by Omesha N. Perera, Alexander P. Sobinoff, Erdahl T

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Telomerase promotes formation of a telomere protective complex in cancer cells by Omesha N. Perera, Alexander P. Sobinoff, Erdahl T. Teber, Ashley Harman, Michelle F. Maritz, Sile F. Yang, Hilda A. Pickett, Anthony J. Cesare, Jonathan W. Arthur, Karen L. MacKenzie, and Tracy M. Bryan Science Volume 5(10):eaav4409 October 2, 2019 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 1 A noncanonical function of hTERT in telomere protection. A noncanonical function of hTERT in telomere protection. (A) Relative TERT mRNA expression measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) after siRNA treatment of HT1080 cells for 48 hours (mean ± SE; n = 3 independent experiments). Normalized to control siRNA (siSc). *P = 0.011, **P = 0.0018. (B) Left: Representative images from the meta-TIF analysis of HT1080 cells depleted of hTERT for 48 hours. γ-H2AX immunofluorescence in red, telomere FISH in green, and chromosomes in blue. Right: Quantitation of γ-H2AX–associated telomeres from meta-TIF assays in HT1080 cells (mean ± SE; n = 3 independent experiments). Normalized to control siRNA (siSc). **P = 0.0064, ***P = 0.0002. (C) Fluorescence intensity of telomeric signals as a measure of telomere length in HT1080 cells, analyzed using the TFL-TELO program (60); 15 metaphase spreads were analyzed for each sample. One-way analysis of variance (ANOVA) was performed, P = 0.9996. (D) Left: Cell cycle profile using flow cytometry of HT1080 cells treated with control and hTERT siRNAs. Representative experiment quantifying >15,000 cells per condition. The x axis (PI-A) represents the propidium iodide intensity, while the y axis represents the cell count. Right: Quantitation of the proportion of cells in G1, S, and G2-M phases of the cell cycle (mean ± SE; n = 3 independent experiments). Two-tailed unpaired Student’s t tests were conducted on just the proportion of cells in G1. **P < 0.01, ****P < 0.0001. (E) hTERT Western blot using whole-cell extracts from HT1080 cells showing overexpression of sR hTERT (127 kDa) for 48 hours. Actin (42 kDa) is used as a loading control. (F) Relative TERT mRNA expression after siRNA treatment of HT1080 cells for 48 hours using qRT-PCR with primers specific for endogenous hTERT (mean ± SE; n = 3 independent experiments). Normalized to control siRNA + vector control (siSc Vec). **P = 0.0018, ***P < 0.001, ****P < 0.0001, n.s., not significantly different. (G) Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in HT1080 cells expressing sR hTERT (mean ± SE; n = 3 independent experiments). **P = 0.0022, ***P = 0.0008. (H) Western blot using whole-cell extracts from GM639 cells, showing transient overexpression of WT and catalytically inactive (D712A) hTERT for 24 hours. Actin was used as a loading control. (I) Left: Representative images from meta-TIF assays using GM639 cells overexpressing either WT or D712A hTERT for 24 hours. γ-H2AX immunofluorescence in red, telomere FISH in green, and chromosomes in blue. Right: Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in GM639 cells (mean ± SE; n = 3 independent experiments). **P = 0.0021, ***P = 0.0003. (J) Fluorescence intensity of telomere signals obtained from telomere FISH in GM639 cells, as a measure of telomere length, was analyzed using the TFL-TELO program (60); 15 metaphase spreads were analyzed for each sample. One-way ANOVA was performed, P = 0.8123. See also the Supplementary Materials, figs. S1 to S3. Omesha N. Perera et al. Sci Adv 2019;5:eaav4409 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 2 hTERT induces expression of heat shock protein genes through HSF1 and interacts with Hsp70-1. hTERT induces expression of heat shock protein genes through HSF1 and interacts with Hsp70-1. (A) hTERT Western blot using whole-cell extracts from GM639 cells showing overexpression of WT or catalytically inactive (D712A) hTERT for 24 hours in triplicate samples used for RNA sequencing. Actin is used as a loading control. (B) Venn diagram showing differential gene expression resulting from transient overexpression of WT and catalytically inactive (D712A) hTERT compared to the vector control (Vec). Only five genes were up-regulated >2-fold, including TERT. For full details of gene names and fold changes, refer to fig. S4A. (C) Validation using qRT-PCR of the up-regulation of Hsp70-1 genes HSPA1A and HSPA1B upon transient hTERT overexpression for 24 hours in GM639 cells. mRNA levels are normalized to vector control (mean ± SE; n = 3 independent experiments). *P < 0.05, ***P < 0.001, ****P < 0.0001. (D) Validation using qRT-PCR of the up-regulation of Hsp70-1 genes HSPA1A and HSPA1B upon transient hTERT overexpression for 24 hours in HT1080 cells. mRNA levels are normalized to vector control (mean ± SE; n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001. (E) hTERT (127 kDa) and Hsp70-1 (70 kDa) Western blots using whole-cell extracts from GM639 cells transiently overexpressing WT and D712A hTERT for 24 hours. Actin is used as a loading control. (F) Relative mRNA expression of HSPA1A and HSPA1B using qRT-PCR, in GM639 cells overexpressing hTERT with simultaneous depletion of HSF1, 24 hours after transfection (mean ± SE; n = 3 independent experiments). Normalized to Vec + control siRNA (Vec siSc). *P < 0.05, ****P < 0.0001. (G) Immunoprecipitation using antibodies against hTERT (sheep), Hsp70-1 (mouse), and immunoglobulin G (IgG) control [mouse (top) or sheep (bottom)] from lysates of 293T cells overexpressing hTERT. Input (2 × 104 cell equivalents) and eluates were visualized on Western blots probed for hTERT (goat antibody) and Hsp70 (rabbit antibody). Volumes of eluate loaded correspond to the following cell equivalents: 2 × 105 (hTERT eluate on hTERT blot), 4 × 105 (Hsp70 eluate on Hsp70 blot), 1 × 106 (IgG control), and 4 × 106 (hTERT eluate on Hsp70 blot and Hsp70 eluate on hTERT blot). (H) Representative fluorescent micrographs of GM639 cells transiently overexpressing WT hTERT for 24 hours, with immunofluorescence for Hsp70-1 (red) and GFP-tagged hTERT (green), and 4′,6-diamidino-2-phenylindole (DAPI) staining showing the nucleus. Scale bars, 10 μm. The two images shown are displaying primarily cytoplasmic localization of hTERT. (I) PLA for hTERT and Hsp70-1 (red), in HT1080 cells with and without hTERT overexpression, combined with hTERT immunofluorescence (green) and DAPI staining showing the nucleus (blue). A negative PLA control was performed by leaving out the Hsp70-1 primary antibody (−ve control). (J) 3D reconstruction of image in Fig. 2I, using the Imaris software (Bitplane), to further validate the nuclear localization of hTERT-Hsp70-1 PLA foci (red), with DAPI staining showing the nucleus (blue). See also the Supplementary Materials, fig. S4. Omesha N. Perera et al. Sci Adv 2019;5:eaav4409 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 3 Hsp70-1 is necessary for full telomere protection, but its overexpression is insufficient. Hsp70-1 is necessary for full telomere protection, but its overexpression is insufficient. (A) Left: Relative mRNA expression using qRT-PCR, showing knock-down of the indicated genes 48 hours after siRNA transfection of HT1080 cells (mean ± SE; n = 3 independent experiments). ****P < 0.0001. Middle: Representative images from the meta-TIF analysis of HT1080 cells with depletion of HSPA1A and HSPA1B for 48 hours. γ-H2AX immunofluorescence in red, telomere FISH in green, and chromosomes in blue. Right: Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in HT1080 cells with depletion of the indicated Hsp70-1 mRNAs (mean ± SE; n = 3 independent experiments). *P = 0.035, **P = 0.0049. (B) Left: Representative hTERT and Hsp70-1 Western blot using whole-cell extracts from GM639 cells, with transient overexpression of hTERT and simultaneous depletion of Hsp70-1 for 24 hours. Actin is used as a loading control. Right: Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in GM639 cells with and without transient overexpression of D712A hTERT and simultaneous depletion of the indicated Hsp70 mRNAs (mean ± SE; n = 3). **P < 0.005. (C) Left: hTERT Western blot using whole-cell extracts from GM639 cells, showing transient overexpression of GFP-tagged WT and R3E/R6E hTERT for 24 hours, following sorting for GFP fluorescence-positive cells. Actin is used as a loading control. Right: Relative mRNA expression of HSPA1A (left) and HSPA1B (right) in GM639 cells overexpressing WT and R3E/R6E mutant hTERT, using qRT-PCR, following sorting for GFP fluorescence-positive cells (mean ± SE; n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001. (D) Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in GM639 cells transfected with WT or R3E/R6E mutant hTERT (mean ± SE; n = 3 independent experiments). *P = 0.0125. (E) hTERT and Myc Western blots using whole-cell extracts from GM639 cells, showing transient overexpression of D712A hTERT and Myc-tagged ZNF827 (119 kDa). Actin is used as a loading control. (F) Relative mRNA expression of HSPA1A (left) and HSPA1B (right) in GM639 cells overexpressing either D712A hTERT or ZNF827 using qRT-PCR (mean ± SE; n = 3 independent experiments). *P = 0.011, **P < 0.01. (G) Left: Hsp70-1 Western blot using whole-cell extracts from GM639 cells, showing an up-regulation of Hsp70-1 following transient overexpression of D712A hTERT and Myc-tagged ZNF827. Actin is used as a loading control. Right: Quantitation of Hsp70-1 protein expression following transient overexpression of D712A hTERT and Myc-tagged ZNF827 (mean ± SE; n = 3 independent experiments). **P = 0.004, ***P = 0.0006. (H) Left: Representative images from meta-TIF assays using GM639 cells transiently overexpressing either D712A hTERT or ZNF827. γ-H2AX immunofluorescence in red, telomere FISH in green, and chromosomes in blue. Right: Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in GM639 cells (mean ± SE; n = 3 independent experiments). *P = 0.0189. See also the Supplementary Materials, fig. S5. Omesha N. Perera et al. Sci Adv 2019;5:eaav4409 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 4 hTERT induces telomeric localization of heat shock protein Hsp70-1. hTERT induces telomeric localization of heat shock protein Hsp70-1. (A) Representative fluorescent micrographs of HT1080 cells treated with two separate siRNAs targeting hTERT, with immunofluorescence for Hsp70-1 (red) and FISH for telomeres (green), and DAPI staining showing the nucleus (blue). Scale bars, 10 μm. White arrowheads indicate colocalizations between Hsp70-1 and telomeres (yellow), one of which is shown in the zoomed image on the far right. (B) Top: Relative hTERT mRNA expression measured by qRT-PCR after hTERT siRNA treatment of HT1080 cells (mean ± SE; n = 3 independent experiments). ***P < 0.001. Bottom: Quantitation of the colocalizations of Hsp70-1 and telomeres in HT1080 cells depleted of hTERT (mean ± SE; n = 3 independent experiments). ****P < 0.0001. (C) Representative fluorescent micrographs of GM639 cells sorted for cells overexpressing GFP-tagged WT hTERT or R3E/R6E hTERT, with immunofluorescence for Hsp70 (red), FISH for telomeres (green), and DAPI staining showing the nucleus (blue). Scale bars, 10 μm. White arrowheads indicate colocalizations between Hsp70 and telomeres (white), one of which is shown in the zoomed image on the far right. (D) Quantitation of colocalizations of Hsp70-1 and telomeres in GM639 cells overexpressing WT or R3E/R6E mutant hTERT (mean ± SE; n = 3 independent experiments). ****P < 0.0001. (E) Quantitation of colocalizations of Hsp70-1 and telomeres in GM639 cells overexpressing either D712A hTERT or ZNF827 (mean ± SE; n = 3 independent experiments). ****P < 0.0001. (F) PLA for detection of Hsp70-1 and TRF2 interactions (red) in HT1080 cells with and without hTERT overexpression. See also the Supplementary Materials, fig. S6. Omesha N. Perera et al. Sci Adv 2019;5:eaav4409 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 5 hTERT promotion of Hsp70-1 telomere localization is dependent on Apollo and assists the telomere protective function of Apollo. hTERT promotion of Hsp70-1 telomere localization is dependent on Apollo and assists the telomere protective function of Apollo. (A) Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in GM639 cells (mean ± SE; n = 3 independent experiments) with or without overexpression of TRF2. ***P = 0.0002. (B) Left: Representative Western blot showing transient overexpression of Myc-DDK–tagged Apollo (60 kDa) in HT1080 cells. Actin is used as a loading control. Right: PLA conducted for detection of Apollo-TRF2 interactions (red) in HT1080 cells with myc-Apollo overexpression. An anti-Myc tag antibody was used for Apollo detection. (C) PLA for detection of Apollo-Hsp70 interactions (red) in HT1080 cells overexpressing Apollo with and without hTERT overexpression. An anti-Myc tag antibody was used for Apollo detection. (D) Immunoprecipitation using antibodies against Myc tag (mouse antibody), Hsp70-1 (mouse), and IgG control (mouse) from lysates of 293T cells overexpressing Myc-DDK (FLAG)–tagged Apollo. Input (2 × 104 cell equivalents) and eluates were visualized on Western blots probed with antibodies against DDK (FLAG) tag (rabbit) and Hsp70 (rabbit). Volumes of eluate loaded correspond to the following cell equivalents: 5 × 105 (Apollo eluate on Apollo blot and Hsp70 eluate on Hsp70 blot), 1 × 106 (IgG control), 3 × 106 (Hsp70 eluate on Apollo blot), and 4 × 106 (Apollo eluate on Hsp70 blot). (E) Representative fluorescent micrographs (left) and quantitation of normalized colocalizations per cell (right) (mean ± SE; n = 3 independent experiments) of HT1080 cells overexpressing Myc-DDK–tagged Apollo and the indicated siRNAs, with immunofluorescence for Myc tag (red) and TRF2 (green), and DAPI staining showing the nucleus (blue). Scale bars, 10 μm. White arrowheads indicate colocalizations between Apollo and TRF2 (white). *P = 0.038, **P < 0.01. (F) Quantitation of the percentage of γ-H2AX–associated telomeres from meta-TIF assays in GM639 cells with knock-down of DCLRE1B (Apollo), with and without overexpression of D712A hTERT (mean ± SE; n = 3). **P = 0.0056. Note that the depletion of Apollo in GM639 cells, which have a high basal level of meta-TIFs, did not further increase telomere deprotection, suggesting that this cell line is unable to tolerate higher levels of telomere dysfunction. (G) Representative fluorescent micrographs of GM639 cells overexpressing WT hTERT with immunofluorescence for Hsp70-1 (red) and TRF2 (green), and DAPI staining showing the nucleus (blue). Scale bars, 10 μm. White arrowheads indicate colocalizations between Hsp70-1 and TRF2 (yellow), one of which is shown in the zoomed image on the right. (H) Quantitation of the colocalizations of Hsp70-1 and TRF2 immunofluorescence in GM639 cells shown in (G) (mean ± SE; n = 3). ****P < 0.0001. (I) Left: Examples of normal or aberrant telomere staining on chromosomes from metaphase spreads of HT1080 cells, with FISH against telomeres (green) and centromeres (red). Right: Quantitation of numbers of “fragile telomeres” per metaphase in HT1080 cells depleted of Hsp70 (siHSPA1A + siHSPA1B), hTERT, or Apollo (mean ± SE; n = 40 metaphases counted from each of three independent experiments). *P = 0.02, ****P < 0.0001. See also the Supplementary Materials, fig. S7. Omesha N. Perera et al. Sci Adv 2019;5:eaav4409 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 6 Model for the telomere protective function of hTERT and Hsp70-1. Model for the telomere protective function of hTERT and Hsp70-1. hTERT and Hsp70-1 engage in a positive feedback loop, in which hTERT induces expression of Hsp70-1, resulting in its own stabilization and proper folding. We have demonstrated that hTERT is also able to promote the localization of Hsp70-1 to deprotected telomeres, where it stabilizes the Apollo-TRF2 complex. This allows Apollo to engage in its known function of facilitating telomere replication. In this way, hTERT provides a protective function to telomeres and thereby avoids telomere damage-induced cell cycle arrest. Omesha N. Perera et al. Sci Adv 2019;5:eaav4409 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).