Inter-locus as well as intra-locus heterogeneity in LINE-1 promoter methylation in common human cancers suggests selective demethylation pressure at specific CpGs

Nicole Nüsgen,Rolf Fimmers,Albertas Dauksa,Wolfgang A Schulz,Johannes Oldenburg,Holger Fröhlich,Antanas Gulbinas,Wolfgang Goering,Amit Sharma,Osman El-Maarri,Arijit Biswas,Muhammad Ahmer Jamil,Ioanna Dimitriou,Heike Singer

doi:10.1186/s13148-015-0051-y

Abstract

BackgroundHypomethylation of long interspersed element (LINE)-1 has been observed in tumorigenesis when using degenerate assays, which provide an average across all repeats. However, it is unknown whether individual LINE-1 loci or different CpGs within one specific LINE-1 promoter are equally affected by methylation changes. Conceivably, studying methylation changes at specific LINE-1 may be more informative than global assays for cancer diagnostics. Therefore, with the aim of mapping methylation at individual LINE-1 loci at single-CpG resolution and exploring the diagnostic potential of individual LINE-1 locus methylation, we analyzed methylation at 11 loci by pyrosequencing, next-generation bisulfite sequencing as well as global LINE-1 methylation in bladder, colon, pancreas, prostate, and stomach cancers compared to paired normal tissues and in blood samples from some of the patients compared to healthy donors.ResultsMost (72/80) tumor samples harbored significant methylation changes at at least one locus. Notably, our data revealed not only the expected hypomethylation but also hypermethylation at some loci. Specific CpGs within the LINE-1 consensus sequence appeared preferentially hypomethylated suggesting that these could act as seeds for hypomethylation. In silico analysis revealed that these CpG sites more likely faced the histones in the nucleosome. Multivariate logistic regression analysis did not reveal a significant clinical advantage of locus-specific methylation markers over global methylation markers in distinguishing tumors from normal tissues.ConclusionsMethylation changes at individual LINE-1 loci are heterogeneous, whereas specific CpGs within the consensus sequence appear to be more prone to hypomethylation. With a broader selection of loci, locus-specific LINE-1 methylation could become a tool for tumor detection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0051-y) contains supplementary material, which is available to authorized users.

Highlights

Hypomethylation of long interspersed element (LINE)-1 has been observed in tumorigenesis when using degenerate assays, which provide an average across all repeats
Principle component analysis (PCA) used at a false discovery rate of 5% visualizes the separation between different normal tissues by the methylation levels of specific LINE-1 loci (Figure 1B)
(See figure on previous page.) Figure 1 Tissue-specific methylation at specific LINE-1 loci. (A) Heat map of methylation means at different LINE-1 loci in all tissues and in blood as measured by bisulfite pyrosequencing

Summary

Introduction

Hypomethylation of long interspersed element (LINE)-1 has been observed in tumorigenesis when using degenerate assays, which provide an average across all repeats. It is unknown whether individual LINE-1 loci or different CpGs within one specific LINE-1 promoter are affected by methylation changes. Studying methylation changes at specific LINE-1 may be more informative than global assays for cancer diagnostics. DNA methylation changes at LINE-1 elements, in particular, have been reported for almost all tumor types [2,3,4,5,6,7,8,9,10,11]. In addition to changes in cancer tissues, LINE-1 methylation in blood and other body fluids has been suggested as a biomarker for monitoring cancer risk [14]

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