Regulation of the Human Telomerase Gene TERT by Telomere Position Effect—Over Long Distances (TPE-OLD): Implications for Aging and Cancer
(A) Schematic map of the human 5p chromosome containing the hTERT locus and design of probes. The sub-telomeric 5p probe (far, red, grey color) stains the end of chromosome 5p that is located 75 Kb from the 5p telomeric repeats (red color). The hTERT probe (green) stains a specific genomic region containing the hTERT gene, which is 1.1 Mb from the 5p telomere. (B) BJ human fibroblasts at PD60 was stained with indicated fluorescent probes against the hTERT locus, sub-telomeric region 5p, and the telomere. A representative deconvolved image was selected. Scale bar represents 5 uM (C) Graph shows the distance between the hTERT locus and the closest telomere depends on the distance between the hTERT locus and the sub-telomeric 5p. (D) Percentage of adjacent allele (A) pairs versus separated allele (S) pairs was determined by 3D-FISH in normal BJ cells. BJ fibroblasts at different PDs were analyzed as indicated in the Materials and Methods. Cas9-mediated transient perturbation at the sub-telomeric 5p region was performed in BJ cells at PD25. (E) Percentage of adjacent allele pairs (A) versus separated allele pairs (S) was determined by 3D-FISH in BJ fibroblasts. BJ clones were transfected with a floxable hTERT, followed by excision with Cre recombinase at different time points. Two clones with different lengths of telomeres were analyzed at the same number of population doublings in culture. Indicated telomere lengths were measured by TRF (terminal restriction fragment) Southern blot shown in Fig 1C. (F) Telomere-DNA damage induced foci (TIF) analysis performed in BJ long (13 kb) and short cells (9 kb) (TRF shown in Fig 1C for matched cells). Cells were stained with telomere PNA and gamma-H2AX antibody. At least 81 nuclei were quantified. Scale bar is 5 μM. Western blotting shows lack of induction of γ-H2AX in BJ cells with different telomere lengths. β-Actin was used as a loading control. (G) 3C analysis shows distal genomic interactions between the 5p telomere and hTERT. 3C libraries were generated from BJ cells at PD20 and PD70, followed by ddPCR amplification of genomic interactions between indicated regions. Proximity control amplification of 3C libraries from BJ cells at PD20 and PD70 was performed with ddPCR. t test revealed a significant effect. *p < 0.05, n.s. = no significant. Data associated with this figure can be found in the supplemental data file (S1 Data).