Genetic Rearrangements Modify Nearby Chromatin Figure 5. Different stability of reactivation after sequence rearrangement. (A) Hygromycin resistance in later generations of mutant RD1. (B) Progressive methylation in later generations of mutant RD1. Analysis as in Figure 4. (C) Effect of drug treatment (mock, 40 mM zebularine or 2 mM DZNep) on P1 transcript levels determined by qRT-PCR. Normalization to S; nd: not detectable, reference gene EIF4a (At3g13920); error bars represent standard deviation of triplicate measurements; wild type (W), line S (S) and cis-mutant RD1 in generation S6. doi:10.1371/journal.pgen.1002331.g005 After mutagenesis, we identified several hygromycin-resistant plants in which mutations in the epiallele sequence downstream of the HPT coding region had reactivated the previously silenced epiallele. Combining DNA and RNA sequence analysis and characterization of chromatin modifications, we found that these structural changes of the DNA sequence caused substantial upstream changes in chromatin and transcriptional activity. Beyond the complex and mutually dependent interplay of chemical modifications of the DNA and the associated histones, and longer and small, coding and non-coding RNAs described in numerous cases, the results presented here have shown that even small and non-overlapping modifications of the genomic template, outside of the promoter and open reading frame, can modify transcription and chromatin states in the vicinity. These changes are not minor: the bacterial gene HPT coding for hygromycin phosphotransferase is a selectable marker gene applied in numerous plant transformation experiments [32], but plants need a significant amount of HPT transcript to produce enough protein to detoxify the antibiotic. Minor reactivation in the background of some epigenetic mutants tested in a reverse genetic approach (data not shown) was not sufficient. Therefore, the stringent assay for restored hygromycin resistance required a substantial change, as in the case of the trans-acting mutants from the same screen that revealed a double lock of two simultaneous chromatin modifica- tions [22]. HPT expression levels are indeed similar between cis- and trans-acting mutants. Although the transgenic marker allowed this convenient selection for drastic epigenetic switches, without affecting plants under non-selective conditions, it could have been considered not representative for other, plant-endogenous or general cases. However, a recent publication [33] describes an interesting mutation that affects expression of the gene for nodulation factor SUNN in Medicago truncatula. The mutation is closely linked to the SUNN gene, acts only in cis but does not change the DNA sequence of the SUNN gene itself. Although the nature of this mutation is not yet identified, it could exert its effect in a similar way to the cis-mutants described here, especially since the ‘like sunn supernodulator’ mutant phenotype is occasionally unstable, like the hygromycin resistance in RD1, 5, and 6. Other examples may be found upon further inspection of natural transcript level PLoS Genetics | www.plosgenetics.org 8 October 2011 | Volume 7 | Issue 10 | e1002331