1. Volume 55, Issue 3, Pages 361-371 (August 2014)
Regulation of Active DNA Demethylation by an α-Crystallin Domain Protein in Arabidopsis 
Weiqiang Qian, Daisuke Miki, Mingguang Lei, Xiaohong Zhu, Huiming Zhang, Yunhua Liu, Yan Li, Zhaobo Lang, Jing Wang, Kai Tang, Renyi Liu, Jian-Kang Zhu 
Molecular Cell 
Volume 55, Issue 3, Pages (August 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 55, 361-371DOI: (10.1016/j.molcel.2014.06.008)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Identification of idm2 Mutants, Their Genetic Interactions with ros1, rdr2, and nrpe1 Mutations, and Their Effects on DNA Methylation, Histone Modification, and Transcriptional Silencing
(A) Analysis of DNA methylation level at the At1g26400 locus by Chop PCR. Because HhaI digestion is sensitive to CG DNA methylation, DNA hypermethylation results in increased levels of the PCR product. Undigested DNA is shown as a control.
(B) Bisulfite sequencing data showing the effects of idm2 mutations on At1g26400 DNA methylation in different sequence contexts and genetic interactions with ros1, rdr2, or nrpe1.
(C) Effect of idm1 and idm2 mutations on 35S-SUC2 transgene-mediated root growth suppression. Seedlings were grown on plates with MS medium plus 2% sucrose for 3 weeks before they were photographed.
(D) Real-time PCR analysis of the expression levels of SUC2 and HPTII transgenes in the different genotypes. TUB8 was used as an internal control. Error bars represent standard error (n = 3).
(E) ChIP assay of H3K4me2 and K3H9me2 in the wild-type, idm1-9, and idm2-3. Ten-day-old seedlings were used for ChIP assay with antibodies against H3K9me2 or H3K4me2. 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.
(F) The DNA hypermethylation phenotype of idm2-1 plants is rescued by transgenic expression of wild-type IDM2 but not by expression of the D49E, G246D, or G260D mutant. Chop PCR results for At1g26400 are shown for two representative transgenic lines generated from each construct.
See also Figures S1 and S4 and Tables S4 and S5.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Analysis of DMRs Identified in idm2 Mutant
(A) The number of DMRs identified in idm2 and the overlap of hyper-DMRs between different mutant plants.
(B) Composition of the hypermethylated loci in idm2-1, idm1-1, ros1-4, and rdd mutants. TE, transposable element.
(C) Enrichment of small RNAs (sRNAs) in the 1,218 hypermethylated regions identified in idm2-1. Small RNA reads were generated from the wild-type and rdd mutant plants by Lister et al. (2008). Black dots indicate the sRNA densities from randomly selected regions in the genome, and red dots indicate the sRNA densities in the hypermethylated regions in idm2-1. Horizontal lines indicate the 99th percentile of sRNA density from 1,000 simulated runs.
(D and E) Overlap of hyper-DMRs between idm2 and idm1 (D) and between idm2 and rdd (E). Boxplots represent methylation levels of each class of DMRs.
See also Figure S2 and Tables S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Gene Expression Level in idm1 and idm2 Mutants
(A) Expression of the hypermethylated genes or genes near DMRs in 2-week-old idm1-1, idm2-1, and ros1-4 seedlings. Error bars represent standard error (n = 3).
(B) Overlap of differentially expressed genes between the different comparison groups.
See also Figure S3 and Tables S3 and S4.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Protein-Protein Interactions between IDM2 and IDM1
(A) Yeast two-hybrid analysis of IDM1 and IDM2 interaction. AD, activating domain; BD, binding domain.
(B) LCI assays showing that IDM1 can interact with IDM2 in Arabidopsis protoplasts. 1, IDM1-nLUC+IDM2-cLUC; 2, IDM1-nLUC+cLUC; 3, nLUC+IDM2-cLUC; 4, nLUC+cLUC. Three biological replicates were performed, and very similar results were obtained. Standard errors were calculated from three technical repeats.
(C) Co-IP of IDM1 and IDM2 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.
(D) The interaction between Flag-IDM2 and IDM1-HA as determined by co-IP. Transgenic plants expressing Flag-IDM2 or IDM1-HA under their native promoters and their F1 offspring were used for co-IP.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Subnuclear Localization of IDM2 and Its Colocalization with IDM1
(A) Detection of IDM2 (red) in the wild-type and idm2-1 mutant nuclei by immunostaining using anti-iDM2. Scale bars represent 10 μm.
(B) Dual immunolocalization of IDM2 (red) and IDM1-HA (green). DNA was stained with DAPI (blue). The frequency of nuclei displaying each interphase pattern is shown on the right. Scale bars represent 10 μm. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 The Effect of idm2 Mutation on Histone Modification Marks
H3K18ac and H3K23ac levels at the DMRs and 35S promoter and control regions were determined by ChIP on wild-type, idm1-9, and idm2-3 plants with anti-H3K18ac or anti-H3K23ac antibodies. 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.
See also Figure S6 and Table S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions