extents, indicating several independent mutational changes of the sequence. An extended northern blot analysis, with either total RNA or poly(A)-enriched RNA, showed that the P1 transcript in all lines besides RD6 is polyadenylated (Figure S6), likely due to a flanking sequence with some similarity to a polyA signal. While no P2 transcript from the second promoter is detectable in RD1, RD2, RD4, and RD6, there is a signal in RD3 and RD5, including in the poly(A) fraction (Figure S6C, S6D). To characterize the P1 transcripts, and to identify the transcriptional termination sites in the cis-mutants, we performed 39-RACE. We also analyzed the genomic DNA of all cis-mutants after amplification of the transgenic insert from genomic DNA and aligned DNA and RNA sequences (Figure 3D). This verified six different sequence rearrangements within the 39 region: mainly deletions, but also one case of an inserted plant DNA fragment (RD3). The mutants RD1 and RD2 have both lost the duplicated promoter P2 and the NC sequence. The vector duplication was partially (RD1) or completely (RD2) deleted, as was part of the flanking plant sequence. The deletions in RD4, RD5, and RD6 did not or only partially affect the P2 promoter, and two of them maintain also the NC sequence. The rearrangement in RD3 is most complex: here, a 1243 bp plant DNA sequence derived from a position 1.2 kb upstream of the transgene location was inserted between the P1 transcript and the downstream vector fragment. In the mutants RD1, RD2, RD3, and RD4, the P1 transcripts are terminated at the (first) site of rearrangement, while the transcripts go beyond the breakpoints in RD5 and RD6. Only RD3 and RD5 are able to produce the P2 transcript, as in these cases, the P2 promoter is complete and the heterologous sequence downstream was only slightly affected by mutagenesis (Figure 3D). Nevertheless, the P2 transcript levels are much lower than in the R line (Figure 3B). Interestingly, there is no overlap between the deletions in all individual cis-mutants, but the rearrangements had either affected the second promoter copy (RD1, RD2, RD6), or the DNA template for the P2 transcript (RD1, RD2 and RD4), or the connection between both sequences (RD3, RD5). All cis-mutants were tested for effects outside of the epiallele by analyzing the degree of genome-wide methylation at endogenous repeats and by introgressing a transcriptionally silent marker gene coding for b-glucuronidase from line L5, shown to be affected by other epigenetic mutations [27–28]. None of the cis-mutants changed the modification or expression of these markers (Figure S7). Therefore, it is unlikely that they have an effect outside of the epiallele. Due to the hygromycin selection in the screen, all cis-mutants were expected to have a functional resistance marker gene. Indeed, the upstream promoter P1 and the HPT coding region were intact and identical in RD1-6 and hence potential new epialleles of the resistance gene. Therefore, we compared the chromatin state in this region. We found reduced DNA methylation levels in cis-mutants compared to S (Figure 4A), and a detailed bisulfite methylation analysis confirmed an overall reduction of DNA methylation in the promoter region of cis-mutants (Figure 4B, 4C). However, the degree of hypomethylation, and the distribution of the remaining methylated cytosine residues, do not support a direct and linear correlation with expression levels. Although RD2, RD3, and RD4 show the strongest reduction of CG methylation, especially at the transcription factor binding sites (Figure 4B, asterisk), and have expression levels comparable to R (Figure 3B), methylation in RD5 is similar to RD3 and RD4, although P1 transcript expression is much lower. Also, RD3 and RD4 have even gained CHH methylation in the 59 region. Genetic Rearrangements Modify Nearby Chromatin Concomitant with the loss of DNA methylation, the modifica- tion specific for the silent state (H3K9me2) was changed in favor of the active mark (H3K4me3) in P1 and P1-transcribed regions, as demonstrated by ChIP (Figure 4D). One mutant (RD1) maintained a high level of H3K9me2 similar to that of the silent epiallele. Nonetheless, it also acquired a remarkable amount of H3K4me3, although less than other cis-mutants. Independent of the modifications, and similar to the resistant line, cis-mutants showed a decreased level of H3 association, indicating that the sequence rearrangements had also affected the nucleosome density (Figure 4E). On the whole, the cis-mutants demonstrate that structural rearrangements can cause significant changes in transcriptional activation and chromatin configuration at the previously silent epiallele. These changes are surprisingly divergent and reflect specific effects of similar but not overlapping deletions. Stability of Silencing after Sequence Rearrangement The extreme stability of R and S epialleles through many generations and in callus cultures raised the question of expression stability in the cis-mutants. Most structurally rearranged deriva- tives displayed similar stability and provided comparable hygro- mycin resistance over several generations of homozygous cis- mutants (S4 to S6 tested). RD2, RD3, and RD4 produced resistant progeny in consecutive generations. Resistance in RD5 and RD6 was lower in S4 (56% and 61%, respectively), but maintained this level up to S6. In contrast, RD1 plants that were clearly hygromycin-resistant in S4 (84%) generated partially sensitive S5 and fully sensitive S6 progeny (Figure 5A). This correlates well with the loss of unmethylated sites at the transgenic insert (Figure 5B), similar to gradual loss of resistance over 5 generations described for another marker gene [29]. The instability in RD1 does not correspond with additional sequence changes, as the same rearranged structure (Figure 3D) is maintained in subsequent generations. Rather, it correlates with the epigenetic state, since RD1 was characterized by the bivalent histone modifications (Figure 4D). The re-silencing in generation S6 of RD1 allowed us to compare silencing maintenance at promoter 1 between this line and the S epiallele. We tested plants of both lines after growth in the presence of zebularine [reducing DNA methylation, 30] or DZNep [reducing histone methylation and also DNA methylation via SAHH-inhibition, 22,31]. Zebularine alone did not reactivate promoter P1 in line S, but in RD1S6, and DZNep-induced activation was twice as high in RD1S6 compared to S (Figure 5C). This indicates that S and RD1S6 differ in the stringency of silencing, either due to presence or absence of the P2 promoter and transcript, or to the lineage history of RD1S6 from a recently active state. The presence of the P2 promoter in RD3 - 6 and the expression of the P2 transcript in RD3 and 5, which do not cause re-silencing in later generations, make the latter explanation more likely. Discussion The thorough analysis of the HPT transgene in its two opposite expression states has revealed sequence identity over the full length of the insertion, significant differences in chromatin modifications and few, but silencing-specific, small RNA molecules. Chromatin differences are restricted to the affected sequence, with no hint of genome-wide changes or modified localization of the genomic region within the nucleus. Together with heritability of the expression states over many generations, and their maintenance even upon de-differentiation, the data prove the transcriptionally PLoS Genetics | www.plosgenetics.org 6 October 2011 | Volume 7 | Issue 10 | e1002331