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

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.

Non-canonical bases differentially represented in the sex chromosomes of the dioecious plant Silene latifolia

“…On the other hand, 5fC is associated with hypomethylation and gene expression, and with transposon activation in oocytes ( Inoue et al , 2012 ; Raiber et al , 2012 ; Neri et al , 2015 ). Our data suggest a common behaviour for 5mC and 5fC that could be interpreted as silencing if we look at the main function of methylation, and this is supported by transposon silencing in fungi ( Ličytė et al , 2022 ). Likewise, the low correlation between 5hmC and 5mC agrees with previous research published in rye ( Kalinka et al , 2023 ).…”

5-Hydroxymethylcytosine: the many faces of the sixth base of mammalian DNA

“…71 Due to the rarity of fC and caC and sometimes hmC, special care is required during sample processing 72 and base-specific derivatization could be used to increase the sensitivity (by MS or optical detection) and enable enrichment of the target fragments from bulk genomic DNA. 73,74 A particularly useful analytical modification of hmC is its enzymatic glucosylation to a much bulkier and distinctive residue, 5-glucosyloxy-methylcytosine, using T4 b-glucosyltransferase (BGT) and the uridine-5 0 -diphospho-D-glucose (UDP-glc) cofactor or its analogs (see below). The BGT reaction is robust and highly selective for the 5-hydroxymethyl group of hmC (or hmU).…”

Alkyne-Substituted Deoxybenzoins as Precursors to Cycloaddition Chemistry and the Preparation of Low-Flammability Polymers and Blends

“…Pure (additive-free) PET was obtained from Eastman. Bishydroxydeoxybenzoin (BHDB) 20 and 1,4-bis(azidomethyl)benzene (diazide) 39 were prepared according to literature procedures. Tetrahydrofuran (THF) was purchased from Fisher Scientific and dried and distilled over sodium/benzophenone ketyl before use.…”