Targeted Long-Read Bisulfite Sequencing Identifies Differences in the TERT Promoter Methylation Profiles between TERT Wild-Type and TERT Mutant Cancer Cells

SIMPLE SUMMARY: TERT promoter methylation is enriched in cancers lacking TERT genetic alterations (wild-type cancers), but its functional impact on TERT transcription remains elusive. We developed a long-read bisulfite-sequencing platform to characterize the TERT promoter methylation profile at a single-molecule level. In wild-type cancer cell lines, both epialleles were hypermethylated symmetrically on the TERT distal promoter. In the core and proximal promoter, by contrast, the transcribed epialleles were significantly more hypomethylated than the silent epialleles. Decitabine-therapy reduced the core and proximal (not the distal) promoter methylation and reactivated the silent allele. We showed that TERT allele-specific expression is amenable to in vitro epigenetic manipulation in wild-type cancers. ABSTRACT: Background: TERT promoter methylation, located several hundred base pairs upstream of the transcriptional start site, is cancer specific and correlates with increased TERT mRNA expression and poorer patient outcome. Promoter methylation, however, is not mutually exclusive to TERT activating genetic alterations, as predicted for functionally redundant mechanisms. To annotate the altered patterns of TERT promoter methylation and their relationship with gene expression, we applied a Pacific Biosciences-based, long-read, bisulfite-sequencing technology and compared the differences in the methylation marks between wild-type and mutant cancers in an allele-specific manner. Results: We cataloged TERT genetic alterations (i.e., promoter point mutations or structural variations), allele-specific promoter methylation patterns, and allele-specific expression levels in a cohort of 54 cancer cell lines. In heterozygous mutant cell lines, the mutant alleles were significantly less methylated than their silent, mutation-free alleles (p < 0.05). In wild-type cell lines, by contrast, both epialleles were equally methylated to high levels at the TERT distal promoter, but differentially methylated in the proximal regions. ChIP analysis showed that epialleles with the hypomethylated proximal and core promoter were enriched in the active histone mark H3K4me2/3, whereas epialleles that were methylated in those regions were enriched in the repressive histone mark H3K27me3. Decitabine therapy induced biallelic expression in the wild-type cancer cells, whereas the mutant cell lines were unaffected. Conclusions: Long-read bisulfite sequencing analysis revealed differences in the methylation profiles and responses to demethylating agents between TERT wild-type and genetically altered cancer cell lines. The causal relation between TERT promoter methylation and gene expression remains to be established.