Distribution and regulatory roles of oxidized 5-methylcytosines in DNA and RNA of the basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea.

Janina Ličytė,Audronė Rukšėnaitė,Edita Kriukienė,Saulius Klimašauskas,Eglė Godliauskaitė,Viktoras Masevičius,Gražina Petraitytė,Kotryna Kvederavičiūtė,Vita Tomkutė,Povilas Gibas

doi:10.1098/rsob.210302

Abstract

The formation of three oxidative DNA 5-methylcytosine (5mC) modifications (oxi-mCs)-5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)-by the TET/JBP family of dioxygenases prompted intensive studies of their functional roles in mammalian cells. However, the functional interplay of these less abundant modified nucleotides in other eukaryotic lineages remains poorly understood. We carried out a systematic study of the content and distribution of oxi-mCs in the DNA and RNA of the basidiomycetes Laccaria bicolor and Coprinopsis cinerea, which are established models to study DNA methylation and developmental and symbiotic processes. Quantitative liquid chromatography-tandem mass spectrometry revealed persistent but uneven occurrences of 5hmC, 5fC and 5caC in the DNA and RNA of the two organisms, which could be upregulated by vitamin C. 5caC in RNA (5carC) was predominantly found in non-ribosomal RNA, which potentially includes non-coding, messenger and small RNA species. Genome-wide mapping of 5hmC and 5fC using the single CG analysis techniques hmTOP-seq and foTOP-seq pointed at involvement of oxi-mCs in the regulation of gene expression and silencing of transposable elements. The implicated diverse roles of 5mC and oxi-mCs in the two fungi highlight the epigenetic importance of the latter modifications, which are often neglected in standard whole-genome bisulfite analyses.

Highlights

5-Methylcytosine (5mC) is a widespread DNA modification found in organisms ranging from bacteria to mammals
We measured the global amounts of oxidized 5mC modifications (oxi-mCs) in DNA extracted from the mycelium of L. bicolor and C. cinerea by western dot–blot and HPLC-MS/MS analysis
In each of the two fungi, the amounts of 5fC and 5hmC were similar, in contrast to mammalian DNA, in which 5hmC is the most abundant of oxi-mCs, while levels of 5caC were the lowest of all oxi-mCs. These results contrasted with the western dot–blot analyses using the antibodies raised against 5caC, which showed a high 5caC signal in C. cinerea DNA

Summary

Introduction

5-Methylcytosine (5mC) is a widespread DNA modification found in organisms ranging from bacteria to mammals. Besides widely explored 5mC oxidation in DNA by TET proteins in vitro and in vivo, these enzymes are able to oxidize 5mC in RNA (5mrC) to all three oxi-mC forms (5hmrC, 5frC, 5carC) in vitro [18,19]. The distribution of individual oxi-mCs in the genomes of L. bicolor and C. cinerea and the impact of individual oxi-mCs on gene expression and transposon regulation has not been explored in detail. Both fungi belong to the order Agaricales, a large group of Basidiomycota. Because of the essential role of the fungi for tree growth and in the cycling of essential nutrients, ectomycorrhizal symbiosis is of global ecological and economic importance [34]

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Conclusion