Title: <scp>ATRX</scp>loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization
Abstract: Article27 August 2019free access Source DataTransparent process ATRX loss induces telomere dysfunction and necessitates induction of alternative lengthening of telomeres during human cell immortalization Fei Li orcid.org/0000-0002-7818-6525 Department of Neurosurgery, Southwest Hospital, Chongqing, China Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Zhong Deng The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Ling Zhang Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Department of Pathophysiology, Norman Bethune Medical School at Jilin University, Changchun, China Search for more papers by this author Caizhi Wu orcid.org/0000-0002-2449-2818 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Ying Jin Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Inah Hwang Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Olga Vladimirova The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Libo Xu Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Department of Pathophysiology, Norman Bethune Medical School at Jilin University, Changchun, China Search for more papers by this author Lynnie Yang Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Bin Lu Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Javaraju Dheekollu The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Jian-Yi Li Department of Pathology and Lab Medicine, North Shore University Hospital and Long Island Jewish Medical Center, Northwell Health, Lake Success, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, USA Search for more papers by this author Hua Feng Department of Neurosurgery, Southwest Hospital, Chongqing, China Search for more papers by this author Jian Hu Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Christopher R Vakoc Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Haoqiang Ying Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Jihye Paik Corresponding Author [email protected] orcid.org/0000-0002-5481-2202 Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Paul M Lieberman Corresponding Author [email protected] orcid.org/0000-0002-3935-9921 The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Hongwu Zheng Corresponding Author [email protected] orcid.org/0000-0002-9781-7935 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Fei Li orcid.org/0000-0002-7818-6525 Department of Neurosurgery, Southwest Hospital, Chongqing, China Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Zhong Deng The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Ling Zhang Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Department of Pathophysiology, Norman Bethune Medical School at Jilin University, Changchun, China Search for more papers by this author Caizhi Wu orcid.org/0000-0002-2449-2818 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Ying Jin Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Inah Hwang Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Olga Vladimirova The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Libo Xu Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Department of Pathophysiology, Norman Bethune Medical School at Jilin University, Changchun, China Search for more papers by this author Lynnie Yang Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Bin Lu Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Javaraju Dheekollu The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Jian-Yi Li Department of Pathology and Lab Medicine, North Shore University Hospital and Long Island Jewish Medical Center, Northwell Health, Lake Success, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, USA Search for more papers by this author Hua Feng Department of Neurosurgery, Southwest Hospital, Chongqing, China Search for more papers by this author Jian Hu Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Christopher R Vakoc Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Search for more papers by this author Haoqiang Ying Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Jihye Paik Corresponding Author [email protected] orcid.org/0000-0002-5481-2202 Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Paul M Lieberman Corresponding Author [email protected] orcid.org/0000-0002-3935-9921 The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Hongwu Zheng Corresponding Author [email protected] orcid.org/0000-0002-9781-7935 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA Search for more papers by this author Author Information Fei Li1,2,‡, Zhong Deng3,‡, Ling Zhang2,4,‡, Caizhi Wu2, Ying Jin2, Inah Hwang5, Olga Vladimirova3, Libo Xu2,4, Lynnie Yang2, Bin Lu2, Javaraju Dheekollu3, Jian-Yi Li6, Hua Feng1, Jian Hu7, Christopher R Vakoc2, Haoqiang Ying8, Jihye Paik *,5, Paul M Lieberman *,3 and Hongwu Zheng *,2,5 1Department of Neurosurgery, Southwest Hospital, Chongqing, China 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA 3The Wistar Institute, Philadelphia, PA, USA 4Department of Pathophysiology, Norman Bethune Medical School at Jilin University, Changchun, China 5Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA 6Department of Pathology and Lab Medicine, North Shore University Hospital and Long Island Jewish Medical Center, Northwell Health, Lake Success, Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, USA 7Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA 8Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 212 746 6151; E-mail: [email protected] *Corresponding author. Tel: +1 215 898 9491; E-mail: [email protected] *Corresponding author. Tel: +1 212 746 6620; E-mail: [email protected] EMBO J (2019)38:e96659https://doi.org/10.15252/embj.201796659 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Loss of the histone H3.3-specific chaperone component ATRX or its partner DAXX frequently occurs in human cancers that employ alternative lengthening of telomeres (ALT) for chromosomal end protection, yet the underlying mechanism remains unclear. Here, we report that ATRX/DAXX does not serve as an immediate repressive switch for ALT. Instead, ATRX or DAXX depletion gradually induces telomere DNA replication dysfunction that activates not only homology-directed DNA repair responses but also cell cycle checkpoint control. Mechanistically, we demonstrate that this process is contingent on ATRX/DAXX histone chaperone function, independently of telomere length. Combined ATAC-seq and telomere chromatin immunoprecipitation studies reveal that ATRX loss provokes progressive telomere decondensation that culminates in the inception of persistent telomere replication dysfunction. We further show that endogenous telomerase activity cannot overcome telomere dysfunction induced by ATRX loss, leaving telomere repair-based ALT as the only viable mechanism for telomere maintenance during immortalization. Together, these findings implicate ALT activation as an adaptive response to ATRX/DAXX loss-induced telomere replication dysfunction. Synopsis Alternative lengthening of telomeres (ALT) in human cancers is frequently associated with loss of the histone H3.3 chaperone ATRX or its cofactor DAXX. This study shows that ATRX/DAXX do not function as immediate repressor of ALT, but that ALT is rather an an adaptive response to ATRX depletion-induced telomere replication dysfunction. ATRX loss induces telomere replication dysfunction irrespective of endogenous telomerase activity. ATRX-DAXX histone H3.3 chaperone activity is required for telomere maintenance. ATRX depletion progressively induces chromatin de-compaction at telomeres. ATRX loss dictates the telomere maintenance program during ATRX mutant cell immortalization. Introduction Telomeres are specialized structures located at chromosomal ends of eukaryotes that function to protect against promiscuous DNA repair activities and nucleolytic degradation (Cech, 2004; Blackburn, 2005; de Lange, 2009; Sarek et al, 2015). In the absence of de novo telomere elongation, telomeres shorten with each cell division, ultimately leading to cellular senescence or apoptosis (Harley et al, 1990). Hence, the development of an active telomere length maintenance system is essential for sustaining replicative immortality and tumorigenesis. While a majority of human malignancies achieve this by upregulation of telomerase that adds telomere repeats to the chromosomal ends (Kim et al, 1994), a remaining 10–15% of human cancers adopt a telomerase-independent mechanism, termed alternative lengthening of telomeres (ALT), to potentiate their replicative immortality (Bryan et al, 1997; Henson et al, 2005). Phenotypically, the ALT cells are characterized by a unique set of features consistent with hyperactive homologous recombination at telomeres, including the generation of extrachromosomal telomere repeats, the presence of ALT-associated PML bodies (APBs), and frequent intra- and inter-chromosomal telomere exchange (Londono-Vallejo et al, 2004; Muntoni et al, 2009; Cho et al, 2014; Pickett & Reddel, 2015). In addition, ALT is also associated with massive genome rearrangement, defective G2/M checkpoint control, and altered DNA damage repair (Lovejoy et al, 2012). Besides its occurrence in human cancers, ALT also presents in a subset of in vitro immortalized human cell lines that emerge from telomere crisis at very low frequency (Shay & Wright, 1989; Yeager et al, 1999). Likewise, ALT has been often considered as a survival mechanism in response to critical telomere shortening (Cesare & Reddel, 2010; Hu et al, 2012; Napier et al, 2015), even though the molecular basis behind of its activation remains poorly understood. Over the past decade, high-throughput exome and whole-genome sequencing efforts across a wide spectrum of cancer types have identified a specifically high incidence of ALT activation in tumors that harbor genetic alterations of genes encoding histone chaperone protein ATRX (α-thalassemia/mental retardation syndrome X-linked) or DAXX (death-domain-associated) (Heaphy et al, 2011; Schwartzentruber et al, 2012; Ceccarelli et al, 2016). Frequent ATRX (and less commonly DAXX) loss-of-function mutations were identified in a variety of cancer types including pediatric and adult gliomas (Jiao et al, 2012; Schwartzentruber et al, 2012; Brennan et al, 2013; Zhang et al, 2013; Wu et al, 2014; Brat et al, 2015), pancreatic neuroendocrine tumor (Jiao et al, 2011), neuroblastoma (Cheung et al, 2012; Molenaar et al, 2012), and multiple sarcomas (Chen et al, 2014) (Cancer Genome Atlas Research Network. Electronic address & Cancer Genome Atlas Research, 2017, Chudasama et al, 2018). Importantly, mutations of ATRX or DAXX in human tumors were found to be mutually exclusive with TERT promoter mutations (Killela et al, 2013; Brat et al, 2015; Eckel-Passow et al, 2015; Ceccarelli et al, 2016; Barthel et al, 2017), suggesting a telomere maintenance-related function of ATRX-DAXX complex. Indeed, a survey of 22 immortalized human ALT cell lines reported that ~90% of them exhibit abnormal ATRX and/or DAXX protein expression (Lovejoy et al, 2012), further substantiating the connection between ATRX/DAXX status and ALT activation. ATRX is a SWI/SNF2 type of chromatin remodeling factor that is implicated in many nuclear biological activities, such as gene expression, DNA replication, and chromatin state and composition maintenance (Gibbons et al, 1995; Ratnakumar et al, 2012; Sarma et al, 2014; He et al, 2015; Ramamoorthy & Smith, 2015; Voon et al, 2015; Danussi et al, 2018; Juhasz et al, 2018). Structurally, ATRX consists of a C-terminal ATP-dependent helicase domain and an N-terminal cysteine-rich ADD (ATRX-DNMT3-DNMT3L) motif that acts to specify its chromatin location (Gibbons et al, 2008; Eustermann et al, 2011; Iwase et al, 2011). Functionally, ATRX and its binding partner DAXX are known to form a histone variant H3.3-specific chaperone complex that orchestrates replication-independent nucleosome assembly at repetitive heterochromatic DNA regions including telomeres (Drane et al, 2010; Goldberg et al, 2010; Lewis et al, 2010; Elsasser et al, 2012; Voon et al, 2015). Besides its histone H3.3 depositing function, ATRX has also been implicated in the process of telomere DNA replication by facilitating resolution of telomere cohesion, R-loops, and G-quadruplex structures (Law et al, 2010; Clynes et al, 2015; Ramamoorthy & Smith, 2015; Nguyen et al, 2017). Given the fact that telomere length maintenance is essential for cell immortalization and tumorigenesis, the high concordance of ATRX deficiency with ALT activation has hence prompted the speculation that ATRX may exert its tumor suppressor function by directly repressing ALT (Pickett & Reddel, 2015; Maciejowski & de Lange, 2017). Indeed, restoration of ATRX expression in ATRX-negative ALT lines suppresses many ALT-associated phenotypes (Clynes et al, 2015; Napier et al, 2015). Along this line, a variety of ATRX nuclear functions have been invoked to explain its participation in ALT suppression, including its involvement in telomeric H3.3 loading (Wong et al, 2010), telomere cohesion resolution (Ramamoorthy & Smith, 2015), guarding the DNA replication against replication stress or G-quadruplex structure formation (Law et al, 2010; Leung et al, 2013; Clynes et al, 2015), and telomeric R-loop suppression (Nguyen et al, 2017). But despite the extensive efforts, the crucial demonstration of a causal role between the ATRX or DAXX loss and ALT activation has not been reported. Particularly, knockdown of ATRX or DAXX expression in either mortal or telomerase-positive cell lines has largely failed to activate ALT and the reason remains unclear (Lovejoy et al, 2012; O'Sullivan et al, 2014; Flynn et al, 2015). In this study, we adopt multiple cell-based models to analyze the functional role of ATRX-DAXX on telomere maintenance and to determine the impact of their loss on human cell immortalization. Our findings reveal that ATRX and DAXX do not act as direct ALT suppressor. Instead, we found that their depletion provokes a delayed onset of telomere replication stress that activates ALT-associated DNA repair pathway, while at the same time, it compromises mutant cell growth. We show that this process is independent of telomere length and pre-existing endogenous telomerase activity. Furthermore, we demonstrate that ATRX inactivation disrupts H3.3-mediated telomere nucleosome assembly and triggers a process of progressive telomere de-compaction that culminates in the inception of telomere replication dysfunction. Finally, we show that endogenous telomerase activity cannot overcome ATRX loss-associated telomere dysfunction. As a consequence, the mortal ATRX-deficient cells are obligated to adopt the homology-directed telomere DNA repair ALT pathway as an alternative telomere maintenance mechanism during immortalization process. These findings have implications for the understanding of ATRX or DAXX deficiency-associated ALT activation, cell immortalization, and tumorigenesis. Results ATRX loss induces telomere dysfunction and ALT-associated features We applied genome editing with the CRISPR/Cas9 nickase system as a strategy to investigate the role of ATRX-DAXX histone chaperone complex in telomere maintenance (Ran et al, 2013). Independent pairs of sgRNAs targeting human ATRX exon 16 or 21 region were transiently transfected into TP53 wild-type and telomerase-positive U87 glioma cells. Individual clones from the sgATRX-transfected cells were isolated and verified for their ATRX protein expression using immunofluorescence. Surprisingly, although we were able to clonally identify ATRX-deleted cells and grow them initially, the isolated mutant U87 cells A-1 and A-2 would eventually undergo cell cycle arrest and senescence as evidenced by the accumulation of β-galactosidase-positive senescent cell population (Fig 1A and B). Notably, this late onset of cell cycle checkpoint control was also observed in TP53-mutant LN464 glioma cells following CRISPR-induced ATRX abrogation (Fig EV1A and B). Given that cell cycle checkpoint was activated long after ATRX depletion (~10 cell doubling), we reason that the phenotype is unlikely caused by ATRX depletion directly. Figure 1. Depletion of ATRX induces growth arrest and telomere dysfunction in human cells A. Growth curves show proliferation reduction in ATRX-depleted U87 cells. Data are expressed as means ± standard deviation (SD), N = 2. 500 cells/well were plated. B. β-gal staining revealed the increased senescent cell population in ATRX-deleted U87 cells. Upper panel, micrographs of control and ATRX-deleted U87 cells processed for ATRX immunofluorescence (IF) and β-gal senescence assay. Scale bar, 200 μm. D, DAPI. Lower panel, percentage of senescent cells. Data are expressed as means ± SD, N = 3, unpaired t-test. C, D. PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87 cells (D). Scale bar, 10 μm. (D) Percentages of APB-positive cells are expressed as means ± SD, N = 3, unpaired t-test. E. C-circle assays revealed enhanced C-circle formation in ATRX-deleted U87 cells. Reactions lacking DNA or ɸ29 serve as negative controls. 30 ng of indicated template DNA was used in each reaction. F, G. 53BP1/TelG immuno-FISH (F) shows increased TIF formation in ATRX-deleted U87 cells (G). Arrows denote telomeres positive for 53BP1 signals. Scale bar, 10 μm. (G) Percentages of cells containing ≥ 4 TIFs are expressed as means ± SD, N = 3, unpaired t-test. H. Growth curves revealed increase in cell proliferation in ATRX-deleted U87 cells (A-1 and A-2) after hTERT transduction. Data are expressed as means ± SD, N = 2. I. Western blots show depletion of ATRX protein expression in hTERT-transduced and ATRX-deleted A-1 and A-2 U87 cells. Source data are available online for this figure. Source Data for Figure 1 [embj201796659-sup-0003-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. ATRX depletion induces telomeric dysfunction, ALT-like features, and cell growth arrest A. Growth curves show proliferation reduction in ATRX-depleted LN464 cells. Data are expressed as means ± standard deviation (SD), N = 2. 500 cells/well were plated. B. Immunofluorescence shows depletion of ATRX protein (green) in ATRX-deleted A-L1 and A-L2 LN464 cells. DAPI staining of nucleus is shown in blue. Scale bar, 25 μm. C, D. PML/TelG immuno-FISH (C) revealed increased APB formation in ATRX-deleted LN464 cells (D). Images are maximum-intensity projections of ˜10 stacks captured with a 63× lens with 2× zoom. Scale bar, 10 μm. (D) Percentages of APB-positive cells are expressed as means ± SD, N = 3, unpaired t-test. E. Growth curves revealed increase in cell proliferation in ATRX-deleted LN464 cells (A-L1 and A-L2) after transduction of hTERT. Data are expressed as means ± SD, N = 2. F. Western blots show depletion of ATRX protein expression in ATRX-deleted A-L1 and A-L2 LN464 cells transduced with hTERT. Download figure Download PowerPoint It has become increasingly clear that ATRX loss is associated with human cancers or cell lines that employ ALT for telomere maintenance (Heaphy et al, 2011; Lovejoy et al, 2012; Schwartzentruber et al, 2012). To determine whether ATRX abrogation activates ALT, we examined ALT-associated features in those clonally isolated ATRX-deleted cells. Indeed, telomere fluorescence in situ hybridization (FISH) analysis of both ATRX-deleted U87 (Fig 1C and D) and LN464 cells (Fig EV1C and D) revealed robust co-localization of TTAGGG foci with promyelocytic leukemia (PML) bodies, a pattern indistinguishable from the ALT-associated PML bodies (APBs) (Yeager et al, 1999). The activation of ALT-associated pathway was further substantiated by the detection of partial single-stranded telomeric (CCCTAA)(n) DNA circles (C-circles) (Fig 1E), another feature typical for ALT cells (Henson et al, 2009). In addition to the ALT-associated features, we also observed a significantly elevated telomeric DNA damage response (DDR) in the ATRX-deleted U87 A-1 and A-2 cells, as indicated by their increased formation of 53BP1-associated telomere dysfunction-induced foci (TIF) (Fig 1F and G). This suggests that ATRX is required for telomere DNA damage protection. To test whether the observed telomere dysfunction is responsible for the ATRX deletion-induced cell cycle checkpoint activation, we transduced the ATRX-deficient U87 or LN464 cells with a lentiviral-encoded telomerase catalytic subunit hTERT. Indeed, ectopic hTERT overexpression rescued the growth defects of the mutant cells (Figs 1H and I, and EV1E and F), suggesting telomere dysfunction as the likely cause of ATRX loss-induced growth phenotype. TERT overexpression alleviates ATRX deletion-associated telomere DNA damage response U87 and LN464 cells are telomerase-positive tumor cell lines. Considering the observation that their endogenous telomerase activities are insufficient to suppress ATRX deletion-induced cell cycle arrest, we questioned whether the ATRX loss-associated telomere dysfunction was caused by progressive telomere shortening. To test this, we next generated clonally derived ATRX-deleted cells from U87-T or LN464-T cells that were pre-transduced with a floxed version of TERT expression construct. Notably, this lox-TERT-lox system enables inducible deletion of the exogenously transduced TERT by Cre-mediated recombination. As expected, abrogation of ATRX in the TERT-overexpressed U87-T or LN464-T cells had only minor effect on their proliferation (Figs 2A and EV2A and B). In comparison, further removal of the exogenous floxed TERT by adenovirus-encoded Cre (Ad-Cre) provoked a rapid onset of cell cycle arrest in those ATRX-deficient cells, further indicating that their endogenous telomerase activities are not sufficient to suppress ATRX loss-induced telomere dysfunction. Figure 2. Exogenous TERT expression mitigates ATRX depletion-induced telomeric dysfunction A. Growth curves revealed that deletion of ATRX had minor effect on proliferation of TERT-overexpressed U87-T cells, but further removal of the exogenous floxed TERT by Ad-Cre induced rapid cell cycle arrest. Data are expressed as means ± SD, N = 3. B. Telomere restriction fragment (TRF) analysis shows increased telomeric length heterogeneities in ATRX-deleted U87-T and the further enhancement following Ad-Cre treatment. Genomic DNAs (EtBr staining, left panel) prepared from control and ATRX-deleted U87-T cells at day 9 post-control or Ad-Cre infection (middle panel) were assayed by hybridization with 32P-labeled (TTAGGG)4 probe, followed by re-hybridization with an oligonucleotide probe specific for centromere region (right panel). Genomic DNA from U2OS cells was used as an ALT-positive control. C, D. PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87-T cells (D) at day 9 post-control or Ad-Cre infection. Images are maximum-intensity projections of ˜10 stacks captured with a 63× lens with 2× zoom. Scale bar, 10 μm. (D) Percentages of APB-positive cells are expressed as means ± SD, N = 3, unpaired t-test. E. 2D gel analysis revealed increased T-circle formation in ATRX-deleted U87-T cells without or with Ad-Cre infection. Shown are gels stained with EtBr and blots hybridized with a TelG probe. Indicated are linear (lin) and open (oc) T-circle forms of telomeric DNA. F. C-circle assays show increased C-circle formation in ATRX-deleted U87-T cells at day 9 post-control or Ad-Cre infection. G, H. 53BP1/TelG immuno-FISH (G) shows increased TIF formation in ATRX-deleted U87-T cells at day 9 post-control or Ad-Cre infection (H). Scale bar, 10 μm. (H) Percentages of cells containing ≥ 4 TIFs are expressed as means ± SD, N = 3, unpaired t-test. Source data are available online for this figure. Source Data for Figure 2 [embj201796659-sup-0004-SDataFig2.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Ablation of exogenous TERT expression in ATRX-deleted LN464-T cells enhances ALT-like activity A. Western blots show depletion of ATRX protein expression in hTERT-transduced and ATRX-deleted A-d#1 and A-d#2 LN464 cells. B. Growth curves revealed that ATRX deletion had minor effect on proliferation of TERT-overexpressed LN464-T cells, but further removal of the floxed exogenous TERT by Ad-Cre induced rapid cell growth arrest. Data are expressed as means ± SD, N = 3. C. TRF analysis shows increased telomeric length heterogeneities in ATRX-deleted LN464-T and the further enhancement following Ad-Cre treatment. Genomic DNAs (EtBr staining, left panel) prepared from control and ATRX-deleted LN464-T cells at day 9 post-control or Ad-Cre infection were assayed by hybridization with 32P-labeled (TTAGGG)4 probe (middle panel), followed by re-hybridization with an oligonucleotide probe specific for centromere region (right panel). D, E. PML/TelG immuno-FISH (D) revealed increased APB formation in ATRX-deleted LN464-T cells (E) at day 9 post-control or Ad-Cre infection. Images are maximum-intensity projections of ˜10 stacks captured with a 63× lens with 2× zoom. Scale bar, 5 μm. (E) Percentages of APB-positive cells are expressed as means ± SD, N = 3, unpaired t-test. F, G. C-circle assays (F) show increased C-circle formation in ATRX-deleted LN464 cells at day 9 post-control (−) or Ad-Cre (+) infection (G). (G) Relative fold change was calculated by normalizing to the signal of U2OS cells. Data are expressed as means ± SD, N = 3, paired t-test. H, I. Exogenous TERT expression suppresses ATRX loss-associated telomere DNA damage response. 53BP1/TelG immuno-FISH (H) was performed in ATRX-deleted LN464-T cells at day 9 following control (−) or Ad-Cre (+) infection. Scale bar, 5 μm. (I) Percentages of TIF-positive cells are expressed as means ± SD, N = 3, unpaired t-test. J. Western blots show increased DNA damage response in ATRX-deleted U87-T cells after Ad-Cre treatment. Protein extracts from control and ATRX-deleted U87-T cells were collected at day 9 post-control (−) or Ad-Cre (+) infection and processed against indicated antibodies. Download figure Download PowerPoint ALT cells are known to adopt a unique homology-directed telomere maintenance mechanism that results in telomere length heterogeneity (Cesare & Reddel, 2010; Pickett & Reddel, 2015). To test whether deletion of ATRX in those TERT-overexpressed cells would also activate ALT-associated activities, we next performed telomere restriction fragment (TRF) analysis of ATRX-deleted mutant U87-T and LN464-T cells. Indeed, Southern blot analysis of both cells revealed enhanced telomeric length heterogeneities, compared to respective control cells (Figs 2B and EV2C). By contrast, the length distribution of centromeric DNA in the same ATRX-depleted cells did not show significant alterations, indicative of a telomere-specific effect. Consistently, fluorescence telomere FISH assay of ATRX-deleted mutant U87-T (Fig 2C and D) and LN464-T cells (Fig EV2D and E) also showed increased APB formation. The activation of ALT-associated activities was further confirmed by two-dimensional (2D) gel assays of ATRX-abrogated U87-T A-d#1 and A-d #2 cells that exhibited markedly elevated extrachromosomal telomeric t-circle signals when compared to control U87-T cells (Fig 2E). Finally, the ATRX-deleted U87-T and LN464-T cells also showed prominent C-circle formation, a feature not observed in the control U87-T or LN464-T cells (Figs 2F and EV2F and G). The di