Abstract

The telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. CpG methylation in the TERT promoter (TERTp) was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to facilitate binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and chromatin accessibility) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin samples and in melanoma cell lines (n = 61). We unexpectedly observed that the methylation of TERTp 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, TERT mRNA was only expressed in the melanoma cell lines with either high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and TERT mRNA 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 which TERT expression requires either a widely open chromatin state in TERTp-WT samples due to high methylation throughout the promoter or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T or C250T mutations.

Highlights

  • 90% of all human cancers share a transcriptional alteration: reactivation of the telomerase reverse transcriptase (TERT) gene [1, 2]

  • Since the cutaneous melanoma originates from melanocytes of the skin, we found the difference in methylation fraction (MF) between normal melanocytes and cutaneous melanoma cells quite remarkable

  • In order to validate the TERT promoter (TERTp) MF obtained through next-generation sequencing (NGS) in a quantitative manner, we have developed a droplet digital PCR (ddPCR) assay (Fig 2A) using methylation-sensitive restriction enzymes (MSREs) HgaI and AvaI, which recognise the CpG on position 1,295,737 and 1,295,731 in hg19, respectively

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Summary

Introduction

90% of all human cancers share a transcriptional alteration: reactivation of the telomerase reverse transcriptase (TERT) gene [1, 2]. TERT encodes the catalytic subunit of the ribonucleoprotein telomerase and is capable of extending the repetitive, non-coding DNA sequence on terminal ends of chromosomes, the telomeres. As the single-stranded 5’ ends of chromosomes are shortened with each cellular division, telomeres prevent loss of coding chromosomal DNA [3,4,5,6].

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