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Detection of Oxidation Products of 5-Methyl-2′-Deoxycytidine in Arabidopsis DNA
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-29-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-29-deoxycytidine (5-FodC), 5-carboxyl-29-deoxycytidine (5-CadC), 5-hydroxymethyl-29-deoxyuridine (5-
HmdU), and the (59S) diastereomer of 8,59-cyclo-29-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 Tetfamily
dioxygenase enzymes in Arabidopsis
Arabidopsis HDA6 Regulates Locus-Directed Heterochromatin Silencing in Cooperation with MET1
Heterochromatin silencing is pivotal for genome stability in eukaryotes. In
Arabidopsis, a plant-specific mechanism called
RNA–directed DNA methylation (RdDM) is involved in heterochromatin
silencing. Histone deacetylase HDA6 has been identified as a component of such
machineries; however, its endogenous targets and the silencing mechanisms have
not been analyzed globally. In this study, we investigated the silencing
mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the
loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent
24-nt siRNAs, however their transcript levels were mostly unaffected in the
rdr2 mutant. Strikingly, we observed significant overlap of
genes silenced by HDA6 to those by the CG DNA methyltransferase MET1.
Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted
in loss of heterochromatic epigenetic marks and aberrant enrichment for
euchromatic marks at HDA6 direct targets, along with ectopic expression of these
loci. Acetylation levels increased significantly in the hda6
mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16.
Interestingly, we observed two different CG methylation statuses in the
hda6 mutant. CG methylation was sustained in the
hda6 mutant at some HDA6 target loci that were surrounded
by flanking DNA–methylated regions. In contrast, complete loss of CG
methylation occurred in the hda6 mutant at the HDA6 target loci
that were isolated from flanking DNA methylation. Regardless of CG methylation
status, CHG and CHH methylation were lost and transcriptional derepression
occurred in the hda6 mutant. Furthermore, we show that HDA6
binds only to its target loci, not the flanking methylated DNA, indicating the
profound target specificity of HDA6. We propose that HDA6 regulates
locus-directed heterochromatin silencing in cooperation with MET1, possibly
recruiting MET1 to specific loci, thus forming the foundation of silent
chromatin structure for subsequent non-CG methylation