Table 1.
Mass-spectrometric analysis of IDM1, IDM2 and IDL1 co-purifying proteins.
Fig 1.
Protein-protein interactions between IDM1, IDM2, IDL1 and MBD7.
(A) Yeast two-hybrid assays. Yeast cells carrying different fusion protein combinations are listed on the left. Yeast cells expressing the indicated proteins from the pGBK-T7 (BD) and pGAD-T7 (AD) vectors were plated onto medium lacking Leu and Trp (SD-LT) (left) or medium lacking Leu, Trp, Ade and His (SD-LTHA) (right). (B) Pull-down assays showing that IDM2, IDL1 and MBD7 interact with each other. (C) Split-luc assays showing that IDL1 and MBD7 can interact with IDM2 in N. benthamiana leaves. Three biological replicates were performed, and similar results were obtained. (D) Co-immunoprecipitation of MBD7 with IDM2 or IDL1 in tobacco leaves. MYC-tagged IDM2 and GFP-tagged IDM1 were transiently expressed in N. benthamiana leaves. Anti-GFP was used for immunoprecipitation (IP); anti-MYC and anti-GFP were used for immunoblotting; Input, total protein before immunoprecipitation. Transgenic plants expressing MBD7-Myc or IDM1-HA-YFP under their native promoters and their F1 offspring were used for co-IP.
Fig 2.
Methylome analysis of mbd7-1 mutant.
(A) Snapshot in the Integrated Genome Browser showing DNA methylation levels of the FAD-binding berberine gene family in WT, idm1-1, ros1-4 and mbd7-1. Hypermethylated regions in mbd7-1 were highlighted with red boxes. DT-77, which was hypermethylated in idm1-1 or ros1-4 but not in mbd7-1, was highlighted with green box. (B) Confirmation the whole genome bisulfite sequencing results by chop PCR. (C) Overlap of differentially methylated loci between mbd7-1 and ros1-4 and idm1-1. Boxplots represent methylation levels of each class of differentially methylated loci.
Fig 3.
MBD7 prevents the silencing of a report gene and some endogenous genes.
(A) The mbd7-1 mutation causes the silencing of 35S-NPTII but not the RD29A-LUC transgene. Seedlings grown in MS plates were imaged after cold treatment at 4°C for 24 h. For kanamycin resistance test, the seeds were planted on MS medium supplemented with 50 mg/L kanamycin and incubated for 2 weeks before being photographed. The reporter genes were introduced to mbd7-1 mutant plants by crossing. (B) Real-time PCR analysis of the expression level of LUC and NPTII reporter genes in the different genotypes. (C-E) Effect of mbd7 mutation on the expression of endogenous genes. Left column: Snapshot in the IGB showing DNA methylation levels in ros1-4, idm1-1, mbd7-1 mutant plants and wild type control; right column: real-time PCR analysis of the expression level of the hypermethylated genes or nearby genes in different genotypes. TUB8 was used as an internal control. Error bars represent standard error (n = 3).
Fig 4.
Histone acetylation marks and MBD7 association with chromatin.
(A) H3K18 acetylation (ac) and H3K23ac levels at the hyper-DMR loci and control regions. ChIP was performed with antibodies against H3K18ac and H3K23ac. The ChIP signal was quantified relative to input DNA. The no-antibody precipitates served as negative control. Two biological replicates were performed, and very similar results were obtained. Standard errors were calculated from three technical repeats, *P < 0.05. (B) Association of MBD7 protein with hyper-DMR loci. ChIP was performed in WT and MBD7-Myc transgenic plants with antibody against Myc.
Fig 5.
Working model for the IDM1-IDM2-IDL1-MBD7 complex functioning in ROS1 mediated active DNA demethylation at some loci in Arabidopsis.
MBD7 forms a complex with IDM1, IDM2 and IDL1, and recognizes methylated DNA through methyl-CpG-binding domains. Then IDM1 is recruited to specific loci and acetylates histone H3 at K18 and K23, facilitating active DNA demethylation by ROS1.