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

• Type of Material: Article
• Authors: Seungjae Lee, St. Jude Children's Research Hospital; Ti-Cheng Chang, St. Jude Children's Research Hospital; Patrick Schreiner, St. Jude Children's Research Hospital; Yiping Fan, St. Jude Children's Research Hospital; Neeraj Agarwal, Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA 90048, USA.; Charles Owens, University of Colorado Anschutz Medical Campus; Reinhard Dummer, University Hospital Zurich; John M. Kirkwood, University of Pittsburgh; Raymond L. Barnhill, Institut Curie; Dan Theodorescu, Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute; Gang Wu, St. Jude Children's Research Hospital; Armita Bahrami, Emory University
• Language: English
• Date: 2022-08-19
• Publisher: MDPI
• Publication Version: Final Published Version
• Copyright Statement: © 2022 by the authors.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.3390/cancers14164018
• Title of Journal or Parent Work: Cancers (Basel)
• Volume: 14
• Issue: 16
• Grant/Funding Information: This work was supported in part by ALSAC and by the National Cancer Institute grant P30 CA021765 and W81XWH-19-1-0144/CA180175 (DT). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
• Keywords: methylation; proximal and distal promoters; allele-specific methylation; TERT promoter; bisulfite sequencing
• Research Categories: Health Sciences, Oncology; Biology, Cell

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.