Abstract
Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-2′-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5-HmdC, 5-formyl-2′-deoxycytidine (5-FodC), 5-carboxyl-2′-deoxycytidine (5-CadC), 5-hydroxymethyl-2′-deoxyuridine (5-HmdU), and the (5′S) diastereomer of 8,5′-cyclo-2′-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 106 nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tet-family dioxygenase enzymes in Arabidopsis.
Highlights
DNA methylation at the C5 position of cytosine is a conserved epigenetic mark for transcriptional gene silencing in diverse organisms [1]
The identical elution times in selectedion chromatograms (SICs) and similar MS3 spectra for the analytes and their stable isotope-labeled standards confirmed the identities of the modified nucleosides and allowed for their reliable quantification
Since the recent discoveries of Tetmediated oxidation of 5-mdC to 5-HmdC, 5-FodC, and 5-CadC, many biophysical and biochemical techniques have been employed for their detection, including LC-MS analysis [26,27,28,29], thin layer chromatography [30], chemical derivatization followed by sequencing analysis [31,32], single molecule detection [33], antibody-based dot-blot analysis [11,20,34], etc
Summary
DNA methylation at the C5 position of cytosine is a conserved epigenetic mark for transcriptional gene silencing in diverse organisms [1]. In addition to the primary methylation at CG sites, cytosine in plants can be methylated in CHG and, less frequently, in CHH sequences (‘H’ represents A, C or T) [3]. The plant methylation patterns are established by different methyltransferase activities. De novo domains rearranged methyltransferases (DRMs) transfer methyl groups to completely unmethylated duplex DNA in all sequence contexts, and chromomethylase 3 (CMT3) can convert cytosine to 5-mC at non-CG sites [4]. The CG methylation is propagated during mitotic cell divisions by a group of maintenance methyltransferases (MET1 in Arabidopsis) [5]
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