Genome-wide chromatin accessibility analysis in Tert promoter WT and mutant isogenic BLM cells (ATAC-seq)

Transcriptional re-activation of hTERT is the limiting step in tumorigenesis. While mutations in hTERT promoter seen in 19% of all cancers are recognized as key drivers of hTERT reactivation, mechanisms by which the wildtype hTERT (WT-hTERT) promoter is reactivated, as observed in the majority of cancers, remain unknown. We report that unlike with the mutant-hTERT promoters, a T-INT2 (Tert INTeracting region 2) region located ~120kb upstream of the hTERT proximal promoter is essential to uniquely reactivate the WT-hTERT promoter, via long-range chromatin interactions. Unlike mutant-hTERT promoters, which are driven by GABPa/b tetramers tethered between T-INT1 (Tert-INTeracting-region 1) and de-novo ETS sites created by promoter mutations, WT-hTERT promoter firing is initiated by 2 events a) elevated JunD mediated recruitment of CTCF and b) β-catenin and CBP mediated recruitment of Sp1 tetramers between T-INT2 and WT-hTERT proximal-promoter, leading to chromatin-openness, pol2 recruitment and productive transcription. The BLM melanoma cell line which originally harbors TERT promoter mutation was corrected to WT using Crispr editing. Furthermore, mutant Tert promoter specific chromatin interaction region (T-INT1) was knocked out by CRISPR-cas9 method. Tert promoter WT/mutant isogenic lines with or without the T-INT1 region were analyzed for ATAC-seq. Two replicates were used for each clones.