Robert M. Erdmann, Amanda L. Souza, Clary B. Clish and Mary Gehring

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.

See G3: Genes, Genomes, Genomics January 1, 2015 vol. 5 no. 1 1-8

http://www.g3journal.org/content/5/1/1.abstract?etoc

Figure 2: A 5-hmC antibody recognizes plant DNA. (A) Slot blot of 400 ng of genomic DNA from the indicated species and 20 ng of synthetic DNA containing 10 pmol of C, 5-mC, or 5-hmC as controls probed with a 1:500 dilution of a rat monoclonal 5-hmC antibody (top) or a 1:500 dilution of a mouse monoclonal 5-mC antibody (bottom). (B) IP-chip of 5-hmC (green track) and 5-mC (blue track) from A. thaliana genomic DNA. A 2-Mb representative region on chromosome 1 from 7000000 to 9000000 (top) and a 80-kb region from 5222870 to 5294010 (bottom) is shown.