Interplay betweenTERTpromoter mutations and methylation culminates in chromatin accessibility andTERTexpression

Abstract

AbstractThetelomerase reverse transcriptase(TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. Contrary to common concepts, CpG methylation in theTERTpromoter (TERTp), was correlated withTERTmRNA expression. Furthermore, two hotspot mutations inTERTp, dubbed C228T and C250T, have been revealed to assist binding of transcription factor ETS/TCF and subsequentTERTexpression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and higher-order chromatin structure) and genetic (promoter mutations) mechanisms in regulatingTERTgene expression in healthy skin and in melanoma cell lines (n=61). We unexpectedly observed that the methylation ofTERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples,TERTmRNA was only expressed in the melanoma cell lines with high methylation or intermediate methylation in combination withTERTmutations.TERTp methylation was positively correlated with chromatin accessibility and expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in whichTERTexpression requires either a widely open chromatin state throughout the promoter inTERTp-WT samples due to high methylation or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T/C250T mutations.Author summaryPvdV and RvD formulated research goals and aims and supervised the overall progress. Wet-lab experiments, preparation of the manuscript and statistical analysis were performed by CS and CR. CS designed the novel assays. RN was involved in the experimental setup. RvD, NG and PvdV were responsible for funding acquisition. CR, RN, NG, RvD and PvdV critically reviewed the manuscript.