variation between regions with very similar gene sequences in plants [e.g. 8] or in the connection between chromatin structure and trinucleotide repeat expansion in mammals [for review 34]. Transcriptional gene silencing is often associated with the presence of homologous sequences in the genome [e.g. 35–37], and intentional rearrangements from complex inserts to single copies by site-specific recombinase eliminate silencing [e.g. 38]. Therefore, when we started the analysis of the sequence changes in the cis-mutants, we were expecting a clear dependence of reactivation on loss of the duplicated region. This is not the case, since all cis-mutants, with the exception of RD2, still retain some duplicated regions. Also against expectation, a loss of the non- coding sequence homologous to the most abundant small RNAs is not a prerequisite for reactivation (RD3, RD5, and RD6). Furthermore, a loss of the small transcript starting from the P2 promoter is not necessary (RD3 and RD5), although its level in these mutants is not as high as in R plants. It should be kept in mind that neither the tandem sequence duplications, nor either of the two transcripts, are sufficient to initiate silencing, since R plants (with the complete insert and substantial transcription from P1 and P2) are fully resistant and stable. This is distinct from the FWA gene where tandem repeats are necessary and sufficient for silencing and DNA methylation [39]. Considering the lack of DNA methylation and small RNAs at the HPT insert in R plants, it is possible that the initial steps of silencing do not occur, are not efficient enough to start the reinforcing mechanism [39], or are inhibited by efficient transcription [40]. However, such conditions must have been overruled on the rare occasions that produced the silent epiallele in the first place. The deletions in the different cis-mutants do not overlap in a specific region, and the smallest change is the loss of just 65 bp (RD5). Apparently, rather than affecting a specific sequence, the rearrangements change the overall organization at this locus. These changes can have variable consequences for the upstream promoter, causing either decisive, stable epigenetic switches (RD2, RD3, RD4) or leading to ambivalent states that can later fall back into silencing (RD1). How such small genetic heterogeneity, that does not affect coding or regulatory regions, can cause extreme epigenetic diversity at a promoter elsewhere remains an open question. The sequence changes could exert their effect by modifying the distance to flanking regulatory regions, the nucleosome arrangement or density, the association with DNA- binding molecules, or any higher order structure within the DNA. It is clearly different from the ‘spreading’ effect of silencing often associated with RdDM [41–42]: it causes activation (not silencing), goes against (not along with) the direction of transcription, and the most abundant of the relatively few small RNAs does not match the affected sequence of the upstream promoter. The results emphasize the mutual dependence between genetic and epigenetic factors, while indicating that these do not necessarily act at overlapping genomic sites. Similar effects might explain some of the associated changes in gene expression in the vicinity of small or large sequence modifications by transposon or recombination events. One example at a similar distance might be the transposon-dependent loss and gain of DNA methylation and inverse gene expression regulating sex determination in melon, at a site just 1.5 kb away from the insertion/excision site [43]. The relatively high number of cis-mutants in the screen was plausible in retrospective: mutations outside of the epiallele released silencing only if they reduce two epigenetic marks simultaneously. This is achieved by a few special mutations [22] or theoretically by rare double mutations and explains the low number of trans-acting mutants. In the study here, the genetic changes were found after mutagenesis by Agrobacterium-mediated Genetic Rearrangements Modify Nearby Chromatin T-DNA transformation [22], although none of the cis-mutations was connected with an integrated fragment of the incoming T- DNA. T-DNA transformation is also known to create mutations unlinked, or independent, from the site of integration [44] and can cause complex chromosome rearrangements [45–46]. Successful, and possibly also attempted, integrations occur at sites of microhomologies between T-DNA and plant DNA [47– 48]. The incoming T-DNA [49] has some homology with the terminator sequences in the epiallele (DT), and in fact, the deletion sites in two cis-mutants (RD2, RD3) are near, or in, this sequence. The other deletions are close to promoter copy P2 that has no homology with the T-DNA, but potentially reflect a recombination hotspot in the 35S promoter sequence [50]. Alternatively, the double strand breaks connected with completed or aborted integration might stimulate repair via homologous recombination between the duplicated sequences of the epiallele (RD3). This would indeed have selected for 39 rearrangements since those affecting the upstream copy are likely to lose the functional HPT cassette. All together, the R and S epialleles described here provide an example of identical DNA sequences with converse expression states and specific epigenetic configuration that are faithfully transmitted to progeny. However, sequence changes in the vicinity of the silent epiallele can induce an epigenetic switch to the opposite state. These can have different degrees of stability, depending on the complex interplay between the nature of the sequence alteration, the consequences for transcription and transcripts, and the chromatin organization (Figure 6). This also illustrates a tight dependence of epigenetic regulation on local structures and makes it likely that DNA rearrangements can potentially change or induce new epialleles outside the affected region. Materials and Methods Plant Material, Growth, and Chemical Treatments Arabidopsis thaliana lines with R and S epialleles in accession Z u¨rich and mutagenesis of line S were described previously [20,22]. Stratified seeds were surface-sterilized with 5% sodium hypochlorite and 0.05% Tween-80 for 6 min, washed and air- dried overnight. Sterilized seeds were germinated and grown in Petri dishes containing agar-solidified germination medium (GM) in growth chambers under 16 h light/8 h dark cycles at 21uC. For drug treatments, seeds were sown and plants grown on GM plates with hygromycin (10 mg/ml, Calbiochem), zebularine (40 mM, Sigma) or 3-deazaneplanocin (DZNep, 2 mM, donated by Dr. Victor Marquez) under the conditions described above. Nucleic Acid Isolation and Gel Blot Analysis Genomic DNA was isolated from 3 week-old seedlings using either DNeasy Plant Mini Kit (Qiagen) or Phytopure (Amersham), following the manufacturers’ protocols, except that genomic DNA was eluted in sterile water. Total RNA extraction from 3 week-old seedlings was performed with RNeasy Plant Mini Kit (Qiagen) including an on-column DNase I digest (Qiagen). For Southern blot analysis, 10 mg of genomic DNA were digested overnight with 20 U restriction enzymes. For methylation-specific Southern blot analysis, the methylation-sensitive restriction enzymes (HpaII, blocked by mCG and mCHG, and MspI, blocked only by mCHG) were used. Digested samples were electrophoretically separated on 1.2% TAE agarose gels, depurinated for 10 min in 250 mM HCl, denaturated for 30 min in denaturation solution containing 0.5 M NaOH and 1.5 M NaCl and neutralized twice in 0.5 M Tris, 1.5 M NaCl and 1 mM EDTA at pH7.2 for 15 min. For northern PLoS Genetics | www.plosgenetics.org 9 October 2011 | Volume 7 | Issue 10 | e1002331