Figure 2. RNA-directed DNA Methylation and Heterochroma- tinization at the MPF. (A) Genomic structure of the FLC locus and flanking regions examined by bisulfite sequencing (B1, B2, B3 and B4) or ChIP (C1, C2 and C3). Green box represents the hAT element; pink boxes represent exons; the gray arrow represents the promoter; the orange box represents the TE insertion in Ler. (B) Small RNA tags matched to MPF found from the MPSS (green) or 454 sequencing data (red), and the LNA probe used for small RNA hybridization (blue) are represented with their length indicated by numbers. The color coding of the cytosines in (B) matches the legend in (C). (C) Bisulfite sequencing result of the MPF at the B1 region in Ler. The bars with red stars represent sites that were detected by Southern blot (Figure 1) and n indicates the number of the sequenced clones. (D) Small RNA Northern blots probed with the LNA probe (B) in Ler and Col; tRNA and other RNA bands stained with ethidium bromide (EtBr) were used to indicate the amount of loaded RNA. (E) Chromatin Immunoprecipitation (ChIP) to detect H3K9 mono-, di-, and tri-methylation (represented as H3K9me1, H3K9me2, and H3K9me3, respectively) at MPF (C1) in Ler and Col. Input is saved before immunoprecipitation and ‘‘No AB’’ refers to the sample without antibody. Ta3 served as an internal control for heterochromatic loci. doi:10.1371/journal.pgen.1000056.g002 matching to this methylated region in Ler. Because no methylation was found at the MPF in Col, we speculated that there would be no small RNAs matching to this region. However, four 17 nt tags with very low abundances (approx- imately two transcripts per quarter-million, TPQ) were found in the Col-derived small RNA massively parallel signature Small RNA-Directed Epigenetic Natural Variation sequencing (MPSS) datasets [12]. These small RNAs precisely matched both strands of the highly asymmetrically methylated region within MPF (Figure 2B). We performed a small RNA Northern blot hybridization to verify these small RNA in Col and Ler. By using an LNA (locked nucleic acid) modified oligonucleotide probe (Figure 2B) and a large amounts of RNA enriched for small RNAs (see materials and method for more details), we found that siRNAs complementary to this probe (MPF-siRNAs) were more abundant in Ler than in Col (Figure 2D). Published high-throughput small RNA 454 sequencing datasets from Ler [15] confirmed our RNA gel blot results. In those data, six unique 23 to 24 nt small RNAs were found matching to a region of ,50 bp at the MPF, in exactly the same region as the Col-derived MPF-siRNAs (Figure 2B). Analyses of additional Col-derived 454 small RNA data [16,27] didn’t identify any MPF-matching small RNAs, possibly due to lower sequencing depth compared to that of the MPSS data. We performed chromatin immunoprecipita- tion (ChIP) experiments and demonstrated that the MPF in Ler was enriched in H3K9me2, a characteristic of heterochromatin, in comparison to Col (Figure 2E). These data suggest that the high levels of MPF-siRNAs in Ler could trigger DNA methylation and heterochromatinization at MPF whereas the lower levels in Col might not be sufficient. Methylation at MPF Is Sensitive to Deficiency in RdDM Next, we investigated methylation at the MPF using silencing pathway mutants in either a Ler background or in lines that had been backcrossed to Ler to have the homozygote FLC-Ler allele. These mutants included hen1-1, cmt3-7, ago4-1, kryptonite-2 (kyp, a histone H3K9 methyltransferase, also known as SUVH4, can affect the DNA methylation at some loci[28–30], and drm2 56Ler (homozygous drm2 backcrossed five times to Ler). Methylation at MPF was sensitive to the deficiency in the RdDM machinery: all mutants tested, with the exception of kyp- 2, completely relieved methylation in all three sequence contexts at MPF (Figure 3A and Figure S2A). Although KYP has been reported to control CNG methylation together with CMT3 [26,30], the methylation at MPF was independent of its function, perhaps because MPF at several hundred base pairs is too small for KYP to maintain the positive feed back between DNA methylation and chromatin modification [30]. Alternatively, in addition to KYP, the heterochromatic feature of this region might be redundantly controlled by other two histone H3K9 methyltransferases, SUVH5 and SUVH6 [31]. In addition, methylation of the nearby TE insertion (Figure 3B and Figure S2C) was also sensitive to ago4-1 and hen1-1 (Figure 3B). However, none of these mutants released all DNA methylation at AtSN1, a retroelement which also undergoes RdDM [26] (Figure 3C). Moreover, AGO4 complementation [15] could not restore DNA methylation at the MPF in ago4-1 (data not shown). This situation resembles the FWA locus whose methylation, once lost in ddm1(decrease in DNA methylation 1) mutant, is not recovered again even in the presence of wild type DDM1 [32]. The MPF in hen1-4, a strong hen1 allele in the Col background, had an identical methylation pattern to Col (Figure 1). Also, the identical methylation pattern of the miRNA deficient mutant dcl1-9 [7] to Ler at MPF (Figure S2B) ruled out the possibility that the restricted methylation at MPF is directed by miRNAs [33]. These observations were substantially different from prior analyses of silenced loci, at which DNA methylation was often affected in certain but never all sequence contexts by mutants in the RdDM pathway [26]. PLoS Genetics | www.plosgenetics.org 3 April 2008 | Volume 4 | Issue 4 | e1000056