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) ß-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. Overall design: 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.