Homologous Chromosomes Make Contact at the Sites of Double-Strand Breaks in Genes in Somatic G0/G1-Phase Human Cells

Homologous chromosomes make contact at the sites of double-strand breaks in genes in somatic G0/G1-phase human cells

Manoj Gandhia,1, Viktoria N. Evdokimovaa,1, Karen T.Cuencob,c, Marina N. Nikiforovaa, Lindsey M. Kellya, James R. Stringerd, Christopher J. Bakkenistd,e, and Yuri E. Nikiforova,2

aDepartment of Pathology and Laboratory Medicine, University of Pittsburgh, Pittsburgh, PA 15213; bDepartment of Oral Biology, School of Dental Medicine and cDepartment of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15219; dDepartment of Molecular Genetics, University of Cincinnati, Cincinnati, OH 45267; and eDepartments of Radiation Oncology and Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213

Edited by James E. Cleaver, University of California, San Francisco, CA, and approved May 1, 2012 (received for review April 5, 2012)

Double-strand DNA breaks (DSBs) are continuously induced in cells Results by endogenously generated free radicals and exogenous geno- Homologous Chromosomes Form Arm-Specific Contact Spontaneously toxic agents such as ionizing radiation. DSBs activate the kinase and After Exposure to Ionizing Radiation. We used four-color 3D- activity in sensor proteins such as ATM and DNA-PK, initiating FISH and confocal microscopy (11, 12) to analyze the pattern a complex DNA damage response that coordinates various DNA and frequency of interaction between homologous chromosomes repair pathways to restore genomic integrity. In this study, we in untreated and irradiated primary cultured human epithelial report the unexpected finding that homologous chromosomes thyroid cells and fibroblasts. Cells were highly enriched in G0/G1 contact each other at the sites of DSBs induced by either radiation by plating at high density in the absence of serum and other or the endonuclease I-PpoI in human somatic cells. Contact involves growth factors (84% of thyroid cells and 91% of fibroblasts were short segments of homologous chromosomes and is centered on in G0/G1 phase by FACS). For each chromosome, arm-specific a DSB in active genes but does not occur at I-PpoI sites in intergenic

paints were used to visualize the p and q arm separately, and CELL BIOLOGY DNA. I-PpoI-induced contact between homologous genes is abro- a centromeric probe was used to monitor the location and du- gated by the transcriptional inhibitors actinomycin D and α-amanitin A fi plication of centromeres (Fig. 1 ). Contact between homologous and requires the kinase activity of ATM but not DNA-PK. Our nd- chromosomes was detected by using intensity-based image seg- ings provide documentation of a common transcription-related and mentation analysis (Fig. S1). S/G2-phase cells, which were ATM kinase-dependent mechanism that induces contact between identified by the presence of centromere duplication that occurs allelic regions of homologous chromosomes at sites of DSBs in in S phase (13), were excluded from further analysis. The accuracy human somatic cells. of the exclusion criterion was confirmed by combining PCNA (marker of S phase) or cyclin A (marker of S and G phase) (14) homologous chromosome interaction | homologous recombination 2 immunostaining and FISH analysis in selected experiments (Fig. 1 E and F). ouble-strand DNA breaks (DSBs) are continuously induced Arm-specific contact between homologous chromosomes was Din eukaryotic cells by the free radicals generated by endog- readily identified for all six chromosomes studied in epithelial enous metabolic processes and by exposure to environmental cells and all three chromosomes studied in fibroblasts. The genotoxic agents such as ionizing radiation (IR) and are con- common patterns of contact were between two homologous q – sidered to be the most dangerous DNA lesions (1 3). DSBs in- arms (Fig. 1B), between two homologous p arms (Fig. 1C), and duce a complex DNA damage response that activates DNA repair simultaneous contact between two p arms and two q arms of pathways and cell cycle checkpoints and induces chromatin homologous chromosomes, i.e., p:p and q:q contact (Fig. 1D and remodeling and apoptosis. ATM and DNA-PK, two phosphati- Fig. S2). dylinositol 3-kinase–like kinases, are primary mediators of the Analysis performed on primary thyroid epithelial cells from two DNA damage response to DSBs. Although ATM phosphorylates donors revealed that 15–26% (mean, 19%) of homologous chro- hundreds of substrates that are believed to regulate multiple mosomes had arm-specific contact in untreated cells (Fig. 1G). DNA repair pathways including DSB repair by homologous Fifteen minutes after exposure to 5-Gy IR, the frequency of arm- recombination (HR) (4, 5), DNA-PK primarily regulates a limited specific contact between homologous chromosomes doubled to group of effectors that mediate DSB repair by nonhomologous 28–45% (mean, 38%). Similar findings were observed in un- end-joining (NHEJ) (3, 6). Misrepair of DSBs can result in the treated and irradiated human fibroblasts (Fig. 1H). The frequency intrachromosomal and interchromosomal rearrangements that of contact between homologous chromosomes was ∼20-fold generate oncogenic gene fusions. Previously, we (7, 8) and others lower than the predicted frequency of DSBs induced by 5 Gy of IR (9, 10) have shown that cancer-specific chromosomal rear- (150–200 DSBs per cell; 6–8 DSBs per average-size chromo- rangements commonly arise as a result of exchange between some), indicating that if contact is due to formation of DSBs, chromosomal loci that are located in close spatial proximity at the time of DSB formation. While exploring the role of nuclear architecture and gene Author contributions: Y.E.N. and C.J.B. designed research; M.G., V.N.E., K.T., and L.M.K. topology in the generation of chromosomal rearrangements in performed research; Y.E.N., M.G., V.N.E., K.T., M.N.N., J.R.S., and C.J.B. analyzed data; and human somatic cells, we observed an unexpected finding that Y.E.N., M.G., V.N.E., M.N.N., J.R.S., and C.J.B. wrote the paper. homologous chromosomes frequently contact each other at the The authors declare no conflict of interest. sites of DSBs induced in G0/G1 cells by either IR or the re- This article is a PNAS Direct Submission. striction enzyme I-PpoI. Further characterization demonstrated 1M.G. and V.N.E. contributed equally to this work. that this contact between homologous chromosomes is initiated 2To whom correspondence should be addressed. E-mail: [email protected]. by DSBs in genes but not by DSBs in intergenic DNA, requires This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. active gene transcription, and depends on ATM kinase activity. 1073/pnas.1205759109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1205759109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 cells (range 0.6–1.6%) for all chromosomes, whether cells were irradiated or not irradiated, which likely represents a background level of random contact.

Homologous Chromosomes Form Contacts at the Sites of DSBs Induced by Homing Endonuclease I-PpoI in Gene Regions but Not Outside of Genes. To determine whether contact between the corresponding arms of homologous chromosomes was induced by a DSB, and to find whether it involves the same allelic region on both homologs, we induced DSBs at specific chromosomal sites by expressing the homing endonuclease I-PpoI (15, 16) in the TPC-1 human thyroid cancer cell line (17). To introduce the I-PpoI gene, cells were transduced with the HA-ER-I-PpoI retrovirus (gift of M. Kastan Duke Cancer Institute, Durham, NC) by using either a single infection or three sequential infections. To induce DSBs, the infected cells were treated with 4-hydroxytamoxifen (4-OHT), which causes I-PpoI to enter the nucleus (16, 18). I-PpoI induction for 6 h produced multiple γH2AX foci in ∼55% and ∼75% of cells after single and triple infections, respectively (Fig. 2A). Using site-specific DNA probes, we analyzed six chromosomal regions, all diploid in TPC-1 cells (Table S1). Two of the regions have an I-PpoI site located within genes (DAB1 on 1p and GRIP1 on 12q). Two other studied regions (2qIG and 5qIG) are intergenic loci containing an I-PpoI site. Two regions that lack an I-PpoI site (16pNC and 16qNC) were also analyzed. Quantitative PCR (qPCR) analysis revealed that between 9 and 16% of the analyzed target sites were cut at 6 h after I-PpoI induction (Fig. 2B), as expected based on previous observations showing that in human cells I-PpoI cleaves approximately 10% of the 200–300 target sites (16, 18). FISH analysis with site-specific probes combined with cyclin A staining revealed that, in G0/G1-phase cells, I-PpoI cleavage induced contact between gene regions on homologous chromosomes, but did not induce contact between homologous intergenic regions or regions that do not contain an I-PpoI site (Fig. 2 C and D). For both genes (DAB1 and GRIP1), the proportion of cells showing contact at the I-PpoI site corresponded to the proportion of cells with expected cleavage at that site, estimated based on the known efficiency of cutting at each site and number of infected cells after single and triple infection. By contrast, the two intergenic regions containing an I-PpoI site exhibited no increase in allelic contact above the level observed in cells before I-PpoI entering the nucleus and in DNA regions lacking an I-PpoI site. Additionally, the frequency of contact between heterologous gene pairs, i.e., between DAB1 and GRIP1 genes, was low and unchanged when studied before (0.93 ± 0.24%) and after (0.90 ± 0.21%) DSB induction by I-PpoI. To confirm that contact between homologous genes is a general phenomenon and not restricted to specific chromosomes, we Fig. 1. Arm-specific contact between homologous chromosomes in G0/G1- phase human thyroid cells (HT) and fibroblasts (HF). (A) Color scheme of expanded the analysis to include three additional genes that probes for FISH. (B) q arm contact. (C) p arm contact. (D) p:p and q:q contact. contain endogenous I-PpoI cleavage sites, all located on differ- (E)G0/G1 nuclei on the right show either p:p and q:q contact (upper) or ent chromosomes: ARID5B (10q), ERC2 (3q), and PDE1A (2q) p arm contact (lower), whereas S/G2-phase nucleus on the left is cyclin A (Table S2). Induction of I-PpoI for 6 h in TPC-1 cells stably positive (orange). (F) S-phase nucleus in Upper shows a duplicated centro- transfected with the HA-ER-I-PpoI retrovirus resulted in the mere (arrows) and is positive for PCNA (orange), whereas G0/G1 nucleus in increased contact among all three pairs of homologous genes Lower shows q arm contact. (G and H) Frequency of contact between homologous chromosomes: Gray bars represent p:p or/and q:q contact in un- (Fig. S3). treated cells; black bars represent p:p or/and q:q contact after IR; white bars represent contact between opposite arms (p:q) of homologous chromo- Contact Between Homologous Chromosomes Involves a Relatively somes. Data based on 300 cells analyzed from each of two donors shown as Small Region Flanking the Break. Experiments illustrated in Fig. 1 mean ± SEM. The differences between frequency of contact before and showed that when contact involved single arms of homologous after IR were highly statistically significant (P < 0.0002 for all chromosomes, chromosomes, most of that arm and all of the other arm were two sample t test). (Scale bars: 5 μm.) apart. This finding suggested that contact between homologous chromosomes involved only a limited section of the chromosomes. To estimate the length of the contact between homologous a fraction of induced DSBs are participating in contact at any chromosomes, we used an array of DNA probes extending up to given time. Contact between opposite arms of homologous 3 Mb centromeric and telomeric from the I-PpoI site in the chromosomes (i.e., p:q contact) was observed in only ∼1% of DAB1 gene (Table S3). In a series of high-resolution four-color

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1205759109 Gandhi et al. Downloaded by guest on September 30, 2021 Fig. 2. Contact between homologous chromosomes at the sites of DSBs induced by endonuclease I-PpoI in genes. (A) Examples of multiple γH2AX foci (orange) formed 6 h after I-PpoI induction in human TPC-1 cells, indicating the efﬁciency of I-PpoI in generating DSBs in these cells. (B) Time course and percentage of DNA cleavage at four I-PpoI sites detected by real-time qPCR. (C) FISH visualization of the I-PpoI site in the DAB1 gene showing contact be-

tween DAB1 signals (orange) in one cell (arrow), which is in G0/G1 phase because it lacks cyclin A staining (green), compared with a cell on the left in S/G2 CELL BIOLOGY phase. (D) Frequency of contact between homologous regions with an I-PpoI site located in gene regions and intergenic regions and between homologous regions lacking an I-PpoI site. Data based on 1,000 cells scored in each of two experiments shown as mean ± SEM. **P < 0.001, *P < 0.01, two sample t test. (Scale bars: 5 μm.)

3D-FISH analyses (Fig. 3 A and B), we observed nuclei in which which inhibits RNA polymerases II and III (20). Treatment with probes spanning the I-PpoI site were colocalized, whereas probes either of the transcription inhibitors resulted in abrogation of the located 300 kb and further from the I-PpoI site were at a distance contact between each of five homologous gene pairs (Fig. 4B). (Fig. 3C). These data demonstrated that contact formed at the site of a DSB can involve less than 300 kb on either side of the Break-Induced Contact Between Homologous Chromosomes Requires break. In addition, in this experiment, we observed nuclei that Function of ATM, but Not DNA-PK. We proceeded to investigate had p arm territories of both chromosome homologs positioned whether the site-specific contact between homologous chromo- near each other, and aligned in the same direction, but not in somes depends on the kinase activities of either ATM or DNA-PK, contact (Fig. 3D). Such mutual positioning of homologous arms which are required for DSB recognition and repair by various of chromosome 1p was seen more often than contact at the site pathways including HR (ATM) and classical NHEJ (DNA-PK) of I-PpoI cleavage (∼5:1) and may represent stages of approxi- (6, 21). We observed that inhibition of the kinase activity of mation and alignment of homologous regions before the initia- ATM using the ATM inhibitor KU55933 abrogated the I-PpoI– tion of contact at a DSB. induced contact between gene regions in TPC-1 cells, whereas the DNA-PK inhibitor Nu7441 had no effect (Fig. 5 A and B). To Frequency of Contact Is Higher After I-PpoI Cleavage of Multiple confirm the ATM kinase dependence of contact, we knocked Sites. Experiments with I-PpoI using chromosome 1p paint, il- down ATM expression by using a selective shRNA and showed lustrated in Fig. 3, also allowed us to evaluate contact between that knockdown abrogated contact (Fig. 5 C and D). These any regions of homologous 1p arms after the induction of I-PpoI results indicate that contact between homologous gene regions at for 6 h. At least four I-PpoI cleavage sites are located on 1p, the site of a DSB requires functional ATM kinase. including three sites in gene regions and one in intergenic DNA (Table S4). The frequency of contact between homologous 1p Discussion arms was found to be 24.23 ± 1.21%, significantly higher than Homologous chromosome pairing is known to occur during that seen at the DAB1 site alone (7.10 ± 0.57%). This finding meiosis, where it is essential for high levels of recombination and demonstrates that induction of multiple I-PpoI cleavage sites the correct segregation of homologs into haploid germ cells (22). on one chromosome arm results in the higher frequency of ho- In this study, we used high-resolution confocal microscopy and mologous arm contact, which becomes comparable to that seen chromosome arm paints or gene-specific probes to demonstrate after 5 Gy of IR. that contact between allelic regions of homologous chromosomes frequently occurs in somatic human cells and can be induced by Break-Induced Contact Between Homologous Chromosomes Requires ionizing radiation or DNA cleavage by a site-specificendonucle- Active Gene Transcription. Because all five genes that exhibited ase. The contact was observed between homologous chromosome contact between homologous gene pairs upon induction of arms and homologous genes on multiple different chromosomes I-PpoI cleavage are expressed in TPC-1 cells (Fig. 4A), we tested and, therefore, appears to represent a general phenomenon oc- whether active transcription is required for contact. Cells were curring in human somatic cells. treated with actinomycin D, which blocks transcription elonga- However, in contrast to homologous chromosome pairing in tion by all three RNA polymerases (19) and with α-amanitin, meiosis, which occurs by multiple interstitial interactions along

Gandhi et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 Fig. 3. Contact between homologous chromosomes is centered on a DSB and extends less than 300 Kb telomeric and centromeric from the break. (A) Scheme of the FISH probes used to map the extent of contact between chromosome 1 homologs at the I-PpoI site in the DAB1 gene region 6 h after I-PpoI induction in TPC-1 cells. (B) Representative nucleus with two 1p territories located at a distance. (C) Representative nucleus with full colocalization of the I-PpoI site probes (yellow), whereas ﬂanking regions (red and aqua) remain at a distance. (D) Representative nucleus with 1p arm territories (green) near each other and aligned with signals in the same order. (Scale bars: 5 μm.)

the entire length of homologs, contact in somatic cells observed that nucleotide excision repair proceeds more actively on the in this study typically involved a limited section of the chromosome, transcribed strands of expressed genes, removing the damage by centering on a site of DSB. A more extensive pairing was rarely excising a short DNA fragment containing DNA lesion and filling seen and likely reflected juxtaposition of homologs in response to the gap by using the undamaged strand as a template (27, 28). The several breaks occurring along both arms of the chromosome. results of our study raise the possibility that a specialized DSB Importantly, the I-PpoI–induced contact was restricted to repair pathway operates on DSBs in expressed genes and, con- DSBs in coding regions, and we were unable to observe contact ceivably, uses a homologous chromosome as a repair template. at cleavage sites in intergenic DNA. Moreover, contact between However, it is also possible that contact between homologous homologous active genes was reversed after RNA polymerase chromosomes at the site of DSBs does not directly provide inhibition and arrest of RNA synthesis by actinomycin D and a template for repair of broken DNA molecule but contributes to α-amanitin. Interestingly, it has been shown that recruitment of other aspects of DSB recognition and repair. Another possibility some DNA repair factors such as 53BP1 to the sites of DNA is that contact is not associated with repair at all and is a product damage requires direct interaction with RNA molecules, and of other gene functions, such as of sharing transcription factories, treatment with RNase results in dissociation of 53BP1 from the which has been suggested to exist between allelic genes on ho- IR-induced nuclear foci (23). Moreover, homologous chromo- mologous chromosomes (24) and between active heterologous somes that pair in meiosis are transcriptionally active, and it has genes (29). This possibility is, however, unlikely, because it would been proposed that RNA:RNA base pairing involving sense and be expected to result in pairing along the full length of homol- antisense transcripts tethered through their polymerases may be ogous chromosomes as seen in meiosis, would not depend on the a driver of homolog pairing in meiosis (24). In light of these induction of DSBs and ATM kinase, or would be expected to reports, the results of our study raise at least a theoretical pos- bring together active heterologous genes, such as DAB1 and sibility that sequence-specific RNA molecules may be involved in GRIP1, which was not seen in this study. homology search and/or establishing contact between allelic gene In summary, our findings demonstrate that homologous gene loci in somatic human cells. regions in human somatic cells make contact at the sites of DSBs. Although our findings that contact centered on a break and As such, we also demonstrate that cells possess a mechanism that required functional ATM kinase suggest that the contact may uses sequence homology to bring otherwise distant DNA mole- have a role in repair of DSBs, the DNA repair pathway involved cules into contact. Our data suggest that this process may involve remains unknown. ATM is known to regulate multiple DNA RNA molecules or active gene transcription, although the details repair pathways including HR (25, 26). It is also well established of this mechanism remain unknown. Nevertheless, the data

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1205759109 Gandhi et al. Downloaded by guest on September 30, 2021 CELL BIOLOGY

Fig. 4. Contact between homologous genes upon DSB induction requires Fig. 5. Contact between homologous genes upon DSB induction requires active transcription. (A) Expression of five studied genes in TPC-1 cells rela- the kinase activity of ATM but not that of DNA-PK. (A) Western blot showing tive to expression of GAPDH. NC, a gene known to be not expressed in TPC-1 selective inhibition of ATM autophosphorylation by KU55933 (Upper) and of cells. (B) Frequency of contact between homologous gene regions after selective inhibition DNA-PK autophosphorylation by NU7441 (Lower)inTPC- I-PpoI induction and influence of transcription inhibition by actinomycin D 1 cells. (B) Contact frequency between gene regions and intergenic regions and α-amanitin for 6 h. Data based on 1,000 cells scored in each of two after I-PpoI induction with and without KU55933 and NU7441. Data based experiments depicted as mean ± SEM. The difference between contact be- on 1,000 cells scored in each of two experiments shown as mean ± SEM. *P < fore and after transcription inhibition with either agent was highly statis- 0.0001, **P > 0.55, two sample t test. (C) Western blot showing sequence tically significant (P < 0.0001 for all genes, two sample t test). specific knockdown of ATM protein by an shRNA that targets the ATM mRNA but not a scrambled shRNA sequence. (D) Frequency of contact between homologous gene regions after I-PpoI induction and influence of described here provide documentation of a common transcription- ATM knockdown by using shRNA. Data for DAB1 and GRIP1 from 1,000 cells related and ATM kinase-dependent mechanism that induces scored in each of two experiments are combined and shown as mean ± SEM. contact between homologous chromosomes at sites of DSBs in *P < 0.0001, two sample t test. human somatic cells.

Materials and Methods Pretreatment and hybridization were as described (8). For the simultaneous fi Cell Culture and Irradiation. Primary cultures of human thyroid cells were detection of speci c DNA targets and nuclear proteins, FISH was combined established from surgically removed thyroid glands as described (7) by fol- with immunostaining (31). Mouse monoclonal antibodies for PCNA (Sigma- lowing the University of Pittsburgh Institutional Review Board approval. Aldrich; P-8825, 1:1,500) and cyclin A (Santa Cruz Biotechnology; sc 56299, Cells were cultured in RPMI medium 1640 with 15% FBS and 10 mU/mL of 1:100) were used. Immunostaining was immediately followed by 3D-FISH. thyroid stimulation hormone (Sigma) for 1–2 wk. Primary fibroblast cultures were established from fragments of perithyroidal fibrous tissue. Cells were Image Acquisition and Analysis of Chromosome Contact. Confocal microscopy cultured for 3–4 wk in DMEM supplied with 10% FBS, nonessential amino was performed by using a Leica SP5 TCS 4D confocal laser scanning fluo- acids, L-glutamine, and 4 ng/mL basic fibroblast growth factor (Sigma). To rescence microscope using a 63×, 1.4 N.A. oil PlanApo objective. For most

enrich cell populations for cells that are in the G0/G1 phase of the cell cycle, experiments, 300 nuclei from each of two different donors were scanned cells were plated at high density and cultured without FBS and growth and analyzed. Nuclear boundaries were identified by DAPI staining, and factors for 72 h. When irradiation was used to induce DNA breaks, a single image stacks were acquired with z steps of 0.13 μm. The digital image stacks dose of γ-irradiation from a cesium-137 source was delivered at a dose rate were reconstructed by using Volocity software (PerkinElmer). Image stacks of 301.66 cGy/min. Induction of DSBs was monitored by immunofluorescence were subjected to uniform noise reduction by using the fine filter algorithm with anti-γH2AX antibody (05–636; Upstate Biotechnology) (30). provided in the software. The analysis of spatial contact between chromosome arms was performed by using the intensity-based image segmentation Three-Dimensional Cell Fixation, FISH, and Immuno-FISH. Cells plated in technique (32). chamberslidesweresubjectedto3Dfixation in 4% paraformaldehyde followed by repeated freeze-thaw cycles in liquid nitrogen (11). Centromeric enu- Induction of DSBs by I-PpoI. HA-ER-I-PpoI retrovirus (gift of M. Kastan, Duke meration probes prelabeled with either SpectrumAqua or SpectrumOrange Cancer Institute, Durham, NC) was propagated in Phoenix Ampho packaging (pseudocolored in blue for illustration) (Abbott Laboratories) and prelabeled cell line as described (33). Retroviral transductions of TPC-1 thyroid cancer arm-specific chromosome painting probes (p arm, FITC; q arm, Texas red) cell line were carried out by using ViraDuctin Retrovirus Transduction Kit (Metasystems) were used. BAC clones for region-specific DNA probes (BAC/ (Cell Biolabs). To increase transduction efficacy, three sequential transductions PAC Resources, Children’s Hospital, Oakland, CA) were labeled by nick of cells were used. 4-hydroxytamoxifen (4-OHT) was added to a final con- translation using d-UTP tagged Spectrum dyes (Abbott Laboratories). centration of 1 μMfor6–48 h to induce DNA digestion by I-PpoI (16, 18). To

Gandhi et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 establish a cell line stably expressing HA-ER-I-PpoI, retrovirus was generated cells stably expressing HA-ER-I-PpoI retrovirus were plated in chamber slides, as described and used to infect TPC-1 cells, then selected with 1 μg/mL pu- grown overnight, and treated with actinomycin D (5 μg/mL) or α-amanitin romycin for 2 wk. Induction of DSBs was monitored by γH2AX staining (30). (50 μg/mL) (Sigma-Aldrich) for 6 h with or without presence of 4-OHT, washed Cells with >5 γH2AX foci per nucleus were considered positive for I-PpoI– with PBS, and fixed. induced cleavage. ATM and DNA-PK Inhibitors and ATM Knockdown Using shRNA. ATM kinase Identification of I-PpoI Sites in the Human Genome and qPCR Assay for inhibitor KU55933 and DNA-PK kinase inhibitor NU7441 were reconstituted Cleavage Efficiency. The I-PpoI site located in the DAB1 gene has been in DMSO and used at 10 μM and 5 μM, respectively (34). I-PpoI was induced in characterized by Berkovich et al. (16), whereas other I-PpoI cleavage sites TPC-1 cells infected with HA-ER-I-PpoI retrovirus by using 4-OHT for 6 h in fi were mapped by us (Tables S1, S2, and S4). The ef ciency of cleavage at the presence of vehicle, KU55933, or NU7441. In a separate experiment, fl these sites was assessed by quantitative real-time PCR with primers anking ATM was disrupted in TPC-1 cells by using a short hairpin RNA (shRNA) that fi each I-PpoI site (Table S5) using ampli cation of the GAPDH gene that does targeted the ATM sequence 5′-TGATGGTCTTAAGGAACATCT-3′. Stable cell not contain an I-PpoI site for normalization as described by Berkovich et al. lines expressing both the shRNA and the puromycin-resistance gene were (16, 18). selected in 1 μg/mL puromycin for five cell doublings after lentivirus transduction (University of Pittsburgh Cancer Institute lentiviral core). ATM fi Analysis of Contact Between Speci c Regions of Homologous Chromosomes knockdown and the efficacy of KU55933 and NU7441 in TPC-1 cells was After I-PpoI Induction. I-PpoI was induced for 6 h in TPC-1 cells after single and determined by immunoblotting. Mouse monoclonal anti-ATM antibody triple infection with HA-ER-I-PpoI retrovirus followed by 3D fixation and FISH (MAT3-4G10/8; Sigma-Aldrich), rabbit monoclonal anti-phosphoserine-1981 with region-specificprobes(Tables S1 and S2). For each experimental condi- ATM (EP1890Y; Epitomics), rabbit polyclonal anti-DNA-PK (4062; Cell Sig- tion, 1,000 nuclei of TPC-1 cells were visually assessed by using a microscope naling), and mouse monoclonal anti-phosphoserine-2056 (18192; Abcam) with 63×, 1.4 N.A. oil PlanApo objective. Site-specificprobesineachnucleus were used. were identified by using the appropriate filter cube, and signals were scored as positive for contact when the space between them was smaller than size of one signal. The experiment was performed in duplicate. ACKNOWLEDGMENTS. We thank M. Kastan for providing HA-ER-I-PpoI retrovirus; J. Esplen, J. Murphy, and M. B. Durso for technical assistance; and W. Saunders for thoughtful comments on the manuscript. This work was Detection of Gene Expression and Transcription Inhibition. Expression levels of supported by National Institutes of Health Grants R01 CA88041 (to Y.E.N.) studied genes were detected by using qRT-PCR with CyberGreen (ABI Bio- and R01 CA148644 (to C.J.B.) and University of Pittsburgh Cancer Center systems) with primers shown in Table S6. For transcription inhibition, TPC-1 Support Grant P30 CA047904.

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