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

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.

Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing.

BACKGROUND: Massively parallel sequencing of maternal cell-free DNA (cfDNA) is widely used to test fetal genetic abnormalities in non-invasive prenatal testing (NIPT). However, sequencing-based approaches are still of high cost. Building upon previous knowledge that placenta, the main source of fetal circulating DNA, is hypomethylated in comparison to maternal tissue counterparts of cfDNA, we propose that targeting either unmodified or 5-hydroxymethylated CG sites specifically enriches fetal genetic material and reduces numbers of required analytical sequencing reads thereby decreasing cost of a test. METHODS: We employed uTOPseq and hmTOP-seq approaches which combine covalent derivatization of unmodified or hydroxymethylated CG sites, respectively, with next generation sequencing, or quantitative real-time PCR. RESULTS: We detected increased 5-hydroxymethylcytosine (5hmC) levels in fetal chorionic villi (CV) tissue samples as compared with peripheral blood. Using our previously developed uTOP-seq and hmTOP-seq approaches we obtained whole-genome uCG and 5hmCG maps of 10 CV tissue and 38 cfDNA samples in total. Our results indicated that, in contrast to conventional whole genome sequencing, such epigenomic analysis highly specifically enriches fetal DNA fragments from maternal cfDNA. While both our approaches yielded 100% accuracy in detecting Down syndrome in fetuses, hmTOP-seq maintained such accuracy at ultra-low sequencing depths using only one million reads. We identified 2164 and 1589 placenta-specific differentially modified and 5-hydroxymethylated regions, respectively, in chromosome 21, as well as 3490 and 2002 Down syndrome-specific differentially modified and 5-hydroxymethylated regions, respectively, that can be used as biomarkers for identification of Down syndrome or other epigenetic diseases of a fetus. CONCLUSIONS: uTOP-seq and hmTOP-seq approaches provide a cost-efficient and sensitive epigenetic analysis of fetal abnormalities in maternal cfDNA. The results demonstrated that T21 fetuses contain a perturbed epigenome and also indicated that fetal cfDNA might originate from fetal tissues other than placental chorionic villi. Robust covalent derivatization followed by targeted analysis of fetal DNA by sequencing or qPCR presents an attractive strategy that could help achieve superior sensitivity and specificity in prenatal diagnostics.