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Unformatted text preview: A structural-maintenance-of-chromosomes hinge domain–containing protein is required for RNA-directed DNA methylation Tatsuo Kanno 1,5 , Etienne Bucher 1,4,5 , Lucia Daxinger 1 , Bruno Huettel 1 , Gudrun Bo ¨hmdorfer 1 , Wolfgang Gregor 2 , David P Kreil 3 , Marjori Matzke 1 & Antonius J M Matzke 1 RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region 1,2 . To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region. A screen for mutants defective in meristem silencing and enhancer methylation retrieved six dms complementation groups, which included the known factors DRD1 (ref. 3; a SNF2-like chromatin-remodeling protein) and Pol IVb subunits 4,5 . Additionally, we identified a previously unknown gene DMS3 (At3g49250), encoding a protein similar to the hinge-domain region of structural maintenance of chromosomes (SMC) proteins. This finding implicates a putative chromosome architectural protein that can potentially link nucleic acids 6 in facilitating an RNAi-mediated epigenetic modification involving secondary siRNAs and spreading of DNA methylation. In plants, small RNAs directed to promoter regions can trigger promoter methylation and transcriptional gene silencing 1 . In the two-component transgene system used here, this strategy has been modified to target an upstream enhancer that is active in meristems for methylation by a homologous hairpin RNA, which is encoded at an unlinked silencer locus ( Fig. 1a ). In the absence of the silencer locus, GFP expression can be observed in the root apical meristem of seedlings, whereas fluorescence is abolished in the presence of the silencer ( Fig. 1b ). Through analysis of DNA methylation using bisulfite sequencing, we found that the target enhancer completely lacks cytosine methyla- tion in the absence of the silencer locus (data not shown) but acquires heavy methylation of cytosines in all sequence contexts in the presence of the silencer ( Fig. 2 , T + S (target + silencer)). Unexpectedly, we also detected a high degree of methylation in the DNA sequence down- stream of the targeted enhancer region in silenced plants ( Fig. 2 , T + S). This transcriptional silencing system therefore differs from several other systems in which methylation does not infiltrate sig- nificantly into nontargeted sequences 3,7 . The spreading of methylation beyond the originally targeted region suggests the presence of secondary siRNAs, which form adjacent to the target sites of primary siRNAs during RNAi in plants and Caenorhab- ditis elegans 8,9 . Secondary siRNAs depend on RNA-dependent RNA polymerase (RDR) for their biogenesis. Indeed, we detected 24-ntpolymerase (RDR) for their biogenesis....
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This note was uploaded on 08/01/2009 for the course 1 1 taught by Professor Maz during the Spring '01 term at Abilene Christian University.
- Spring '01