Mechanistic Studies Of Quinones Induced DNA Hydroxymethylation

5-methylcytosine (5mC) is an important epigenetic mark playing critical roles in various biological processes in mammals, including regulation of gene transcription and silencing of retrotransposon. The formation of5mC is catalysed by DNA methyltransferases (DNMTs), and can be "read" by DNA methyl-CpG-binding proteins. The5mC modification is highly dynamic and can be oxidized to5-hydroxymethylcytosine (5hmC) by the TET protein family. Various chemicals can severely impact human health by inducing DNA mutations or changes in the epigenetic landscape. Quinones, a class of redox-active compounds, are ubiquitous in nature and humans are exposed to quinones in the diet, but also via cancer therapeutics and environment pollutants. Quinones can stimulate the formation of highly reactive oxygen species (ROS). We speculate that redox-active quinones may change the homeostasis of iron (Ⅱ) and/or α-ketoglutarate (α-KG), thereby affecting the catalytic activity of TET dioxygenases, and hence influence the5mC to5hmC conversion.In this study, we wanted to characterise the effect of quinones on cellular5hmC levels and understand the molecular mechanism of DNA hydroxymethylation. To this end, we first developed an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method of high sensitivity, accuracy and reliability to identify5hmC in hydrolysis products from both poly (5hmdC) standards and cellular genomic DNA. We demonstrated that tetrachloro-1,4-benzoquinone (TCBQ) and tetrachloro-1,4-hydroquinone (TCHQ) treatment induces5hmC formation in mammalian cells in a dose and time dependent manner. A more thorough examination using UHPLC-MS/MS showed that quinone induced5hmC generation depends on the TET1and TET2proteins in mouse embryonic stem cells (mESCs). The α-KG competitor, dimethyloxalylglycine (DMOG), as well as the iron (Ⅱ) deliver and chelator, ammonium ferric citrate (FAC) and1,10-phenanthroline all significantly influenced quinones induced5hmC formation. Furthermore, quinones treatment elevated both the cytoplasmic and nuclear level of ferritin (FTL) significantly, indicating an elevation of cellular labile iron (Ⅱ).In addition to TCBQ and TCHQ, a number of other quinoid compounds also promoted the generation of5hmC, including2,5-dichloro-1,4-benzoquinone (2,5-DCBQ),2,6-dichloro-1,4-benzo-quinone (2,6-DCBQ),2-chloro-1,4-benzoquinone (2-CBQ), tetrabromo-1,4-benzoquinone (TBrBQ) and O-chloranil.Taken together, these results support our notion that quinones may stimulate the catalytic activity of TET proteins by increasing the availability of its cofactor iron (Ⅱ) and hence promote the conversion of5mC to5hmC.To investigate the global DNA methylation changes induced by quinones, we analysed changes in the genome-wide5hmC distribution upon TCBQ treatment by deep-sequencing of chemically labelled5hmC. We identified many new5hmC modification sites associated with gene-rich regions. Interestingly, we observed a significant enrichment of5hmC in gene bodies as well as in proximal upstream and downstream regions relative to transcription start sites (TSSs) and transcription end sites (TESs) following quinone treatment. KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathway and GO (Gene Ontology) analyses revealed that genes showing enrichment of5hmC after quinone treatment were involved in a wide variety of biological processes. We validated5hmC enrichment and gene expression levels in a number of genes using RT-qPCR. By Combining our previous results with RNA-seq data, we could showed that quinone treatment induced5hmC-mediated gene expression changes of genes belonging to several functional pathways, including protein catabolic process, apoptosis, cell localization and transport, and RNA processing. This suggests that quinones-dependent5hmC formation may influence a broad range of cellular functions.In summary, in this study we established a regulatory relationships between quinones, TET proteins, and DNA hydroxymethylcytosine and demonstrated that quinones induced epigenetic changes affect various aspects of cellular life and hence are of great biological significance.