5-hydroxymethylcytosine is not present in appreciable quantities in Arabidopsis DNA.
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.
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
Robert M
http://orcid.org/0000-0002-4325-4196
RM
Erdmann
Broad Institute, Cambridge, Massachusetts 02142.
Amanda L
AL
Souza
Broad Institute, Cambridge, Massachusetts 02142.
Clary B
CB
Clish
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 mgehring@wi.mit.edu.
Mary
M
Gehring
D044503:5-Methylcytosine / Q000031:analogs & derivatives; D017360:Arabidopsis / Q000235:genetics*; D002855:Chromatography, Thin Layer; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D018744:DNA, Plant / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D060888:Real-Time Polymerase Chain Reaction
D018744:DNA, Plant; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine
5-hydroxymethylcytosine; 5-methylcytosine; Arabidopsis; DNA demethylation; epigenetics