Fig 1: TRF2 binding on hTERT promoter is independent of telomeres(A) Scheme showing insertion of Gaussia luciferase downstream ofthe hTERT promoter (+33 to –1,267 bp) at CCR5 locus using CRISPR/Cas9-mediated editing in HEK293T cells (see Supplemental information for characterization of cells). Position of primers designed for ChIP-qPCR indicated by arrows.(B) Effect of TRF2 silencing or expression of full-length TRF2 or TRF2-deletion mutants TRF2-DelB, TRF2-DelM, and TRF2-DelB-DelM on hTERT-promoter Gaussia luciferase activity relative to untreated control cells. Normalized using total protein in each case.(C) qPCR following TRF2 ChIP at the inserted-hTERT promoter at CCR5 locus using primers shown in scheme above (A); normalized over mock (IgG). GAPDH promoter was used as negative control for TRF2 occupancy.(D and E) qPCRfollowing ChIPforTRF1, POT1, and RAP1: at the CCR5-locus-inserted-hTERT promoterand the endogenous hTERT promoter (+38to -237 bp) in HEK293T cells(D) and spanningthe endogenous hTERT promoter in HT1080cells (E). Chromosome 5p region 100 kb downstream ofthehTERT locus reported for physical association with telomeres by looping was used as positive control and GAPDH as negative control.All error bars represent ± SDsfrom mean values; pvalues calculated by paired/unpairedttest; for(B), (D), and (E) two-wayANOVAwas used (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001).