Transcriptional regulation of 7q TERRA. (A) Total RNA from ALT, telomerase-positive (Telomerase+), or primary (Pr.) cells was analyzed by northern blotting using probes for the 7q/12q subtelomere or UUAGGG repeats (total TERRA). GAPDH and U6 RNAs were used as loading controls. (B) Total and nuclear RNA from HCT116 and DKO cells was analyzed by northern blotting as in A. U2OS RNA was included in the same blots for comparison with an ALT cell line. (C) Northern blot analysis of eGFP expression in HeLa cells transfected with the p7q promoter reporter. An empty vector plasmid devoid of promoter sequences (pEV) and the p3.5 plasmid containing a 29 bp repeat promoter were used as negative and positive controls, respectively. Twenty-four hours after transfection, cells were selected using puromycin and 48 h later harvested for RNA preparation. The bar graph shows the quantification of eGFP signals normalized through the corresponding U6 signals. Bars and error bars are the means and SDs from three independent experiments. P values (Student's t-test) are indicated. (D) Representative images of eGFP fluorescence in HeLa cells treated as in C. eGFP is shown in green, DAPI-stained DNA in blue. The bar graph shows the total eGFP fluorescence per cell. Bars and error bars are the means and SDs from two independent experiments. P values (Student's t-test) are indicated. (E) Heatmap representation of 7q CpG island methylation for the indicated cell lines. The CpG island coordinates are chr7:159,335,203–159,335,443 in the GRCh38/hg38 reference genome. The position of 7q TERRA TSS gauged from TERRA ONTseq is indicated.
