Telomeres control human telomerase (TERT) expression through non-telomeric TRF2

  1. Antara Sengupta
  2. Soujanya Vinayagamurthy
  3. Dristhi Soni
  4. Rajlekha Deb
  5. Ananda Kishore Mukherjee
  6. Subhajit Dutta
  7. Jushta Jaiswal
  8. Mukta Yadav
  9. Shalu Sharma
  10. Sulochana Bagri
  11. Shuvra Shekhar Roy
  12. Priya Poonia
  13. Ankita Singh
  14. Divya Khanna
  15. Amit Kumar Kumar Bhatt
  16. Akshay Sharma
  17. Suman Saurav
  18. Rajender K Motiani
  19. Shantanu Chowdhury  Is a corresponding author
  1. Integrative and Functional Biology Unit, CSIR-Institute of Genomics and Integrative Biology, India
  2. Academy of Scientific and Innovative Research (AcSIR), India
  3. CSIR-Institute of Genomics and Integrative Biology, India
  4. Laboratory of Calciomics and Systemic Pathophysiology (LCSP), Regional Centre for Biotechnology (RCB), India
12 figures, 1 table and 1 additional file

Figures

Graphical representation of the Telomere Sequestration Partitioning (TSP) model.

The binding of TRF2 outside of telomeric regions (non-telomeric) is dependent on telomere length. When telomeres shorten, the occupancy of TRF2 at telomeres diminishes, resulting in increased TRF2 binding at non-telomeric sites. This shift in TRF2 distribution triggers epigenetic modifications at promoters showing telomere-dependent gene regulation. Schematic reused from Supplementary Information in Mukherjee et al, eLife, 2025.

Figure 2 with 1 supplement
Telomere-dependent non-telomeric TRF2 binding at the telomerase reverse transcriptase (TERT) promoter controls TERT expression.

(A) Telomere length in isogenic cancer cell lines with short telomeres (ST, in grey) or long telomeres (LT, in blue) namely, HT1080-ST/LT, MDA-MB-231-ST/LT, and HCT116- ST/LT (Telomere trimming-Cas9 and TERC knockdown) as determined by Flow cytometry (FACS). MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models were generated by telomerase-independent mode of telomere length (TL) alteration (See Methods). Relative fold change is shown in insets with FACS plots. (B–C) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 (B) and H3K27me3 (C) in respective ST/LT cells as mentioned in (A); occupancy normalised to respective IgG or total Histone H3 (for H3K27me3). qPCR on the GAPDH promoter was used as the negative control in all cases. (D) TERT mRNA expression by qRT-PCR in ST/LT cell line pairs as mentioned in (A) normalised to GAPDH or 18 S mRNA levels. Primers specific to 3'UTR for endogenous TERT were used for the HT1080-ST/LT system where telomerase was overexpressed for telomere elongation; primers for functional (reverse transcriptase domain) (exon7/8) and full-length (exon 15/16) transcript were used for all other systems. MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models have been analysed as paired samples in each biological replicates. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. P-values are calculated by an unpaired t-test for all data except mRNA for MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models where paired t-test was performed. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 2—source data 1

Source data for all plots in Figure 2, related to HT1080 ST/LT cell line model.

https://cdn.elifesciences.org/articles/104045/elife-104045-fig2-data1-v1.xlsx
Figure 2—source data 2

Source data for all plots in Figure 2, related to MDA MB 231 ST/LT cell line model.

https://cdn.elifesciences.org/articles/104045/elife-104045-fig2-data2-v1.xlsx
Figure 2—source data 3

Source data for all plots in Figure 2, related to HCT116 ST/LT Telomere trimming-Cas9 model.

https://cdn.elifesciences.org/articles/104045/elife-104045-fig2-data3-v1.xlsx
Figure 2—source data 4

Source data for all plots in Figure 2, related to HCT116 ST/LT TERC knockdown model.

https://cdn.elifesciences.org/articles/104045/elife-104045-fig2-data4-v1.xlsx
Figure 2—figure supplement 1
Telomere-dependent non-telomeric TRF2 binding at the telomerase reverse transcriptase (TERT) promoter controls TERT expression (continued).

(A) Telomere length in isogenic cancer cell lines with short telomeres (ST, in grey) or long telomeres (LT, in blue) namely, HT1080- ST/LT, MDA-MB-231-ST/LT, and HCT116- ST/LT (Telomere trimming-Cas9 and TERC knockdown) as determined by qRT-PCR based assay (See Methods). (B) Telomerase activity in isogenic cancer cells with short telomeres (ST) or long telomeres (LT) namely, HT1080- ST/LT, MDAMB 231-ST/LT, HCT116-ST/LT and HCT116 p53 null -ST/LT, determined using telomerase-repeat-amplification protocol (TRAP) followed by ELISA (see Methods); (C) Scheme showing the telomeric specific sgRNA used to generate short telomere versions in HCT116, HEK293T CCR5 promoter insert cells and iPSCs using transient expression of Cas9- telomere targeting sgRNA plasmid. Error bars represent ± SDs from the mean from two independent biological replicates. p values are calculated by unpaired t-test. (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001).

Telomere length dictates chromatin accessibility at the telomerase reverse transcriptase (TERT) promoter.

(A, B) ChIP followed by qRT-PCR at the TERT promoter for REST (A) and EZH2 (B) in HT1080-ST/LT, MDA-MB-231-ST/LT, and HCT116- ST/LT (Telomere trimming-Cas9 and hTERC knockdown) cells; occupancy normalised to respective IgG. qPCR on the GAPDH promoter was used as the negative control in all cases. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. P-values are calculated by unpaired t-test (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Artificially inserted telomerase reverse transcriptase (TERT) promoter shows telomere length–dependent regulation.

(A) Scheme depicting CRISPR modified HEK293T cells with 1300 bp TERT promoter driving Gaussia luciferase (Gaussia Luc) construct inserted at the CCR5 safe harbour locus. Scheme denotes ChIP primers used to study chromatin occupancy of 0–300 bp TERT promoter region inserted at the exogenous locus. (B) Relative fold change in telomere length in TERT promoter insert cells following telomere shortening determined by FACS; quantification in the right panel. (C,D) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) insert at CCR5 locus for TRF2 (C), and H3K27me3 (D) in ST/LT cells. Occupancy normalised to respective IgG or total histone H3 (for H3K27me3). (E) TERT promoter-Gaussia luciferase activity in ST cells over LT cells from inserted exogenous with TERT promoter. Reporter activity is presented as luminescence (arbitrary units, a.u.) normalised to respective total protein levels. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. P-values are calculated by an unpaired t-test for all data except mRNA for MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models where paired t-test was performed. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 5 with 1 supplement
Temporal telomere length elongation followed by shortening shows telomere-sensitive transcriptional regulation of telomerase reverse transcriptase (TERT) in HT1080 cells.

(A) Scheme depicting the protocol followed for doxycycline (Dox) inducible TERT overexpression in HT1080 cells (Dox-HT1080). ++/-- denotes the presence/ absence of dox at the indicated day points. (B) Relative fold change in telomere length at Day 0,10, and 24 determined by FACS in Dox-HT1080 cells; quantification in right panel. (C–F) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 (C) REST (D) EZH2 (E) or H3K27me3 (F) in Dox-HT1080 cells at Day 0,10, and 24; occupancy normalised to respective IgG or total Histone H3 (for H3K27me3). (G) TERT mRNA expression by qRT-PCR using TERT-specific 3'UTR primers in Dox- HT1080 cells at day intervals (as indicated); normalised to GAPDH mRNA levels. Fold changes were calculated independently for each biological replicate, as the three conditions represent paired samples (uninduced, induced with doxycycline, and post-doxycycline withdrawal). Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. One-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across time points in Figs C-G (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 5—figure supplement 1
Temporal telomere length elongation followed by shortening shows telomere-sensitive transcriptional regulation of telomerase reverse transcriptase (TERT) in HT1080 cells (continued).

(A) Relative fold change in telomere length at multiple day intervals (as indicated) in Dox-HT1080 cells as determined by qPCR- based telomere length detection method Telomeric signal normalised over single copy gene, 36B4 for qPCR-based analysis. ++/-- denotes the presence/ absence of dox at the indicated day points. (B) Telomerase activity at multiple-day intervals (as indicated) in Dox-HT1080 cells determined using telomerase-repeat-amplification-protocol (TRAP) followed by ELISA (see Methods); ++/-- denotes the presence/ absence of dox at the indicated day points. All error bars represent ± SDs from the mean from two independent biological replicates. One-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across time points in figures (A,B). (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 6 with 1 supplement
Temporal telomere length elongation followed by shortening shows telomere-sensitive transcriptional regulation of telomerase reverse transcriptase (TERT) in MDAMB 231 cells.

(A) Scheme depicting protocol followed for doxycycline (Dox) inducible TERT overexpression in MDA-MB-231 (Dox-MDA-MB-231). ++/-- denotes the presence/ absence of dox at the indicated day points. (B) Relative fold change in telomere length at Day 0,10, and 14 determined by Flow cytometry in Dox-MDA-MB-231; quantification in right panel. (C–F) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 (C) REST (D) EZH2 (E) or H3K27me3 (F) in Dox-MDA-MB-231 cells at Day 0,10, and 14; occupancy normalised to respective IgG or total Histone H3 (for H3K27me3). (G) TERT mRNA expression by qRT-PCR using TERT-specific 3'UTR primer in Dox- MDA-MB-231 cells at day intervals (as indicated); normalised to GAPDH mRNA levels. Fold changes were calculated independently for each biological replicate, as the three conditions represent paired samples (uninduced, induced with doxycycline, and post-doxycycline withdrawal). Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. One-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across time points in Figs C-G (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 6—figure supplement 1
Temporal telomere length elongation followed by shortening shows telomere-sensitive transcriptional regulation of telomerase reverse transcriptase (TERT) in MDAMB 231 cells (continued).

(A) Relative fold change in telomere length at multiple day intervals (as indicated) in Dox-MDAMB231 as determined by qPCR- based telomere length detection method Telomeric signal normalised over single copy gene, 36B4 for qPCR-based analysis. ++/-- denotes the presence/ absence of dox at the indicated day points. (B) Telomerase activity at multiple-day intervals (as indicated) in Dox-MDAMB231 cells determined using telomerase-repeat-amplification-protocol (TRAP) followed by ELISA (see Methods); ++/-- denotes the presence/ absence of dox at the indicated day points. All error bars represent ± SDs from the mean from two independent biological replicates. One-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across time points in figures (A,B). (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 7 with 1 supplement
Telomere length-sensitive telomerase reverse transcriptase (TERT) regulation in in vivo tumour xenografts (A) Scheme depicting the generation of tumour xenograft with HT1080-ST or HT1080-LT cells.

(B) Relative fold change in telomere length in xenograft samples determined by qRT- PCR-based telomere length detection method as reported earlier (O’Callaghan et al., 2008 and Cawthon, 2002). Telomeric signal normalised over single copy gene, 36B4. (C) TERT (exon 15/16 full-length transcript) and hTERC mRNA expression in xenograft tissues by qRT-PCR; normalised to 18 S mRNA levels. (D, E) ChIP followed by qPCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 (D) and H3K27me3 (E); occupancy normalised to respective IgG and total H3 (for H3K27me3). Error bars represent ± SDs across individual values of n=5 xenograft tumour samples in each group. P-values are calculated by unpaired t-test with Welch’s correction (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 7—figure supplement 1
Telomere length-sensitive telomerase reverse transcriptase (TERT) regulation in in vivo tumour xenografts (continued).

(A) Telomerase activity in xenograft tissues of HT1080-ST and HT1080-LT cells.

G-quadruplex-mediated TRF2 binding is essential for telomere-dependent telomerase reverse transcriptase (TERT) regulation.

(A) Scheme depicting CRISPR-modified HEK293T cells with 1300 bp TERT promoter with G>A substitution at –124 or –146 bp (upstream of TSS) driving Gaussia luciferase (Gaussia Luc) construct at the CCR5 safe harbour locus. Scheme denotes ChIP primers used to study chromatin occupancy of 0–300 bp TERT promoter region inserted at the exogenous locus. (B) Relative fold change in telomere length in two independent pairs of cells with short or long telomeres containing TERT promoter G4 disrupting mutations (–124G>A or –146 G>A) at CCR5 locus TERT promoter insert as determined by Flow cytometry; quantification in right panel. (C, D) ChIP followed by qRT-PCR at the inserted TERT promoter (0–300 bp upstream of TSS) for TRF2 (C), and H3K27me3 (D) in pairs of cells generated with –124G>A or –146 G>A mutation with long/ short telomeres, along with WT promoter ST/LT pair (as in Figure 4C and D). Occupancy normalised to respective IgG or total histone H3 (for H3K27me3). (E) TERT promoter-Gaussia luciferase activity in short or long telomere cells with - 124G>A and –146 G>A mutated TERT promoter sequence, along with WT promoter ST/LT pair (as in Figure 4E). Reporter activity presented as luminescence (arbitrary units, a.u.) normalised to respective total protein levels. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test for all pairwise comparisons. For planned comparisons between each parental and short-telomere cell line, unpaired t-tests were used. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 9 with 1 supplement
TRF2 R17 residue is required for the repression of telomerase reverse transcriptase (TERT) expression.

(A) TERT full-length transcript (exon 15/16) mRNA expression levels by qRT-PCR; normalised to GAPDH mRNA levels upon stable doxycycline induction of wild-type (WT) TRF2 or R17H TRF2 mutants in HT1080, HCT116, or MDA-MB-231 cells. (B) TERT mRNA FISH upon TRF2 WT or TRF2 R17H overexpression (untransfected, UT as control) in HT1080 cells; quantification shown in right panel. FLAG-tagged TRF2 overexpression was confirmed by Immunofluorescence microscopy. (C–F) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for FLAG-tagged TRF2 (C), REST (D), EZH2 (E), or H3K27me3 (F) in HT1080 cells upon expression of WT TRF2 or TRF2 R17H. Occupancy normalised to respective IgG and total Histone H3 (for H3K27me3); qRT-PCR on the GAPDH promoter was used as the negative control in all cases. (G) In vitro methyltransferase activity of the reconstituted PRC2 complex resulting in H3K27 trimethylation in the presence or absence of TRF2 WT or TRF2 R17H protein. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. Unpaired t-tests were conducted to assess the significance of each condition individually within the same cell line, and to compare the two conditions TRF2-WT or TRF2-R17H in (A); one-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across the three conditions in (B); P-values are calculated by unpaired t-test in (C–F); and two-way ANOVA followed by post-hoc tests (Tukey’s HSD) in (G). (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 9—figure supplement 1
TRF2 R17 residue is required for the repression of telomerase reverse transcriptase (TERT) expression(continued).

(A) TRF2 mRNA levels in HT1080 cells as TRF2 PTM variants are overexpressed under endogenous TRF2 silenced conditions using shRNA (B) telomerase reverse transcriptase (TERT) full-length mRNA transcript (exon 15/16) levels by RT-PCR in HT1080 cells upon overexpression of TRF2 PTM variants as depicted in (A) under endogenous TRF2 knockdown condition (B). (C) TRF2 protein induction with Dox treatment in HT1080, HCT116, and MDAMB 231 TRF2 inducible lentiviral stable cells, confirmed by Western blot analysis Mol. Wt. ladder used in HT1080 and MDAMB 231 is Puregene 4 colour Prestained Protein Ladder,10–180 kDa and that of HCT116 is G Biosciences PAGEmark Tricolour PLUS. (D) Dose-dependent Dox induction of TRF2-WT and R17H variant (Left graph) in stable inducible TRF2 HT1080 cells to check TERT full-length mRNA transcript (exon 15/16) levels by RT-PCR (right graph). (E) Purified TRF2 WT and TRF2 R17H protein from HEK 293T cells as developed by anti-TRF2 and anti-FLAG antibodies (left panel) and representative Coomassie Brilliant Blue (CBB) gel for protein purification protocol. The lower band in the anti-FLAG blot is of bead-bound FLAG peptide. (F) H3K27 trimethylation levels in in vitro histone methyltransferase assay with empty (TRF2 unbound) FLAG beads. Error bars represent ± SDs from the mean from two independent biological replicates. P-values are calculated by an unpaired t-test in 4B. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Figure 9—figure supplement 1—source data 1

Source data of all plots in Figure 9—figure supplement 1.

https://cdn.elifesciences.org/articles/104045/elife-104045-fig9-figsupp1-data1-v1.xlsx
Figure 9—figure supplement 1—source data 2

PDF file containing original western blot for Figure 9C indicating the relevant bands and treatment.

https://cdn.elifesciences.org/articles/104045/elife-104045-fig9-figsupp1-data2-v1.pdf
Figure 9—figure supplement 1—source data 3

Original image files for western blot for Figure 9C.

https://cdn.elifesciences.org/articles/104045/elife-104045-fig9-figsupp1-data3-v1.zip
Figure 9—figure supplement 1—source data 4

PDF file containing original western blot for Figure 9E indicating the relevant bands.

https://cdn.elifesciences.org/articles/104045/elife-104045-fig9-figsupp1-data4-v1.pdf
Figure 9—figure supplement 1—source data 5

Original image files for western blot for Figure 9E.

https://cdn.elifesciences.org/articles/104045/elife-104045-fig9-figsupp1-data5-v1.zip
Telomere length influences telomerase reverse transcriptase (TERT) regulation in fibroblasts and derived iPSCs.

(A) Scheme showing generation of induced pluripotent stem cells (iPSCs) from foreskin fibroblast (FS Fibroblast) cells by overexpressing Yamanaka factors (Oct4, Sox2, Klf4, Myc). (B) Characterization of iPSCs (Upper panel, bright field image) generated from FS Fibroblast cells by immunofluorescence using Oct-4, SSEA-4, Sox-2, and TRA-1–60 antibodies as stemness markers. (C) mRNA levels for TERT (full-length exon 15/16 transcript), TERC (RNA component), and stemness marker genes Nanog, Klf4 in FS fibroblast and derived iPSC, analysed in pairs in each biological replicates. (D) Telomerase activity in FS Fibroblast cells and derived iPSCs determined using telomerase-repeat-amplification-protocol (TRAP) followed by ELISA (see Methods). (E) Relative fold change in telomere length in primary FS Fibroblast cells and derived iPSC, determined by qPCR-based telomere length detection method. (F–I) ChIP followed by qPCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 (F), REST (G), EZH2 (H), and H3K27me3 (I) up to 300 bp upstream of transcription start site (TSS); occupancy normalized to respective IgG or total Histone H3 (for H3K27me3). qPCR on the GAPDH promoter was used as a negative control in all cases. All error bars represent ± SDs from the mean of two independent biological replicates of each experiment. P-values are calculated by an unpaired t-test (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Telomere shortening in induced pluripotent stem cellss (iPSCs) increases TRF2 binding and represses TERT.

(A) Scheme depicting generation of induced pluripotent stem cells (iPSCs) with shortened telomeres, (iPSC-ST) using telomere-specific sgRNA-guided CRISPR-Cas9 to trim telomeres. (B) Relative fold change in telomere length in iPSC-ST cells with respect to unaltered iPSC, determined by qPCR-based telomere length detection method. (C–D) ChIP followed by qPCR at the 0–300 bp TERT promoter (upstream of TSS) in iPSC-ST cells in comparison to unaltered iPSC, for TRF2 (L) and H3K27me3 (M); occupancy normalized to respective IgG or total Histone H3 (for H3K27me3). qPCR on the GAPDH promoter was used as a negative control in all cases. (E) mRNA levels for TERT (full-length exon 15/16 transcript), TERC (RNA component), and TRF2 in iPSC-ST over unaltered iPSC. All error bars represent ± SDs from the mean of 2 independent biological replicates of each experiment. p values are calculated by unpaired t-test (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001).

Telomere length controls human telomerase (TERT) expression through non-telomeric TRF2.

Illustration of the telomere-dependent epigenetic modification of chromatin structure at the TERT promoter, resulting in upregulation or downregulation of TERT expression due to altered non-telomeric TRF2 binding at the promoter. Relatively long telomeres cells (top panel) have lower TRF2 binding at the TERT promoter, promoting permissive chromatin and upregulation of TERT transcription. Conversely, shorter telomere cells (bottom panel) with increased TRF2 binding at the TERT promoter recruit more REST-PRC2 epigenetic complex causing increased repressor histone H3K27 trimethylation deposition. This leads to a more closed chromatin state at the TERT promoter, suppressing its transcription.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell Line (Homo sapiens)HT0180ATCCATCC -CCL-121
RRID:CVCL_0317
Fibrosarcoma
Cell Line (Homo sapiens)MDAMB231ATCCATCC HTB-26
RRID:CVCL_0062
Breast cancer
Cell Line (Homo sapiens)HCT116ATCCATCC-CCL-247
RRID:CVCL_0291
Colorectal carcinoma
Cell Line (Homo sapiens)HEK293TNCCS Cell RepositoryRRID:CVCL_0063Embryonic kidney derived
AntibodyTRF2 (rabbit polyclonal)Novus(Novus NB110-57130)
RRID:AB_844199
ChIP (1:100), IP (1:100), WB (1:1000)
AntibodyRESTMillipore#17–641
RRID:AB_1977463
ChIP (1:100)
AntibodyHistone H3 (rabbit polyclonal)Abcam(Abcam ab1791)- RRID:AB_302613ChIP (1:100)
AntibodyGAPDH (mouse monoclonal)Santa-cruz# sc-32233,
RRID:AB_627679
WB (1:1000)
AntibodyAnti-Rabbit IgG (rabbit polyclonal)Millipore(Millipore 12–370)
RRID:AB_145841
isotype control (1:100)
AntibodyAnti-mouse IgG (mouse polyclonal)Millipore(Millipore 12–371)
RRID:AB_145840
isotype control (1:100)
AntibodyH3K27me3 (rabbit monoclonal)Abcamab6002, RRID:AB_305237ChIP (1:100)
AntibodyEZH2 (rabbit monoclonal)Cell Signal Technology# 5246
RRID:AB_10694683
ChIP (1:100)
AntibodyDDK/FLAG (mouse monoclonal)Merck#F3165
RRID:AB_259529
ChIP (1:100), WB (1:1000)
AntibodyAnti-FLAG M2 Magnetic BeadsMilliporeM8823
RRID:AB_2637089
for protein purification
Recombinant DNA ReagentpENTR11InvitrogenK253520
Plasmid (Shuttle vector for Gateway Cloning)
Recombinant DNA ReagentpCW57.1Addgene41393Plasmid (Tet. Inducible lentiviral system - gateway cloning), used for generating TERT and TRF2 PTM induced expression system
Recombinant DNA ReagentTRF2 shRNAorigeneTL308880Plasmid
Recombinant DNA ReagentpSpCas9(BB)–2A-Puro (PX459) V2.0Addgene#62988
RRID:Addgene_62988
Plasmid
Recombinant DNA ReagentControl shRNA Plasmid-ASanta-cruzsc-108060Plasmid
Recombinant DNA ReagentTERC shRNASanta-Cruzsc-106994-SHPlasmid
Recombinant DNA ReagentpCMV6-myc-DDK(FLAG)-TRF2 vector (TRF2 WT, R17H)origeneRC223601Plasmid
Sequence-Based ReagentGTR OLIGOHuman telomeric sequencesynthesised by Sigma(TTAGGG)4
Peptide, Recombinant Proteinhistone H3 N-terminal peptideActive Motif kitCatalog No. 56100used as template for in vitro histone methyltransferase assay
Peptide, Recombinant ProteinEZH2 /EED/SUZ12/RbAp48/AEBP2 humanSigma-Aldrichcat no.SRP0381used for in vitro histone methyltransferase assay
Commercial Assay Or KitHistone H3 (tri-methyl K27) Quantification Kit (Colorimetric)abcamab115072used for in vitro histone methyltransferase assay
Commercial Assay Or KitHistone H3 (K27) Methyltransferase Activity Quantification Assay Kitabcamab113454histone assay buffer
Commercial Assay Or KitROCHE TeloTAGGG Telomerase PCR ELISARocheCat. No. 11 854 666 910Telomeric Repeat Amplification Protocol Kit
Chemical Compound, DrugDoxycyclineSigma AldrichD98911 µg/mL
Chemical Compound, DrugPuromycinGIBCOCatalog number A11138031 µg/mL
Software, AlgorithmUTRdb 2.0http://212.189.202.211
/utrdb/index_107.html
Used to design TERT 3'
UTR specific primers
OtherTetracycline-free
Fetal Bovine Serum (FBS)
Tet System Approved
FBS Clontech Laboratories, Inc.
#631106
OtherStellaris FISH Probes,
Human TERT
with Quasar 670 Dye
LGC BiosearCh
Technologies
VSMF-2411–5

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  1. Antara Sengupta
  2. Soujanya Vinayagamurthy
  3. Dristhi Soni
  4. Rajlekha Deb
  5. Ananda Kishore Mukherjee
  6. Subhajit Dutta
  7. Jushta Jaiswal
  8. Mukta Yadav
  9. Shalu Sharma
  10. Sulochana Bagri
  11. Shuvra Shekhar Roy
  12. Priya Poonia
  13. Ankita Singh
  14. Divya Khanna
  15. Amit Kumar Kumar Bhatt
  16. Akshay Sharma
  17. Suman Saurav
  18. Rajender K Motiani
  19. Shantanu Chowdhury
(2025)
Telomeres control human telomerase (TERT) expression through non-telomeric TRF2
eLife 14:RP104045.
https://doi.org/10.7554/eLife.104045.3