Simultaneous sequencing of oxidized methylcytosines produced by TET/JBP dioxygenases in Coprinopsis cinerea

Abstract

TET/JBP enzymes oxidize 5-methylpyrimidines in DNA. In mammals, the oxidized methylcytosines (oxi-mCs) function as epigenetic marks and likely intermediates in DNA demethylation. Here we present a method based on diglucosylation of 5-hydroxymethylcytosine (5hmC) to simultaneously map 5hmC, 5-formylcytosine, and 5-carboxylcytosine at near-base-pair resolution. We have used the method to map the distribution of oxi-mC across the genome of Coprinopsis cinerea, a basidiomycete that encodes 47 TET/JBP paralogs in a previously unidentified class of DNA transposons. Like 5-methylcytosine residues from which they are derived, oxi-mC modifications are enriched at centromeres, TET/JBP transposons, and multicopy paralogous genes that are not expressed, but rarely mark genes whose expression changes between two developmental stages. Our study provides evidence for the emergence of an epigenetic regulatory system through recruitment of selfish elements in a eukaryotic lineage, and describes a method to map all three different species of oxi-mCs simultaneously.T he discovery that oxidative modifications of DNA bases are catalyzed by the TET/JBP family of 2-oxoglutarate and irondependent dioxygenases (1-4) opened a major area of research into the epigenetics of various eukaryotic lineages (reviewed in refs. 5-7). In metazoans, the TET/JBP family is represented by TET proteins, which are present in all animals that are known to possess DNA cytosine methylation (3,8,9). The three TET paralogs of vertebrates have been shown to catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) (2), which is progressively oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (10-12). The discovery of these oxidized methylcytosine (oxi-mC) modifications triggered a flurry of studies on the mammalian TET paralogs; the reports increasingly point toward important roles for these oxidative modifications as intermediates in the enigmatic process of DNA demethylation, and also as epigenetic marks in their own right (13, 14) (reviewed in refs. 5-7). TET proteins have roles in diverse biological processes, including epigenetic regulation of gene transcription, embryonic development, stem cell function, and cancer (5), but the mechanisms underlying their biological activities are still poorly defined.Several methods have been developed to profile individual oxi-mC species at base resolution in genomic DNA (reviewed in ref. 5). However, none of these methods can simultaneously map all three oxi-mC species-5hmC, 5fC, and 5caC-at the same time; rather, they rely on chemical or enzymatic conversion of individual oxi-mCs followed by bisulfite sequencing. Two recent sequencing technologies, single-molecule, real-time (SMRT) sequencing and protein nanopore sequencing, are capable of recognizing modified bases in unamplified genomic DNA (15-18). In SMRT sequencing, 5mC and 5hmC are barely detectable in unmodified DNA, whereas 5fC and 5caC yield a robust kinetic signature (16). As 5hmC is an abundant oxi-mC modification in ...

Biochemical characterization of a Naegleria TET-like oxygenase and its application in single molecule sequencing of 5-methylcytosine

“…Among them, JBP1 and JBP2 have been shown to possess T-hydroxylation activity, whereas multiple paralogous genes in mammals, mushroom (Coprinopsis cinerea), and honey bee (Apis mellifera) have been shown to possess 5m C-oxygenase activity (1,6,22,23). Our results demonstrate that NgTET1 and the evolutionarily distant mTET1CD possess both 5m C-and T-oxygenase activities, which may initially have been shared by a single ancestral enzyme.…”

“…Many other species, from simple to complex, maintain DNA methylation machinery throughout their life cycle that may contribute to epigenetic regulation. Therefore, an interesting perspective is to examine shared and distinct features of TET oxygenases in diverse eukaryotes (5,6).…”

Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification

“…DmTET is closely related to vertebrate TETs, and retains all features of the active site of the latter enzymes which accommodate a pyrimidine rather than a purine 15, 49. A diversity of characterized eukaryotic TET/JBP enzymes have been observed only to modify 5‐methylpyrimidines rather than purines 49, 50, 51, 104, 105. This is further confirmed by contextual analysis of bacterial and phage TET/JBP family members, which are predicted to primarily modify pyrimidines 15, 90.…”

“…Renewed focus on m 6 A in eukaryotic DNA has added it to the growing list of epigenetic marks in DNA 15, 37, 49, 51, 105. However, there are still several biochemical questions needing careful consideration in the future: (i) which members of the AlkB family are involved in demethylation of m 6 A?…”

Compilation of Modern Technologies To Map Genome‐Wide Cytosine Modifications in DNA

“…[48b, 49] SMRT sequencing has different sensitivities for different modifications, and thus, each modification type has a specific signal-to-noise ratio. Detection of DNA methylation requires ah igh sequencing depth, and additional labeling steps can help to improve the signal confidence.B yu sing Te t1 to convert 5mC into 5caC [50] and two T-evenb acteriophage enzymest oc onvert 5hmC into ad iglucosylated adduct, [51] the signal-to-noise ratios for the detection of theset wo important epigenetic markers can be improved.…”