E regulation of DNA methylation and epigenetic gene silencing at heterochromatic
E regulation of DNA methylation and epigenetic gene silencing at heterochromatic regions (Woo et al., 2007, 2008). Additionally, a current genome-wide DNA methylome analysis revealed that CG and CHG methylation was strongly decreased inside the vim1 vim2 vim3 triple mutant (hereafter designated vim1/2/3) (Stroud et al., 2013). However, the roles of the VIM proteins in histone modification have not been investigated. Studies involving Arabidopsis VIM proteins EGFR/ErbB1/HER1 review enhanced our understanding on the mechanistic basis for VIMmediated epigenetic gene silencing. The VIM proteins recognize methylcytosine in any sequence context, with preferential affinity for hemi-methylated CG web-sites (Bostick et al., 2007; Kinesin-14 Accession Johnson et al., 2007; Woo et al., 2007; Yao et al., 2012). UHRF1 binds each 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) web sites with equivalent affinity, whereas VIM1 binds to 5hmC web-sites with significantly lower affinity than it binds to 5mC sites (Frauer et al., 2011; Yao et al., 2012). It was also reported that VIM1 possesses E3 ubiquitin protein ligase activity (Kraft et al., 2008). VIM1 is linked with NtSET1, a tobacco SU(VAR)3 protein, indicating that VIM1 could recruit H3K9 methyltransferases for the duration of heterochromatin formation (Liu et al., 2007). Nevertheless, endogenous targets of your VIM proteins for epigenetic gene silencing have not been analyzed making use of a genomewide screen. Additionally, the mechanisms by which the VIM proteins coordinate maintenance of DNA methylation and epigenetic gene silencing are largely unknown. In this study, a genome-wide expression microarray analysis was performed inside the vim1/2/3 triple mutant to identify the targets on the VIM proteins. We identified 544 derepressed loci in vim1/2/3, which includes 133 genes encoding proteins of known function or those equivalent to known proteins. VIM1 bound to each the promoter and transcribed regions of the derepressed genes in vim1/2/3. In addition, VIM deficiency resulted in sturdy DNA hypomethylation in all sequence contexts at the direct targets of VIM1, in addition to a clear reduction in H3K9me2 was observed at condensed heterochromatic regions in the vim1/2/3 mutant. The vim1/2/3 mutation also led to significant modifications in transcriptionally active and repressive histone modification at the VIM1 targets. VIM1-binding capacity to its target genes was substantially decreased by the met1 mutation, suggesting that VIM1 binds its targets primarily by way of recognition of CG methylation. Taken with each other, these data strongly recommend that the VIM proteins regulateGenome-Wide Epigenetic Silencing by VIM ProteinsMolecular Plantup-regulated genes in vim1/2/3 a drastically higher proportion of genes had been positioned close to TEs (within two kb) in comparison to the all annotated Arabidopsis genes (Figure 1E). This observation implies that proximity to TE could possibly be a crucial determinant on the derepression of gene expression in vim1/2/3. Nearly half from the loci up-regulated in vim1/2/3 (298 of 544, 53.six ) have been strongly silenced (signal intensity one hundred) in WT plants (Figure 1F and Supplemental Table 1), indicating that massive reactivation of silenced genes occurred in vim1/2/3. In addition, 66 loci that were highly expressed in WT plants (11.9 ; signal intensity 1000) were up-regulated inside the vim1/2/3 mutant. We then asked whether the transcriptional activation observed in vim1/2/3 depends upon DNA methylation. The information from a genome-wide DNA methylation analysis of Arabidopsis indicated that 20.2 and 56.0 o.
