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Published OnlineFirst December 17, 2014; DOI: 10.1158/1541-7786.MCR-14-0381

Cell Cycle and Senescence Molecular Cancer Research DNA-Directed Polymerase Subunits Play a Vital Role in Human Telomeric Overhang Processing Raffaella Diotti, Sampada Kalan, Anastasiya Matveyenko, and Diego Loayza

Abstract

Telomeres consist of TTAGGG repeats bound by the shelterin could also show that the CST, shelterin, and polymerase com- complex and end with a 30 overhang. In humans, telomeres plexes interact, revealing contacts occurring at telomeres. We shorten at each cell division, unless telomerase (TERT) is found that the polymerase complex could associate with telome- expressed and able to add telomeric repeats. For effective telomere rase activity. Finally, depletion of p180 by siRNA led to increased maintenance, the DNA strand complementary to that made by overhang amounts at telomeres. These data support a model in telomerase must be synthesized. Recent studies have discovered a which the polymerase complex is important for proper telomeric link between different activities necessary to process telomeres overhang processing through ﬁll-in synthesis, during S phase. in the S phase of the cell cycle to reform a proper overhang. These results shed light on important events necessary for efﬁcient Notably, the human CST complex (CTC1/STN1/TEN1), known to telomere maintenance and protection. interact functionally with the polymerase complex (POLA/primase), was shown to be important for telomere processing. Here, Implications: This study describes the interplay between DNA focus was paid to the catalytic (POLA1/p180) and accessory replication components with proteins that associate with chro- (POLA2/p68) subunits of the polymerase, and their mechanistic mosome ends, and telomerase. These interactions are proposed to roles at telomeres. We were able to detect p68 and p180 at be important for the processing and protection of chromosome telomeres in S-phase using chromatin immunoprecipitation. We ends. Mol Cancer Res; 13(3); 402–10. 2014 AACR.

Introduction suggesting coordination between telomerase and the C-strand synthesis machinery. In humans, the mechanism of C-strand Telomeres are structures located at the end of linear chromo- processing is tightly regulated, as demonstrated by the observa- somes, essential for their stability and integrity. They consist of 0 0 0 tion that the 5 ends in AATC-5 80% of the times and in AATCC-5 stretches of repetitive DNA sequences and the protein complex 0 15% of the times (6). The terminal 5 nucleotide, however, bound to them, known in mammals as shelterin. Their role is to becomes randomized upon the depletion of POT1 (7), the protect the chromosome ends from being inappropriately overhang binding protein in the shelterin complex. Different recognized by the DNA damage machinery (1). The ends of 0 models for the creation of the 3 G-strand overhang have been mammalian telomeres exhibit a 30 overhang of 50 to 300 proposed and, in general, they invoke an interplay of leading and nucleotides, produced by competing actions of telomerase, the lagging strand processing due to the intrinsic properties of extending it by repeat additions, by ExoI and Apollo, which DNA replication and telomerase activity (see for example ref. 8). create the 50 resected end (2), and by C-strand fill-in processing. Recently, it has been elegantly shown that the two strands are in Another complex, called the CST complex, is known to limit fact differentially processed during replication (9). The leading telomerase activity in S phase (3), and to associate with the overhang is initially a blunt end that is processed later in S phase RNA primer synthesizing complex Pola–primase (4), thereby for the creation of the overhang. The lagging strand overhang contributing an additional activity likely to participate in over- length is first determined by the placement of the last RNA primer, hang processing. which is then removed before further processing (9). Final over- In human cells, telomeres shorten at each cell division, unless hang processing occurs in late S–G . This study establishes the telomerase is present and active in adding TTAGGG repeats. 2 basis for a differential processing of the two strands without During active telomere elongation in telomerase-positive cells, excluding other additional processing events for the C-strand. no significant change is observed in mean overhang length (5), Additional indication of differential metabolism for the two terminal strands was described in the mouse system, in which the exonucleases Apollo and ExoI, were found to be important for Department of Biological Sciences, Hunter College and CUNY Grad- overhang production through 50 end resection at the leading and uate Center, New York, New York. lagging strands, respectively (2). This work also elucidated an Note: Supplementary data for this article are available at Molecular Cancer important role for POT1b in both leading and lagging strand Research Online (http://mcr.aacrjournals.org/). overhang formation through the coordination of the nucleases Corresponding Author: Diego Loayza, Hunter College, City University of New action and the C-strand fill-in through association with the CST York, 695 Park Avenue, New York, NY 10065. Phone: 212-772-5312; Fax: 212-772- complex. 5227; E-mail: [email protected] In Saccharomyces cerevisiae, as well as in most other eukar- doi: 10.1158/1541-7786.MCR-14-0381 yotes, the CST complex, composed by three RPA-like proteins, 2014 American Association for Cancer Research. Cdc13-Stn1-Ten1 (CTC1-OBFC1-Ten1 in humans), was found

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to be important for telomere protection and replication into rabbits, as per the protocol set by the manufacturer (BioSyn- (10, 11). The role of the CST complex in the coordination of thesis). The peptides were: NH2-GCKGRQEALERLKKAKAGEK- telomere elongation in humans was corroborated by the find- OH for p180, and NH2-GCRLYLRRPAADGAERQSP-OH for p68. ings that it contributes to limiting telomerase activity at extend- The peptide for FEN1 NH2-GCSTKKKAKTGAAGKFKRGK–OH, ing telomeres through complex interactions with telomerase for TRF1 NH2-GCGSIEKEHDKLHEEIQNLI-OH (as described itself and TPP1/POT1 (3). In addition, it was recently shown in ref. 24), for POT1 NH2-CYGRGIRVLPESNSDVDQLKKDLES that CST plays a role in both general telomere replication and (as described in ref. 26), for TPP1 NH2-GCTGPRAGRPRA- overhang processing, particularly for longer telomeres that QARGVRGR-OH, and for OBFC1 NH2-GCKTKIEIGDTIRVRG- represent a challenge for the replication machinery. Specifically, SIRT-OH. The p53BP1 antibody was purchased from Novus C-strand fill in was affected in OBFC1-depleted cells, delaying (NB100-304). The TRF2 antibody used for immunofluorescence the processing that leads to the final, mature G-overhang was purchased from Millipore, clone 4A794 (05-521). The Chk1- (12, 13). The human CST complex, in addition, was isolated p-Ser345 antibody was purchased from Cell Signaling Technol- as a set of accessory factors for the POLa–primase complex (4). ogy (#2348). The p68 and p180 antibodies used for Western blot In S. cerevisiae, Cdc13 is able to interact with POL1, the POLa analyses and TRAP assays were purchased from Abcam (Ab57002 D homolog, and STN1 interacts with POL12, the regulatory and Ab65009). The POT1 OB and FLAG-TRF1 constructs and cell subunit of Pola–primase, which is named POLA2 in humans lines are described in refs. 24 and 26. (14). The POLa–primase complex is believed to be important for replication of the lagging strand and the C-strand fill in for Plasmids both strands after resection. The complex is composed by the The cDNA for p68 (gene name POLA2) was purchased as a full- catalytic subunit, Pola, also termed p180, two primase sub- length clone from the EST collection maintained by Invitrogen. units, and the regulatory subunit POLA2, or p68 (see ref. 15). The full-length cDNA was amplified by PCR using primers with Although it has recently been established that telomeres, not- appropriate cloning sites (50 BamHI and 30 EcoRI) and cloned into withstanding their reported nature as fragile sites of DNA pLPC-MYC (25) to generate a MYC-tagged version driven by the replication on chromosomes (16), do not undergo a telo- CMV promoter. The PCR oligonucleotides were: 50-TGCTTAG- mere-specific replication program, but mainly a chromo- GATCCGCATCCGCCCAGCAGCTG-30 and 50-TGGAGAGAATT- some-specific one (17), it has been found that partial altering CTCAGATCCTGACGACCTGCACAG-30 corresponding to target of the replication machinery can have telomere-specific effects. sites for codons 2–7 at the 50 end and the last 7 codons of the Mouse cells with a temperature-sensitive p180 exhibit overhang cDNA, including the stop codon. The OBFC1 cDNA was PCR extension and concomitant overall telomere elongation (18). cloned from a complete EST purchased from ATCC as a template, Therefore, although defects in DNA replication and in partic- and with the following two oligonucleotides: 50-ATAACACA- ular in Pola–primase activity are predicted to affect general GATCTCAGCCTGGATCCAGCCGGTGTG-30 and 50-TTCACCTC- chromosomal DNA replication, it is possible that this activity TCGAGTCAGAACGCTGTGTAGTAGTGC-30, yielding a BglII-XhoI displays noncanonical, telomere-specificroles,inparticularin fragment as a PCR product. The TPP1 EST was purchased from overhang processing. Invitrogen and PCR cloned with the following two oligonucleo- In addition, telomerase was found physically associated with tides 50-AGGAGGATCCCCTGGCCGCTGTCAGAGTGACG-30 and primase and the lagging strand replication machinery in the ciliate 50-GAGGACTCGAGTCACATCGGAGTTGGCTCAGAC-30,yielding Euplotes crassus (19). This association appeared to be develop- a BamHI-XhoI fragment as a PCR product. mentally regulated as it occurred specifically in mated cells, and not in vegetatively growing cells. Genetic evidence in fission and budding yeast also implicates primase subunits in telomere Depletions by siRNA or shRNA replication (14, 20, 21), although the mechanisms at play remain HeLaII cells were maintained in DMEM (Invitrogen) supple- unclear. mented with 1% penicillin and streptomycin and 10% FBS. The Our study investigates these possible roles at human telo- siRNAs used were synthesized by Dharmacon RNA Technologies. meres, and focuses on p68 (POLA2) and p180 (POLA1). We For p68 RNAi, double-stranded siRNA were designed to target the show that they are present at telomeres in S phase, interact following sequences: p68-1 siRNA 50-UGGAAGAAGAAGAG- with the shelterin complex and with the CST subunit OBFC1, GAAAUUU-30 (target in the 50 UTR, exon 3) and p68-2 siRNA as well as with telomerase itself, and that they are important 50-UAUCUGAGCUUAAGGAAUAUU-30 (coding sequence, exon for the regulation of telomeric overhang amounts in human 7). For p180: p180-1 siRNA 50-CUGAGUACUUGGAAGUUAA-30 cells. (coding sequence, exon 13); p180-2 siRNA 50-CAGAUCAUGU- GUGAGCUAA-30 (coding sequence, exon 21); p180-3 siRNA 50- GAGAGUAGCUGGAAUGUAA-30 (30UTR, exon 37). HeLaII cells Materials and Methods were transfected using Lipofectamine (Invitrogen) according to Cell lines and antibodies the manufacturer's instructions. Briefly, cells at a confluency of The HeLaII line is a subclone of HeLa S3 (ATCC CCL-2.2), with approximately 50% to 60% were plated in a 6-well plate 18 to 24 telomere length in the 3- to 6.5-kb range (22), and used in ref. hours before transfection. Transfections were done once and cells (23). The HTC75 cell line is a HT1080 derivative described in were processed 48 hours after transfection for protein extraction ref. 24. The cells were grown in DMEM/10% BCS, and the or immunofluorescence. As a control, siGFP (Dharmacon) was retroviral transduction protocol was identical to that described used. For shRNA, the LMP vector from Open Biosystems was used, in ref. 25. Cells were selected for the plasmid with 2 mg/mg which is based on the miR30 miRNA. The target sequences were puromycin, where applicable. All rabbit sera used were generated PCR cloned according to the manufacturer's protocol based on the against a peptide conjugated to KLH and used for immunization NM_002689.2 sequence for the p68 cDNA. The target sequences

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were: sh50UTR, 50-CTCTGCCACCGTCACTGAGAAGTAGTGAA- the untreated sample signal. For the coimmunoprecipitation GCCACAGATGTACTTCTCAGTGACGGTGGCAGAA-30; sh664, experiments, the enrichment values were obtained by subtracting 50-CCCTCTTGAACTCTTACACCACTAGTGAAGCCACAGATGTA- the signal from the preimmune from the serum signal for each GTGGTGTAAGAGTTCAAGAGGA-30; sh1088, 50-ACCTGTCAC- antibody. TCTGCTGGGCCAGTAGTGAAGCCACAGATGTACTGGCCCAG- 0 0 0 CAGAGTGACAGGC-3 ; sh3 UTR, 5 -AATGCTCCGTGTCCAGAA- TRAP assays GTAATAGTGAAGCCACAGATGTATTACTTCTGGACACGGAGC- 0 The TRAP assays were performed as per the manufacturer's ATG-3 . protocol (Millipore, S7710). For the IP-TRAP assays, the immunoprecipitations were performed as described above, and the Immunofluorescence beads were washed six times in cold lysis buffer. The beads were Immunostaining for p53BP1 and TRF2 was performed for then resuspended in 40 mL of lysis buffer, and 2 mL of the HeLaII cells plated onto glass coverslips and processed for RNAi. resuspended beads were used as input for the TRAP assay. For After the 48-hour transfection period, cells were extracted with Tx RNAse-treated controls, 20 mL of resuspended beads were buffer (0.5% Triton X-100, 20 mmol/L Hepes-KOH, pH 7.9, 50 removed and treated with 10 mg of RNAse A at 37 C for 30 mmol/L NaCl, 3 mmol/L MgCl2, 300 mmol/L sucrose) for 10 minutes and washed twice with cold lysis buffer. minutes at room temperature (RT). After two PBS washes, the cells were fixed with PBS/3% paraformaldehyde, 2% sucrose for 10 Chromatin immunoprecipitations minutes at RT. After two PBS washes, cells were permeabilized The chromatin immunoprecipitations (ChIP) were performed, with Tx buffer for 10 minutes at RT, washed twice with PBS, and as described in ref. 26, on HeLaII cells synchronized by double blocked with PBG (PBS/0.2% fish gelatin. 0.5% BSA) for 30 thymidine block as described. minutes. Coverslips were then incubated with the rabbit anti- p53BP1 antibody (Novus NB100-304A-1), at a concentration of In-gel hybridization 1:500 in PBG overnight. Cover slips were then rinsed three times Genomic DNA was isolated from cells as described in ref. 25 with PBG solution and incubated with secondary TRITC-conju- and processed for in-gel hybridization. The DNA was digested gated goat anti-rabbit antibody (Jackson Immunoresearch) in with AluI and MboI, and control samples were further digested PBG at a concentration of 1:500 for 45 minutes at RT. Cover 0 with ExoI to digest the signal derived from the 3 overhang. Of slips were rinsed two times with PBG. Coverslips were then note, 4 mg of DNA for each sample was loaded on a 0.7% agarose incubated with PBG and DAPI at 100 ng/mL to visualize the gel in 0.5X TBE. Following electrophoresis, the gels were dried at nuclei. Coverslips were mounted onto slides with embedding RT for at least 3 hours, and then prehybridized with in Church mix media. Images were collected with an Olympus BX61 fluorescence (0.5 mol/L Na2PO4, pH 7.2, 1 mmol/L EDTA, pH 8.0, 7& SDS, 1% microscope using a 60 objective connected to a Hamamatsu BSA) for 30 minutes at 50 C and hybridized overnight with end ORCA-ER CCD camera, controlled by the SlideBook 5.1 image labeled (CCCTAA) or (TTAGGG) oligonucleotides at 50 C. capture software. The telomere FISH shown in Supplementary Fig. 4 4 After hybridization, the gels were washed three times with 4 S1C was performed as described in ref. 2. SCC and one time with 4 SCC, 0.1% SDS at 55 C, and exposed overnight to a PhosphorImager screen. To detect the total telo- Cell synchronization and FACS mere signal, the gels were then denatured in 0.5 mol/L NaOH and HeLaII were plated 106 cells in 10-cm plates. After 24 hours, 1.5 mol/L NaCl for 30 minutes and neutralized (3 mol/L NaCl, fi thymidine to a nal concentration of 2 mmol/L was added to the 0.5 mol/L Tris-HCl, pH 7.0) twice for 15 minutes, rinsed with dH2 media. The cells were treated with thymidine for 14 hours, and O and hybridized with the end-labeled oligonucleotides. The gels then they were rinsed three times with warm PBS and fresh were then exposed overnight to a PhosphorImager screen, and the medium without thymidine. The cells were released for 11 hours G-overhang signal was calculated by dividing the native fi and then thymidine, 2 mmol/L nal concentration, was added to (CCCTAA)4 signal by that obtained with the denatured gel. the medium again for the second block. After 14 hours the cells, were released as above and collected at the appropriate time Telomeric 30overhang analysis by double-strand specific points for ChIP and FACS. For FACS, cells were collected and nuclease (DSN) rinsed twice in cold PBS, resuspended in 0.2 mL of PBS/2 mmol/L The DSN reaction was performed as previously described (27). EDTA, 2 mL of cold 70% ethanol was added dropwise and the cells Four micrograms of genomic DNA in 1 DSN buffer was digested fi were kept at 4 C 24 hours for xation. The cells were then spun with 0.2 U/mg of DNA of DSN (Axxora cat. EVN-EA001-KI01) at down and resuspended in 0.5 mL of PBS/2 mmol/L EDTA. Of 37C for 2 hours. As a control, 10 U of ExoI were added to the m m note, 10 L of heat-inactivated RNase A (10 mg/mL) and 25 Lof genomic DNA before DSN and incubated at 37C for 1 hour to propidium iodide (1 mg/mL) were added and the cells were digest terminal single-stranded DNA. The digestions were stopped incubated at 37 C for 30 minutes. The samples were then ana- with 0.5 mL of 0.5 mol/L EDTA and an equal volume of form- lyzed using a FACSCalibur Flow Cytometer. amide before heating the samples at 65C for 5 minutes. The samples were run on a 6% denaturing polyacrylamide gel contain- Cell extracts and immunoprecipitations ing 8 mol/L urea. Electrophoresis was performed at 15V/cm in Cell extracts and immunoprecipitations were performed as 0.5 TBE. The gel was then electroblotted onto a Hybond N described in ref. (26). The quantitations shown in Fig. 4 were membrane (Amersham-GE) in 0.5 TBE buffer at 4C and 30V done using the ImageJ analysis software. For siRNA and shRNA for 90 minutes. The membrane was then air dried, UV cross- Western blot analyses, the relative ratios were obtained by nor- linked, and hybridized to a C-rich telomeric probe at 42C. The malizing the p180 and p68 signal to GAPDH and by dividing by membrane was washed with 0.2 mol/L wash buffer (0.2 mol/L

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more significant reduction of Pola activity. Knockdown of p180 generated a broad DNA damage response in the cell, as judged by the induction of p53BP1 foci (Fig. 2A). This effect was not observed upon depletion of p68. The average number of nuclei with three or more p53BP1 foci went from below 8% in control or p68 siRNA, to 35% with our best p180 siRNA targets (Fig. 2C). This observation likely corresponds to a broad induction of DNA damage, and not telomere deprotection, because no colocaliza- tion with telomeres was observed in this case (Supplementary Fig. S1C). The DNA damage response seen with p180 depletions is compatible with replication stress, as a significant activation of Chk1 was observed (Fig. 2B), known to result from induction of ATR. Upon quantitation of the signal detected for Chk1-Ser-345 phosphorylation, the levels of activated Chk1 increased by at least 5-fold upon p180 depletion (Fig. 2D). We did not detect a significant effect with p68 depletion, as the signal obtained was not significantly different from the siGFP-negative control (Fig. 2B and D). Thus, disruption of Pola activity led to an apparent ATR- dependent DNA damage response as well as a mild S phase delay in the case of p180 depletion, while no overt effects on cell-cycle progression or induction of DNA damage in the case of our p68 knockdown experiments were observed.

POLa–primase components can be detected at telomeres by chromatin immunoprecipitation To determine whether p180 and p68 could be detected at telomeres, we performed ChIP, a technique widely used to study proteins localization at telomeres. The assay was carried out on Figure 1. Depletion of p68 (POLA2) or p180 (POLA) by siRNA in HeLaII cells. A, Western asynchronous as well as synchronized cell lines using anti-peptide blot analysis showing p180 depletion with three different target sites. B, rabbit sera against p180, p68, and OBFC1, the latter being a depletion of p68, with two different target sites. C, quantitation of the subunit of the CST complex. ChIP done on asynchronous HTC75 depletion of p180. D, quantitation of the depletion of p68. The signal obtained control cell lines yielded a low but reproducible signal for both for p180 or p68 was normalized to GAPDH, and divided by the signal detected p68 and p180 (Fig. 3A and B). We were able to visualize shelterin for the untreated control. The values shown are averages and SE on three components at telomeres in these cells, such as TRF1 and POT1, as independent experiments. previously reported, as well as FEN1, with a yield of 4% of the total telomeric signal, likely representing an S phase population (28, 29). In these cells, we were able to detect a low but repro- Na2HPO4 pH 7.2, 1 mmol/L EDTA, and 2% SDS). The overhang ducible signal for p68, with a yield of 3%. The p180 subunit was signal was calculated by subtracting the ExoI signal from the DSN not detected at telomeres in this setting. We also performed the signal, and by calculating the ratio of each sample over the ChIP on HTC75 cells with normal or elongated telomeres D untreated condition. (through POT1 OB expression) and found that OBFC1 and p68 were also present at elongated telomeres (not shown). As the roles Results of Pola–primase are primarily related to DNA replication in S phase, we sought to determine whether a telomeric association Depletion of p180 by siRNA, but not of p68, leads to a DNA would be occurring for p68, and perhaps also p180, during this damage response phase, as has been reported for FEN1 (28). HeLaII cells were To study the roles of p180 and p68, we performed depletions of synchronized through a double thymidine block, and the cells both proteins by siRNA and p68 by shRNA in HeLaII cells (Fig. 1 were collected at 0, 2, 4, 6, and 8 hours after release (Fig. 3C and D and Supplementary Fig. S4). Among the three siRNA targets used and Supplementary Fig. S2). We found that, in staged S phase for p180, the depletion was consistent down to about 30% of cells, both p68 and p180 could be reliably detected at telomeres, control endogenous levels (Fig. 1A and C). For p68, the ﬁrst target albeit at low levels. Although p180 peaked in the early S phase and site resulted in slightly over 50% depletion, and the second one in late S–G , p68 associated with telomeres more stably through- yielded a higher level of depletion, of 65% (Fig. 1B and D). These 2 out S phase. These results could indicate a possible role for Pola at levels of depletion correlated with mild effects on cell-cycle telomeres speciﬁcally during S phase. progression, in the case of p180, with a slight delay in S/G2–M (Supplementary Fig. S1A and S1B), and no obvious effect was seen in the case of p68. The depletions we report in this study are The POLa–primase complex interacts with shelterin milder than those previously reported by others (15), and may We then sought to determine whether an association between explain our capacity to detect the telomeric effects reported here the Pola complex and the telomeric shelterin complex could be without major inhibition of cell-cycle progression elicited with detected. We also examined possible interactions with OBFC1,

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Figure 2. Depletion of p180, but not p68, leads to formation of p53BP1 foci and Chk1 activation. A, detection of p53BP1 in HeLaII cells 48 hours after siRNA treatment for p68 or p180, with the indicated target sites. Blue, DAPI; Red, p53BP1; Green, TRF2. B, Western blot analysis showing pChk1-Ser345 for the control and siRNA against p68 or p180. The top two panels represent different exposures of the same blot. The GAPDH loading control is shown below. C, quantitation of p53BP1 foci in HeLaII cells treated with p68 and p180 siRNAs as indicated. A hundred nuclei were counted for each sample, and the averages and SE were calculated from three independent experiments. D, quantitation of Chk1 phosphorylation, as shown in B. The relative ratio was obtained by normalizing the pChk1- Ser345 signal to GAPDH and dividing by the siGFP control (mean SEM for three experiments).

given the known relationship with the Pola–primase complex interaction. MYC-TPP1 was found to coimmunoprecipitate with (4). To that end, we used HTC75 cells expressing tagged versions p180 (Fig. 4B), suggesting an interaction between the Pola and of OBFC1, POT1, TRF1, or TPP1 and asked whether each com- shelterin complexes. Similarly, MYC-p68 could be immunopre- ponent could individually be immunoprecipitated with anti-p68 cipitated in HTC75 and HeLa 1.2.1.1 with POT1 or TRF2 anti- or anti-p180 antibodies. bodies (Supplementary Fig. S3A and S3B). We confirmed these We were able to detect an association between shelterin sub- finding by immunoprecipitating endogenous p68 in HeLaII cells units POT1 and TPP1, and OBFC1 (Fig. 4A). In addition, we with antibodies against POT1, TRF1, TRF2, and TPP1 (Fig. 4C). In confirmed the association between OBFC1 and p180 reported addition, we found that FLAG-TRF1 could be pulled down with previously (ref. 4; Fig. 4A). We then extended our studies to p68 or p180 antibodies, confirming the interactions between possible interactions between p180 and p68 with shelterin com- shelterin and Pola in a different cell line. Therefore, it is possible ponents. HTC75 cells overexpressing MYC-TTP1 (Fig. 4B) and that the amounts of p180 and p68 observed at telomeres by ChIP both HTC75 and HeLa 1.2.1.1 overexpressing MYC-p68 (Sup- (Fig. 3) result from interactions at the protein level with the CST plementary Fig. S3A and S3B) were used to test the potential and shelterin complexes. The protein interactions found did not

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Figure 3. Association of p68 and p180 with telomeres by ChIPs. A, ChIP in unsynchronized HTC75 cells. The Alu probe was used as a non-telomere control. The antibodies used are indicated on top; PI, preimmune serum. B, quantitation of the DNA yields for the HTC75 ChIP. The yield for each sample was divided by the total DNA value after subtraction of the preimmune background value. C, ChIP in synchronized HeLaII cells. Cells were subjected to a double thymidine block, and processed for ChIP at 2-hour intervals after release. Probes are indicated at the bottom of the blots. D, quantitation of the yields for p180, p68, and OBFC1 at indicated time points after release. STY, values obtained with the TTAGGG (telomeric) probe. An Alu probe was used as a control.

require cell synchronization as the ChIPs did. Our results there- down telomerase activity. As controls, no TRAP activity was either fore demonstrate interactions between three known complexes, detected with preimmune serum, or with RNase-treated material the shelterin, CST, and Pola–primase complexes, in three differ- (Fig. 5A and Supplementary Fig. S4B). We found similar yields for ent human cell lines, which are likely to reflect associations FEN1, which was previously shown to associate with telomerase occurring at telomeres. activity (30), and could precipitate TRAP activity with p68 antibodies as well (Supplementary Fig. S4A). Such an interaction was The POLa–primase complex interacts with telomerase. also described by others for TPP1 (31). OBFC1, in our hands, did Our observation that Pola–primase is associated with telo- not pull down TRAP activity (Supplementary Fig. S4A). We could meres and shelterin places p180 and p68 in prime position to confirm the association between Pola–primase and telomerase by interact with the telomerase complex. We thus asked whether an coimmunoprecipitation between p180 and hTERT, the catalytic association could be detected. To this end, a HeLaII extract was subunit of telomerase (Fig. 5B): p180 antibodies could precipitate prepared for immunoprecipitations with p180 antibodies, and MYC-hTERT from a HTC75 extract, whereas OBFC1 antibodies the resulting pulled down material was used as input for TRAP did not. Thus, we are able to show the telomerase–Pola–primase activity assay to assess a possible association with telomerase. interaction in two ways, by the IP-TRAP assay and by In Fig. 5A, we show that p180 antibodies can specifically pull coimmunoprecipitation.

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We conclude that a decrease in p180 leads to increased overhang amounts, suggesting a role in the regulation of the length of the telomeric overhang without affecting the resection process creating the telomeric 50 end. It is possible that p68 is also involved in this process, although the effect appears weaker.

Discussion The results presented here provide a link between the Pola– primase complex, and two other telomeric complexes, shelterin and the CST complex. Although shelterin and CST have well- documented roles in telomere function, pertaining to the protection and maintenance of telomeres, the implication of Pola– primase in these processes poses a question: are its roles at Figure 4. telomeres related to conventional chromosome DNA replication, Interactions between Pola and shelterin components detected by IP- or are there telomere-specific, noncanonical roles for Pola–pri- Western. A–C, extracts prepared from HTC75 cells expressing tagged mase at telomeres? Although the final answer requires additional constructs, indicated on the right, were used for immunoprecipitations with work, we would argue based on our findings that the observed the antibodies indicated at the top of the lanes. The input fractions and effect on overhang processing reflects a telomere-specific role for amounts precipitated with beads only (no antibody) are shown on the a leftmost lanes of each blot. The numbers below the blots represent the fold Pol not related to origin-initiated DNA replication. First, we enrichment for a specific antibody as compared with its preimmune serum. report here for the first time an interaction between p180, and p68 The antibodies used to probe the Western blot analyses were anti-MYC 9E10 with shelterin. These types of associations were described in antibody (A and B) and an anti-POLA2 serum (C). The antibodies used for budding and fission yeast (20, 21), but never, to our knowledge, immunoprecipitations are indicated at the top of the lanes. The blot was in human cells. Second, we did not observe major defects in the probed with a commercial mouse anti-p68 antibody. cell-cycle profiles of p180- or p68-depleted cells in our study, arguing for mild effects, if any, in chromosomal DNA replication Depletion of p180 leads to increased telomeric overhang (Fig. 2 and Supplementary Fig. S2). These observations could be amounts simply explained by arguing that the remaining amounts of p180 As both shelterin and CST play important roles in telomere or p68 in our depletion experiments are sufficient to support DNA protection through overhang processing, we looked at the telo- replication, but limiting for effective overhang processing. And, meric overhang amounts in conditions where p180 or p68 were depleted. We examined both short-term depletion (48 hours) via siRNA for both p68 and p180 (Fig. 6) and long-term depletion using shRNAs for p68 (Supplementary Fig. S4). This assay involves separating restricted genomic DNA by size on an agarose gel and probing the gel in native conditions with a labeled TTAGGG oligonucleotide (Fig. 6A, left). The amounts of overhang detected for each cell line are normalized to the total telomeric DNA detected in denatured conditions (Fig. 6A, right). A role for p180 in limiting overhang length was observed in our siRNA experiments (Fig. 6A and B). Of the three targets sites used against p180, two showed an overhang increase of 35% (target site 2) and 24% (target site 3) representing values of significance below P ¼ 0.05 on three independent experiments. No significant effect was observed with the siRNAs against p68. We also performed this assay on long-term depletions for p68. The p68 shRNAs were introduced in HeLaII cells through retroviral transduction; p68 was knocked down using four different shRNAs targeting different sites on the predicted mRNA (Supple- mentary Fig. S4). In this case, we detected a mild effect for one of the target site (sh644) with a statistically significant P value (Supplementary Fig. S4C). The increase in overhang amounts averaged 15% in this case. No obvious effect on telomere length was observed. To confirm these results with a different technique, we applied the DSN assay (27) to p180-depleted HelaII cells Figure 5. (Supplementary Fig. S6). The DSN assay allows the detection of Interaction between p180 and telomerase. A, TRAP assay performed on HelaII undigested single-stranded telomeric overhangs by hybridization extract (left lane), and on immunoprecipitated material with p180 serum (S) to a labeled C-rich oligonucleotide. We found that the telomeric or a p180 monoclonal purchased from Abcam. Controls include RNase treated overhangs increased in amounts in p180-depleted cells (Supple- lysates, as well as beads only, as indicated. B, IP-Western showing immunoprecipitation of hTERT with p180 serum. The lysate was prepared mentary Fig. S6A), to an extent similar to that seen with the native from HTC75 cells expressing MYC-tagged hTERT. The blot was probed with hybridization assay (Supplementary Fig. S6B). the 9E10 anti-MYC antibody.

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Pola and Human Telomeres

negative approaches could allow for a finer analysis of telomere-specific functions for Pola–primase. An important future direction will be to understand the mechanism of action of Pola–primase at telomere, if different from or more complex than mere association with the replication fork progressing through the telomere. At present, we think it valuable to interpret these results in light of recent work showing that the CST complex, in particular OBFC1, limits telomerase activity, thereby participating in telomere length homeostasis (3) and has a specific role in C-strand fill-in (12). Knowing that OBFC1 is part of an "alpha activating factor" complex (4), but has no obvious role in the recruitment of the complex to telomeres, leads us to suggest that Pola–primase is recruited through telomere-specific interactions, perhaps involving shelterin, to lay down the terminal RNA primer and restrict the length of the overhang. Depleting amounts of Pola–primase would render this step limiting and result in increasing overhang length overall. It will be interesting to analyze the nature of the potential direct contacts between shelterin and Pola–primase, as well as between Pola–primase and the telomerase complex. In addition, although we did not observe any significant change in the terminal 50 nucleotide in our conditions using the STELA assay (not shown), 50 end resection may be altered upon stronger inactivation of the Pola complex. This would present an interesting synergy with POT1, which does influence the terminal 50 nucleotide in human cells (7). As a result of the interactions reported here, the overhang phenotype we describe is predicted to be dependent on telomerase activity, and it would thus be interesting and important to perform the depletion experiments for p68 and p180 in telomerase-negative primary cells. In this context, targeting Pola–primase, or the specific interactions with shelterin, would possibly result in immediate or premature senescence in primary cells, thereby activating an important tumor suppressor system. It is relevant to note that in mouse cells, POT1b is the recruiting activity for the CST complex, itself mediating fill- a– Figure 6. in synthesis, likely through Pol primase recruitment (2). Our Increased overhang amounts upon siRNA depletion of p180. A, in-gel data are compatible with these findings, and it would be valuable hybridization for genomic DNA from HeLaII cells treated with siRNA for p68 to test the possibility that POT1 is the recruiting factor for CST and and p180. Left, native gel probed with the labeled oligonucleotide (CCCTAA)4 Pola in human cells. Two issues remain of note in our opinion. hybridizing to the telomeric overhang. Right, same gel reprobed after First, as we did not detect obvious telomere deprotection (by denaturation to detect all telomeric sequences. Samples treated with ExoI looking at telomeric p53BP1 foci), this pathway for overhang were run alongside to control for detection of single-strand DNA in the native gel. B, overhang intensity is indicated as the ratio of the native signal over the processing is not expected to lead to immediate effects such as denatured, normalized to the siGFP control. Error bars represent the SE for apoptosis or premature senescence. However, one could hypoth- three independent experiments. The two-tailed Student t test for the two esize that lack of effective fill-in synthesis would exacerbate the so- significant values (<0.05) is shown. called "end-replication problem" and limit the proliferative potential of cells, and perhaps even of telomerase-positive tumor finally, we documented an association between telomerase and cells. Longer term experiments than those reported here in pri- the p180 and p68 subunits of Pola–primase. This association can mary human cells would be required to examine this possibility. be detected at the protein level with hTERT, the catalytic subunit of The second issue is whether the overhang phenotype requires cells telomerase, and likely reflects a functional role because we can to be passing through S phase, or whether the specific function of precipitate telomerase activity with primase subunits p68 and Pola–primase suggested here is independent of actual DNA p180. This idea is in our view interesting in that it could uncover a replication. Even though we did detect an increase in p68 and telomere-specific set of events implicating Pola–primase, which p180 at telomeres in S phase, it remains possible that some of could be targeted in tumor cells, for instance, to limit their these interactions occur outside of the context of chromosomal proliferation. Such a role could be highly conserved in evolution, DNA replication and be important for telomere protection. and be related to the finding that a special mutant allele in the Therefore, it would be valuable to assess the possible effects of POL12 budding yeast gene, which represents the p68 ortholog, Pola–primase in cells that are in G0, which do not experience displays dysregulated telomere function, including longer telo- progression through the cell cycle, S phase, or proliferation. mere length, and telomere deprotection (14). In addition, Our view, based on the results presented here, is that telo- the yeast POL12 gene interacts genetically with STN1, the OBFC1 mere function relies on multiple interactions among three ortholog in this organism (14). In human cells, dominant- important complexes, shelterin, CST, and Pola–primase. In

www.aacrjournals.org Mol Cancer Res; 13(3) March 2015 409

Diotti et al.

addition, Pola–primase appears to associate with the telomerase Analysis and interpretation of data (e.g., statistical analysis, biostatistics, complex, presumably at telomeres. Shelterin is known to be computational analysis): R. Diotti, S. Kalan, A. Matveyenko, D. Loayza quantitatively associated with telomeres, whereas the CST and Writing, review, and/or revision of the manuscript: R. Diotti, S. Kalan, a– A. Matveyenko, D. Loayza Pol primase complexes could be viewed as telomere-associated Study supervision: D. Loayza factors, acting only transiently at telomeres in S phase. We provide a– evidence here for Pol primase as an important complex to Acknowledgments consider in the context of telomere protection and maintenance, The authors thank the D.L. laboratory for help and advice, especially S. Uddin which potentially offers additional targets to counteract cell and C. Vuong for excellent technical support, and for comments on the article. transformation and proliferation during tumorigenesis. Grant Support ﬂ Disclosure of Potential Con icts of Interest This work was funded by a SC3 score award # 1SC3GM094071-01A1 from No potential conﬂicts of interest were disclosed. the NIGMS. The costs of publication of this article were defrayed in part by the payment of Authors' Contributions page charges. This article must therefore be hereby marked advertisement in Conception and design: R. Diotti, D. Loayza accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Development of methodology: R. Diotti, D. Loayza Acquisition of data (provided animals, acquired and managed patients, Received July 8, 2014; revised November 10, 2014; accepted December 5, provided facilities, etc.): R. Diotti, S. Kalan, A. Matveyenko 2014; published OnlineFirst December 17, 2014.

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410 Mol Cancer Res; 13(3) March 2015 Molecular Cancer Research

DNA-Directed Polymerase Subunits Play a Vital Role in Human Telomeric Overhang Processing

Raffaella Diotti, Sampada Kalan, Anastasiya Matveyenko, et al.

Mol Cancer Res 2015;13:402-410. Published OnlineFirst December 17, 2014.

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