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.

Telomere-dependent non-telomeric TRF2 binding at the hTERT promoter controls hTERT 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 hTERC 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 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 hTERT 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) hTERT mRNA expression by qRT-PCR in ST/LT cell line pairs as mentioned in (A), normalised to GAPDH or 18S mRNA levels. Primers specific to 3’UTR for endogenous hTERT 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.

(E, F) ChIP followed by qRT-PCR at the hTERT promoter for REST (E) and EZH2 (F) in respective ST/LT cells as mentioned in (A); occupancy normalised to respective IgG. qPCR on the GAPDH promoter was used as the negative control in all cases.

(G) Scheme depicting CRISPR modified HEK293T cells with 1300 bp hTERT promoter driving gaussia luciferase (Gluc) construct inserted at the CCR5 safe harbour locus. Scheme denotes ChIP primers used to study chromatin occupancy of 0-300 bp hTERT promoter region inserted at the exogenous locus.

(H) Relative fold change in telomere length in hTERT promoter insert cells following telomere shortening determined by FACS; quantification in the right panel.

(I, J) ChIP followed by qRT-PCR at the 0-300 bp hTERT promoter (upstream of TSS) insert at CCR5 locus for TRF2 (J), and H3K27me3 (K) in ST/LT cells. Occupancy normalised to respective IgG or total histone H3 (for H3K27me3).

(K) hTERT promoter-gaussia luciferase activity in ST cells over LT cells from inserted exogenous with hTERT promoter. Reporter activity normalised to respective total protein levels.

Error bars represent ± SDs from the mean of 3 independent biological replicates of each experiment. p values are calculated by unpaired t-test (with Welch’s correction in (G)) (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001).

Temporal telomere length elongation followed by shortening shows telomere-sensitive transcriptional regulation of hTERT.

(A) Scheme depicting protocol followed for doxycycline (Dox) inducible hTERT 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 hTERT 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) hTERT mRNA expression by qRT-PCR using hTERT specific 3’UTR primers in Dox-HT1080 cells at day intervals (as indicated); normalised to GAPDH mRNA levels.

(H) Scheme depicting protocol followed for doxycycline (Dox) inducible hTERT overexpression in MDA-MB-231 (Dox-MDA-MB-231). ++/-- denotes the presence/ absence of dox at the indicated day points.

(I) 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.

(J-M) ChIP followed by qRT-PCR at the 0-300 bp hTERT promoter (upstream of TSS) for TRF2 (J), REST (K), EZH2 (L) or H3K27me3 (M) in Dox-MDA-MB-231 cells at Day 0,10, and 14; occupancy normalised to respective IgG or total Histone H3 (for H3K27me3).

(N) hTERT mRNA expression by qRT-PCR using hTERT specific 3’UTR primer in Dox-MDA-MB- 231 cells at day intervals (as indicated); normalised to GAPDH mRNA levels.

Error bars represent ± SDs from the mean of 3 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, J,-N (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001).

Telomere length sensitive hTERT 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 et al 2002). Telomeric signal normalised over single copy gene, 36B4.

(C) hTERT (exon 15/16 full-length transcript) and hTERC mRNA expression in xenograft tissues by qRT-PCR; normalised to 18S mRNA levels.

(D, E) ChIP followed by qPCR at the 0-300 bp hTERT 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).

G-quadruplex mediated TRF2 binding is essential for telomere-dependent hTERT regulation.

(A) Scheme depicting CRISPR modified HEK293T cells with 1300 bp hTERT promoter with G>A substitution at -124 or -146 bp (upstream of TSS) driving gaussia luciferase (Gluc) construct at the CCR5 safe harbour locus. Scheme denotes ChIP primers used to study chromatin occupancy of 0-300 bp hTERT 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 hTERT promoter G4 disrupting mutations (-124G>A or - 146G>A) at CCR5 locus hTERT promoter insert as determined by Flow cytometry; quantification in right panel.

(C, D) ChIP followed by qRT-PCR at the inserted hTERT promoter (0-300 bp upstream of TSS) for TRF2 (C), and H3K27me3 (D) in pairs of cells generated with -124G>A or -146G>A mutation with long/ short telomeres. Occupancy normalised to respective IgG or total histone H3 (for H3K27me3).

(E) hTERT promoter-gaussia luciferase activity in short or long telomere cells with - 124G>A and - 146G>A mutated hTERT promoter sequence. Reporter activity normalised to respective total protein levels. Fold changes have been calculated over unmutated promoter-gluc LT cells (Fig 2L) for all cases.

Error bars represent ± SDs from the mean of 3 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).

TRF2 R17 residue is required for the repression of hTERT expression.

(A) hTERT full-length transcript (exon 15/16) mRNA expression levels by qRT-PCR; normalised to GAPDH mRNA levels upon stable doxycycline induction of wildtype (WT) TRF2 or R17H TRF2 mutants in HT1080, HCT116 or MDA-MB-231 cells

(B) hTERT mRNA FISH upon TRF2 WT or TRF2 R17H overexpression (un-transfected, UT as control) in HT1080 cells; quantification shown in right panel. FLAG-tagged TRF2 overexpression was confirmed by Immuno-fluorescence microscopy.

(C-F) ChIP followed by qRT-PCR at the 0-300 bp hTERT 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 methyl transferase 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 3 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).

Telomere length regulates hTERT expression in iPSC.

(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 immuno-fluorescence using Oct-4, SSEA-4, Sox-2 and TRA-1-60 antibodies as stemness markers.

(C) mRNA levels for hTERT (full-length exon 15/16 transcript), TERC (RNA component) and stemness marker genes Nanog, Klf4 in FS fibroblast and derived iPSC.

(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 hTERT 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.

(J) Scheme depicting generation of induced pluripotent stem cells (iPSCs) with shortened telomeres, (iPSC-ST) using telomere-specific sgRNA guided CRISPR-Cas9 to trim telomeres.

(K) Relative fold change in telomere length in iPSC-ST cells with respect to unaltered iPSC, determined by qPCR-based telomere length detection method

(L-M) ChIP followed by qPCR at the 0-300 bp hTERT promoter (upstream of TSS) in iPSC-ST ells 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.

(N) mRNA levels for hTERT (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 (hTERT) expression through non-telomeric TRF2.

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