Mass spectrometry reveals the presence of specific set of epigenetic DNA modifications in the Norway spruce genome

Igor A Yakovlev,Konstantin V Krutovsky,Marta Starczak,Marcos Viejo,Alexey Ruzov,Carl Gunnar Fossdal,Abdulkadir Abakir,Ryszard Olinski,Daniel Gackowski

doi:10.1038/s41598-019-55826-z

Abstract

5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene expression in metazoans and plants. Iron-(II)/α-ketoglutarate-dependent dioxygenases can oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although these oxidized forms of 5mC may serve as demethylation intermediates or contribute to transcriptional regulation in animals and fungi, experimental evidence for their presence in plant genomes is ambiguous. Here, employing reversed-phase HPLC coupled with sensitive mass spectrometry, we demonstrated that, unlike 5caC, both 5hmC and 5fC are detectable in non-negligible quantities in the DNA of a conifer, Norway spruce. Remarkably, whereas 5hmC content of spruce DNA is approximately 100-fold lower relative to human colorectal carcinoma cells, the levels of both - 5fC and a thymine base modification, 5-hydroxymethyluracil, are comparable in these systems. We confirmed the presence of modified DNA bases by immunohistochemistry in Norway spruce buds based on peroxidase-conjugated antibodies and tyramide signal amplification. Our results reveal the presence of specific range of noncanonical DNA bases in conifer genomes implying potential roles for these modifications in plant development and homeostasis.

Highlights

Epigenetic mechanisms are instrumental in plant development and adaptation to environmental stress[1,2]
An early study based on chemical derivatization coupled with liquid chromatography/tandem mass spectrometry reported the presence of all three oxi-mCs in a number of plant species[17], and, according to another study, 5hmC was found to be localized in transcriptionally silent transposable elements in rice[18], there are conflicting reports on the prevalence of this DNA modification in Arabidopsis[15,16,17]
To determine the prevalence of 5mC, its oxidized derivatives as well as a thymine base modification, 5-hydroxymethyluracil (5hmU)[22] together with the products of DNA base damage[23,24,25] in our samples, we employed a sensitive two dimensional ultra-performance liquid chromatography tandem mass-spectrometry (2D-UPLC–MS/MS) that we previously successfully used for quantification of these DNA modifications in cancer cell lines and tumor tissue[26,27] (Supplementary Fig. S1 and Table S1)

Summary

Introduction

Epigenetic mechanisms are instrumental in plant development and adaptation to environmental stress[1,2]. In contrast with www.nature.com/scientificreports animal systems, in plants, 5mC can be directly recognized and excised from DNA by specific DNA glycosylases, such as ROS1 and DME, without the preceding enzymatic oxidation[3,13] In line with these differences in the mechanisms of DNA demethylation, TET/JBP dioxygenases have not been identified in plants to date[14], and experimental evidence supporting the presence of the oxidized forms of 5mC (referred together as oxi-mCs) in the plant genomes is very limited and ambiguous[15,16,17,18]. We wanted to compare the abundance of these DNA modifications in spruce with those of human colorectal carcinoma (HCT 116) and human embryonic stem cells (hESCs), which are well-studied regarding DNA modifications[21]

Objectives

Methods

Results