Epigenetic splicing regulation of hTERT mediated by hTAPAS

Abstract

Telomerase activity is primarily controlled by expression of the catalytic subunit hTERT, yet the mechanisms coordinating its transcriptional and post-transcriptional regulation remain incompletely understood. Paradoxically, hypermethylation of a CpG-rich region upstream of the hTERT transcription start site, termed the TERT Hypermethylated Oncological Region (THOR), is strongly associated with hTERT activation. Recent studies suggest that THOR methylation influences the expression of hTAPAS, a long non-coding RNA transcribed antisense to the hTERT promoter that represses hTERT expression. Here, we investigated the relationship between DNA methylation and alternative splicing of hTERT. Using CRISPR-Cas9-mediated targeted enrichment combined with Nanopore sequencing, we generated high-resolution DNA methylation maps across several kilobases surrounding the hTERT promoter and intronic regions encompassing exons 6-8 in multiple human cell lines. These analyses revealed distinct methylation signatures that correlate with specific hTERT splice isoform profiles. Functional perturbation experiments further demonstrated that altering the epigenetic state of the locus influences hTERT splicing. Overexpression of the antisense lncRNA hTAPAS, as well as treatment with the DNA demethylating agent 5'-Azacytidine, reduced CpG methylation at the hTERT promoter and induced a shift in hTERT splice isoform distribution. Together, our findings identify DNA methylation as an upstream regulator of hTERT alternative splicing and implicate the antisense lncRNA hTAPAS in shaping this regulatory landscape. The results point to an epigenetic mechanism linking hTAPAS to hTERT promoter methylation and splice isoform selection at the hTERT locus and provide new insight into how telomerase regulation may be remodeled during development and in cancer.