Pervasive promoter hypermethylation of silencedTERTalleles in human cancers

In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded byTERT. Yet, most cancers show only modest levels of telomerase gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating telomerase gene expression in cancer cells is not fully understood. Here, we have carried out the most comprehensive characterization to date ofTERTpromoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the coreTERTpromoter in 96 different human cell lines. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250TTERTpromoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally observe hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observedTERTexpression in cancer cells. Our findings suggest that hypermethylation of theTERTpromoter alleles signals transcriptional repression of those alleles, leading to the attenuation ofTERTactivation in cancer cells.SIGNIFICANCEHypermethylation of theTERTpromoter alleles to attenuateTERTactivation in cancer cells may account for the modest activation ofTERTexpression in most cancers.