LAUSR

Significance Mutations in hTERT promoter are seen in over 19% of human cancers, irrespective of the cancer type. Understanding the molecular players that regulate Mut-hTERT promoters may help the design of effective targeting strategies to inhibit telomerase reactivation specifically in cancer cells. Our work uses genome-wide functional screens to identify 30 specific regulators of Mut-hTERT promoters. These candidates identified from the screening serve as an excellent resource to understand how telomerase is reactivated and as targets for making inhibitors to telomerase, a key driver of cancer. Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.