1. Volume 142, Issue 2, Pages 230-242 (July 2010)
TRF2 and Apollo Cooperate with Topoisomerase 2α to Protect Human Telomeres from Replicative Damage 
Jing Ye, Christelle Lenain, Serge Bauwens, Angela Rizzo, Adelaïde Saint-Léger, Anaïs Poulet, Delphine Benarroch, Frédérique Magdinier, Julia Morere, Simon Amiard, Els Verhoeyen, Sébastien Britton, Patrick Calsou, Bernard Salles, Anna Bizard, Marc Nadal, Erica Salvati, Laure Sabatier, Yunlin Wu, Annamaria Biroccio, Arturo Londoño-Vallejo, Marie-Josèphe Giraud-Panis, Eric Gilson 
Cell 
Volume 142, Issue 2, Pages (July 2010)
DOI: /j.cell
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1 TRF2 Stimulates Apollo 5′-3′ Exonuclease Activity
(A) Denaturing PAGE analysis of DNA products obtained after digestion of ssTelT (left), AH41 (right) by Apollo and Artemis in the presence of TRF2. Increasing amounts of TRF2 (molar ratio TRF2/nuclease: 0.1, 0.5, 1, 5, 15) were added to 4 ng TEV1-digested Apollo or 20 ng Artemis prior to incubation with DNA.
(B) Quantification of Apollo, RecJf, and Artemis activities as a function of the molar ratio between TRF1 or TRF2 and the various nucleases. Cleavage stimulation was calculated as the ratio of the fraction of DNA cleaved in the presence and in the absence of TRF proteins. The data are represented as mean ± SD.
(C) Same experiment as in (A), with Apollo and TRF1 or RecJf and TRF2.
(D) Denaturing PAGE analysis of DNA products obtained after digestion of dsTelT labeled on the top strand by 4 ng TEV1-digested Apollo in the absence or presence of a 5-fold molar excess of TRF2.
(E) Left: denaturing PAGE analysis of DNA products obtained after digestion of dsTelT labeled on the bottom strand by 4 ng TEV1-digested Apollo in the presence of increasing amounts of TRF2 (molar ratio TRF2/Apollo: 0.1, 0.5, 1, 5, 15). Right: quantification of Apollo activity as a function of the molar ratio between TRF2 and Apollo. Cleavage stimulation was calculated as in (C). The data are represented as mean ± SD.
See also Figure S1.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 2
Induction of DNA Damage Signals and Telomere Aberrations in Apollo Compromised Cells
(A) Representative images of confocal sections of 53BP1 foci (red) and TRF1 (green) in nuclei of HT1080 cells expressing either wild-type (Apwt-G) or mutant (Apm1-G) forms of Apollo or GFP and transfected with CL2 siRNA. White arrows indicate colocalization between 53BP1 and TRF1. The magnification of the images is shown as a scale bar (5 μm).
(B) Quantification of TIFs shown in (A) as well as in nuclei of cells retrovirally transduced for a shRNA directed against the coding region of Apollo. The data are represented as mean ± SD. The p value corresponds to the comparison of Apwt-G and three Apollo mutants.
(C) Representative images of the main telomeric aberration encountered in Apollo-compromised cells (STD, single-telomere deletion; DTD, double-telomere deletion; TD, telomere doublets; STA, sister telomere association).
(D, E, and G) Quantification of telomeric aberrations in metaphase spreads of cells expressing Apwt-G and three Apollo mutants as percentage of events per chromosome. The telomere deletions were determined by eye inspection of the metaphases. The numbers in brackets on the x axis indicate the numbers of chromosomes counted in each cell type. The data are represented as mean ± SD.
(F) Enlarged images of chromosomes with STD or DTD. The circles indicate the remaining faint signals.
(G) The data are represented as mean ± SD. The p value corresponds to the comparison between Apwt-G and three Apollo mutants.
(H) Representative images of telomere Q-FISH showing chromosomes with STD affecting both leading and lagging telomeres.
(I) Quantitative analysis of STD on lagging and leading sister chromatids after replication. The data are represented as mean ± SD.
∗ indicates a p value of < See also Figures S2, S3, and S4.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 3
Accelerated Onset of Senescence and Increase in Nucleoplasmic Bridges in Cells Expressing Nuclease-Inactive Alleles of Apollo
(A) Cell proliferation arrests in response to the expression of nuclease-inactive Apm1 mutant in primary fibroblasts.
(B) Representative microscopic images of β-galactosidase staining in primary cells.
(C) Representative images of one normal binucleate cell with counterstaining of β-tubulin (red), which shows the cytoskeleton between the binucleated cells and DAPI stain (blue).
(D) Typical image of one narrow nucleoplasmic bridge.
(E) The relatively wide nucleoplasmic bridges in one binucleate cell in HT1080 cells expressing Apm1-G.
(F) The percentage of nucleoplasmic bridges was scored in at least 1000 binuclei in each indicated cell condition. The data are represented as mean ± SD.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 4
Apollo Dysfunction Triggers a Replication Delay in TRF1-Enriched Telomeric Chromatin
(A) FACS analysis of cells released from G1/S block at the time points indicated.
(B) Total BrdU incorporation in the genome. The data are represented as mean ± SD.
(C) The amount of telomeric DNA in the total DNA immunoprecipitated with anti-BrdU antibodies was assayed by slot blot. The data are represented as mean ± SD.
(D) ChIP experiments on synchronized cells incubated with BrdU. Precipitations were performed with antibodies against TRF1. The precipitated DNA was analyzed by slot blot with telomeric and Alu probes.
(E) Densitometric evaluation of the experiment shown in (D) and two other independent experiments. The data are represented as mean ± SD.
(F) Detection of BrdU on the slot blot of TRF1 ChIP by hybridization and exposure to anti-BrdU antibodies.
(G) Densitometric evaluation of the experiment shown in (F) and three other independent experiments. The data are represented as mean ± SD.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

7. Figure 5
The Nuclease Domain of Apollo Protects Interstitial Telomeric DNA from Breakage
(A) Schematic representation of the 800 bp long interstitial telomeric sequence (4qITS) artificially integrated in the middle of chromosome 4q in SNG28 cells. Comparative staining of metaphase chromosomes with a specific 4qITS green probe and a red-labeled chromosome 4 painting probe. ChIPs were revealed by qPCR of fragments located 0.2 kb and 1.5 kb from the 4qITS and of sequences in the ANT1 and FRG1 genes located approximately 4.8 and 0.4 Mb from the starting position of telomeric DNA at the end of chromosome 4q.
(B) Representative images of confocal sections using FISH staining of the 4qITS region (green) and IF staining of 53BP1 (red). The magnification of the images is shown as a scale bar (20 μm for HT1080 cells expressing empty and TRF2ΔBΔM nuclei; 5 μm for Apwt-G, Apm1-G, shCtl and shPOT1 nuclei).
(C) The percentage of colocalizing signals using the 4qITS probe and 53BP1 antibodies in (B). The data are represented as mean ± SD.
(D) Representative images of confocal sections using FISH staining of the 4qITS and 4qITS regions and IF staining of 53BP1 in HT1080 cells treated with 3 μg/ml ICRF-193, 1 μM RHPS4, and 1 μM hydroxyurea for 24 hr. 4qST indicates a probe specific of the 4q subtelomeric region. The magnification of the images is shown as a scale bar (5 μm). The data are represented as mean ± SD.
(E) The percentage of colocalizing signals using the 4qITS probe and 53BP1 antibodies in (D).
∗ indicates a p value of < See also Figures S5 and S6.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

8. Figure 6 Apollo and TRF2 Assist TOP2α to Protect Telomeres
(A) Images of confocal sections show reduced expression of TOP1 or TOP2α (red) in HT1080 cells processed 3 days after their transfection with siRNA targeting TOP1, TOP2 α and mock gene.
(B) Bar graph showing the mean number of TIFs per nucleus. The p values indicate the comparison between Apm1-G and the GFP nuclei. The data are represented as mean ± SD.
(C) Overexpression of Apwt-G or TRF2 rescues the telomere dysfunction induced by the treatment with ICRF-193 or siRNA TOP2α, as examined by IF. The bar graph shows the mean number of TIFs per nucleus. The p values indicate the comparison between Apwt-G and the GFP nuclei. The data are represented as mean ± SD.
(D) Telomeric ChIPs were performed using HT1080 cells infected with the indicated viral vector. Duplicate slot blots were probed for telomeric or Alu DNA.
(E) Quantification of the data in (D). The relative association of various topoisomerases with telomeric or Alu DNA was calculated by normalizing the percentage of telomeric DNA recovered in each ChIP to that recovered in cells transfected with the control vectors. The data are represented as mean ± SD.
(F) Representatives images of nuclei with high (left) and low (right) level of TOP2α in HT1080 cells overexpressing wild-type Apollo (Apwt-G) or control cells overexpressing GFP. The rectangle indicates an area of the nucleus, which is shown enlarged at the top of the panel. White arrows indicate foci with a colocalization of TOP2α and telomeric PNA. Images were generated by projection of optical sections acquired at 0.3 μM intervals with the LSM-500 confocal imaging system from Zeiss. The entire nucleus is presented. The scale bar represents 5 μm.
(G) Representative image of a field of cells costained with PCNA and TOP2a antibodies as well as with PNA Telo probe. The magnification of the image is shown as a scale bar (20 μm).
(H) Bar graph showing the average number of colocalized TOP2α and telomeric PNA foci in various subsets of cells. The data are represented as mean ± SD.
∗ indicates a p value of < See also Figure S7.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

9. Figure 7
A Topology-Based Model for TRF2-Mediated Coupling of the Exonuclease Activity of Apollo to Telomere Replication
(A) Left: bandshift experiment showing the binding of TRF2 on a 3200 bp linear DNA containing 650 bp of human telomeric repeat (pTelo2 Lin). 5′-labeled pTelo2 Lin (0.1 nM) was incubated with increasing quantities of TRF2. Lane 1, the sample did not contain protein. Lanes 2–11, samples containing 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, 40, and 100 nM of TRF2. Middle: agarose gel migrations of the negatively supercoiled (−SC), positively supercoiled (+SC), relaxed (R), and linear pTelo2 plasmids used in the bandshift and competition experiments in (A) and (B). Right: Competition assay results. 5′-labeled pTelo2 Lin (0.1 nM) was mixed with increasing quantities of cold +SC (lanes 3–8), −SC (lanes 9–14), and R (lanes 15–20) pTelo2. Ratios between competitors and labeled DNA were as follows: 0.5, lanes 3, 9, and 15; 1, lanes 4, 10, and 16; 2, lanes 5, 11, and 17; 5, lanes 6, 12, and 18; 10, lanes 7, 13, and 19; and 15, lanes 8, 14, and 20. The DNA mixture was incubated with 10 nM purified TRF2. Lane 1 contains only the labeled pTelo2 Lin probe, and the same DNA in lane 2 was incubated with TRF2 in the absence of competitor.
(B) Competition assay results. 5′-labeled pTelo2 Lin (0.1 nM) was mixed with increasing quantities of cold +S (lanes 2–5), −SC (lanes 7–10), and R (lanes 11–14) pTelo2. Ratios between competitors and labeled DNA were as follows: 2, lanes 2, 7, and 11; 5, lanes 3, 8, and 12; 10, lanes 4, 9, and 13; and 15, lanes 5, 10, and 14. The DNA mixture was incubated with 20 nM purified TRF1. Lane 6 contained only the labeled pTelo2 Lin probe, and the same DNA in lane 1 was incubated with TRF1 in the absence of competitor.
(C) ChIP experiments were performed on cells treated with 3 μg/ml of ICRF-193 for 24 hr. The slot blot membrane was hybridized with telomeric DNA probe and then with the Alu probe. IgG antibody was used as a negative control.
(D) Model for the cooperation between TRF2–Apollo and topoisomerases 2 to solve the topological stress that arises during telomere replication.
See also Figure S1.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

10. Figure S1
TRF2 Does Not Exhibit a 5′ Exonuclease Activity, Apollo Mutants Do Not Harbour Exo- nor Endo- Nucleolytic Activities and TRF2 Prefers Positively Supercoiled DNA in a Non-telomeric Context, Related to Figures 1 and 7
(A) Denaturing PAGE analysis of the DNA products obtained after digestion of ssTelT by Apollo in the presence of TRF2. Increasing amounts of TRF2 (molar ratio TRF2/nuclease: 0.1and 15) were added to 4 ng of TEV1 digested Apollo prior to incubation with DNA. In the last lane the DNA probe is incubated with TRF2 only at the highest concentration used in the experiment.
(B) Denaturing PAGE analysis of the DNA products obtained after digestion of ssTelT by TEV1 digested wild-type Apollo (ApWT) or increased amount of Apm1, Apm2, Apm3 mutants and control sample (60ng, 100 ng, 200 ng and 400 ng).
(C) Sequence alignment of a region of the β-lactamase domain of Apollo and the Apollo mutants. Underlined residues are conserved in hSNM1, Apollo and Artemis. Boxed residues correspond to those mutated in the various Apollo mutants.
(D) Sequence design of anApollo gene resistant to the siRNA CL2.
(E) Agarose gel migrations of the positively supercoiled (+SC), negatively supercoiled (-SC), relaxed (R), and linear pUC19 plasmids used in the competition experiments in panels B and C.
(F) Competition assay results. 5′-labeled pUC19 Lin (0.1 nM) was mixed with increasing quantities of cold +SC, −SC and R pUC19. Ratios between competitors and labeled DNA are noted above the gel. The DNA mixture was incubated with 100 nM purified TRF2.
(G) Competition assay results. 5′-labeled pTelo2 Lin (0.1 nM) was mixed with increasing quantities of cold +SC pUC19. Ratios between competitors and labeled DNA are noted above the gel. The DNA mixture was incubated with 10 nM purified TRF2.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

11. Figure S2
Expression and Phenotypes of Apollo Nuclease-Dead Mutants, Related to Figures 2 and 3
(A) Indirect immunofluorescence (IF) showing the colocalization of GFP-tagged Apollo proteins (green) together with TRF1 (red). The magnification of the images is shown as the scale bar (5 μm).
(B) Quantification of the percentage of wild-type or mutant foci colocalizing with TRF1 in the experiment shown in panel A. ∗ indicates p<0.05.
(C) Slot-blotting analyses of immunoprecipitates from cross-linked chromatin preparations of cells expressing GFP-tagged Apollo proteins using anti-GFP antibodies. The same filter was first hybrized with a telomeric DNA probe (Telo probe) and then, after a complete dehybridation, to an Alu probe (Alu probe).
(D) Images of confocal sections of 53BP1 foci (green) and TRF1 (red) in HT1080 nuclei. White arrows show the 53BP1 foci colocalized with TRF1 and named TIF for telomere damage Induced Foci. The histogram next to the images indicates the quantification of TIFs shown in panel D. The bar graph shows the percentage of nucleus with more than 6 TIFs. p values indicate the comparison between Apollo-GFP and three mutant Apollo-GFP nucleus, respectively; ∗ indicates p < The magnification of the images is shown as the scale bar (10 μm).
(E) Cell cycle analysis by flow cytometry.
(F) A representative image shows that cells exhibiting the highest (green arrows) and lowest (white arrows) levels of ectopic Apwt-G have a similar range of TIF. The histogram indicates that the increased number of TIFs is not due to the expression level of Apollo but to the nuclease-dead mutations.
(G) The histogram indicates the marked decrease of transcription level of exogeneous and endogeneous allels of Apollo due to the expression of shAp. FACS analysis also shows the severe reduction of Apollo-G level as expressed by the MEAN and MEDIAN on the right side of the graph due to the expression of shAp.
(H) Schematic representation of sequential transduction using the pWPIR TRF2ΔBΔM lentivirus and the pBABE Apm1-GFP retrovirus'and transfection of SiCL2 in HT1080 Cells.
(I) Quantification of the frequency of telomere fusions for 100 chromosomes. n indicates the numbers of chromosomes counted for each experiment.
(J) The expression of the nuclease-dead Apm1 exacerbates the percentage of telomere fusion in TRF2ΔBΔM cells and causes the appearance of chromosome trains when cells are further depleted of the endogenous Apollo protein by the SiCL2 RNAi in the same background. Telomere fusions are visualized in metaphase spreads stained with a FITC-PNA probe (CCCTAA). The arrows show examples of telomere fusions.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

12. Figure S3
Apollo Dysfunction Does Not Activate the ATR Pathway of DNA Damage Response and Does Not Change the Length of Telomeric Single-Stranded DNA, Related to Figure 2
(A) hTERT immortalized fibroblasts (T11hT) were transfected with Apollo siRNA CL2. This treatment leads to an inhibition of the Apollo mRNA of roughly 88% and triggers a significant increase in the amount of TIFs (data not shown). 3 days after transfection, cells were processed for immunofluorescence with antibodies to ATR and RPA. To visualize ssDNA foci, Apollo siRNAs transfected T11hT cells were grown for one population doubling in the presence of BrdU and immunostained in native conditions with an anti-BrdU antibody. Cells treated with 5 mM Hydroxyurea (HU) for 2h were used as a positive control of replication stress. By contrast to HU-reated cells, we failed to find large foci of ATR after the inspection of a large number of nuclei. For RPA and native BrdU, we counted the number of foci and failed to detect an increase in Apollo knock-down cells. The values above the mRNA levels correspond to the number of counted nuclei.
(B) same condition as in (A) except that the RPA and native BrdU immunofluorescence experiments were combined with antibodies against TRF1. Although the treatment with HU increases the number of foci showing a colocalization between native BrdU and TRF1, we failed to detect any significant increases of RPA or BrdU staining in Apollo-compromised cells.
(C) Combined immunofluorescence against ATR or RPA34 with PNA FISH against telomeric DNA.
(D) Immunoblotting against the phophorylated form of CHK1.
(E) G-strand overhang analysis by in-gel hybridization. HinfI/RsaI digested genomic DNA isolated from HT1080 cells was separated on agarose gel. The native gel was then processed for hybridization to (CCCTAA)4 labeled probe (left panel). The gel was subsequently denatured and hybridized to the same probe (right panel).
(F) Quantitation of the G-strand overhang signals. The relative amount of the 3′overhang was calculated by normalizing the signal before and after denaturation. siCL2 and siCL15 target Apollo.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

13. Figure S4
The Expression of Nuclease-Dead Alleles of Apollo Leads to a Decreased Proportion of Long Telomeres, Related to Figure 2
(A) A Southern-blot experiment of telomere restriction fragments performed 7 days post selection on HT1080 cells expressing wt-Apollo-GFP and the three nuclease-dead alleles. Genomic DNA was digested with HinfI and RsaI and telomeric DNA revealed using the TTAGGG 32P labeled probe.
(B) Profiles and quantification of the DNA bands on the Southern-blot shown in A. The percentages refer to the area of the long (above the average length) or short (below the average length) telomeres. Dot lines show the peak of DNA signal intensity for each lane and the numbers below the corresponding molecular weight.
(C) Cells were grown in the presence of BrdU/C for one round of replication before metaphase preparation. A first hybridization was performed with a telomere-specific C-rich PNA probe (red), thus revealing the G-rich strands of both sister chromatids on metaphase chromosomes (top panel). At least 25 metaphases were captured and saved along with their position coordinates. Next, the same slides were treated for CO-FISH, which produces single stranded chromatids through degradation of the neosynthesized (base-substituted) strands. Preparations were hybridized again with both C-rich (red) and G-rich (green) probes to reveal the parental strands (middle panel). The same metaphases captured after the first procedure were retrieved and captured again to distinguish the mechanism of replication (leading = C-strand; lagging = G-strand).
(D) Using this information, the fluorescence intensities corresponding to lagging and leading sister chromatids were measured on the initial images (top panel). Only extremities that could be unambiguously assigned to a particular replication mechanism were included in the calculation of mean fluorescence intensity (bottom panel). The number of extremities analyzed per sample is indicated within the bars.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

14. Figure S5
Analysis of Interstitial Telomeric Sequences, Related to Figure 5
(A) The qPCR experiments were performed with the same Chip DNA samples as in Figure 5A with primers targeting either non-telomeric region (beta-actin or histone H4 loci) or natural ITS regions (chromosome 10q, 13q, Yq).
(B) TRF1(red) colocalized with a specific ITS FISH probe (green, schematic shown in Figure 5A) in SNG 28 cells bearing the 4qITS insertion. Cells were harvested either 5 days after overexpression of pWPIR TRF2ΔBΔM or pWPIR empty vector as a control, or 21 days post selection after pBabe Apollo-GFP or pBabe Apollo mt1-GFP transduction. Cells were first extracted with Triton X-100 and dehydrated to remove the GFP signal as seen by fluorescence microscopy. DNA was stained with DAPI (blue).
(C) Percentage of ITS probe colocalizing with TRF1 in the experiment shown in panel B.
(D) TRF2 (red) also colocalized with a specific ITS FISH probe (green) in cells after pBabe Apollo-GFP or pBabe Apollo mt1-GFP transduction. Cells were processed 7 days after selection. DNA was stained with DAPI. The magnification of the images is shown as the scale bar (10 μm).
(E) Percentage of ITS probe colocalizing with TRF2 in the experiment shown in panel D.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

15. Figure S6
Pot1 Knockdown Triggers DNA Damage Signal at Chromosome Ends Not at the 4qITS, Related to Figure 5
(A) Representative confocal section images of 53BP1 and TRF1 foci. Cells were processed 21 days post selection.
(B) The slot blots were performed using the same ChIP DNA samples as for the qPCR in Figure 5A and in Figure S7. IgG antibody was used as a negative control, and histone H3 antibody as a positive control. The intensity of the signal was measured and quantified by the software Fuji Multi Gauge. The bar graphs show the enrichment of telomeric DNA in precipitated bulk DNA. Values were obtained by first calculating the ratio between the ChIP signals in the various conditions and the ChIP signal obtained for histone H3 separately in TELO or in ALU and then by dividing TELO versus ALU. The magnification of the images is shown as the scale bar (10 μm).
(C and D) Pot1 knockdown, as well as RHPS4 treatment (1uM, 24 hr) increases the telomere dysfunction at telomeres. The bar graph shows the percentage of indicated cells with more than 4 TIFs.
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions

16. Figure S7
Topoisomerase 2 Dysfunction Induces DNA Damage at Telomeres, Related to Figure 6
(A) Representative images of confocal sections used to quantify the TIFs shown in Figure 6B. The scale bar represents 10 μm.
(B) Mean number of 53BP1 foci corresponding to the TIF quantification of the ICRF-193 (3 μg/ml) experiments shown in Figure 6C.
(C and D) In contrast to ICRF-193, which induces DNA damages at telomeres (TIF) without a marked increase in the number of 53BP1 foci, bleomycin (1 μg/ml, 24hs) triggers a global increase of DNA damage (C) but not an enhanced frequency of TIF (D).
Cell  , DOI: ( /j.cell )
Copyright © 2010 Elsevier Inc. Terms and Conditions