DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase IV (Pol IV) transcribes target sequences to initiate 24-nt siRNA production and action. The putative DNA binding protein DTF1/SHH1 of Arabidopsis has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. DTF1 interacts directly with the chromatin remodeling protein CLASSY 1 (CLSY1), and both DTF1 and CLSY1 are associated in vivo with Pol IV but not Pol V, which functions downstream in the RdDM effector complex. DTF1 and DTF2 (a DTF1-like protein) contain a SAWADEE domain, which was found to bind specifically to histone H3 containing H3K9 methylation. Taken together, our results show that DTF1 is a core component of the RdDM pathway, and suggest that DTF1 interacts with CLSY1 to assist in the recruitment of Pol IV to RdDM target loci where H3K9 methylation may be an important feature. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci.histone modifications | small RNA | gene silencing | transposon D NA cytosine methylation is a conserved epigenetic mark that plays important roles in maintaining genome stability, transcriptional gene silencing, and developmental regulation (1, 2). In plants, DNA methylation occurs in three sequence contexts: CG, CHG, and CHH (H = A, C, T). CG and CHG methylation are symmetric in sequence and are maintained through a semiconservative mechanism that requires the DNA methyltransferases (METHYLTRANSFERASE 1) (MET1) and CHROMOMETHYLASE 3 (CMT3), respectively. In contrast, the asymmetric CHH methylation needs to be established during each cell cycle (1, 2). In plants a 24-nt small interfering RNA (siRNA)-dependent DNA methylation pathway is involved in recruiting the de novo DNA methyltransferase DOMAINS REARRANGED METHYLASE 2 (DRM2) and is responsible for DNA methylation at many transposable elements and repetitive sequences (3, 4).Two plant-specific homologs of RNA polymerase II play important roles in the RNA-directed DNA methylation (RdDM) pathway (5). RNA polymerase IV presumably initiates 24-nt siRNA biogenesis by specifically transcribing RdDM target loci to produce single-stranded RNA (ssRNA) transcripts, which serve as templates for RNA-dependent RNA polymerase 2 (RDR2) to generate double-stranded RNAs (dsRNAs). CLASSY 1 (CLSY1), a putative ATP-dependent chromatin remodeling protein, is prop...

RNA-directed DNA methylation (RdDM) is an important de novo