Figure 1.

IBM1 downregulates H3K9me2 at genes. (A, B) Overexpression of FLAG-IBM1 in tobacco leaf cells induced reduction in H3K9me2 and H3K9me1. Nuclei transfected with FLAG-IBM1 (A) or a mutated construct, FLAG-IBM1m (B), were mixed with control nuclei without the transfection (see Materials and methods for details). The transfected nuclei and control nuclei could be distinguished by immunostaining with anti-FLAG (shown in red). Histone methylation was visualized by immunostaining with rabbit polyclonal methylation-specific antibodies (green). Nuclei were visualized by DAPI staining (blue). Arrows indicate nuclei transfected with FLAG-IBM1 or FLAG-IBM1m. Quantification of these signals, together with those from other samples, is shown in Supplementary Figure S1A and B. (C, D) Representative examples of H3K9me2 analysed by tilling array for a gene-rich region (C, position 20 829 000–20 934 000 on chromosome 1) and a transposon-rich region (D, positions 16 494 000–16 592 000 on chromosome 1). Profile of DNA methylation (Miura et al, 2009) is also shown. Blue and red boxes indicate genes and transposons, respectively. They are oriented 5′–3′ on the top and 3′–5′ on the bottom. Each vertical bar represents the log₂ ratio of the immunoprecipitated DNA to input control for the DNA methylation (DNAme) and H3K9me2, respectively. Each brown bar represents difference of signals for each comparison. (E) H3K9me2 level in each gene (blue dot) and transposon (red dot), compared between wild type and ibm1. (F) Comparison between the effect of ibm1 mutation on DNA methylation (DNAme) and H3K9me2 in each gene (blue dot) and transposon (red dot). The changes in DNA methylation and H3K9me2 levels were not proportional, most likely because each gene differs in the CG methylation level, which is not affected by the ibm1 mutation (Miura et al, 2009).