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Telomerase-Dependent 39 G-Strand Overhang Maintenance
Facilitates GTBP1-Mediated Telomere Protection from
Misplaced Homologous Recombination
C W
Yong Woo Lee and Woo Taek Kim1
Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea
At the 39-end of telomeres, single-stranded G-overhang telomeric repeats form a stable T-loop. Many studies have focused
on the mechanisms that generate and regulate the length of telomere 39 G-strand overhangs, but the roles of G-strand
overhang length control in proper T-loop formation and end protection remain unclear. Here, we examined functional
relationships between the single-stranded telomere binding protein GTBP1 and G-strand overhang lengths maintained by
telomerase in tobacco (Nicotiana tabacum). In tobacco plants, telomerase reverse transcriptase subunit (TERT) repression
severely worsened the GTBP1 knockdown phenotypes, which were formally characterized as an outcome of telomere
destabilization. TERT downregulation shortened the telomere 39 G-overhangs and increased telomere recombinational
aberrations in GTBP1-suppressed plants. Correlatively, GTBP1-mediated inhibition of single-strand invasion into the doublestrand telomeric sequences was impaired due to shorter single-stranded telomeres. Moreover, TERT/GTBP1 double knockdown
amplified misplaced homologous recombination of G-strand overhangs into intertelomeric regions. Thus, proper G-overhang
length maintenance is required to protect telomeres against intertelomeric recombination, which is achieved by the balanced
functions of GTBP1 and telomerase activity.
INTRODUCTION
Telomeres, the ends of linear eukaryotic chromosomes, are
composed of double-stranded repeated DNA with terminal 39 Gstrand overhangs of a single-stranded repeated DNA sequence
(Blackburn, 1991; Griffith et al., 1999). Telomeric DNA is shielded
by numerous telomere-specific binding proteins, resulting in
functional nucleoprotein structures (de Lange, 2005). The functions of the binding proteins include telomere length regulation,
G-overhang processing, T-loop formation, and distinguishing
telomeres from DNA damage sites (Bailey et al., 2001; de Lange,
2002, 2005; Sfeir and de Lange, 2012). Several proteins that
have telomeric single-strand-specific binding affinity have been
studied, but only a few have been shown to be functionally
associated with telomeres. PROTECTION OF TELOMERES
PROTEIN1 (POT1) is the most thoroughly characterized singlestranded telomeric DNA binding protein. As a subunit of the
shelterin complex, POT1 participates in the protection of telomeric ends from DNA damage responses (Loayza and De
Lange, 2003; de Lange, 2005; Denchi and de Lange, 2007; Gong
and de Lange, 2010). Another single-stranded DNA binding
protein, human heterogeneous nuclear ribonucleoprotein A1
(hnRNPA1), regulates telomere length through an interaction with
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Woo Taek Kim (wtkim@
yonsei.ac.kr).
C
Some figures in this article are displayed in color online but in black and
white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.112.107573
telomerase (LaBranche et al., 1998). Association of hnRNPA1
prevents replication protein A (RPA) from binding to telomeres in
late S phase (Flynn et al., 2011). After S phase, POT1 replaces
hnRNPA1 and further inhibits RPA binding to 39 G-overhangs
under the regulation of telomeric repeat–containing RNA (TERRA).
TERRA competes with G-overhangs by binding to hnRNPA1,
removes human heterogeneous nuclear ribonucleoproteins from
G-overhangs, and enables POT1 to bind at 39 telomeric ends
(Flynn et al., 2011).
Most 39 G-overhang telomeric ends form protective T-loop
structures through strand invasion to upstream duplex telomeric
sequences (Griffith et al., 1999). Although the details are not yet
fully understood, G-overhang generation is regulated by cell
cycle–dependent mechanisms involving C-strand resection and
fill-in (Wellinger et al., 1993; Jacob et al., 2003; Larrivée et al.,
2004; Bonetti et al., 2009; Dai et al., 2010). In mammalian cells,
the lengths of 39 G-overhangs were affected by a number of
telomere-associated proteins, including telomerase (Stewart
et al., 2003; Masutomi et al., 2003; Hockemeyer et al., 2006;
Dimitrova and de Lange, 2009; Wu et al., 2010). In cultured
human cells, telomerase overexpression prevents G-strand erosion
during continuous rounds of cell division (Stewart et al., 2003),
and telomerase disruption causes instability of G-overhang
maintenance (Masutomi et al., 2003). On the other hand, G-overhang
generation is unchanged in telomerase-negative transgenic
mice (Hemann and Greider, 1999). This raised the possibility that
telomerase may not be a major factor for overhang processing;
rather, telomerase affects G-overhang stability. Decreases in
G-overhang length correlate with cellular senescence in normal
human cells (Stewart et al., 2003; Masutomi et al., 2003). However, it is still unclear how proper G-overhang length maintenance
contributes to telomere stability.
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Figure 1. TERT Repression Intensifies GTBP1-Knockdown Phenotypes in Tobacco.
(A) Schematic representation of hnRNPA1 and tobacco GTBP1. Amino acid sequence identities between hnRNPA1 and GTBP1 are indicated in each
domain. RRM1 and RRM2, RNA recognition motifs. aa, amino acid residue.
Telomere 39 G-Strand Overhang Protection
In this article, we examined functional relationships between
single-stranded telomere binding protein GTBP1, a tobacco
(Nicotiana tabacum) putative ortholog of hnRNPA1, and Gstrand overhang lengths maintained by telomerase in tobacco.
Our data indicate that telomerase reverse transcriptase subunit
(TERT) and GTBP1 double knockdown amplified misplaced homologous recombination (HR) of G-strand overhangs into telomeric regions, resulting in genomic instability. GTBP1 binding to
single-stranded telomeric ends was impaired due to shortening
of the G-overhangs, which explains why TERT repressioninduced G-overhang reduction increased telomeric HR in GTBP1
knockdown plants. Although plant development is generally
plastic in response to genome instability, G-overhang reduction
due to the TERT knockdown with an insufficient GTBP1 level
could trigger undesirable telomeric HR that leads to abnormal
growth arrest in transgenic tobacco plants. These results suggest that proper G-overhang length maintenance is required
to protect telomeres against aberrant intertelomeric recombination and to prevent developmental damage to the plants,
which is achieved by the balanced functions of the singlestranded telomere binding protein GTBP1 and telomerase
activity.
RESULTS
TERT Repression Intensified GTBP1-Knockdown
Phenotypes in Tobacco
GTBP1 is a tobacco putative ortholog of hnRNPA1 that plays
protective roles in telomere G-overhangs (Figure 1A). GTBP1suppressed RNA interference (RNAi) transgenic tobacco plants
(35S:RNAi-GTBP1) display aberrant telomere recombination
and genome instability (Lee and Kim, 2010). To examine the
functional correlation between GTBP1 and telomerase activity in
telomere stability, the 35S:RNAi-TERT construct was introduced
into the T0 35S:RNAi-GTBP1 transgenic line using Agrobacterium tumefaciens–mediated T-DNA delivery methods and
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telomerase/GTBP1 double-knockdown RNAi transgenic tobacco
plants (35S:RNAi-TERT/35S:RNAi-GTBP1) were subsequently
produced (Figure 1B; see Supplemental Figures 1A and 1B
online). Telomerase single knockdown (35S:RNAi-TERT) transgenic lines were also generated. Genomic DNA gel blot analysis
showed that the 35S:RNAi-TERT and 35S:RNAi-TERT/35S:RNAiGTBP1 transgenic plants used in this study are independent
lines (see Supplemental Figure 1C online). Real-time quantitative
RT-PCR (qRT-PCR) analysis demonstrated significant downregulation of TERT mRNA in 35S:RNAi-TERT and 35S:RNAiTERT/35S:RNAi-GTBP1 transgenic tobacco plants (Figure 1C,
left panel). Telomerase enzyme activities were also reduced in
RNAi knockdown transgenic callus relative to wild-type callus
determined by telomere repeat amplification (TRAP) assays
(Figure 1D). As reported previously (Fitzgerald et al., 1996; Riha
et al., 1998; Yang et al., 2002), telomerase activities were very
low in mature leaves of both wild-type and transgenic plants
(see Supplemental Figure 1D online). In addition, GTBP1 was
effectively suppressed in 35S:RNAi-GTBP1 and 35S:RNAiTERT/35S:RNAi-GTBP1 plants (Figure 1C, right panel). Transgenic 35S:RNAi-TERT T0 plants showed no visible phenotypic
defects compared with wild-type plants (Figure 1E, left panel;
see Supplemental Figure 1E online). This is consistent with
previous results that showed that the telomerase-deficient
Arabidopsis thaliana tert mutants were normal for up to five
generations (Riha et al., 2001). By contrast, T0 35S:RNAi-TERT/
35S:RNAi-GTBP1 plants exhibited severe phenotypic anomalies. The morphological abnormalities of the 35S:RNAi-TERT/
35S:RNAi-GTBP1 double-RNAi plants became progressively
more serious as the plants grew. The 2-month-old plants exhibited markedly retarded growth and premature senescence,
failed to develop normal reproductive organs, and died without
producing functional seeds (Figure 1E, middle panel). Furthermore, their leaves were smaller than wild-type leaves and were
unable to mature to full size (Figure 1F). Reduced internode
length was evident in 35S:RNAi-TERT/35S:RNAi-GTBP1 plants,
even though their leaf emerging rates were normal (Figure 1G).
The phenotypes of 35S:RNAi-GTBP1 single knockdown plants
Figure 1. (continued).
(B) Schematic structures of TERT and GTBP1 RNAi binary vector constructs. The 35S:RNAi-TERT vector includes the inverted-repeat sequence of two
different regions (660 to 984 bp and 984 to 1478 bp) of TERT cDNA (see Supplemental Figure 1B online). The 35S:RNAi-GTBP1 vector contains the
inverted-repeat sequence of the 726- to 1070-bp region of GTBP1 cDNA. LB, left border; OCS ter, octopine synthase terminator; NOS ter, nopaline
synthase terminator; NPTII, neomycin phosphotransferase II; HPTII, hygromycin phosphotransferase II; RB, right border.
(C) Suppression of TERT (left panel) and GTBP1 (right panel) mRNAs in transgenic tobacco plants. Total leaf RNA isolated from wild-type (WT), T0 35S:
RNAi-GTBP1, and four independent T0 35S:RNAi-TERT and 35S:RNAi-GTBP1/35S:RNAi-TERT transgenic lines was used for qRT-PCR. Expression
levels of TERT and GTBP1 were normalized to that of the EF1a gene. Error bars represent 6 SE from three independent experiments.
(D) Telomerase activities in wild-type and T0 35S:RNAi-TERT, 35S:RNAi-GTBP1, and 35S:RNAi-GTBP1/35S:RNAi-TERT transgenic tobacco plants.
The telomerase activities were examined in callus tissue by a TRAP assay.
(E) Morphology of GTBP1- and TERT-repressed transgenic tobacco plants. Representative 2-month-old (left and middle panels) and 3-month-old (right
panel) wild-type and T0 RNAi transgenic plants. All four independent 35S:RNAi-GTBP1/35S:RNAi-TERT transgenic lines died after 2 months, whereas
the 35S:RNAi-GTBP1 lines remain alive after 3 months. One-month-old RNAi transgenic plants that displayed mild phenotypic defects are shown in
Supplemental Figure 1E online.
(F) Morphological comparison of leaves from 2-month-old wild-type and RNAi transgenic plants. Genotypes are a combination of those indicated in the
row and column. Leaf positions are indicated at the bottom of the figure.
(G) Leaf number and stem lengths of 2-month-old wild-type and T0 RNAi-suppressed transgenic tobacco plants.
[See online article for color version of this figure.]
Figure 2. Reduction of 39 G-Overhang Lengths and Enhanced t-Circle Formation in TERT/GTBP1 Double-Knockdown Telomeres.
Telomere 39 G-Strand Overhang Protection
were intermediate between wild-type and 35S:RNAi-TERT/35S:
RNAi-GTBP1 plants (Figures 1E to 1G). Three-month-old 35S:
RNAi-GTBP1 plants survived (Figure 1E, right panel), but most
of their floral organs failed to produce seeds (Lee and Kim,
2010). Thus, suppression of TERT did not affect the growth of
wild-type plants but synergistically amplified abnormal phenotypes of GTBP1-suppressed plants, resulting in the early growth
arrest of the 35S:RNAi-TERT/35S:RNAi-GTBP1 plants.
Reduction of 39 G-Overhang Lengths and Enhanced t-Circle
Formation in TERT/GTBP1 Double-Knockdown Telomeres
Because 35S:RNAi-TERT/35S:RNAi-GTBP1 and most 35S:RNAiGTBP1 transgenic plants were sterile, T0 tobacco plants were
used for subsequent experiments. To address the telomere status
of the RNAi transgenic plants, restricted genomic DNA was subjected to in-gel hybridization with telomere probes following pulsefield gel electrophoresis. It was previously reported that denatured
pulse-field gel electrophoresis could be used to measure the
lengths of telomeres (>50 kb) in tobacco chromosomes (Fajkus
et al., 1995; Yang et al., 2004), while relatively shorter 39 G-overhang
single-stranded telomeric signals were effectively quantified by
native in-gel hybridization analysis (Heacock et al., 2007). In native
gels, the levels of 39 G-overhang signals in 35S:RNAi-TERT and
35S:RNAi-TERT/35S:RNAi-GTBP1 plants were reduced to 32 to
46% and 30 to 40%, respectively, compared with those in wildtype plants (Figures 2A, left panel, and 2B). The 39 G-overhang
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signal of 35S:RNAi-GTBP1 also decreased by ;25% relative to
the wild-type signal. This native gel signal was markedly reduced
following Exo I treatment determined by pulse-field (Figure 2C, left
panel) and standard agarose (Figure 2C, right panel) gel electrophoresis, confirming that the signal resulted from 39 G-overhang
single-stranded DNA. By contrast, total telomere lengths in all
RNAi plants examined were not significantly altered but mostly
remained within wild-type telomere length ranges (Figures 2A,
right panel, and 2C). Loss of TERT decreases telomere length by
;500 bp per generation in Arabidopsis (Riha et al., 1998). This
decrease in telomere length is relatively subtle in comparison with
long tobacco telomeres (15 to 50 kb); thus, it is still possible that
total telomere lengths in RNAi knockdown tobacco plants were
slightly changed, and this could be undetectable by our pulsefield gel electrophoresis system. Overall, decreases in TERT and
GTBP1 mRNA levels reduced 39 G-overhang single-stranded
telomere lengths, whereas double-stranded telomere lengths
were relatively constant in T0 RNAi transgenic plants.
HR in telomeres is typified by the formation of extrachromosomal telomeric circles (t-circles) (Wang et al., 2004; Wu et al.,
2006; Zellinger et al., 2007). Two-dimensional (2-D) pulse-field gel
electrophoresis indicated that t-circle formation in wild-type and
35S:RNAi-TERT plants was negligible (Figure 2D). By contrast,
telomeres from 35S:RNAi-TERT/35S:RNAi-GTBP1 plants showed
significantly enhanced t-circle formation. The degree of t-circle
formation in 35S:RNAi-GTBP1 plants was between those of 35S:
RNAi-TERT and 35S:RNAi-TERT/35S:RNAi-GTBP1 plants. These
Figure 2. (continued).
(A) Measurements of single-stranded 39 G-overhangs and double-stranded telomere signals in wild-type (WT), 35S:RNAi-TERT, 35S:RNAi-GTBP1, and
35S:RNAi-GTBP1/35S:RNAi-TERT transgenic lines using pulse-field gel electrophoresis followed by in-gel hybridization. TaqI-restricted leaf genomic
DNA from each transgenic plant was subjected to in-gel hybridization with a telomere (CCCTAAA)4 probe under native pulse-field gel conditions to
measure the single-stranded 39 G-overhang telomere signal (left panel). Restricted leaf genomic DNA was rehybridized under denatured pulse-field gel
conditions to measure double-stranded telomere signals (right panel).
(B) Single-stranded 39 G-overhang telomere signals in wild-type, 35S:RNAi-TERT, 35S:RNAi-GTBP1, and 35S:RNAi-GTBP1/35S:RNAi-TERT transgenic plants. Genomic DNA was purified and used for in-gel hybridization after native agarose gel electrophoresis. Quantified G-overhang signals were
normalized to the levels of denatured total telomere signals determined by a telomere repeat fragment assay.
(C) Pulse-field (left panel) and standard agarose (right panel) gel electrophoresis under native or denatured conditions followed by in-gel hybridization
experiments in the presence (+) or absence (2) of Exo I. Quantified G-overhang signals were normalized to the levels of denatured total telomere
signals.
(D) Extrachromosomal t-circle formation in wild-type and RNAi-downregulated plants. Restricted leaf genomic DNA isolated from wild-type, 35S:RNAiTERT, 35S:RNAi-GTBP1, and 35S:RNAi-GTBP1/35S:RNAi-TERT transgenic plants was subjected to 2-D pulse-field gel electrophoresis followed by ingel hybridization with a 32P-labeled (CCCTAAA)4 probe. Linear telomeric DNA and extrachromosomal t-circles are indicated by arrows and arrowheads,
respectively.
(E) The TCA assay. Restricted tobacco leaf genomic DNA was treated with exonuclease V to remove linear DNAs. The TCA reaction was performed with
enzyme-treated DNA mixtures and f29 DNA polymerase. The reaction mixtures were separated on 1% agarose gels and subjected to in-gel hybridization with a 32P-labeled (CCCTAAA)4 probe. Quantified t-circle signals were normalized to the levels of denatured total telomere signals determined by a telomere repeat fragment assay.
(F) Downregulation of TERT in cultured tobacco BY-2 suspension cells. BY-2 cells were transfected with the 35S:RNAi-TERT construct. The levels of
TERT mRNA and telomerase activity in wild-type and two independent transfected BY-2 cell lines were determined by qRT-PCR and TRAP assays,
respectively. Error bars represent 6 SE from three independent experiments.
(G) Double-stranded telomere lengths in vector control and RNAi-transfected BY-2 cells during three rounds of subculture. Genomic DNA was purified
and used for in-gel hybridization after denatured pulse-field gel electrophoresis.
(H) Single-stranded 39 G-overhang telomere signals in vector control and RNAi-transfected BY-2 cells during three rounds (1st, 2nd, and 3rd) of
subculture. Genomic DNA was purified and used for in-gel hybridization after native agarose gel electrophoresis with (+) or without (2) Exo I treatment.
Quantified G-overhang signals were normalized to the levels of denatured total telomere signals.
[See online article for color version of this figure.]
Figure 3. Strand Invasion and ChIP Assays.
Telomere 39 G-Strand Overhang Protection
results were further confirmed by the t-circle amplification (TCA)
assay as described by Zellinger et al. (2007) in connection with
exonuclease V. The results indicated that t-circle formation increased approximately two to threefold in 35S:RNAi-TERT/35S:
RNAi-GTBP1 lines compared with that in 35S:RNAi-TERT plants
(Figure 2E). Thus, t-circle generation correlates with the phenotypic
abnormalities of RNAi transgenic plants. Therefore, double knockdown of TERT and GTBP1 resulted not only in 39 G-overhang
shortening, but also in t-circle formation that may be due to aberrant HR within the telomeres. Both of these events, in turn, were
possibly associated with the impaired development observed in
35S:RNAi-TERT/35S:RNAi-GTBP1 tobacco plants.
In human fibroblasts, a lack of telomerase activity causes disruption of G-overhang maintenance during prolonged cell population growth (Masutomi et al., 2003). To further examine whether
decreased telomerase activity reduces 39 G-overhang singlestranded telomere lengths, cultured tobacco Bright Yellow-2 (BY-2)
suspension cells were transfected with the 35S:RNAi-TERT
construct. Transfected BY-2 cells have repressed TERT mRNA
levels that lead to decreased telomerase activity, as determined
by qRT-PCR and TRAP assays, respectively (Figure 2F). Total telomere length in 35S:RNAi-TERT BY-2 cells was largely unchanged
over three rounds of subculture (Figure 2G). However, G-overhang
signals from the same subculture of telomerase-repressed BY-2
cells were progressively reduced and reached ;70 to 80% and
40 to 60% of the G-overhang signals in wild-type cells after two
and three rounds of subculture, respectively (Figure 2H). These
results are consistent with the notion that telomerase activity is
responsible for 39 G-overhang maintenance. Although telomerase
activity seems to be the most likely explanation, it is still possible
that TERT has a nonenzymatic role in G-overhang maintenance.
GTBP1 Binding to Single-Stranded Telomeric Ends Was
Reduced Due to Shortening of the G-Overhangs That
Resulted from TERT Downregulation
Invasion of a 39 G-overhang single-stranded telomeric sequence into double-stranded telomeric DNA is a prerequisite
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for interchromosomal telomeric HR (Cesare and Reddel, 2010).
The increased telomere recombination rates in TERT/GTBP1suppressed transgenic plants prompted us to investigate the
relationship between G-overhang length and telomere recombination. Gel retardation assays revealed that binding of bacterially
expressed maltose binding protein (MBP)-GTBP1 fusion protein to
telomere single-stranded probes progressively increases between
(TTTAGGG)3 and (TTTAGGG)8 repeats (Figure 3A). Consequently,
MBP-GTBP1 more effectively inhibited the invasion of singlestranded DNA into double-stranded DNA in a length-dependent
manner (Figure 3B). Approximately 40% of the single-strand invasion was inhibited by a saturating amount of GTBP1 with
(TTTAGGG)5 repeats, whereas >75% of the invasion was inhibited
with (TTTAGGG)8 repeats. Intriguingly, C-terminal deletion mutants
of GTBP1 (GTBP1DC11-194 and GTBP1DC21-179) bound equally
well to the different lengths of single-stranded telomere repeats
(Figure 3C). This suggests that the C-terminal region of GTBP1 is
responsible for the length-dependent interactions between GTBP1
and single-stranded telomere repeats. These results are in agreement with those in Figure 2; namely, that double-suppression
of TERT and GTBP1 caused shortening of the 39 G-overhang
length accompanied by a reduction in cellular GTBP1 levels,
which resulted in aberrant telomeric HR and abnormal growth
arrest in tobacco plants. On the other hand, MBP alone failed
to interact with all of the different lengths of single-stranded
telomere repeats examined (see Supplemental Figure 2 online).
This view was further supported by the results of chromatin
immunoprecipitation (ChIP) assays. The 35S:HA-GTBP1 construct was transfected into wild-type or 35S:RNAi-TERT BY-2
suspension cells. The nucleoprotein complexes from the transfected cells were immunoprecipitated using anti-HA antibody.
The coimmunoprecipitated DNA was visualized by hybridization
with a 32P-labeled (TTTAGGG)70 probe. Figure 3D shows that
telomeric DNA was pulled down by the anti-HA antibody in 35S:
HA-GTBP1 BY-2 cells. However, the telomere-specific ChIP
signal was reduced to 58 to 66% in 35S:HA-GTBP1/35S:RNAiTERT BY-2 cells, indicating that GTBP1 binding to single-
Figure 3. (continued).
(A) Schematic representation of GTBP1 and binding activities of GTBP1 to single-stranded telomere sequences. Different concentrations (0, 75, 150,
and 300 nM) of bacterially expressed MBP-GTBP1 recombinant protein were subjected to gel mobility shift assays with radiolabeled, single-stranded
(TTTAGGG)3, (TTTAGGG)4, (TTTAGGG)5, (TTTAGGG)6, or (TTTAGGG)8 telomeric repeats. The “-” lanes contain single-stranded probes only. The relative
binding activity was determined by the shifted band intensity. Asterisk indicates 32P-labeled 59 nucleotide end. aa, amino acids.
(B) Strand invasion assay with different single-stranded telomeric repeats. GTBP1 (0, 75, 150, and 300 nM) was incubated with various 32P-labeled
single-stranded (TTTAGGG)n repeat probes (n = 3, 4, 5, 6, or 8) with T-vector plasmid containing a double-stranded (TTTAGGG)70 telomere repeat. The
relative level of invasion of the single-stranded telomeric probe into the plasmid was determined by the shifted band intensity. Asterisk indicates 32Plabeled 59 nucleotide end.
(C) Binding activities of C-terminal deletion mutants of GTBP1 to single-stranded telomere sequences. MBP-GTBP1△C11-194 and MBP-GTBP1△C21-179
mutant proteins were subjected to gel mobility shift assays as described in (A). The relative binding activity was determined by the shifted band
intensity. Asterisk indicates 32P-labeled 59 nucleotide end.
(D) Telomere ChIP assay. The genomic DNA-protein complexes from wild-type (WT), 35S:HA-GTBP1, and 35S:HA-GTBP1/35S:RNAi-TERT BY-2 cells
were fragmented by sonication and subjected to immunoprecipitation (IP) with an anti-HA antibody. The coimmunoprecipitated DNA was hybridized
with 32P-labeled (TTTAGGG)70 or HRS60 repeated tobacco DNA sequences. The “-” lane indicates a negative control without anti-HA antibody.
Quantified immunoprecipitation signals were normalized to 5% input signals. This experiment was independently repeated five times. Error bars
represent 6 SE.
[See online article for color version of this figure.]
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The Plant Cell
Figure 4. TERT/GTBP1 Double Knockdown Amplified Aberrant Intertelomeric HR.
Telomere 39 G-Strand Overhang Protection
stranded telomeric ends was significantly reduced due to
shortening of the G-overhangs that resulted from TERT downregulation. Therefore, the levels of telomerase activity and GTBP1
are critically associated with 39 G-overhang maintenance and
telomere stability in tobacco.
TERT/GTBP1 Double Knockdown Amplified Aberrant
Intertelomeric HR
To investigate 39 G-overhang-mediated telomeric HR due to the
downregulation of GTBP1 and TERT, we employed a DNA-tag
intertelomeric integration assay (Dunham et al., 2000). Wild-type
tobacco plants were transformed with a chimeric construct
(telomere:DNA-tag) that contains a DNA-tag positioned next to
the 39 end of the telomere repeats (490 bp) without a functional
promoter (Figure 4A). Chromosomal integration of the telomererepeat:DNA-tag construct was detected by in situ PCR followed
by fluorescence in situ hybridization (FISH). Three copies of the
construct were nonspecifically inserted into the chromosomes
as evidenced by randomly distributed FISH signals in Telomere:
DNA-tag #17 nuclei (Figure 4B, left panel) and genomic DNA
gel blotting with a DNA-tag-specific probe (see Supplemental
Figure 3 online). These DNA-tag insertion spots were located
independently of the internal telomere spots. Subsequently, the
35S:RNAi-TERT construct was transformed into Telomere:DNAtag #17 transgenic plants (see Supplemental Figures 4A and 4D
online). In Telomere:DNA-tag/35S:RNAi-TERT nuclei, the DNAtag insertion spots did not overlap with the telomere signals
(Figure 4B, middle and right panels), indicating that downregulation of TERT alone did not cause telomeric HR.
Next, the 35S:RNAi-GTBP1 construct was transformed into
Telomere:DNA-tag plants (see Supplemental Figures 4B and 4E
online). FISH analysis detected multicopy integrations of the
telomere-repeat:DNA-tag construct in the Telomere:DNA-tag/
35S:RNAi-GTBP1 chromosomes (Figure 4C). Moreover, localization of the DNA-tag significantly overlapped the telomeres in
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these chromosomes. More than 75% of the Telomere:DNA-tag/
35S:RNAi-GTBP1 nuclei displayed overlapping signals between
the DNA-tag and the telomeres. Approximately 25% of the nuclei
showed at least three overlapping DNA-tag spots merged with telomeres. Finally, the 35S:RNAi-TERT construct was transformed
into Telomere:DNA-tag/35S:RNAi-GTBP1 plants (see Supplemental
Figures 4C to 4E online). The Telomere:DNA-tag/35S:RNAi-GTBP1/
35S:RNAi-TERT nuclei contained even more overlapping signals
between the DNA-tag and the telomeres. Specifically, 62 to 76%
of the nuclei contained more than three DNA-tag spots integrated
into telomere regions (Figure 4C). These results indicate that
downregulation of GTBP1 increased telomeric HR, and GTBP1/
TERT double knockdown induced even more aberrant telomeric
HR, which closely correlated with the degree of phenotypic
anomalies of these RNAi transgenic plants. Figures 4B and 4C
also show that Telomere:DNA-tag/35S:RNAi-GTBP1 and Telomere:DNA-tag/35S:RNAi-GTBP1/35S:RNAi-TERT nuclei contained increased copy numbers of the telomere-repeat:DNA-tag
in their chromosomes compared with the Telomere:DNA-tag
and Telomere:DNA-tag/35S:RNAi-TERT plants. Thus, the DNAtag construct was amplified by intertelomeric HR (Figure 4D).
This notion was confirmed by genomic PCR that indicated that
downregulation of GTBP1 and TERT, but not of TERT alone,
increased the amount of DNA-tag in Telomere:DNA-tag transgenic plants (Figure 4E). Overall, these results argue that double
suppression of GTBP1 and TERT results in the deregulation of 39
G-overhang maintenance, which induces G-overhang-mediated
intertelomeric HR and anomalous growth arrest in tobacco
plants.
DISCUSSION
In this study, RNAi-mediated downregulation of TERT significantly reduced single-stranded telomere 39 G-overhang length
in transgenic tobacco plants (Figures 2A and 2B). However, 35S:
Figure 4. (continued).
(A) Schematic representation of the telomere (TTTAGGG)70:DNA-tag construct. LB, left border; RB, right border; BAR, herbicide Basta (glufosinate
ammonium) resistant gene.
(B) DNA-tag intertelomeric integration assay. The telomere-repeat:DNA-tag construct was transformed into tobacco plants. Chromosomal integration
of the construct was detected by in situ PCR followed by FISH in Telomere:DNA-tag #17 and two independent Telomere:DNA-tag/35S:RNAi-TERT
(lines a and b) transgenic chromosomes. The DNA-tag signal was amplified by in situ PCR with DNA-tag-specific primers. Chromosomal DNA was
denatured and incubated with an Alexa Fluor 488–labeled DNA-tag-specific probe and a Texas red-dUTP–incorporated (TTTAGGG)70 telomeric probe.
The chromosomes were counterstained with 49,6-diamidino-2-phenylindole (DAPI) and observed using fluorescence microscopy. The green signals
indicate the DNA-tag sequence, whereas red signals indicate internal telomere sequences. The DNA-tag sequences merged with telomere sequences
were counted. At least 50 nuclei from each T0 transgenic plant were observed. Bars = 5 mm.
(C) DNA-tag intertelomeric integration assays were conducted with Telomere:DNA-tag/35S:RNAi-GTBP1 and two independent Telomere:DNA-tag/
35S:RNAi-GTBP1/35S:RNAi-TERT (lines a and b) transgenic chromosomes. At least 50 nuclei from independent T0 transgenic plants were observed.
Arrowheads indicate the DNA-tag sequences that overlap with telomeric signals. Bars = 5 mm.
(D) Schematic representation of possible HR between the telomere (TTTAGGG)70:DNA-tag and internal telomere sequences in Telomere:DNA-tag/35S:
RNAi-GTBP1/35S:RNAi-TERT transgenic chromosomes. Red bars indicate telomere repeat sequences, and green bars indicate a DNA-tag sequence.
As a result of HR, chromosomal copy numbers of telomere:DNA-tag increased in the Telomere:DNA-tag/35S:RNAi-GTBP1/35S:RNAi-TERT chromosomes.
(E) Genomic PCR analysis. Leaf genomic DNA was isolated from Telomere:DNA-tag #17, Telomere:DNA-tag/35S:RNAi-TERT (independent lines a and
b), Telomere:DNA-tag/35S:RNAi-GTBP1, and Telomere:DNA-tag/35S:RNAi-GTBP1/35S:RNAi-TERT (independent lines a and b) transgenic plants.
DNA was analyzed by PCR using DNA-tag-specific primers. BAR is a loading control.
[See online article for color version of this figure.]
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Figure 5. A Working Model of Possible Roles of GTBP1 and Telomerase Activity against Aberrant Telomeric Recombination
GTBP1 binds to single-stranded telomere 39 G-overhang ends and plays a role in inhibiting abnormal telomeric HR. When GTBP1 was repressed,
aberrant telomere recombination occurred and telomere stability was partially disrupted. TERT/GTBP1 double knockdown amplified misplaced HR
of G-strand overhangs into telomeric regions, which is associated with genome instability and early senescence of tobacco plants. A reduction of
G-overhang length, in conjunction with GTBP1 repression, caused G-overhang uncapping from GTBP1-mediated telomere protection, due to the
G-overhang length-dependent binding of GTBP1. Open circles indicate GTBP1.
RNAi-TERT transgenic lines were phenotypically normal (Figure
1E), and their double-stranded telomeric length remained largely
unchanged with undetectable t-circles (Figures 2A and 2D). Our
findings agree with the normal phenotypes of Arabidopsis tert
knockout mutants after up to five generations, which contain no
detectable t-circle formation (Riha et al., 2001; Zellinger et al.,
2007). By contrast, telomerase downregulation, when combined
with repression of the hnRNPA1 homolog GTBP1, resulted in
growth arrest and early senescence (Figures 1E and 1F). The
35S:RNAi-GTBP1/35S:RNAi-TERT plants experienced severe
genomic instability, as frequent t-circle formation and intertelomeric HR were evident in their telomeres. GTBP1/TERT double
knockdown resulted in the telomere-repeat:DNA-tag construct
being integrated into the telomere regions by HR, so that the
copy number of the DNA-tag significantly increased (Figure 4).
Therefore, telomerase downregulation caused G-overhang reduction and subsequently decreased GTBP1 binding to singlestranded telomeres (Figure 3D). The decrease in GTBP1 binding
to 39 G-overhang telomeric ends induced aberrant G-overhangmediated telomeric HR and telomere dysfunction (Figure 5).
It was proposed that telomerase-independent telomere
lengthening (i.e., alternative lengthening of telomeres [ALT]) was
mediated by HR-dependent DNA replication at the 39 telomeric
ends in human ALT cancer cells (Cesare and Reddel, 2010). The
35S:RNAi-GTBP1/35S:RNAi-TERT plants resembled ALT cancer cells in the context of HR-dependent DNA replication, as
shown by their increase in telomere-repeat:DNA-tag integration
into telomeres (Figures 4D and 4E). There are a number of
proteins that affect telomere HR and ALT. Components of the
shelterin complex and other telomere-associated proteins, including
POT1, TRF2, CTC1, and Ku, are involved in the regulation of HR
and ALT processes in telomeres (Wang et al., 2004; Wu et al.,
2006; Cesare and Reddel, 2010). Inappropriate 39 G-overhang
single-strand invasion causes t-circle formation and HR-dependent
DNA replication, which lead to telomere destabilization and entry
into senescence (Lustig, 2003; Wang et al., 2004). Therefore,
there must be a mechanism to repress inappropriate 39 G-overhang
single-strand invasion in normal cells. Recently, it was reported
that TERT along with Ku80 inhibits an HR mechanism of ALT in
Arabidopsis (Kazda et al., 2012). Thus, the synergistic increase
in telomeric HR observed in the GTBP1/TERT doubleknockdown plants may reflect the loss of HR inhibition via GTBP1
coupled with an increased likelihood of telomere HR due to
the loss of TERT. The single tert knockout alone may not induce telomere HR instability or ALT engagement in Arabidopsis
(Zellinger et al., 2007), which suggests that the TERT-mediated
inhibition of HR may be limited to specific conditions, such as
ku80 knockout. TERT downregulation reduces GTBP1 binding
to single-stranded telomeric ends under the GTBP1 knockdown
conditions, which suggests that inhibitory roles of GTBP1 in
aberrant telomeric HR are intimately correlated with the roles of
TERT.
Binding activity of GTBP1 to telomere single-stranded probes
was affected by single-strand probe length (Figure 3A). This
result shows that TERT repression-induced G-overhang reduction increased telomeric HR in GTBP1 knockdown plants.
When the C-terminal region (from 194 to 345 amino acid residues) was removed, GTBP1 bound shorter telomeric repeats
with similar efficiency compared with longer repeats (Figure 3C).
Therefore, the length-dependent binding of GTBP1 to telomeric
Telomere 39 G-Strand Overhang Protection
sequences is not only due to redundancy of the binding site, but
also to the GTBP1-specific characteristics of the C-terminal
region. Since human POT1 binds two or five TTAGGG singlestrand repeats equally well (Loayza et al., 2004), the function of
POT1 may not be inhibited by single-strand length reduction.
Despite the conserved 39 G-overhang structure, POT1 homologs of Brassicaceae plant species have no detectable binding
activity to single-stranded telomeric repeats (Shakirov et al.,
2009), which suggests that POT1 may not be a major singlestrand telomere binding protein in plants. Besides POT1, the
CST (CTC1-STN1-TEN1) complex in human also binds to the
G-overhang with sequence specificity (Miyake et al., 2009; Chen
et al., 2012). Similar to POT1, CST binds with comparable affinity
to longer than three TTAGGG G-strand arrays (Chen et al.,
2012). Therefore, except for the case of GTBP1, G-overhang
length does not appear to significantly affect the binding of
known telomere single-strand binding proteins when the singlestrand length is longer than the minimal binding site.
Telomere G-overhang lengths are tightly and dynamically
regulated. In mammalian cells, G-overhang length increases in
late S/G2 by the process of C-strand resection and fill-in (Dai
et al., 2010). Throughout the cell cycle, G-overhang formation
steps are regulated by the combinatorial action of Apollo,
POT1b, the CST complex, and the 59 exonuclease (Wu et al.,
2012). Occasionally, mutations of telomere-related genes resulted in G-overhang abnormalities. CTC1, one of the CST
complex components, regulates telomere G-overhangs in human and Arabidopsis (Surovtseva et al., 2009). Depletion of
CTC1 causes increased G-overhangs, recombination, and endto-end fusions. Interestingly, ku70 knockout, which increases
G-overhangs, triggers abnormal telomere HR in Arabidopsis
(Zellinger et al., 2007). Indeed, nucleolytic resection of 59 chromosome ends by EXO1 promotes telomere recombination in
ku80 knockout Arabidopsis (Kazda et al., 2012). On the other
hand, our results show that reduction of G-overhang lengths
promoted telomeric HR when GTBP1 level was insufficient. One
possibility is that the length of the 39 G-overhang and cellular
level of GTBP1 both contribute to inhibition of abnormal telomere HR. Overall, these results implicate that proper processing
of G-overhang is important to regulate telomeric HR stability and
thus legitimate T-loop formation.
Telomere attrition during population growth in telomerasenegative cells causes cellular senescence, which provides the
current telomere-directed aging model (Blackburn, 1991). To
decipher the aging process at the molecular level, numerous
telomere/telomerase-associated proteins have been studied
with regard to telomerase regulation. hnRNPA1 was initially proposed as one of the positive telomere length regulators through
an interaction with telomerase (LaBranche et al., 1998). In higher
plants, the relationship between hnRNPA1 orthologs and telomerase was studied. Arabidopsis STEP1, which contains a homologous DNA/RNA binding domain of hnRNPA1, inhibited in
vitro telomerase activity, but its in vivo functions were not elucidated (Kwon and Chung, 2004). Previously, we reported that
GTBP1 inhibited aberrant HR in tobacco telomeres (Lee and
Kim, 2010). In GTBP1 RNAi knockdown plants that displayed
unusual HR, telomerase activity was not significantly altered,
suggesting that a reduction in GTBP1 level did not directly
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decrease telomerase activity. 35S:RNAi-GTBP1/35S:RNAi-TERT
double-knockdown tobacco plants exhibited higher levels of HR
compared with those of 35S:RNAi-GTBP1 plants, suggesting that
TERT is critical for GTBP1-mediated telomere HR stability.
However, hnRNPA1/GTBP1 is a highly abundant protein that has
been shown to affect many aspects of telomere biology (Ford
et al., 2002; He and Smith, 2009). Therefore, we cannot rule out
the possibility that anomalous phenotypes of 35S:RNAi-GTBP1/
35S:RNAi-TERT plants resulted from the loss of other as yet
unidentified GTBP1-related telomere/telomerase functions. One
could argue that telomerase suppression could inhibit telomere
length reconstitution after t-circle excision, which resulted in inefficient healing of short telomeres arising from circle excision and
thus increased severity of 35S:RNAi-GTBP1/35S:RNAi-TERT
plant phenotypes. However, 35S:RNAi-GTBP1/35S:RNAi-TERT
plants contained similar telomere lengths relative to wild-type
plants (Figure 2A).
RPA plays an important role in ataxia telangiectasia and Rad3related checkpoint activation, which triggers HR-mediated repair
of damaged DNA (Jackson and Bartek, 2009). hnRNA1 inhibits
RPA binding to telomeres and distinguishes telomeres from DNA
damage sites (Flynn et al., 2011), thus possibly preventing telomeric HR. These findings are collaterally supportive of our view on
the protective role of GTBP1 against aberrant telomeric HR. Although plant development is generally plastic in response to genome instability, G-overhang reduction with an insufficient GTBP1
level could trigger undesirable telomeric HR that leads to immediate genomic and developmental damage to the plants (Figure
5). In conclusion, our data indicate that telomerase-dependent 39
G-strand overhang maintenance facilitates GTBP1-mediated telomere protection from misplaced HR in tobacco. Therefore, adequate
cellular levels of both telomerase activity and GTBP1 are essential
for telomere stability and function.
METHODS
Plant Materials
Tobacco (Nicotiana tabacum cv Samsun NN) seeds were germinated on
0.8% agar for 7 d on Murashige and Skoog medium in a growth chamber.
Seedlings were transferred to soil and further grown in a growth chamber
under a 16-h-light/8-h-dark photoperiod at 25°C. Tobacco BY-2 suspension cells were cultured in Murashige and Skoog medium on a rotary
shaker (150 rpm) at 25°C in the dark. Stationary-phase cells (5 mL) were
transferred on the seventh day to 45 mL of fresh medium and cultured.
RNA Extraction and qRT-PCR
To obtain partial TERT cDNA, degenerate oligonucleotides corresponding to
the amino acid sequence DVFKAFD for the upstream primer and AMKFHCY
for the downstream primer were synthesized (see Supplemental Table 1
online) and used for RT-PCR. These amino acid sequences are highly
conserved in Arabidopsis and rice (Oryza sativa) TERTs (see Supplemental
Figure 1A online). To obtain a longer TERT cDNA clone, 39 rapid amplification
of cDNA ends was performed as described (Lee and Kim, 2010). The deduced amino acid sequence of the partial TERT is identical to that of
a recently isolated tobacco TERT (Sýkorová et al., 2012). Total RNA was
extracted from mature tobacco leaves and BY-2 cells as described previously (Yang et al., 2004). RNA (2 mg) was used for first-strand cDNA
synthesis with MMLV reverse transcriptase (Promega). RT-PCR was performed
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with gene-specific primers (see Supplemental Table 1 online) and Ex-Taq
polymerase (Takara). cDNA amplification was performed with cycles of 30 s
at 95°C, 30 s at 55°C, and 1 min at 72°C, with 22 cycles for EF1a, 25 cycles
for GTBP1, and 28 cycles for TERT using an automatic thermal cycler
(Perkin-Elmer/Cetus). PCR products were separated by electrophoresis in
1.0% agarose gels containing ethidium bromide and visualized under UV
light. Real time qRT-PCR was performed as described by Cho et al.
(2011). An IQ5 light cycler (Bio-Rad) with SYBR Premix Ex Taq II (Takara)
was used in this study. qRT-PCR data were analyzed with Genex_
Macro_IQ5_conversion_Template and Genex software (Bio-Rad).
Levels of TERT and GTBP1 mRNAs were normalized with those of EF1a
mRNA.
Generation of Transgenic Tobacco Plants
For the construction of the 35S:RNAi-TERT transgenic plants, TERT
cDNA encompassing 325 bp (from 660 to 984 bp) or 495 bp (from 984 to
1478 bp) (see Supplemental Figure 1B online) was inserted in an inverted
orientation into the pCAMBIA vector (CAMBIA) with a 35S promoter and
PDK intron. The telomere-repeat:DNA-tag construct was produced using
the pEarleygate 301 vector by inserting 490 bp of a telomere repeat
(TTTAGGG)70 and partial NgTRF1 cDNA (Yang et al., 2004) into the
corresponding sites. The constructed vectors were transformed into
Agrobacterium tumefaciens strain LBA4404 by electroporation. Tobacco
leaf discs were cocultivated with Agrobacterium as described previously
(Lee and Kim, 2010). Transgenic tobacco plants were generated on
growth medium containing 1 mg/L Basta for Telomere:DNA-tag and
25 mg/L hygromycin for 35S:RNAi-TERT. The 35S:RNAi-GTBP1 plants
were constructed as described previously (Lee and Kim, 2010). The regenerated T0 plants were grown in a growth room under a 16-h-light/8-h-dark
photoperiod at 25°C.
TRAP Assay, Telomere Repeat Fragment Analysis, and 2-D
Pulse-Field Gel Electrophoresis
Telomerase activity was measured using TRAP assays as described
(Yang et al., 2004). For telomere repeat fragment analysis, leaf genomic
DNA (5 mg) was digested with TaqI restriction enzyme and separated on
a CHEF-DR III pulsed-field electrophoresis system (Bio-Rad). Agarose
gels were dried at room temperature and hybridized with a 32P-labeled
(CCCTAAA)4 probe under native conditions (45°C). After autoradiography,
the DNA in the dried agarose gels was denatured with 1.5 M NaCl and
0.5 M NaOH, neutralized with 1.0 M Tris, pH 7.3, and 0.5 M NaOH, and
rehybridized with the telomere probe under denaturing conditions (60°C).
The blots were visualized using a Bio-Imaging Analyzer BAS 2000 (Fuji).
TaqI-restricted DNA was treated with 20 units of Exo I (New England
Biolabs) at 37°C overnight and subjected to telomere repeat fragment
assays. For 2-D pulse-field gel electrophoresis analysis, TaqI-digested
DNA was separated in 0.5% agarose gels at 1 V/cm for 25.5 h at an angle of
120° with switching times ramped from 1 to 6 s at 14°C. Second-dimension
electrophoresis was conducted in 1.1% agarose gels at 6 V/cm for 8.5 h with
the same switch time conditions as described by Hong et al. (2010).
The TCA Assay
The amount of telomeric circles was measured by the TCA assay as described by Zellinger et al. (2007) with minor modifications. TaqI-restricted
tobacco leaf genomic DNA (0.5 mg) was treated with 20 units of exonuclease
V (New England Biolabs) at 37°C for 2 h to remove linear DNAs. Enzymetreated genomic DNA mixtures were resuspended in an annealing buffer
(20 mM Tris, pH 7.5, 20 mM KCl, and 0.1 mM EDTA) with 1 mM (TTTAGGG)3
primer, denatured at 96°C for 5 min, cooled down to 25°C for 30 min, and
then subjected to the TCA assay. The TCA reaction was performed with 10
units of f29 DNA polymerase (MBI Fermentas) at 30°C for 12 h. The reaction
mixtures were separated on 1% agarose gels in 0.53 TBE (44.5 mM Tris,
44.5 mM boric acid, and 1 mM EDTA) at 85 V at room temperature. Gels were
dried and hybridized with a 32P-labeled (CCCTAAA)4 probe.
Gel Retardation and Strand Invasion Assays
Escherichia coli–expressed MBP-fused GTBP1 was purified by affinity
chromatography using amylose resin (New England Biolabs). Different
concentrations (0, 75, 150, and 300 nM) of purified GTBP1 were incubated
with telomere repeat oligomers [(TTTAGGG)3, (TTTAGGG)4, (TTTAGGG)5,
(TTTAGGG)6, or (TTTAGGG)8] for 30 min. Protein-oligomer mixtures were
subjected to 7% acrylamide gel electrophoresis. For strand invasion
assays, pGEM-T Easy plasmid (Promega) containing double-stranded
(TTTAGGG)70 telomeric repeats was incubated with 32P-labled telomere
oligomers according to Amiard et al. (2007). Labeled telomere oligomers
were premixed with GTBP1 in invasion buffer (50 mM HEPES, pH 8.0, 0.1
mg/mL BSA, 1 mM DTT, 100 mM NaCl, and 2% [v/v] glycerol) for 15 min.
After incubation, invasion reactions were stopped by the addition of stop
buffer [10% (w/v) SDS, 6 mg proteinase K, and 25 ng (CCCTAAA)4].
Samples were separated on 1% agarose gels in 0.53 TBE at 85 V at room
temperature. Gels were dried and subjected to autoradiography. The efficiency of invasion of the single-stranded telomeric probes into (TTTAGGG)70
telomeric repeats was determined by the shifted band intensity.
ChIP Assays
ChIP assays were performed as described by Lee and Kim (2010) with
slight modifications. Wild-type, 35S:HA-GTBP1, and 35S:HA-GTBP1/
35S:RNAi-TERT transgenic BY-2 cells were treated with a 1% formaldehyde solution for 15 min to cross-link the nucleoprotein complexes.
After quenching the cross-link with 150 mM Gly, chromosomes were
fragmented by sonication to 0.2- to 1.0-kb fragments in ChIP dilution
buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, and
167 mM NaCl). Sonicated cell lysates were incubated with or without
(negative control) 1:100 diluted anti-HA antibody (New England Biolabs).
Antibody-bound nucleoproteins were collected using protein G agarose
beads (Santa Cruz Biotechnology). Eluted DNA was dot blotted onto
Hybond-N nylon membranes (Amersham) and hybridized with 32P-labeled
(TTTAGGG)70 or HRS 60 (Koukalova et al., 1993) probes.
In Situ PCR and FISH
Mature leaves (10th to 13th leaves from the bottom) from each transgenic
plant were fixed in 1:3 (v/v) acetic acid:ethanol and digested with 2%
cellulase, 1.5% macerozyme, 0.3% pectolyase (Yakult Honsha Co.), and
1 mM EDTA, pH 4.2, for 2 h. After leaves were squashed with 60% acetic
acid on glass slides, digested leaf tissue was dried overnight at room
temperature. Slides were soaked twice in 50 mL of 23 SSC (13 SSC is
0.15 M NaCl and 0.015 M sodium citrate) for 15 min, fixed with 1%
formaldehyde for 15 min at 4°C, and serially dehydrated with 70, 90, and
100% ethanol. In situ PCR was performed with DNA-tag-specific PCR
primers (see Supplemental Table 1 online) as described (Kubaláková et al.,
2001) using Ex-Taq polymerase (Takara). Slides were pretreated at 95°C
for 5 min. PCR reactions consisted of 22 cycles of 30 s at 95°C, 1 min at
55°C, and 2 min at 72°C. Telomeres and amplified DNA-tag were visualized using FISH analysis with Alexa Fluor 488 (Invitrogen) conjugated to
dUTP-labeled DNA-tag probes and Texas Red-dUTP–labeled telomere
(TTTAGGG)70 probes, respectively, as described (Hong et al., 2007).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: tobacco GTBP1 (HM049166) and TRF1 (AF543195), Arabidopsis
Telomere 39 G-Strand Overhang Protection
TERT (NM121691), rice TERT (AAK35007), and human HnRNPA1 (P09651)
and TERT (HM101156).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Construction of 35S:RNAi-TERT and 35S:
RNAi-GTBP1/35S:RNAi-TERT Transgenic Knockdown Tobacco
Plants.
Supplemental Figure 2. Gel Retardation Assay with MBP to SingleStranded Telomere Sequences.
Supplemental Figure 3. Construction of Telomere (TTTAGGG)70:
DNA-tag Transgenic Tobacco Plants.
Supplemental Figure 4. Repression of TERT and GTBP1 mRNA as
Determined by RT-PCR Analysis.
Supplemental Table 1. List of Synthetic Oligonucleotide Sequences.
ACKNOWLEDGMENTS
This work was supported by a grant from the Woo Jang Chun Special
Project (PJ009106) funded by the Rural Development Administration,
Republic of Korea, to W.T.K.
AUTHOR CONTRIBUTIONS
Y.W.L. and W.T.K. designed research, analyzed data, and wrote the
article. Y.W.L. performed research.
Received November 19, 2012; revised February 17, 2013; accepted
March 26, 2013; published April 9, 2013.
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Telomerase-Dependent 3′ G-Strand Overhang Maintenance Facilitates GTBP1-Mediated
Telomere Protection from Misplaced Homologous Recombination
Yong Woo Lee and Woo Taek Kim
Plant Cell; originally published online April 9, 2013;
DOI 10.1105/tpc.112.107573
This information is current as of June 15, 2017
Supplemental Data
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