Telomerase-Dependent 39 G-Strand Overhang MaintenanceFacilitates GTBP1-Mediated Telomere Protection fromMisplaced Homologous RecombinationC 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 focusedon the mechanisms that generate and regulate the length of telomere 39 G-strand overhangs, but the roles of G-strandoverhang length control in proper T-loop formation and end protection remain unclear. Here, we examined functionalrelationships between the single-stranded telomere binding protein GTBP1 and G-strand overhang lengths maintained bytelomerase in tobacco (Nicotiana tabacum). In tobacco plants, telomerase reverse transcriptase subunit (TERT) repressionseverely worsened the GTBP1 knockdown phenotypes, which were formally characterized as an outcome of telomeredestabilization. TERT downregulation shortened the telomere 39 G-overhangs and increased telomere recombinationalaberrations in GTBP1-suppressed plants. Correlatively, GTBP1-mediated inhibition of single-strand invasion into the double-strand telomeric sequences was impaired due to shorter single-stranded telomeres. Moreover, TERT/GTBP1 double knockdownamplified misplaced homologous recombination of G-strand overhangs into intertelomeric regions. Thus, proper G-overhanglength maintenance is required to protect telomeres against intertelomeric recombination, which is achieved by the balancedfunctions of GTBP1 and telomerase activity.

INTRODUCTION

Telomeres, the ends of linear eukaryotic chromosomes, arecomposed of double-stranded repeated DNA with terminal 39 G-strand overhangs of a single-stranded repeated DNA sequence(Blackburn, 1991; Griffith et al., 1999). Telomeric DNA is shieldedby numerous telomere-specific binding proteins, resulting infunctional nucleoprotein structures (de Lange, 2005). The func-tions of the binding proteins include telomere length regulation,G-overhang processing, T-loop formation, and distinguishingtelomeres from DNA damage sites (Bailey et al., 2001; de Lange,2002, 2005; Sfeir and de Lange, 2012). Several proteins thathave telomeric single-strand-specific binding affinity have beenstudied, but only a few have been shown to be functionallyassociated with telomeres. PROTECTION OF TELOMERESPROTEIN1 (POT1) is the most thoroughly characterized single-stranded telomeric DNA binding protein. As a subunit of theshelterin complex, POT1 participates in the protection of telo-meric ends from DNA damage responses (Loayza and DeLange, 2003; de Lange, 2005; Denchi and de Lange, 2007; Gongand de Lange, 2010). Another single-stranded DNA bindingprotein, human heterogeneous nuclear ribonucleoprotein A1(hnRNPA1), regulates telomere length through an interaction with

telomerase (LaBranche et al., 1998). Association of hnRNPA1prevents replication protein A (RPA) from binding to telomeres inlate S phase (Flynn et al., 2011). After S phase, POT1 replaceshnRNPA1 and further inhibits RPA binding to 39 G-overhangsunder the regulation of telomeric repeat–containing RNA (TERRA).TERRA competes with G-overhangs by binding to hnRNPA1,removes human heterogeneous nuclear ribonucleoproteins fromG-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 telomericsequences (Griffith et al., 1999). Although the details are not yetfully understood, G-overhang generation is regulated by cellcycle–dependent mechanisms involving C-strand resection andfill-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 oftelomere-associated proteins, including telomerase (Stewartet al., 2003; Masutomi et al., 2003; Hockemeyer et al., 2006;Dimitrova and de Lange, 2009; Wu et al., 2010). In culturedhuman cells, telomerase overexpression prevents G-strand erosionduring continuous rounds of cell division (Stewart et al., 2003),and telomerase disruption causes instability of G-overhangmaintenance (Masutomi et al., 2003). On the other hand, G-overhanggeneration is unchanged in telomerase-negative transgenicmice (Hemann and Greider, 1999). This raised the possibility thattelomerase may not be a major factor for overhang processing;rather, telomerase affects G-overhang stability. Decreases inG-overhang length correlate with cellular senescence in normalhuman cells (Stewart et al., 2003; Masutomi et al., 2003). How-ever, it is still unclear how proper G-overhang length maintenancecontributes to telomere stability.

1 Address correspondence to wtkim@yonsei.ac.kr.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions 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 andwhite in the print edition.W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.112.107573

The Plant Cell, Vol. 25: 1329–1342, April 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

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 eachdomain. RRM1 and RRM2, RNA recognition motifs. aa, amino acid residue.

In this article, we examined functional relationships betweensingle-stranded telomere binding protein GTBP1, a tobacco(Nicotiana tabacum) putative ortholog of hnRNPA1, and G-strand overhang lengths maintained by telomerase in tobacco.Our data indicate that telomerase reverse transcriptase subunit(TERT) and GTBP1 double knockdown amplified misplaced ho-mologous recombination (HR) of G-strand overhangs into telo-meric regions, resulting in genomic instability. GTBP1 binding tosingle-stranded telomeric ends was impaired due to shorteningof the G-overhangs, which explains why TERT repression-induced G-overhang reduction increased telomeric HR in GTBP1knockdown plants. Although plant development is generallyplastic in response to genome instability, G-overhang reductiondue to the TERT knockdown with an insufficient GTBP1 levelcould trigger undesirable telomeric HR that leads to abnormalgrowth arrest in transgenic tobacco plants. These results sug-gest that proper G-overhang length maintenance is requiredto protect telomeres against aberrant intertelomeric recom-bination and to prevent developmental damage to the plants,which is achieved by the balanced functions of the single-stranded telomere binding protein GTBP1 and telomeraseactivity.

RESULTS

TERT Repression Intensified GTBP1-KnockdownPhenotypes in Tobacco

GTBP1 is a tobacco putative ortholog of hnRNPA1 that playsprotective roles in telomere G-overhangs (Figure 1A). GTBP1-suppressed RNA interference (RNAi) transgenic tobacco plants(35S:RNAi-GTBP1) display aberrant telomere recombinationand genome instability (Lee and Kim, 2010). To examine thefunctional correlation between GTBP1 and telomerase activity intelomere stability, the 35S:RNAi-TERT construct was introducedinto the T0 35S:RNAi-GTBP1 transgenic line using Agro-bacterium tumefaciens–mediated T-DNA delivery methods and

telomerase/GTBP1 double-knockdown RNAi transgenic tobaccoplants (35S:RNAi-TERT/35S:RNAi-GTBP1) were subsequentlyproduced (Figure 1B; see Supplemental Figures 1A and 1Bonline). Telomerase single knockdown (35S:RNAi-TERT) trans-genic lines were also generated. Genomic DNA gel blot analysisshowed that the 35S:RNAi-TERT and 35S:RNAi-TERT/35S:RNAi-GTBP1 transgenic plants used in this study are independentlines (see Supplemental Figure 1C online). Real-time quantitativeRT-PCR (qRT-PCR) analysis demonstrated significant down-regulation of TERT mRNA in 35S:RNAi-TERT and 35S:RNAi-TERT/35S:RNAi-GTBP1 transgenic tobacco plants (Figure 1C,left panel). Telomerase enzyme activities were also reduced inRNAi knockdown transgenic callus relative to wild-type callusdetermined by telomere repeat amplification (TRAP) assays(Figure 1D). As reported previously (Fitzgerald et al., 1996; Rihaet al., 1998; Yang et al., 2002), telomerase activities were verylow in mature leaves of both wild-type and transgenic plants(see Supplemental Figure 1D online). In addition, GTBP1 waseffectively suppressed in 35S:RNAi-GTBP1 and 35S:RNAi-TERT/35S:RNAi-GTBP1 plants (Figure 1C, right panel). Trans-genic 35S:RNAi-TERT T0 plants showed no visible phenotypicdefects compared with wild-type plants (Figure 1E, left panel;see Supplemental Figure 1E online). This is consistent withprevious results that showed that the telomerase-deficientArabidopsis thaliana tert mutants were normal for up to fivegenerations (Riha et al., 2001). By contrast, T0 35S:RNAi-TERT/35S:RNAi-GTBP1 plants exhibited severe phenotypic anoma-lies. The morphological abnormalities of the 35S:RNAi-TERT/35S:RNAi-GTBP1 double-RNAi plants became progressivelymore serious as the plants grew. The 2-month-old plants ex-hibited markedly retarded growth and premature senescence,failed to develop normal reproductive organs, and died withoutproducing functional seeds (Figure 1E, middle panel). Further-more, their leaves were smaller than wild-type leaves and wereunable to mature to full size (Figure 1F). Reduced internodelength 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 twodifferent regions (660 to 984 bp and 984 to 1478 bp) of TERT cDNA (see Supplemental Figure 1B online). The 35S:RNAi-GTBP1 vector contains theinverted-repeat sequence of the 726- to 1070-bp region of GTBP1 cDNA. LB, left border; OCS ter, octopine synthase terminator; NOS ter, nopalinesynthase 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. Expressionlevels 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 (rightpanel) wild-type and T0 RNAi transgenic plants. All four independent 35S:RNAi-GTBP1/35S:RNAi-TERT transgenic lines died after 2 months, whereasthe 35S:RNAi-GTBP1 lines remain alive after 3 months. One-month-old RNAi transgenic plants that displayed mild phenotypic defects are shown inSupplemental 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 therow 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.]

Telomere 39 G-Strand Overhang Protection 1331

Figure 2. Reduction of 39 G-Overhang Lengths and Enhanced t-Circle Formation in TERT/GTBP1 Double-Knockdown Telomeres.

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 mostof their floral organs failed to produce seeds (Lee and Kim,2010). Thus, suppression of TERT did not affect the growth ofwild-type plants but synergistically amplified abnormal pheno-types of GTBP1-suppressed plants, resulting in the early growtharrest of the 35S:RNAi-TERT/35S:RNAi-GTBP1 plants.

Reduction of 39 G-Overhang Lengths and Enhanced t-CircleFormation in TERT/GTBP1 Double-Knockdown Telomeres

Because 35S:RNAi-TERT/35S:RNAi-GTBP1 and most 35S:RNAi-GTBP1 transgenic plants were sterile, T0 tobacco plants wereused for subsequent experiments. To address the telomere statusof the RNAi transgenic plants, restricted genomic DNA was sub-jected to in-gel hybridization with telomere probes following pulse-field gel electrophoresis. It was previously reported that denaturedpulse-field gel electrophoresis could be used to measure thelengths of telomeres (>50 kb) in tobacco chromosomes (Fajkuset al., 1995; Yang et al., 2004), while relatively shorter 39 G-overhangsingle-stranded telomeric signals were effectively quantified bynative in-gel hybridization analysis (Heacock et al., 2007). In nativegels, the levels of 39 G-overhang signals in 35S:RNAi-TERT and35S:RNAi-TERT/35S:RNAi-GTBP1 plants were reduced to 32 to46% and 30 to 40%, respectively, compared with those in wild-type plants (Figures 2A, left panel, and 2B). The 39 G-overhang

signal of 35S:RNAi-GTBP1 also decreased by ;25% relative tothe wild-type signal. This native gel signal was markedly reducedfollowing Exo I treatment determined by pulse-field (Figure 2C, leftpanel) and standard agarose (Figure 2C, right panel) gel electro-phoresis, confirming that the signal resulted from 39 G-overhangsingle-stranded DNA. By contrast, total telomere lengths in allRNAi plants examined were not significantly altered but mostlyremained 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). Thisdecrease in telomere length is relatively subtle in comparison withlong tobacco telomeres (15 to 50 kb); thus, it is still possible thattotal telomere lengths in RNAi knockdown tobacco plants wereslightly changed, and this could be undetectable by our pulse-field gel electrophoresis system. Overall, decreases in TERT andGTBP1 mRNA levels reduced 39 G-overhang single-strandedtelomere lengths, whereas double-stranded telomere lengthswere relatively constant in T0 RNAi transgenic plants.HR in telomeres is typified by the formation of extrachromo-

somal telomeric circles (t-circles) (Wang et al., 2004; Wu et al.,2006; Zellinger et al., 2007). Two-dimensional (2-D) pulse-field gelelectrophoresis indicated that t-circle formation in wild-type and35S:RNAi-TERT plants was negligible (Figure 2D). By contrast,telomeres from 35S:RNAi-TERT/35S:RNAi-GTBP1 plants showedsignificantly enhanced t-circle formation. The degree of t-circleformation 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, and35S:RNAi-GTBP1/35S:RNAi-TERT transgenic lines using pulse-field gel electrophoresis followed by in-gel hybridization. TaqI-restricted leaf genomicDNA from each transgenic plant was subjected to in-gel hybridization with a telomere (CCCTAAA)4 probe under native pulse-field gel conditions tomeasure the single-stranded 39 G-overhang telomere signal (left panel). Restricted leaf genomic DNA was rehybridized under denatured pulse-field gelconditions 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 trans-genic plants. Genomic DNA was purified and used for in-gel hybridization after native agarose gel electrophoresis. Quantified G-overhang signals werenormalized 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 hybridizationexperiments in the presence (+) or absence (2) of Exo I. Quantified G-overhang signals were normalized to the levels of denatured total telomeresignals.(D) Extrachromosomal t-circle formation in wild-type and RNAi-downregulated plants. Restricted leaf genomic DNA isolated from wild-type, 35S:RNAi-TERT, 35S:RNAi-GTBP1, and 35S:RNAi-GTBP1/35S:RNAi-TERT transgenic plants was subjected to 2-D pulse-field gel electrophoresis followed by in-gel 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 withenzyme-treated DNA mixtures and f29 DNA polymerase. The reaction mixtures were separated on 1% agarose gels and subjected to in-gel hy-bridization with a 32P-labeled (CCCTAAA)4 probe. Quantified t-circle signals were normalized to the levels of denatured total telomere signals de-termined 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 ofTERT 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 purifiedand 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) ofsubculture. 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.

Telomere 39 G-Strand Overhang Protection 1333

Figure 3. Strand Invasion and ChIP Assays.

results were further confirmed by the t-circle amplification (TCA)assay as described by Zellinger et al. (2007) in connection withexonuclease V. The results indicated that t-circle formation in-creased 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 phenotypicabnormalities of RNAi transgenic plants. Therefore, double knock-down of TERT and GTBP1 resulted not only in 39 G-overhangshortening, but also in t-circle formation that may be due to ab-errant HR within the telomeres. Both of these events, in turn, werepossibly associated with the impaired development observed in35S:RNAi-TERT/35S:RNAi-GTBP1 tobacco plants.

In human fibroblasts, a lack of telomerase activity causes dis-ruption of G-overhang maintenance during prolonged cell pop-ulation growth (Masutomi et al., 2003). To further examine whetherdecreased telomerase activity reduces 39 G-overhang single-stranded telomere lengths, cultured tobacco Bright Yellow-2 (BY-2)suspension cells were transfected with the 35S:RNAi-TERTconstruct. Transfected BY-2 cells have repressed TERT mRNAlevels that lead to decreased telomerase activity, as determinedby qRT-PCR and TRAP assays, respectively (Figure 2F). Total telo-mere length in 35S:RNAi-TERT BY-2 cells was largely unchangedover three rounds of subculture (Figure 2G). However, G-overhangsignals from the same subculture of telomerase-repressed BY-2cells were progressively reduced and reached ;70 to 80% and40 to 60% of the G-overhang signals in wild-type cells after twoand three rounds of subculture, respectively (Figure 2H). Theseresults are consistent with the notion that telomerase activity isresponsible for 39 G-overhang maintenance. Although telomeraseactivity seems to be the most likely explanation, it is still possiblethat TERT has a nonenzymatic role in G-overhang maintenance.

GTBP1 Binding to Single-Stranded Telomeric Ends WasReduced Due to Shortening of the G-Overhangs ThatResulted from TERT Downregulation

Invasion of a 39 G-overhang single-stranded telomeric se-quence into double-stranded telomeric DNA is a prerequisite

for interchromosomal telomeric HR (Cesare and Reddel, 2010).The increased telomere recombination rates in TERT/GTBP1-suppressed transgenic plants prompted us to investigate therelationship between G-overhang length and telomere recombi-nation. Gel retardation assays revealed that binding of bacteriallyexpressed maltose binding protein (MBP)-GTBP1 fusion protein totelomere single-stranded probes progressively increases between(TTTAGGG)3 and (TTTAGGG)8 repeats (Figure 3A). Consequently,MBP-GTBP1 more effectively inhibited the invasion of single-stranded DNA into double-stranded DNA in a length-dependentmanner (Figure 3B). Approximately 40% of the single-strand in-vasion was inhibited by a saturating amount of GTBP1 with(TTTAGGG)5 repeats, whereas >75% of the invasion was inhibitedwith (TTTAGGG)8 repeats. Intriguingly, C-terminal deletion mutantsof GTBP1 (GTBP1DC11-194 and GTBP1DC21-179) bound equallywell to the different lengths of single-stranded telomere repeats(Figure 3C). This suggests that the C-terminal region of GTBP1 isresponsible for the length-dependent interactions between GTBP1and single-stranded telomere repeats. These results are in agree-ment with those in Figure 2; namely, that double-suppressionof TERT and GTBP1 caused shortening of the 39 G-overhanglength accompanied by a reduction in cellular GTBP1 levels,which resulted in aberrant telomeric HR and abnormal growtharrest in tobacco plants. On the other hand, MBP alone failedto interact with all of the different lengths of single-strandedtelomere repeats examined (see Supplemental Figure 2 on-line).This view was further supported by the results of chromatin

immunoprecipitation (ChIP) assays. The 35S:HA-GTBP1 con-struct was transfected into wild-type or 35S:RNAi-TERT BY-2suspension cells. The nucleoprotein complexes from the trans-fected cells were immunoprecipitated using anti-HA antibody.The coimmunoprecipitated DNA was visualized by hybridizationwith a 32P-labeled (TTTAGGG)70 probe. Figure 3D shows thattelomeric DNA was pulled down by the anti-HA antibody in 35S:HA-GTBP1 BY-2 cells. However, the telomere-specific ChIPsignal was reduced to 58 to 66% in 35S:HA-GTBP1/35S:RNAi-TERT 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 relativebinding 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-labeledsingle-stranded (TTTAGGG)n repeat probes (n = 3, 4, 5, 6, or 8) with T-vector plasmid containing a double-stranded (TTTAGGG)70 telomere repeat. Therelative level of invasion of the single-stranded telomeric probe into the plasmid was determined by the shifted band intensity. Asterisk indicates 32P-labeled 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 bandintensity. 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 cellswere fragmented by sonication and subjected to immunoprecipitation (IP) with an anti-HA antibody. The coimmunoprecipitated DNA was hybridizedwith 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 barsrepresent 6 SE.[See online article for color version of this figure.]

Telomere 39 G-Strand Overhang Protection 1335

Figure 4. TERT/GTBP1 Double Knockdown Amplified Aberrant Intertelomeric HR.

1336 The Plant Cell

stranded telomeric ends was significantly reduced due toshortening of the G-overhangs that resulted from TERT down-regulation. Therefore, the levels of telomerase activity and GTBP1are critically associated with 39 G-overhang maintenance andtelomere stability in tobacco.

TERT/GTBP1 Double Knockdown Amplified AberrantIntertelomeric HR

To investigate 39 G-overhang-mediated telomeric HR due to thedownregulation of GTBP1 and TERT, we employed a DNA-tagintertelomeric integration assay (Dunham et al., 2000). Wild-typetobacco plants were transformed with a chimeric construct(telomere:DNA-tag) that contains a DNA-tag positioned next tothe 39 end of the telomere repeats (490 bp) without a functionalpromoter (Figure 4A). Chromosomal integration of the telomere-repeat:DNA-tag construct was detected by in situ PCR followedby fluorescence in situ hybridization (FISH). Three copies of theconstruct were nonspecifically inserted into the chromosomesas evidenced by randomly distributed FISH signals in Telomere:DNA-tag #17 nuclei (Figure 4B, left panel) and genomic DNAgel blotting with a DNA-tag-specific probe (see SupplementalFigure 3 online). These DNA-tag insertion spots were locatedindependently of the internal telomere spots. Subsequently, the35S:RNAi-TERT construct was transformed into Telomere:DNA-tag #17 transgenic plants (see Supplemental Figures 4A and 4Donline). In Telomere:DNA-tag/35S:RNAi-TERT nuclei, the DNA-tag insertion spots did not overlap with the telomere signals(Figure 4B, middle and right panels), indicating that down-regulation of TERT alone did not cause telomeric HR.

Next, the 35S:RNAi-GTBP1 construct was transformed intoTelomere:DNA-tag plants (see Supplemental Figures 4B and 4Eonline). FISH analysis detected multicopy integrations of thetelomere-repeat:DNA-tag construct in the Telomere:DNA-tag/35S:RNAi-GTBP1 chromosomes (Figure 4C). Moreover, locali-zation of the DNA-tag significantly overlapped the telomeres in

these chromosomes. More than 75% of the Telomere:DNA-tag/35S:RNAi-GTBP1 nuclei displayed overlapping signals betweenthe DNA-tag and the telomeres. Approximately 25% of the nucleishowed at least three overlapping DNA-tag spots merged with te-lomeres. Finally, the 35S:RNAi-TERT construct was transformedinto Telomere:DNA-tag/35S:RNAi-GTBP1 plants (see SupplementalFigures 4C to 4E online). The Telomere:DNA-tag/35S:RNAi-GTBP1/35S:RNAi-TERT nuclei contained even more overlapping signalsbetween the DNA-tag and the telomeres. Specifically, 62 to 76%of the nuclei contained more than three DNA-tag spots integratedinto telomere regions (Figure 4C). These results indicate thatdownregulation of GTBP1 increased telomeric HR, and GTBP1/TERT double knockdown induced even more aberrant telomericHR, which closely correlated with the degree of phenotypicanomalies of these RNAi transgenic plants. Figures 4B and 4Calso show that Telomere:DNA-tag/35S:RNAi-GTBP1 and Telo-mere:DNA-tag/35S:RNAi-GTBP1/35S:RNAi-TERT nuclei con-tained increased copy numbers of the telomere-repeat:DNA-tagin their chromosomes compared with the Telomere:DNA-tagand Telomere:DNA-tag/35S:RNAi-TERT plants. Thus, the DNA-tag construct was amplified by intertelomeric HR (Figure 4D).This notion was confirmed by genomic PCR that indicated thatdownregulation of GTBP1 and TERT, but not of TERT alone,increased the amount of DNA-tag in Telomere:DNA-tag trans-genic plants (Figure 4E). Overall, these results argue that doublesuppression of GTBP1 and TERT results in the deregulation of 39G-overhang maintenance, which induces G-overhang-mediatedintertelomeric HR and anomalous growth arrest in tobaccoplants.

DISCUSSION

In this study, RNAi-mediated downregulation of TERT signifi-cantly reduced single-stranded telomere 39 G-overhang lengthin 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 (glufosinateammonium) resistant gene.(B) DNA-tag intertelomeric integration assay. The telomere-repeat:DNA-tag construct was transformed into tobacco plants. Chromosomal integrationof 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 wasdenatured 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 signalsindicate the DNA-tag sequence, whereas red signals indicate internal telomere sequences. The DNA-tag sequences merged with telomere sequenceswere 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 chro-mosomes.(E) Genomic PCR analysis. Leaf genomic DNA was isolated from Telomere:DNA-tag #17, Telomere:DNA-tag/35S:RNAi-TERT (independent lines a andb), 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.]

Telomere 39 G-Strand Overhang Protection 1337

RNAi-TERT transgenic lines were phenotypically normal (Figure1E), and their double-stranded telomeric length remained largelyunchanged with undetectable t-circles (Figures 2A and 2D). Ourfindings agree with the normal phenotypes of Arabidopsis tertknockout mutants after up to five generations, which contain nodetectable t-circle formation (Riha et al., 2001; Zellinger et al.,2007). By contrast, telomerase downregulation, when combinedwith repression of the hnRNPA1 homolog GTBP1, resulted ingrowth arrest and early senescence (Figures 1E and 1F). The35S:RNAi-GTBP1/35S:RNAi-TERT plants experienced severegenomic instability, as frequent t-circle formation and intertelo-meric HR were evident in their telomeres. GTBP1/TERT doubleknockdown resulted in the telomere-repeat:DNA-tag constructbeing integrated into the telomere regions by HR, so that thecopy number of the DNA-tag significantly increased (Figure 4).Therefore, telomerase downregulation caused G-overhang re-duction and subsequently decreased GTBP1 binding to single-stranded telomeres (Figure 3D). The decrease in GTBP1 bindingto 39 G-overhang telomeric ends induced aberrant G-overhang-mediated telomeric HR and telomere dysfunction (Figure 5).

It was proposed that telomerase-independent telomerelengthening (i.e., alternative lengthening of telomeres [ALT]) wasmediated by HR-dependent DNA replication at the 39 telomericends in human ALT cancer cells (Cesare and Reddel, 2010). The35S:RNAi-GTBP1/35S:RNAi-TERT plants resembled ALT can-cer cells in the context of HR-dependent DNA replication, asshown by their increase in telomere-repeat:DNA-tag integrationinto telomeres (Figures 4D and 4E). There are a number ofproteins that affect telomere HR and ALT. Components of theshelterin complex and other telomere-associated proteins, including

POT1, TRF2, CTC1, and Ku, are involved in the regulation of HRand ALT processes in telomeres (Wang et al., 2004; Wu et al.,2006; Cesare and Reddel, 2010). Inappropriate 39 G-overhangsingle-strand invasion causes t-circle formation and HR-dependentDNA replication, which lead to telomere destabilization and entryinto senescence (Lustig, 2003; Wang et al., 2004). Therefore,there must be a mechanism to repress inappropriate 39 G-overhangsingle-strand invasion in normal cells. Recently, it was reportedthat TERT along with Ku80 inhibits an HR mechanism of ALT inArabidopsis (Kazda et al., 2012). Thus, the synergistic increasein telomeric HR observed in the GTBP1/TERT double-knockdown plants may reflect the loss of HR inhibition via GTBP1coupled with an increased likelihood of telomere HR due tothe loss of TERT. The single tert knockout alone may not in-duce telomere HR instability or ALT engagement in Arabidopsis(Zellinger et al., 2007), which suggests that the TERT-mediatedinhibition of HR may be limited to specific conditions, such asku80 knockout. TERT downregulation reduces GTBP1 bindingto single-stranded telomeric ends under the GTBP1 knockdownconditions, which suggests that inhibitory roles of GTBP1 inaberrant telomeric HR are intimately correlated with the roles ofTERT.Binding activity of GTBP1 to telomere single-stranded probes

was affected by single-strand probe length (Figure 3A). Thisresult shows that TERT repression-induced G-overhang re-duction increased telomeric HR in GTBP1 knockdown plants.When the C-terminal region (from 194 to 345 amino acid resi-dues) was removed, GTBP1 bound shorter telomeric repeatswith similar efficiency compared with longer repeats (Figure 3C).Therefore, the length-dependent binding of GTBP1 to telomeric

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 HRof G-strand overhangs into telomeric regions, which is associated with genome instability and early senescence of tobacco plants. A reduction ofG-overhang length, in conjunction with GTBP1 repression, caused G-overhang uncapping from GTBP1-mediated telomere protection, due to theG-overhang length-dependent binding of GTBP1. Open circles indicate GTBP1.

1338 The Plant Cell

sequences is not only due to redundancy of the binding site, butalso to the GTBP1-specific characteristics of the C-terminalregion. Since human POT1 binds two or five TTAGGG single-strand repeats equally well (Loayza et al., 2004), the function ofPOT1 may not be inhibited by single-strand length reduction.Despite the conserved 39 G-overhang structure, POT1 homo-logs of Brassicaceae plant species have no detectable bindingactivity to single-stranded telomeric repeats (Shakirov et al.,2009), which suggests that POT1 may not be a major single-strand telomere binding protein in plants. Besides POT1, theCST (CTC1-STN1-TEN1) complex in human also binds to theG-overhang with sequence specificity (Miyake et al., 2009; Chenet al., 2012). Similar to POT1, CST binds with comparable affinityto longer than three TTAGGG G-strand arrays (Chen et al.,2012). Therefore, except for the case of GTBP1, G-overhanglength does not appear to significantly affect the binding ofknown telomere single-strand binding proteins when the single-strand length is longer than the minimal binding site.

Telomere G-overhang lengths are tightly and dynamicallyregulated. In mammalian cells, G-overhang length increases inlate S/G2 by the process of C-strand resection and fill-in (Daiet al., 2010). Throughout the cell cycle, G-overhang formationsteps 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 re-sulted in G-overhang abnormalities. CTC1, one of the CSTcomplex components, regulates telomere G-overhangs in hu-man and Arabidopsis (Surovtseva et al., 2009). Depletion ofCTC1 causes increased G-overhangs, recombination, and end-to-end fusions. Interestingly, ku70 knockout, which increasesG-overhangs, triggers abnormal telomere HR in Arabidopsis(Zellinger et al., 2007). Indeed, nucleolytic resection of 59 chro-mosome ends by EXO1 promotes telomere recombination inku80 knockout Arabidopsis (Kazda et al., 2012). On the otherhand, our results show that reduction of G-overhang lengthspromoted telomeric HR when GTBP1 level was insufficient. Onepossibility is that the length of the 39 G-overhang and cellularlevel of GTBP1 both contribute to inhibition of abnormal telo-mere HR. Overall, these results implicate that proper processingof G-overhang is important to regulate telomeric HR stability andthus legitimate T-loop formation.

Telomere attrition during population growth in telomerase-negative cells causes cellular senescence, which provides thecurrent telomere-directed aging model (Blackburn, 1991). Todecipher the aging process at the molecular level, numeroustelomere/telomerase-associated proteins have been studiedwith regard to telomerase regulation. hnRNPA1 was initially pro-posed as one of the positive telomere length regulators throughan interaction with telomerase (LaBranche et al., 1998). In higherplants, the relationship between hnRNPA1 orthologs and telo-merase was studied. Arabidopsis STEP1, which contains a ho-mologous DNA/RNA binding domain of hnRNPA1, inhibited invitro telomerase activity, but its in vivo functions were not elu-cidated (Kwon and Chung, 2004). Previously, we reported thatGTBP1 inhibited aberrant HR in tobacco telomeres (Lee andKim, 2010). In GTBP1 RNAi knockdown plants that displayedunusual HR, telomerase activity was not significantly altered,suggesting that a reduction in GTBP1 level did not directly

decrease telomerase activity. 35S:RNAi-GTBP1/35S:RNAi-TERTdouble-knockdown tobacco plants exhibited higher levels of HRcompared with those of 35S:RNAi-GTBP1 plants, suggesting thatTERT is critical for GTBP1-mediated telomere HR stability.However, hnRNPA1/GTBP1 is a highly abundant protein that hasbeen shown to affect many aspects of telomere biology (Fordet al., 2002; He and Smith, 2009). Therefore, we cannot rule outthe possibility that anomalous phenotypes of 35S:RNAi-GTBP1/35S:RNAi-TERT plants resulted from the loss of other as yetunidentified GTBP1-related telomere/telomerase functions. Onecould argue that telomerase suppression could inhibit telomerelength reconstitution after t-circle excision, which resulted in in-efficient healing of short telomeres arising from circle excision andthus increased severity of 35S:RNAi-GTBP1/35S:RNAi-TERTplant phenotypes. However, 35S:RNAi-GTBP1/35S:RNAi-TERTplants contained similar telomere lengths relative to wild-typeplants (Figure 2A).RPA plays an important role in ataxia telangiectasia and Rad3-

related checkpoint activation, which triggers HR-mediated repairof damaged DNA (Jackson and Bartek, 2009). hnRNA1 inhibitsRPA binding to telomeres and distinguishes telomeres from DNAdamage sites (Flynn et al., 2011), thus possibly preventing telo-meric HR. These findings are collaterally supportive of our view onthe protective role of GTBP1 against aberrant telomeric HR. Al-though plant development is generally plastic in response to ge-nome instability, G-overhang reduction with an insufficient GTBP1level could trigger undesirable telomeric HR that leads to imme-diate genomic and developmental damage to the plants (Figure5). In conclusion, our data indicate that telomerase-dependent 39G-strand overhang maintenance facilitates GTBP1-mediated telo-mere protection frommisplaced HR in tobacco. Therefore, adequatecellular levels of both telomerase activity and GTBP1 are essentialfor telomere stability and function.

METHODS

Plant Materials

Tobacco (Nicotiana tabacum cv Samsun NN) seeds were germinated on0.8% agar for 7 d on Murashige and Skoog medium in a growth chamber.Seedlings were transferred to soil and further grown in a growth chamberunder a 16-h-light/8-h-dark photoperiod at 25°C. Tobacco BY-2 sus-pension cells were cultured in Murashige and Skoog medium on a rotaryshaker (150 rpm) at 25°C in the dark. Stationary-phase cells (5 mL) weretransferred 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 tothe amino acid sequence DVFKAFD for the upstream primer and AMKFHCYfor the downstream primer were synthesized (see Supplemental Table 1online) and used for RT-PCR. These amino acid sequences are highlyconserved in Arabidopsis and rice (Oryza sativa) TERTs (see SupplementalFigure 1Aonline). To obtain a longer TERT cDNA clone, 39 rapid amplificationof cDNA ends was performed as described (Lee and Kim, 2010). The de-duced amino acid sequence of the partial TERT is identical to that ofa recently isolated tobacco TERT (Sýkorová et al., 2012). Total RNA wasextracted from mature tobacco leaves and BY-2 cells as described pre-viously (Yang et al., 2004). RNA (2 mg) was used for first-strand cDNAsynthesiswithMMLV reverse transcriptase (Promega). RT-PCRwasperformed

Telomere 39 G-Strand Overhang Protection 1339

with gene-specific primers (see Supplemental Table 1 online) and Ex-Taqpolymerase (Takara). cDNA amplification was performed with cycles of 30 sat 95°C, 30 s at 55°C, and 1 min at 72°C, with 22 cycles for EF1a, 25 cyclesfor GTBP1, and 28 cycles for TERT using an automatic thermal cycler(Perkin-Elmer/Cetus). PCR products were separated by electrophoresis in1.0% agarose gels containing ethidium bromide and visualized under UVlight. 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 EF1amRNA.

Generation of Transgenic Tobacco Plants

For the construction of the 35S:RNAi-TERT transgenic plants, TERTcDNA encompassing 325 bp (from 660 to 984 bp) or 495 bp (from 984 to1478 bp) (see Supplemental Figure 1B online) was inserted in an invertedorientation into the pCAMBIA vector (CAMBIA) with a 35S promoter andPDK intron. The telomere-repeat:DNA-tag construct was produced usingthe pEarleygate 301 vector by inserting 490 bp of a telomere repeat(TTTAGGG)70 and partial NgTRF1 cDNA (Yang et al., 2004) into thecorresponding sites. The constructed vectors were transformed intoAgrobacterium tumefaciens strain LBA4404 by electroporation. Tobaccoleaf discs were cocultivated with Agrobacterium as described previously(Lee and Kim, 2010). Transgenic tobacco plants were generated ongrowth medium containing 1 mg/L Basta for Telomere:DNA-tag and25 mg/L hygromycin for 35S:RNAi-TERT. The 35S:RNAi-GTBP1 plantswere constructed as described previously (Lee and Kim, 2010). The re-generated T0 plants were grown in a growth roomunder a 16-h-light/8-h-darkphotoperiod at 25°C.

TRAP Assay, Telomere Repeat Fragment Analysis, and 2-DPulse-Field Gel Electrophoresis

Telomerase activity was measured using TRAP assays as described(Yang et al., 2004). For telomere repeat fragment analysis, leaf genomicDNA (5 mg) was digested with TaqI restriction enzyme and separated ona CHEF-DR III pulsed-field electrophoresis system (Bio-Rad). Agarosegels 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 and0.5 M NaOH, neutralized with 1.0 M Tris, pH 7.3, and 0.5 M NaOH, andrehybridized 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 EnglandBiolabs) at 37°C overnight and subjected to telomere repeat fragmentassays. For 2-D pulse-field gel electrophoresis analysis, TaqI-digestedDNA was separated in 0.5% agarose gels at 1 V/cm for 25.5 h at an angle of120° with switching times ramped from 1 to 6 s at 14°C. Second-dimensionelectrophoresis was conducted in 1.1%agarose gels at 6 V/cm for 8.5 h withthe 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 de-scribed by Zellinger et al. (2007) with minor modifications. TaqI-restrictedtobacco leaf genomic DNA (0.5mg) was treatedwith 20 units of exonucleaseV (New England Biolabs) at 37°C for 2 h to remove linear DNAs. Enzyme-treated 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)3primer, denatured at 96°C for 5 min, cooled down to 25°C for 30 min, andthen subjected to the TCA assay. The TCA reaction was performed with 10units off29 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,44.5mMboric acid, and 1mMEDTA) at 85 V at room temperature. Gelsweredried and hybridized with a 32P-labeled (CCCTAAA)4 probe.

Gel Retardation and Strand Invasion Assays

Escherichia coli–expressed MBP-fused GTBP1 was purified by affinitychromatography using amylose resin (New England Biolabs). Differentconcentrations (0, 75, 150, and 300 nM) of purifiedGTBP1 were incubatedwith telomere repeat oligomers [(TTTAGGG)3, (TTTAGGG)4, (TTTAGGG)5,(TTTAGGG)6, or (TTTAGGG)8] for 30 min. Protein-oligomer mixtures weresubjected to 7% acrylamide gel electrophoresis. For strand invasionassays, pGEM-T Easy plasmid (Promega) containing double-stranded(TTTAGGG)70 telomeric repeats was incubated with 32P-labled telomereoligomers according to Amiard et al. (2007). Labeled telomere oligomerswere premixed with GTBP1 in invasion buffer (50 mM HEPES, pH 8.0, 0.1mg/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 stopbuffer [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 roomtemperature. Gels were dried and subjected to autoradiography. The effi-ciency of invasion of the single-stranded telomeric probes into (TTTAGGG)70telomeric repeats was determined by the shifted band intensity.

ChIP Assays

ChIP assays were performed as described by Lee and Kim (2010) withslight modifications. Wild-type, 35S:HA-GTBP1, and 35S:HA-GTBP1/35S:RNAi-TERT transgenic BY-2 cells were treated with a 1% formal-dehyde solution for 15 min to cross-link the nucleoprotein complexes.After quenching the cross-link with 150 mM Gly, chromosomes werefragmented by sonication to 0.2- to 1.0-kb fragments in ChIP dilutionbuffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, and167 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 agarosebeads (Santa Cruz Biotechnology). Eluted DNA was dot blotted ontoHybond-N nylonmembranes (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 transgenicplant were fixed in 1:3 (v/v) acetic acid:ethanol and digested with 2%cellulase, 1.5% macerozyme, 0.3% pectolyase (Yakult Honsha Co.), and1 mM EDTA, pH 4.2, for 2 h. After leaves were squashed with 60% aceticacid on glass slides, digested leaf tissue was dried overnight at roomtemperature. Slides were soaked twice in 50 mL of 23 SSC (13 SSC is0.15 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, and100% ethanol. In situ PCR was performed with DNA-tag-specific PCRprimers (see Supplemental Table 1 online) as described (Kubaláková et al.,2001) using Ex-Taq polymerase (Takara). Slides were pretreated at 95°Cfor 5 min. PCR reactions consisted of 22 cycles of 30 s at 95°C, 1 min at55°C, and 2 min at 72°C. Telomeres and amplified DNA-tag were visu-alized using FISH analysis with Alexa Fluor 488 (Invitrogen) conjugated todUTP-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 GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers: tobacco GTBP1 (HM049166) and TRF1 (AF543195), Arabidopsis

1340 The Plant Cell

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 TobaccoPlants.

Supplemental Figure 2. Gel Retardation Assay with MBP to Single-Stranded Telomere Sequences.

Supplemental Figure 3. Construction of Telomere (TTTAGGG)70:DNA-tag Transgenic Tobacco Plants.

Supplemental Figure 4. Repression of TERT and GTBP1 mRNA asDetermined 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 SpecialProject (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 thearticle. Y.W.L. performed research.

Received November 19, 2012; revised February 17, 2013; acceptedMarch 26, 2013; published April 9, 2013.

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1342 The Plant Cell

DOI 10.1105/tpc.112.107573; originally published online April 9, 2013; 2013;25;1329-1342Plant Cell

Yong Woo Lee and Woo Taek KimTelomere Protection from Misplaced Homologous Recombination

G-Strand Overhang Maintenance Facilitates GTBP1-Mediated′Telomerase-Dependent 3

 This information is current as of June 20, 2018

 

Supplemental Data /content/suppl/2013/03/28/tpc.112.107573.DC1.html

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