5-Hydroxymethylcytosine Is Not Present in Appreciable Quantities in Arabidopsis DNA

Abstract

5-Hydroxymethylcytosine (5-hmC) is an intermediate in active demethylation in metazoans, as well as a potentially stable epigenetic mark. Previous reports investigating 5-hydroxymethylcytosine in plants have reached conflicting conclusions. We systematically investigated whether 5-hmC is present in plant DNA using a range of methods. Using the model organism Arabidopsis thaliana, in addition to other plant species, we assayed the amount or distribution of 5-hydroxymethylcytosine by thin-layer chromatography, immunoprecipitation-chip, ELISA, enzymatic radiolabeling, and mass spectrometry. The failure to observe 5-hydroxymethylcytosine by thin-layer chromatography established an upper bound for the possible fraction of the nucleotide in plant DNA. Antibody-based methods suggested that there were low levels of 5-hmC in plant DNA, but these experiments were potentially confounded by cross-reactivity with the abundant base 5-methylcytosine. Enzymatic radiolabeling and mass spectrometry, the most sensitive methods for detection that we used, failed to detect 5-hydroxymethylcytosine in A. thaliana genomic DNA isolated from a number of different tissue types and genetic backgrounds. Taken together, our results led us to conclude that 5-hmC is not present in biologically relevant quantities within plant genomic DNA.

Expression of the C-Terminal Domain of Mammalian <i>TET</i>3 DNA Dioxygenase in <i>Arabidopsis thaliana</i> Induces Heritable Methylation Changes at <i>rDNA</i> Loci

“…The lack of 5 hmC marks in lines that don't contain or have lost the TET3c transgene supports previous data from a mass spectrometry study in different Arabidopsis thaliana tissue types and genetic backgrounds, which found no evidence for the presence of 5 hmC in Arabidopsis [17]. The absence of 5 hmC may be due to a lack of TET functions in Arabidopsis or it may be an indicator for the efficient processing of 5 hmC by plant enzymes.…”

“…It also remains to be seen if TET3c access is limited to specific loci, and if TET3c− mediated demethylation is retained at some loci and efficiently reverted at others. A genome-wide screen for 5 hmC marks, which are not present in wildtype genomic DNA [17] may help to identify loci with TET3c− induced epi-alleles. TET3c expression may also help to investigate if plant species differ in their susceptibility to methylation changes.…”

“…The oxidation products 5 fC and 5 caC serve as substrates for thymine-DNA glycosylase (Tdg), which mediates their removal and replacement by unmethylated cytosine via the base excision repair (BER) pathway [14] [15]. Plants have no identifiable homologues of TET proteins [16], there is no evidence for 5 hmC presence in plants [17] and it is unclear if oxidation products of 5 mC can be processed by plant glycosylases in vivo.…”

Epigenomics of Plant’s Responses to Environmental Stress

“…Both these unusual modified bases are removed by base-excision repair mechanisms involving thymine-DNA-glycosylases [45,46]. Erdmann et al [47] investigated the presence of 5-hmC in Arabidopsis and other plant species using a range of sensitive methods and failed to detect 5-hmC in different tissues and genetic backgrounds. This suggests that 5-hmC is not present in biologically significant quantity in plant genome.…”

Epigenomics of Plant’s Responses to Environmental Stress

“…Both these unusual modified bases are removed by base-excision repair mechanisms involving thymine-DNA-glycosylases [45,46]. Erdmann et al [47] Methylation of adenine in GATC sequence has been known to be essential for the survival of several bacteria, as Dam methylase creates specific methylation marks important for DNA replication, mismatch repair, segregation, and regulation of gene expression [48,49]. Though N 6 -methyladenine (6-mA) is known to play an important regulatory role in RNA, several earlier studies suggested the presence of 6-mA in eukaryotic genomes.…”