Genetic Rearrangements Modify Nearby Chromatin Figure 1. Epialleles differ in chromatin features. (A) Transgenic insert (identical in lines S and R) with duplicated vector (V1, V2) and CaMV35S promoter (P1, P2) sequences and single copies of the hygromycin phosphotransferase resistance gene (HPT, P1 transcript), a truncated terminator (DT) and a non-coding sequence containing bovine carrier DNA (NC, P2 transcript). Black star: polyadenylation signal-like sequence. (B) Transcript levels determined by qRT-PCR in diploid Arabidopsis ecotype Z u¨rich with (S, R) or without (W, wild type) the transgenic insert. P1 was determined with primers within the HPT sequence, P2 with primers within the NC sequence. Due to the overlap, this might capture also some P1 templates. Normalization to S; reference gene EIF4a (At3g13920); error bars represent standard deviation of triplicate measurements. (C) Methylation analysis in three week-old seedlings with (S, R) or without (W, wild type) the transgenic insert. Genomic DNA was treated with HpaII not cutting mCmCGG, blotted and hybridized to a probe spanning the HPT sequence. Enzyme recognition sites are indicated below the blot. (D, E) Analysis of histone H3 at both promoters (P1, amplicon 133 bp; P2, amplicon 197 bp; primers see Table S4) in lines S and R by chromatin immunoprecipitation. (D) Association with H3K9me2 and H3K4me3; (E) modification-independent precipitation. doi:10.1371/journal.pgen.1002331.g001 the transgenic insert. The library from the R plants had only 59 reads (3 per 1 million reads) with only one sequence with a match in the epiallele (Figure 2A, Table S3). In line S, we found 2661 (129 per 1 million reads) matching the epiallele, with a predominant length of 24 nucleotides (Figure 2A, Table S2 and Table S3), the size class known to be primarily involved in RNA- directed DNA methylation (RdDM). This is significantly more than in R, but still relatively little, compared to an individual miRNA (820 reads per 1 million for miRNA165) or to siRNA from a repetitive sequence (.1000 reads per 1 million for TSI [26]). The reads in S were distributed along the epiallele but mostly outside the HPT coding region. Importantly, among all reads specific for the silent epiallele we found an sRNA peak (671 reads, 476 antisense and 195 sense) covering 61 bp in the middle of the 505 bp non-coding sequence of the P2 transcript (Figure 2B). The most abundant sRNAs overlap with the 54 nucleotides of unknown origin. However, this sequence encompasses 28 nucleotides that are homologous to the most 59 end of the 35S promoter (Figure 2B). In short, these results indicate very stable and completely isogenic epialleles that differ only in their transcriptional activity. DNA methylation, suppressing chromatin marks, and sRNAs, are specifically enriched at the transcriptionally inactive epiallele; while the counterpart produces high transcript levels, lacks DNA methylation and sRNAs, and carries modifications characteristic of open chromatin (Figure 2C). Release of Silencing upon Sequence Rearrangement In addition to the trans-acting mutants identified in a screen for restored HPT expression after mutagenesis of line S [22], we identified six hygromycin-resistant plants in which the mutant phenotype was genetically linked to the resistance gene itself (‘cis- mutations’, RD1-6). All these mutants produced progeny that could grow on hygromycin selection plates (Figure 3A), connected with restoration of variable amounts of P1 and P2 transcripts (Figure 3B). Northern blot analysis of cis-mutant RNA revealed P1 transcripts of smaller size in all cis-mutants compared to those from the active R line (Figure 3C). The length is reduced to different PLoS Genetics | www.plosgenetics.org 3 October 2011 | Volume 7 | Issue 10 | e1002331