A histone acetyltransferase regulates active DNA demethylation in Arabidopsis

Abstract

Active DNA demethylation is an important part of epigenetic regulation in plants and animals. How active DNA demethylation is regulated and its relationship with histone modification patterns are unclear. Here, we report the discovery of IDM1, a regulator of DNA demethylation in Arabidopsis. IDM1 is required for preventing DNA hypermethylation of highly homologous multicopy genes and other repetitive sequences that are normally targeted for active DNA demethylation by Repressor of Silencing 1 and related 5-methylcytosine DNA glycosylases. IDM1 binds methylated DNA at chromatin sites lacking histone H3K4 di- or trimethylation and acetylates H3 to create a chromatin environment permissible for 5-methylcytosine DNA glycosylases to function. Our study reveals how some genes are indicated by multiple epigenetic marks for active DNA demethylation and protection from silencing.

Fig. 1

Identification and characterization of the idm1 mutants. (A)Analysis showing the effects of idm1 mutations on At1g26400 (B) and At4g18650 (C) of DNA methylation level at the At1g26400 locus by chop-PCR (4). Un-DNA methylation in different sequence contexts and genetic interactions digested DNA is shown as acontrol. (B and C) Bisulfite sequencing data with ros1 or nrpd1. WT, wild type.

Fig. 2

Functional analysis of the PHD finger and MBD domains of IDM1. (A) Western blot analysis of the PHD finger of IDM1 in a peptide pull-down assay. Coomassie blue–stained biotinylated histone peptides are shown as a loading control. (B) DNA hypermethylation phenotypes of idm1-1 plants transformed with WT or mutant forms of IDM1. (C) Electrophoretic mobility shift assay (EMSA) of IDM1-N (amino acids 1 to 400) binding to methylated oligonucleotides (fig. S12). -, no competitor; M, methylated; U, unmethylated. (D) EMSA showing IDM1-N (amino acids 1 to 400) binding to a methylated oligonucleotide probe corresponding to the DT-77 locus.

Fig. 3

Histone acetyltransferase activity of IDM1. (A) HAT assays. (Top) Autoradiograph for acetylated histone. (Middle) Coomassie blue–stained membrane picture. (Bottom) Western blot detection of IDM1 with antibody to FLAG. p300-CBP-associated factor (PCAF) was used as a positive control. (B) HAT assay results using recombinant histone H3 mutated at different lysine positions as substrates. (C) HAT assay results using histone H3 as substrate and using WT and mutated forms of IDM1-C1 proteins as enzymes. (D) DNA hypermethylation phenotypes of idm1-1 plants transformed with WT or mutant forms of IDM1.

Fig. 4

Histone acetylation marks and IDM1 and ROS1 association with chromatin. (A) H3K18 acetylation (ac) and H3K23ac levels at the DT loci and control regions. ChIP was performed with antibodies to H3K18ac and H3K23ac. (B) Association of IDM1 protein with DT loci. ChIP was performed in WT, PYR1:3HA, and IDM1:3HA:YFP transgenic plants with antibody to hemagglutinin. (C) Effect of idm1 on ROS1 protein association with DT loci. ChIP was performed in MYC:ROS1/ros1-4 (WT) and MYC:ROS1/idm1-1 plants using antibody to MYC. The ChIP signal was quantified as relative to input DNA. The no-antibody precipitates served as a negative control. Standard errors were calculated from three technical repeats.