mM KCl,
0.5 mM PMSF, 0.5 mM DTT), and were applied to a Spermine-agarose column
(Sigma). The column was washed with 250 mM KCl, and 500 mM KCl in
buffer A. Telomerase activity was eluted with buffer A containing 1 M
KCl. The active fractions were pooled, dialyzed against buffer A-100,
and loaded onto a Heparin Cl-6B column (Pharmacia) equilibrated in
buffer A-100. The column was washed with buffer A containing 220 mM
KCl, and active telomerase was step eluted with 500 mM KCl. Active
fractions were pooled, dialyzed as described above, and applied to a
MonoQ fast protein liquid chromatography column (Pharmacia),
equilibrated with buffer A-100. The column was washed with buffer A
containing 300 mM KCl, and telomerase was eluted with a linear gradient
from 0.3 to 1 M salt in buffer A. The peak of active telomerase eluted at 500 mM KCl. Pooled active fractions showed a specific telomerase activity 100-fold higher than that of the input material as determined by the conventional telomerase assay (34).
Telomerase reactions.
Telomerase reactions were in a
solution containing 25 mM Tris acetate (pH 8.2), 50 mM Na acetate (pH
8.3), 1 mM MgCl2, 1 mM EGTA, 5 mM dATP, 5 mM dTTP, 4.5 µM
cold dGTP, 0.165 to 0.825 µM labelled dGTP (1 to 5 µl of
[
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Control of Human Telomere Length by TRF1 and
TRF2
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C.
Protein concentrations were determined by using the Bradford assay
(Bio-Rad, Hercules, Calif.) with bovine serum albumin as a standard.
80°C. DNA was isolated
as described (15) with inclusion of an RNase A digestion
step, was cleaved with HinfI and RsaI, and was
quantitated by fluorometry using Hoechst 33258. Three micrograms of DNA
was size fractionated on a 0.7% agarose gel in 0.5× Tris-borate-EDTA
(TBE). Gels were processed for genomic blotting and telomeric
restriction fragments were detected with a TTAGGG probe as previously
described (14). The median length of the telomeric
restriction fragments was determined by using ImageQuant software after
scanning with a PhosphorImager (44).
-32P]UTP (800 Ci/mmol, 10 mCi/ml). The template for the hTR RNase protection probe was similarly
prepared by digesting pGRN78 (25) with XbaI, and
the 
-actin probe was obtained from Ambion. The -actin probe was
labeled to a specific activity 200-fold lower than that of the hTERT
and hTR probes by diluting out the radiolabelled UTP with cold UTP.
RNase protection reactions were performed with solutions from the
Direct Protect Lysate Ribonuclease Protection Assay Kit (Ambion) with
20 µg of total RNA (isolated as described above) instead of cell lysates.
32P]dGTP, 3,000 Ci/mmol, 10 mCi/ml; NEN), 0.1 µl of
RNasin, and 1 mM spermidine (optional), in a volume of 20 µl,
typically using 1 to 2 µl of telomerase fractions. Concentration of
primer was 10 nM. Baculovirus-expressed TRF1 or TRF2 protein (5,
17), or bovine serum albumin (BSA) as a control, were incubated
with the DNA in the telomerase reaction buffer for 30 min at room
temperature. Subsequently, telomerase fractions were added, and
reactions were incubated for 1 h at 30°C and stopped by the
addition of 20 µl of H2O and 50 µl of 10 mM Tris (pH
8.0), 5 mM EDTA, and 0.1 mg of RNase/ml and incubation at 45°C for 10 min. Next, 50 µl of 10 mM Tris (pH 8.0) and 1% SDS containing 0.3 mg
of proteinase K/ml were added, and the samples were incubated at 45°C
for 10 min. Samples were extracted with 200 µl of
phenol-chloroform-isoamyl alcohol and were precipitated by the addition
of 50 µl of 3 M Na acetate (pH 5.2), 2.5 µg of tRNA, and 500 µl
of ethanol and incubation on ice for 15 min. Samples were centrifuged
at room temperature for 10 min, and the ethanol was removed. Pellets
were dissolved in 4 µl of formamide loading dye, and all or half of the sample was loaded onto 6% polyacrylamide (19:1
acrylamide-bis-acrylamide) gels containing 7 M urea and 20% (vol/vol)
formamide in 1× TBE. Gels were run at 40 to 55 W, at about 50 to
60°C external temperature. Oligonucleotides were gel purified on
polyacrylamide-7 M urea gels and were annealed in a solution
containing 10 mM Tris (pH 7.5), 1 mM EDTA, and 100 mM NaCl by boiling
for 5 min and slow cooling (over a few hours) from 80°C to room temperature.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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TRF2 affects telomere length. To address the role of TRF2 in telomere length regulation, we employed a previously established tetracyclin-inducible expression system in the human fibrosarcoma cell line HTC75 (45). Although the HTC75 cells express telomerase, the length of their telomeres is stably maintained at a setting that is in part governed by TRF1 (44). Overexpression of TRF1 results in gradual and progressive loss of telomeric DNA. Here we explore the effect of long-term overexpression of TRF2. Four clonal cell lines (called P lines) that can be induced to express high levels of TRF2 by removal of doxycycline from the growth media were established. An initial, short-term (9-day) analysis of these cells revealed no striking phenotypes due to increased TRF2 expression (45), whereas the same study documented a rapid inhibition of cell proliferation by two truncated alleles of TRF2. Long-term growth of the P cell lines allowed us to assess the effect of elevated TRF2 expression on the steady-state length of the telomeres.
The cell lines were maintained with or without doxycycline in the media for 124 PDs, and their telomere length was assessed by genomic blotting. With one exception (P4), the cell lines showed consistent telomere length changes that were dependent on TRF2 induction (Fig. 1). The P4 cell line showed unanticipated fluctuations in telomere length that were not dependent on the presence of doxycycline in the medium (data not shown). The three other P cell lines showed a complex, biphasic pattern of telomere length changes in response to increased TRF2 levels. In the P7 cell line, the telomeres initially shortened at a rate of 82 bp per PD (calculated for the growth period from PD 0 to PD 28) when TRF2 was induced (Fig. 1A). Unexpectedly, around PD 48, the dynamics of the telomeres changed. At this point in the culture, the telomeres lengthened, reaching their original length setting by PD 88. The telomere length of the noninduced culture appeared relatively stable throughout the experiment. A similar course of events was seen in the P33 cell line, where the initial decline in telomere length was 47 bp per PD (growth window, PD 0 to PD 40) and was followed by modest telomere elongation that started at PD 72. The third cell line (P12) showed a telomere-shortening rate of 51 bp/PD in the first 8 PD after TRF2 induction, followed by a rapid increase in telomere length, reaching a final length setting that surpassed the starting value (data not shown). Overall, the overexpression of TRF2 led to an initial decline in telomere length and an eventual cessation of this decline followed by telomere elongation. These results indicate that TRF2, like TRF1, affects telomere maintenance.
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Complex telomere dynamics correlate with loss of TRF2 expression. Although the biphasic nature of the telomere length changes in the P cell lines was unexpected, the fact that the pattern of telomere dynamics was similar in three independent cell lines suggested that this phenotype was an inherent property of HTC75 cells induced to express high levels of TRF2. To further understand the molecular basis of this phenotype, we analyzed the expression levels of TRF2. Immunoblotting showed that even though the initial expression of the exogenous TRF2 protein was very high, the levels of TRF2 declined in each of the cell lines with increasing PD (Fig. 1B, compare lanes at PDs 8 and 124 in the induced state) (data not shown for P12). The decline of the steady-state levels of TRF2 protein detectable by Western analysis was due to a decrease in number of cells that expressed TRF2 as well as in the level of expression in individual cells as deduced from the immunofluorescence staining. For instance, the percentage of immunofluorescence staining-positive cells dropped from 100% at PD 2 to 0% at PD 48 for P12 and from 100% at PD 2 to 21% at PD 76 for P7.
Loss of expression of a transfected gene during long-term growth of the transfected cells has been reported previously and is often ascribed to de novo methylation of the transgene. However, the rapid loss of TRF2 expression in comparison to the stable expression of TRF1 in the same system (see below) suggested that down regulation of TRF2 expression might confer a selective advantage on the cells. In this regard, we noted that the induced cultures contained many cells with an unusual nuclear morphology (multilobulated nuclei) (data not shown). The appearance of the aberrant nuclei tended to coincide with the loss of TRF2 expression. We also noted that each of the induced cultures grew somewhat slower than the uninduced controls. For instance, PD 40 was reached after about 40 days for the controls but 4 days later for the induced cultures. Although we cannot determine whether the altered nuclear morphology is relevant to the gradual disappearance of TRF2-expressing cells, the results are consistent with the possibility that overexpression of TRF2 (perhaps in conjunction with shortened telomeres) inhibits cell growth.Long-term overexpression of full-length TRF1 and TRF166-439. The complex telomere dynamics observed in cells overexpressing TRF2 and the apparent selection against high levels of TRF2 raised the question of whether similar events might be occurring during long-term overexpression of TRF1. Previous analysis of several HTC75 cell lines that overexpressed full-length TRF1 had revealed a gradual and progressive shortening of the telomeres, and no repression of the transfected TRF1 gene was noted (44). However, the initial analysis of these cell lines did not provide information on events after extensive telomere shortening. Thus, it was pertinent to analyze the long-term dynamics of telomeres in cells overexpressing TRF1.
One clonal TRF1-expressing cell line, D4, was selected for this purpose, and its telomere length was monitored over 304 PD. As shown in Fig. 2A, this cell line showed the previously recorded gradual telomere shortening, and this shortening continued until approximately 1.5 kb of telomeric DNA was lost (at PD 200). At that stage, telomere length became stable, and no further changes were observed when the culture was grown for an additional 100 PD. No gross effects on growth rates were observed, and TRF1 expression was unaltered (data not shown). In fact, the overexpression of TRF1 was required to maintain the telomeres at a stable, short, length setting as demonstrated by the rapid telomere lengthening observed when doxycycline was added to the growth media (Fig. 2A).
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Accumulation of TRF1 and TRF2 at chromosome ends correlates with telomere length. The telomere-shortening phenotype associated with high levels of TRF2 expression indicates a role for this factor in telomere length homeostasis. One possibility, similar to what was proposed for TRF1, is that TRF2 measures telomere length by binding to the duplex telomeric repeat tract. Indeed, according to band-shift analysis, TRF2 binds to duplex telomeric repeat arrays (4, 10). However, electron microscope analysis indicated that when TRF2 is associated with telomeric DNA in the t loop configuration, it is preferentially positioned on or near the t loop junction rather than along the telomeric tract (21). This preference for the t loop junction was not observed for TRF1 which displayed extensive binding along the duplex telomeric DNA even when the DNA was in the t loop configuration. It was therefore pertinent to establish whether TRF2 can bind to duplex telomeric DNA in vivo or whether the presence of this protein at chromosome ends reflects a preference for a structural feature of telomeres.
We addressed this question by asking whether TRF2 could associate with telomeric sequences at a chromosome-internal site. Such a situation is represented by certain hamster chromosomes which have sequences related to telomeric repeat DNA associated with their centromeres. Pericentromeric satellite sequences related to TTAGGG repeats are present in several chromosomes in the Syrian hamster cell line, BHK-21, and in the Chinese hamster cell line, CHO-K1, but not in any of the chromosomes of the Armenian hamster cell line, AHL-1 (1, 33) (Fig. 3). Since TRF2 is highly conserved (10), we were able to detect TRF2 in Syrian, Armenian, and Chinese hamster cells with a polyclonal antibody raised against an amino-terminal peptide of human TRF2 (508) (45) (Fig. 3A). By using this antibody in indirect immunofluorescence on metaphase chromosomes from BHK and AHL cells, we detected the expected telomeric staining pattern previously observed for TRF2 in other mammals. In addition, several of the BHK chromosomes showed prominent pericentric signals for TRF2. The use of a second independent polyclonal antibody (647) (X.-D. Zhu and T. de Lange, unpublished data) for indirect immunofluorescence on CHO chromosomes further verified the chromosome-internal accumulation of TRF2 at pericentric sites (Fig. 3B). Such interstitial signals were not seen in AHL cells, consistent with the lack of extensive pericentric telomere-related DNA in these cells. These data indicated that TRF2 has the ability to bind to chromosome-internal telomere-related sequences, suggesting that the binding of TRF2 is not dependent on the DNA being in a t loop configuration. Consistent with the poor conservation of TRF1 (9), we have not been able to detect hamster TRF1 with a polyclonal antibody raised against full-length human TRF1 or with a polyclonal antibody raised against a peptide of mouse Trf1. However, transfection of human TRF1 into BHK cells indicated that TRF1, like TRF2, can locate to the interstitial sites of telomere-related sequences (data not shown).
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Lack of direct effects of TRF1 and TRF2 on telomerase in
vitro.
Previous studies indicated that overexpression or
inhibition of TRF1 and TRF2 does not affect the expression level of
telomerase as detected by the semiquantitative TRAP assay performed
with extracts of cell lines expressing full-length or truncated
proteins (44, 45). Similarly, we have failed to detect a
change in the expression level of the hTERT mRNA or the hTR component
of telomerase in cells overexpressing TRF2 (Fig.
5A). We also failed to detect an
association between telomerase and TRF1 by immunoprecipitation or on
glycerol gradients, and the addition of baculovirus-produced TRF1
protein did not affect telomerase activity in the TRAP assay (data not
shown). Finally, no correlation was noted between expression of TRF1 or
TRF2 and the expression of telomerase activity in immortalized human
cell lines (data not shown).
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]dGTP into the typical 6-nt ladder of telomerase
products (Fig. 5C). We engineered an EcoRI site into the
duplex part of SB2, allowing us to verify that telomerase added TTAGGG
repeats to the correctly folded substrate. Indeed, cleavage of the
telomerase products with EcoRI and subsequent size
fractionation on a denaturing gel indicated the expected downshift of
the product ladder by approximately 40 nt (data not shown).
When TRF1 or TRF2 protein was added to the telomerase reactions with TS
as a substrate, no effect on either the overall incorporation of
labelled precursor or the size distribution of the products was noted
(Fig. 5C). This result was consistent with the observation that the
addition of TRF1 to TRAP assays had no effect on the activity of
telomerase. Similarly, when SB2 was used as a substrate, the products
appeared identical regardless of the presence of saturating amounts of
TRF1 or TRF2 (BSA was used as a control). Because the elongation
products were spread over a large range of molecular weights on the
gel, and because of the presence of a substantial smearing from the
unincorporated [32P-]dGTP, quantitation of the
telomerase products was not attempted. Nevertheless, repetitions of the
experiment (n = 7) allowed us to rule out a major
effect of either telomeric protein on telomerase activity under these
conditions. Only minor variations from experiment to experiment were
observed. Two overhangs, 5'GTTAGGGTT3' and 5'GGTT3', were
tested in the assay with identical results. Telomerase assays were also
performed in the presence of a 25-fold-higher amount of TRF1 and TRF2
(1.25 µM TRF1 and 0.877 µM TRF2). Even though the results showed
greater variability under these conditions, presumably because of
protein aggregation, no clear effect of either protein on telomerase
was observed (data not shown). Thus, under these conditions, binding of
TRF1 or TRF2 near the telomerase substrate site does not interfere with
enzyme activity. Our experiments do not address other
cis-acting mechanisms, and we cannot exclude at this stage
that TRF1 or TRF2 affects telomere length maintenance in
trans through affecting a limiting factor other than telomerase.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The maintenance of human telomeres by telomerase is regulated by a homeostasis mechanism. Previously, TRF1 was identified as a component of a negative feedback loop that limits telomere elongation and results in stable telomere length. Here we report that overexpression of a related telomere binding protein, TRF2, also results in telomere shortening, indicating that TRF2 is a second negative regulator of telomere length. The simplest explanation is that both TRF1 and TRF2 bind along the length of the duplex telomeric repeat array and function to measure telomere length. In agreement, we find that both proteins bind to double-stranded telomeric DNA in vivo and that their accumulation at telomeres is dependent on telomere length.
TRF1 and TRF2 vis-à-vis the protein-counting model of telomere length regulation. Our observations can be interpreted within the context of a protein-counting model for telomere length regulation (31, 40, 44). According to this model, telomeres can exist in two states, an "open" state which allows telomerase to elongate the telomere and a "closed" state in which the enzyme cannot access or extend the telomere terminus. The switching between these two states is proposed to be governed by telomere binding proteins, TRF1 and TRF2 in human cells, which act as negative regulators through promoting the closed state. As telomeres are elongated by telomerase, they will bind a greater number of the negative regulators, increasing the chance of a switch to the closed state. After switching to the closed state, a telomere will gradually lose sequences with each cell division, leading to a smaller number of bound negative regulators and an improved chance of switching back to the open state. As a result, each individual telomere will approach a steady-state length determined by the activity of telomerase, the expression level of TRF1 and TRF2, and other regulatory factors (e.g., tankyrase [42]).
Several predictions of this model are tested here. First, the model predicts that the negative regulators are proteins that bind along the duplex part of the telomere. Although previous results demonstrated the association of TRF1 and TRF2 with human and mouse telomeres, it was not possible to distinguish between their binding to the duplex part of the telomere or to some other feature of the telomeric structure (e.g., the telomere terminus or a structural component of the t loop). By using indirect immunofluorescence, we discovered that TRF2 bound to telomere-related sequences that occur at chromosome-internal sites in some hamster chromosomes. Assuming that these interstitial sequences are present as duplex DNA, this result indicates that TRF2 can bind to duplex TTAGGG repeat tracts in vivo. Indirect evidence indicated that TRF1 also bound interstitial telomeric DNA. A second prediction of the model is that the accumulation of TRF1 and TRF2 at telomeres reflects the length of the duplex telomeric tracts. Indeed, cells with long telomeres had more intense immunofluorescence signals for both proteins. Although a direct quantitative interpretation of immunofluorescence signals is not possible, the data are consistent with the view that longer telomeres attract a greater mass of TRF1 and TRF2, as predicted by the protein-counting model. According to the protein-counting model, very short telomeres would not bind sufficient amounts of TRF1 or TRF2 to create the closed state. At this stage, the telomere elongation would again take place, preventing complete loss of the telomeric DNA and resulting in the stabilization of the telomeres at a short length. This scenario is consistent with the data on long-term overexpression of TRF1 presented here. After an initial period of telomere shortening, the telomeres stabilized at reduced length even though TRF1 levels remained high. In fact, the overexpression of TRF1 continued to dictate the short-length setting as demonstrated by rapid telomere elongation after repression of the exogenous TRF1. In sum, the telomere dynamics in HTC75 cells in response to changes in the levels of TRF1 and TRF2 are consistent with both proteins functioning as negative regulators of telomere length in a protein-counting mode for telomere length regulation.A possible mechanism by which TRF1 and TRF2 regulate telomere length. The protein-counting model of telomere length regulation does not define the nature of the open and closed states nor does it specify by what mechanism TRF1 and TRF2 affect the transition between these states. The only specific prediction of the model is that the events take place in cis, regulating the action of telomerase at individual chromosome ends. In agreement with this prediction, we find that neither the expression of telomerase components nor the global activity of the enzyme are affected by changes in expression of TRF1 or TRF2. We also did not find a direct effect of TRF1 or TRF2 on telomerase when the factors were positioned at or near the 3' end of the substrate.
Recent findings on the structure of human telomeres suggest that t loops may represent the closed conformation. In t loops, the telomere terminus is tucked away in the duplex telomeric repeat array and is therefore likely to be inaccessible to telomerase. TRF2 can remodel telomeric DNA into t loops in vitro, and TRF1 has several biochemical activities that could be expected to promote t loop formation, suggesting that both proteins contribute to the remodelling of telomeres into t loops in vivo. The current data are consistent with this proposal, since both TRF1 and TRF2 act as negative regulators of telomere length as would be expected if both proteins promote the formation of a closed t loop state. The emerging view, based on the current data, is that long telomeres recruit a larger amount of TRF1 and TRF2. The greater abundance of these proteins on longer telomeres is suggested to facilitate remodelling of the telomeres into t loops. In the t loop state, telomerase would no longer be able to elongate the telomere terminus, leading to loss of sequences with cell divisions. Eventually, this sequence loss would result in diminished binding of TRF1 and TRF2 to the telomere, which would consequently form t loops at a lower rate (or at a reduced frequency). The resulting (temporary) persistence of an unfolded telomere, a substrate for telomerase, would then again lead to telomere elongation. This model can be tested by assessing how TRF1 and TRF2 modulate the formation of t loops in vivo and by studying the interaction of telomerase with t loops in vitro and in vivo.Comparison to telomere homeostasis in yeast. A comprehensive view of telomere length control has emerged in the yeast Saccharomyces cerevisiae. Genetic, molecular genetic, and biochemical approaches have revealed the interplay between components of the telomeric chromatin (including Rap1p, Rif1p, Rif2p, and Cdc13p), trans-acting factors (such as telomerase [Est2p/TLC1]), and telomeric chromatin-associated proteins (Est1p and Est3p), as well as additional mediators like the Ku, the Rad50-Mre11-Xrs2 complex (8, 36, 38), Tel1p, and Stn1p (for reviews, see references 37 and 41). In comparison, the dissection of telomere length homeostasis in mammals is in its infancy, with only a few of the players available, most of which show no similarity to the yeast genes. For instance, TRF1 and TRF2 lack significant homology to Rap1p outside the Myb-type DNA binding fold. However, a strong common theme that has emerged is the likely involvement of a protein-counting mechanism in both systems (31, 44). It will be of interest to determine the possible structural similarities between the telomeric complex in yeast and mammals, in particular with regards to the presence of t loops. In this regard, Li and Lustig (29) raised the intriguing possibility of a transient loopback mechanism in combination with strand invasion to explain the rapid deletions they observed at yeast telomeres. Additional hints in favor of t loops in yeast emerged from studies of telomeric silencing (reviewed in reference 22), and Rap1p and TRF2 display intriguing similarities in biochemical activities that are relevant to telomere looping (17, 18, 21). Further parallel analysis of telomere length homeostasis in unicellular and multicellular organisms should reveal the diversity of solutions to this common regulatory problem.
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ACKNOWLEDGMENTS |
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We thank members of the de Lange laboratory, especially Susan Smith and Jan Karlseder, for helpful discussions. Art Lustig is thanked for advice in the revision stage of this manuscript. Jason Lue is thanked for preparation of baculovirus-derived TRF1 and TRF2 proteins. R. A. Weinberg generously provided the hTERT cDNA. G.S. thanks K. Damm and A. Schnapp for useful discussions.
This work was supported by a grant to T.D.L. from the National Cancer Institute (CA76027) and an Irma T. Hirschl Career Scientist Award to T.D.L. T.D.L. is a recipient of the Burroughs Wellcome Fund Scholar Award in Toxicology. A.S. is supported by National Institutes of Health MSTP grant GM07739 to the Cornell/Rockefeller/Memorial Sloan-Kettering Tri-Institutional MD/PhD program. B.V.S. was supported by an HFSP fellowship, and A.B. was the recipient of a Comitato Promotore Telethon graduate fellowship.
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FOOTNOTES |
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* Corresponding author. Mailing address: Box 159, The Rockefeller University, New York, NY 10021. Phone: (212) 327-8146. Fax: (212) 327-7147. Email: delange{at}rockvax.rockefeller.edu.
Present address: Fred Hutchinson Cancer Research Center, Seattle,
WA 98109-1024.
Present address: Department of Molecular Biology, University of
Geneva, Geneva, Switzerland.
§ Present address: Institut für Medizinische Mikrobiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
Present address: Institute for Molecular Pathology, A-1030 Vienna, Austria.
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REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Ashley, T., and D. C. Ward. 1993. A "hot spot" of recombination coincides with an interstitial telomeric sequence in the Armenian hamster. Cytogenet. Cell Genet. 62:169-171[Medline]. |
| 2. | Auffray, C., and G. Rougeon. 1980. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107:303-314[Medline]. |
| 3. |
Barnett, M.,
V. Buckle,
E. Evans,
A. Porter,
D. Rout,
A. Smith, and W. R. A. Brown.
1993.
Telomere directed fragmentation of mammalian chromosomes.
Nucleic Acids Res.
21:27-36 |
| 4. | Bianchi, A. 1999. Characterization of DNA binding activities at vertebrate telomeres. Doctoral dissertation. The Rockefeller University, New York, N.Y. |
| 5. | Bianchi, A., S. Smith, L. Chong, P. Elias, and T. de Lange. 1997. TRF1 is a dimer and bends telomeric DNA. EMBO J. 16:1785-1794[CrossRef][Medline]. |
| 6. | Bianchi, A., R. M. Stansel, L. Fairall, J. D. Griffith, D. Rhodes, and T. de Lange. 1999. TRF1 binds a bipartite telomeric site with extreme spatial flexibility. EMBO J. 18:5735-5744[CrossRef][Medline]. |
| 7. | Bilaud, T., C. Brun, K. Ancelin, C. E. Koering, T. Laroche, and E. Gilson. 1997. Telomeric localization of TRF2, a novel human telobox protein. Nat. Genet. 17:236-239[CrossRef][Medline]. |
| 8. | Boulton, S. J., and S. P. Jackson. 1998. Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J. 17:1819-1828[CrossRef][Medline]. |
| 9. |
Broccoli, D.,
L. Chong,
S. Oelmann,
A. A. Fernald,
N. Marziliano,
B. van Steensel,
D. Kipling,
M. M. Le Beau, and T. de Lange.
1997.
Comparison of the human and mouse genes encoding the telomeric protein, TRF1: chromosomal localization, expression, and conserved protein domains.
Hum. Mol. Genet.
6:69-76 |
| 10. | Broccoli, D., A. Smogorzewska, L. Chong, and T. de Lange. 1997. Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nat. Genet. 17:231-235[CrossRef][Medline]. |
| 11. |
Chong, L.,
B. van Steensel,
D. Broccoli,
H. Erdjument-Bromage,
J. Hanish,
P. Tempst, and T. de Lange.
1995.
A human telomeric protein.
Science
270:1663-1667 |
| 12. | Counter, C. M., A. A. Avilion, C. E. LeFeuvre, N. G. Stewart, C. W. Greider, C. B. Harley, and S. Bacchetti. 1992. Telomere shortening associated with chromosome instability is arrested in immortal cells with express telomerase activity. EMBO J. 11:1921-1929[Medline]. |
| 13. |
Counter, C. M.,
H. W. Hirte,
S. Bacchetti, and C. Harley.
1994.
Telomerase activity in human ovarian carcinoma.
Proc. Natl. Acad. Sci. USA
91:2900-2904 |
| 14. | de Lange, T. 1992. Human telomeres are attached to the nuclear matrix. EMBO J. 11:717-724[Medline]. |
| 15. |
de Lange, T.,
L. Shiue,
R. M. Myers,
D. R. Cox,
S. L. Naylor,
A. M. Killery, and H. E. Varmus.
1990.
Structure and variability of human chromosome ends.
Mol. Cell. Biol.
10:518-527 |
| 16. |
Dignam, J. P.,
R. M. Lebovitz, and R. G. Roeder.
1983.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:1475-1489 |
| 17. | Gilson, E., T. Laroche, and S. M. Gasser. 1993. Telomeres and the functional architecture of the nucleus. Trends Cell Biol. 3:128-134[CrossRef][Medline]. |
| 18. |
Gilson, E.,
T. Muller,
J. Sogo,
T. Laroche, and S. M. Gasser.
1994.
RAP1 stimulates single- to double-strand association of yeast telomeric DNA: implications for telomere-telomere interactions.
Nucleic Acids Res.
22:5310-5320 |
| 19. |
Gossen, M., and H. Bujard.
1992.
Tight control of gene expression in mammalian cells by tetracyclin-responsive promoters.
Proc. Natl. Acad. Sci. USA
89:5547-5551 |
| 20. | Griffith, J., A. Bianchi, and T. de Lange. 1998. TRF1 promotes parallel pairing of telomeric tracts in vitro. J. Mol. Biol. 278:79-88[CrossRef][Medline]. |
| 21. | Griffith, J. D., L. Comeau, S. Rosenfield, R. M. Stansel, A. Bianchi, H. Moss, and T. de Lange. 1999. Mammalian telomeres end in a large duplex loop. Cell 97:503-514[CrossRef][Medline]. |
| 22. | Grunstein, M. 1997. Molecular model for telomeric heterochromatin in yeast. Curr. Opin. Cell Biol. 9:383-387[CrossRef][Medline]. |
| 23. |
Hanish, J. P.,
J. Yanowitz, and T. de Lange.
1994.
Stringent sequence requirements for telomere formation in human cells.
Proc. Natl. Acad. Sci. USA
91:8861-8865 |
| 24. |
Karlseder, J.,
D. Broccoli,
Y. Dai,
S. Hardy, and T. de Lange.
1999.
p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2.
Science
283:1321-1325 |
| 25. |
Kim, N. W.,
M. A. Piatyszek,
K. R. Prowse,
C. B. Harley,
M. D. West,
P. L. C. Ho,
G. M. Coviello,
W. E. Wright,
S. L. Weinrich, and J. W. Shay.
1994.
Specific association of human telomerase activity with immortal cells and cancer.
Science
266:2011-2015 |
| 26. | Kipling, D., and H. J. Cooke. 1990. Hypervariable ultra-long telomeres in mice. Nature 347:400-402[CrossRef][Medline]. |
| 27. |
Lansdorp, P. M.,
N. P. Verwoerd,
F. M. van de Rijke,
V. Dragowska,
M.-T. Little,
R. W. Dirks,
A. K. Raap, and H. J. Tanke.
1996.
Heterogeneity in telomere length of human chromosomes.
Hum. Mol. Genet.
5:685-691 |
| 28. |
Lee, M. S., and E. H. Blackburn.
1993.
Sequence-specific DNA primer effects on telomerase polymerization activity.
Mol. Cell. Biol.
13:6586-6599 |
| 29. |
Li, B., and A. J. Lustig.
1996.
A novel mechanism for telomere size control in Saccharomyces cerevisiae.
Genes Dev.
10:1310-1326 |
| 30. |
Lingner, J., and T. R. Cech.
1996.
Purification of telomerase from Euplotes aediculatus: requirement of a primer 3' overhang.
Proc. Natl. Acad. Sci. USA
93:10712-10717 |
| 31. |
Marcand, S.,
E. Gilson, and D. Shore.
1997.
A protein-counting mechanism for telomere length regulation in yeast.
Science
275:986-990 |
| 32. | Meyerson, M., C. M. Counter, E. N. Eaton, L. W. Ellisen, P. Steiner, S. D. Caddle, L. Ziaugra, R. L. Beijersbergen, M. J. Davidoff, Q. Liu, S. Bacchetti, D. A. Haber, and R. A. Weinberg. 1997. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 90:785-795[CrossRef][Medline]. |
| 33. | Meyne, J., R. J. Baker, H. H. Hobart, T. C. Hsu, O. A. Ryder, O. G. Ward, J. E. Wiley, D. H. Wurster-Hill, T. L. Yates, and R. K. Moyzis. 1990. Distribution of non-telomeric sites of the (TTAGGG)n telomeric sequence in vertebrate chromosomes. Chromosoma 99:3-10[CrossRef][Medline]. |
| 34. | Morin, G. B. 1989. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59:521-529[CrossRef][Medline]. |
| 35. | Morin, G. B. 1991. Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase. Nature 353:454-456[CrossRef][Medline]. |
| 36. | Nugent, C., G. Bosco, L. Ross, S. Evans, A. Salinger, J. Moore, J. Haber, and V. Lundblad. 1998. Telomere maintenance is dependent on activities required for end repair of double-strand breaks. Curr. Biol. 8:657-660[CrossRef][Medline]. |
| 37. |
Nugent, C. I., and V. Lundblad.
1998.
The telomerase reverse transcriptase: components and regulation.
Genes Dev.
12:1073-1085 |
| 38. | Polotnianka, R., J. Li, and A. Lustig. 1998. The yeast Ku heterodimer is essential for protection of the telomere against nucleolytic and recombinational activities. Curr. Biol. 8:831-834[CrossRef][Medline]. |
| 39. | Saltman, D., R. Morgan, M. L. Cleary, and T. de Lange. 1993. Telomeric structure in cells with chromosome end associations. Chromosoma 102:121-128[CrossRef][Medline]. |
| 40. | Shore, D. 1997. Telomerase and telomere-binding proteins: controlling the endgame. Trends Biochem. Sci. 22:233-235[CrossRef][Medline]. |
| 41. | Shore, D. 1997. Telomere length regulation: getting the measure of chromosome ends. Biol. Chem. 378:591-597. |
| 42. |
Smith, S.,
I. Giriat,
A. Schmitt, and T. de Lange.
1998.
Tankyrase, a poly(ADP-ribose) polymerase at human telomeres [see comments].
Science
282:1484-1487 |
| 43. |
Sprung, C. N.,
G. E. Reynolds,
M. Jasin, and J. P. Murnane.
1999.
Chromosome healing in mouse embryonic stem cells.
Proc. Natl. Acad. Sci. USA
96:6781-6786 |
| 44. | van Steensel, B., and T. de Lange. 1997. Control of telomere length by the human telomeric protein TRF1. Nature 385:740-743[CrossRef][Medline]. |
| 45. | van Steensel, B., A. Smogorzewska, and T. de Lange. 1998. TRF2 protects human telomeres from end-to-end fusions. Cell 92:401-413[CrossRef][Medline]. |
| 46. | Wang, H., and E. H. Blackburn. 1997. De novo telomere addition by Tetrahymena telomerase in vitro. EMBO J. 16:866-879[CrossRef][Medline]. |
| 47. | Wright, W. E., M. A. Piatyszek, W. R. Rainey, W. Byrd, and J. W. Shay. 1996. Telomerase activity in human germline and embryonic tissues and cells. Dev. Genet. 18:173-179[CrossRef][Medline]. |
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21: 206-220
[Abstract] [Full Text] -
Lin, X., Gu, J., Lu, C., Spitz, M. R., Wu, X.
(2006). Expression of telomere-associated genes as prognostic markers for overall survival in patients with non-small cell lung cancer.. Clin. Cancer Res.
12: 5720-5725
[Abstract] [Full Text] -
O'Connor, M. S., Safari, A., Xin, H., Liu, D., Songyang, Z.
(2006). A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly. Proc. Natl. Acad. Sci. USA
103: 11874-11879
[Abstract] [Full Text] -
Zhang, D.-H., Zhou, B., Huang, Y., Xu, L.-X., Zhou, J.-Q.
(2006). The human Pif1 helicase, a potential Escherichia coli RecD homologue, inhibits telomerase activity. Nucleic Acids Res
34: 1393-1404
[Abstract] [Full Text] -
Lee, T. H., Perrem, K., Harper, J. W., Lu, K. P., Zhou, X. Z.
(2006). The F-box Protein FBX4 Targets PIN2/TRF1 for Ubiquitin-mediated Degradation and Regulates Telomere Maintenance. J. Biol. Chem.
281: 759-768
[Abstract] [Full Text] -
Opresko, P. L., Mason, P. A., Podell, E. R., Lei, M., Hickson, I. D., Cech, T. R., Bohr, V. A.
(2005). POT1 Stimulates RecQ Helicases WRN and BLM to Unwind Telomeric DNA Substrates. J. Biol. Chem.
280: 32069-32080
[Abstract] [Full Text] -
Shakirov, E. V., Surovtseva, Y. V., Osbun, N., Shippen, D. E.
(2005). The Arabidopsis Pot1 and Pot2 Proteins Function in Telomere Length Homeostasis and Chromosome End Protection. Mol. Cell. Biol.
25: 7725-7733
[Abstract] [Full Text] -
Li, B., Espinal, A., Cross, G. A. M.
(2005). Trypanosome Telomeres Are Protected by a Homologue of Mammalian TRF2. Mol. Cell. Biol.
25: 5011-5021
[Abstract] [Full Text] -
Sabatier, L., Ricoul, M., Pottier, G., Murnane, J. P.
(2005). The Loss of a Single Telomere Can Result in Instability of Multiple Chromosomes in a Human Tumor Cell Line. Mol Cancer Res
3: 139-150
[Abstract] [Full Text] -
Opresko, P. L., Fan, J., Danzy, S., Wilson, D. M. III, Bohr, V. A.
(2005). Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2. Nucleic Acids Res
33: 1230-1239
[Abstract] [Full Text] -
Yang, Q., Zheng, Y.-L., Harris, C. C.
(2005). POT1 and TRF2 Cooperate To Maintain Telomeric Integrity. Mol. Cell. Biol.
25: 1070-1080
[Abstract] [Full Text] -
Moran-Jones, K., Wayman, L., Kennedy, D. D., Reddel, R. R., Sara, S., Snee, M. J., Smith, R.
(2005). hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere. Nucleic Acids Res
33: 486-496
[Abstract] [Full Text] -
Oh, B.-K., Kim, Y.-J., Park, C., Park, Y. N.
(2005). Up-Regulation of Telomere-Binding Proteins, TRF1, TRF2, and TIN2 Is Related to Telomere Shortening during Human Multistep Hepatocarcinogenesis. Am. J. Pathol.
166: 73-80
[Abstract] [Full Text] -
Levy, D. L., Blackburn, E. H.
(2004). Counting of Rif1p and Rif2p on Saccharomyces cerevisiae Telomeres Regulates Telomere Length. Mol. Cell. Biol.
24: 10857-10867
[Abstract] [Full Text] -
Liu, D., O'Connor, M. S., Qin, J., Songyang, Z.
(2004). Telosome, a Mammalian Telomere-associated Complex Formed by Multiple Telomeric Proteins. J. Biol. Chem.
279: 51338-51342
[Abstract] [Full Text] -
Yang, S. W., Kim, S. K., Kim, W. T.
(2004). Perturbation of NgTRF1 Expression Induces Apoptosis-Like Cell Death in Tobacco BY-2 Cells and Implicates NgTRF1 in the Control of Telomere Length and Stability. Plant Cell
16: 3370-3385
[Abstract] [Full Text] -
Karamysheva, Z. N., Surovtseva, Y. V., Vespa, L., Shakirov, E. V., Shippen, D. E.
(2004). A C-terminal Myb Extension Domain Defines a Novel Family of Double-strand Telomeric DNA-binding Proteins in Arabidopsis. J. Biol. Chem.
279: 47799-47807
[Abstract] [Full Text] -
Ye, J. Z.-S., Donigian, J. R., van Overbeek, M., Loayza, D., Luo, Y., Krutchinsky, A. N., Chait, B. T., de Lange, T.
(2004). TIN2 Binds TRF1 and TRF2 Simultaneously and Stabilizes the TRF2 Complex on Telomeres. J. Biol. Chem.
279: 47264-47271
[Abstract] [Full Text] -
Lillard-Wetherell, K., Machwe, A., Langland, G. T., Combs, K. A., Behbehani, G. K., Schonberg, S. A., German, J., Turchi, J. J., Orren, D. K., Groden, J.
(2004). Association and regulation of the BLM helicase by the telomere proteins TRF1 and TRF2. Hum Mol Genet
13: 1919-1932
[Abstract] [Full Text] -
Silverman, J., Takai, H., Buonomo, S. B.C., Eisenhaber, F., de Lange, T.
(2004). Human Rif1, ortholog of a yeast telomeric protein, is regulated by ATM and 53BP1 and functions in the S-phase checkpoint. Genes Dev.
18: 2108-2119
[Abstract] [Full Text] -
Jacob, N. K., Stout, A. R., Price, C. M.
(2004). Modulation of Telomere Length Dynamics by the Subtelomeric Region of Tetrahymena Telomeres. Mol. Biol. Cell
15: 3719-3728
[Abstract] [Full Text] -
Nikitina, T., Woodcock, C. L.
(2004). Closed chromatin loops at the ends of chromosomes. JCB
166: 161-165
[Abstract] [Full Text] -
Ye, J. Z.-S., Hockemeyer, D., Krutchinsky, A. N., Loayza, D., Hooper, S. M., Chait, B. T., de Lange, T.
(2004). POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex. Genes Dev.
18: 1649-1654
[Abstract] [Full Text] -
Blasco, M. A.
(2004). Telomere epigenetics: a higher-order control of telomere length in mammalian cells. Carcinogenesis
25: 1083-1087
[Abstract] [Full Text] -
Mattern, K. A., Swiggers, S. J. J., Nigg, A. L., Lowenberg, B., Houtsmuller, A. B., Zijlmans, J. M. J. M.
(2004). Dynamics of Protein Binding to Telomeres in Living Cells: Implications for Telomere Structure and Function. Mol. Cell. Biol.
24: 5587-5594
[Abstract] [Full Text] -
McMurray, H. R., McCance, D. J.
(2004). Degradation of p53, Not Telomerase Activation, by E6 Is Required for Bypass of Crisis and Immortalization by Human Papillomavirus Type 16 E6/E7. J. Virol.
78: 5698-5706
[Abstract] [Full Text] -
Myung, K., Ghosh, G., Fattah, F. J., Li, G., Kim, H., Dutia, A., Pak, E., Smith, S., Hendrickson, E. A.
(2004). Regulation of Telomere Length and Suppression of Genomic Instability in Human Somatic Cells by Ku86. Mol. Cell. Biol.
24: 5050-5059
[Abstract] [Full Text] -
Fotiadou, P., Henegariu, O., Sweasy, J. B.
(2004). DNA Polymerase {beta} Interacts with TRF2 and Induces Telomere Dysfunction in a Murine Mammary Cell Line. Cancer Res.
64: 3830-3837
[Abstract] [Full Text] -
Armbruster, B. N., Linardic, C. M., Veldman, T., Bansal, N. P., Downie, D. L., Counter, C. M.
(2004). Rescue of an hTERT Mutant Defective in Telomere Elongation by Fusion with hPot1. Mol. Cell. Biol.
24: 3552-3561
[Abstract] [Full Text] -
Loayza, D., Parsons, H., Donigian, J., Hoke, K., de Lange, T.
(2004). DNA Binding Features of Human POT1: A NONAMER 5'-TAGGGTTAG-3' MINIMAL BINDING SITE, SEQUENCE SPECIFICITY, AND INTERNAL BINDING TO MULTIMERIC SITES. J. Biol. Chem.
279: 13241-13248
[Abstract] [Full Text] -
Ohki, R., Ishikawa, F.
(2004). Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats. Nucleic Acids Res
32: 1627-1637
[Abstract] [Full Text] -
Yoshimura, S. H., Maruyama, H., Ishikawa, F., Ohki, R., Takeyasu, K.
(2004). Molecular mechanisms of DNA end-loop formation by TRF2. GENES CELLS
9: 205-218
[Abstract] [Full Text] -
Dantzer, F., Giraud-Panis, M.-J., Jaco, I., Ame, J.-C., Schultz, I., Blasco, M., Koering, C.-E., Gilson, E., Menissier-de Murcia, J., de Murcia, G., Schreiber, V.
(2004). Functional Interaction between Poly(ADP-Ribose) Polymerase 2 (PARP-2) and TRF2: PARP Activity Negatively Regulates TRF2. Mol. Cell. Biol.
24: 1595-1607
[Abstract] [Full Text] -
Iwano, T., Tachibana, M., Reth, M., Shinkai, Y.
(2004). Importance of TRF1 for Functional Telomere Structure. J. Biol. Chem.
279: 1442-1448
[Abstract] [Full Text] -
Xiong, J., Fan, S., Meng, Q., Schramm, L., Wang, C., Bouzahza, B., Zhou, J., Zafonte, B., Goldberg, I. D., Haddad, B. R., Pestell, R. G., Rosen, E. M.
(2003). BRCA1 Inhibition of Telomerase Activity in Cultured Cells. Mol. Cell. Biol.
23: 8668-8690
[Abstract] [Full Text] -
Li, B., de Lange, T.
(2003). Rap1 Affects the Length and Heterogeneity of Human Telomeres. Mol. Biol. Cell
14: 5060-5068
[Abstract] [Full Text] -
Deng, Z., Atanasiu, C., Burg, J. S., Broccoli, D., Lieberman, P. M.
(2003). Telomere Repeat Binding Factors TRF1, TRF2, and hRAP1 Modulate Replication of Epstein-Barr Virus OriP. J. Virol.
77: 11992-12001
[Abstract] [Full Text] -
Incles, C. M., Schultes, C. M., Kelland, L. R., Neidle, S.
(2003). Acquired Cellular Resistance to Flavopiridol in a Human Colon Carcinoma Cell Line Involves Up-Regulation of the Telomerase Catalytic Subunit and Telomere Elongation. Sensitivity of Resistant Cells to Combination Treatment with a Telomerase Inhibitor. Mol. Pharmacol.
64: 1101-1108
[Abstract] [Full Text] -
Igarashi, M., Suda, T., Hara, H., Takimoto, M., Nomoto, M., Takahashi, T., Okoshi, S., Kawai, H., Mita, Y., Waguri, N., Aoyagi, Y.
(2003). Interferon Can Block Telomere Erosion and in Rare Cases Result in Hepatocellular Carcinoma Development with Telomeric Repeat Binding Factor 1 Overexpression in Chronic Hepatitis C. Clin. Cancer Res.
9: 5264-5270
[Abstract] [Full Text] -
Marian, C. O., Bordoli, S. J., Goltz, M., Santarella, R. A., Jackson, L. P., Danilevskaya, O., Beckstette, M., Meeley, R., Bass, H. W.
(2003). The Maize Single myb histone 1 Gene, Smh1, Belongs to a Novel Gene Family and Encodes a Protein That Binds Telomere DNA Repeats in Vitro. Plant Physiol.
133: 1336-1350
[Abstract] [Full Text] -
Karlseder, J., Kachatrian, L., Takai, H., Mercer, K., Hingorani, S., Jacks, T., de Lange, T.
(2003). Targeted Deletion Reveals an Essential Function for the Telomere Length Regulator Trf1. Mol. Cell. Biol.
23: 6533-6541
[Abstract] [Full Text] -
Kanauchi, H., Wada, N., Ginzinger, D. G., Yu, M., Wong, M. G., Clark, O. H., Duh, Q.-Y.
(2003). Diagnostic and Prognostic Value of Fas and Telomeric-Repeat Binding Factor-1 Genes in Adrenal Tumors. J. Clin. Endocrinol. Metab.
88: 3690-3693
[Abstract] [Full Text] -
Yang, S. W., Kim, D. H., Lee, J. J., Chun, Y. J., Lee, J.-H., Kim, Y. J., Chung, I. K., Kim, W. T.
(2003). Expression of the Telomeric Repeat Binding Factor Gene NgTRF1 Is Closely Coordinated with the Cell Division Program in Tobacco BY-2 Suspension Culture Cells. J. Biol. Chem.
278: 21395-21407
[Abstract] [Full Text] -
Opresko, P. L., Cheng, W.-H., von Kobbe, C., Harrigan, J. A., Bohr, V. A.
(2003). Werner syndrome and the function of the Werner protein; what they can teach us about the molecular aging process.. Carcinogenesis
24: 791-802
[Abstract] [Full Text] -
Nakanishi, K., Kawai, T., Kumaki, F., Hiroi, S., Mukai, M., Ikeda, E., Koering, C. E., Gilson, E.
(2003). Expression of mRNAs for Telomeric Repeat Binding Factor (TRF)-1 and TRF2 in Atypical Adenomatous Hyperplasia and Adenocarcinoma of the Lung. Clin. Cancer Res.
9: 1105-1111
[Abstract] [Full Text] -
Gallego, M. E., Jalut, N., White, C. I.
(2003). Telomerase Dependence of Telomere Lengthening in ku80 Mutant Arabidopsis. Plant Cell
15: 782-789
[Abstract] [Full Text] -
Chai, W., Ford, L. P., Lenertz, L., Wright, W. E., Shay, J. W.
(2002). Human Ku70/80 Associates Physically with Telomerase through Interaction with hTERT. J. Biol. Chem.
277: 47242-47247
[Abstract] [Full Text] -
Baumann, P., Podell, E., Cech, T. R.
(2002). Human Pot1 (Protection of Telomeres) Protein: Cytolocalization, Gene Structure, and Alternative Splicing. Mol. Cell. Biol.
22: 8079-8087
[Abstract] [Full Text] -
Meeker, A. K., Hicks, J. L., Platz, E. A., March, G. E., Bennett, C. J., Delannoy, M. J., De Marzo, A. M.
(2002). Telomere Shortening Is an Early Somatic DNA Alteration in Human Prostate Tumorigenesis. Cancer Res.
62: 6405-6409
[Abstract] [Full Text] -
van Heek, N. T., Meeker, A. K., Kern, S. E., Yeo, C. J., Lillemoe, K. D., Cameron, J. L., Offerhaus, G. J. A., Hicks, J. L., Wilentz, R. E., Goggins, M. G., De Marzo, A. M., Hruban, R. H., Maitra, A.
(2002). Telomere Shortening Is Nearly Universal in Pancreatic Intraepithelial Neoplasia. Am. J. Pathol.
161: 1541-1547
[Abstract] [Full Text] -
Opresko, P. L., von Kobbe, C., Laine, J.-P., Harrigan, J., Hickson, I. D., Bohr, V. A.
(2002). Telomere-binding Protein TRF2 Binds to and Stimulates the Werner and Bloom Syndrome Helicases. J. Biol. Chem.
277: 41110-41119
[Abstract] [Full Text] -
Cui, W., Aslam, S., Fletcher, J., Wylie, D., Clinton, M., Clark, A. J.
(2002). Stabilization of Telomere Length and Karyotypic Stability Are Directly Correlated with the Level of hTERT Gene Expression in Primary Fibroblasts. J. Biol. Chem.
277: 38531-38539
[Abstract] [Full Text] -
Bundock, P., Hooykaas, P.
(2002). Severe Developmental Defects, Hypersensitivity to DNA-Damaging Agents, and Lengthened Telomeres in Arabidopsis MRE11 Mutants. Plant Cell
14: 2451-2462
[Abstract] [Full Text] -
Cong, Y.-S., Wright, W. E., Shay, J. W.
(2002). Human Telomerase and Its Regulation. Microbiol. Mol. Biol. Rev.
66: 407-425
[Abstract] [Full Text] -
Dimri, G. P., Martinez, J.-L., Jacobs, J. J. L., Keblusek, P., Itahana, K., van Lohuizen, M., Campisi, J., Wazer, D. E., Band, V.
(2002). The Bmi-1 Oncogene Induces Telomerase Activity and Immortalizes Human Mammary Epithelial Cells. Cancer Res.
62: 4736-4745
[Abstract] [Full Text] -
Bundock, P., van Attikum, H., Hooykaas, P.
(2002). Increased telomere length and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutant. Nucleic Acids Res
30: 3395-3400
[Abstract] [Full Text] -
Marcotte, R., Wang, E.
(2002). Replicative Senescence Revisited. Journals of Gerontology Series A: Biological Sciences and Medical Sciences
57: B257-269
[Abstract] [Full Text] -
Ancelin, K., Brunori, M., Bauwens, S., Koering, C.-E., Brun, C., Ricoul, M., Pommier, J.-P., Sabatier, L., Gilson, E.
(2002). Targeting Assay To Study the cis Functions of Human Telomeric Proteins: Evidence for Inhibition of Telomerase by TRF1 and for Activation of Telomere Degradation by TRF2. Mol. Cell. Biol.
22: 3474-3487
[Abstract] [Full Text] -
Savitsky, M., Kravchuk, O., Melnikova, L., Georgiev, P.
(2002). Heterochromatin Protein 1 Is Involved in Control of Telomere Elongation in Drosophila melanogaster. Mol. Cell. Biol.
22: 3204-3218
[Abstract] [Full Text] -
Mauro, L. J., Foster, D. N.
(2002). Regulators of Telomerase Activity. Am. J. Respir. Cell Mol. Bio.
26: 521-524
[Full Text] -
Karlseder, J., Smogorzewska, A., de Lange, T.
(2002). Senescence Induced by Altered Telomere State, Not Telomere Loss. Science
295: 2446-2449
[Abstract] [Full Text] -
Liu, Y., Kha, H., Ungrin, M., Robinson, M. O., Harrington, L.
(2002). Preferential maintenance of critically short telomeres in mammalian cells heterozygous for mTert. Proc. Natl. Acad. Sci. USA
99: 3597-3602
[Abstract] [Full Text] -
Mergny, J.-L., Riou, J.-F., Mailliet, P., Teulade-Fichou, M.-P., Gilson, E.
(2002). Natural and pharmacological regulation of telomerase. Nucleic Acids Res
30: 839-865
[Abstract] [Full Text] -
Cook, B. D., Dynek, J. N., Chang, W., Shostak, G., Smith, S.
(2002). Role for the Related Poly(ADP-Ribose) Polymerases Tankyrase 1 and 2 at Human Telomeres. Mol. Cell. Biol.
22: 332-342
[Abstract] [Full Text] -
Siriaco, G. M., Cenci, G., Haoudi, A., Champion, L. E., Zhou, C., Gatti, M., Mason, J. M.
(2002). Telomere elongation (Tel), a New Mutation in Drosophila melanogaster That Produces Long Telomeres. Genetics
160: 235-245
[Abstract] [Full Text] -
Verfaillie, C. M., Pera, M. F., Lansdorp, P. M.
(2002). Stem Cells: Hype and Reality. ASH Education Book
2002: 369-391
[Abstract] [Full Text] -
Grossi, S., Bianchi, A., Damay, P., Shore, D.
(2001). Telomere Formation by Rap1p Binding Site Arrays Reveals End-Specific Length Regulation Requirements and Active Telomeric Recombination. Mol. Cell. Biol.
21: 8117-8128
[Abstract] [Full Text] -
Bucholc, M., Park, Y., Lustig, A. J.
(2001). Intrachromatid Excision of Telomeric DNA as a Mechanism for Telomere Size Control in Saccharomyces cerevisiae. Mol. Cell. Biol.
21: 6559-6573
[Abstract] [Full Text]
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