Genetic Rearrangements Modify Nearby Chromatin Figure 3. Sequence rearrangements after mutagenesis cause transcriptional activation of the silent epiallele. (A) Restored hygromycin resistance (percentage of resistant plants within all germinated seeds) of cis-mutants derived from line S after T-DNA mutagenesis, generation S4. (B) P1 and P2 transcript levels in generation S5 of cis-mutants determined by qRT-PCR. 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; nd: not detectable, reference gene EIF4a (At3g13920); error bars represent standard deviation of triplicate measurements. (C) Altered transcript length in cis-mutants (generation S4) compared to line R. Total RNA blot hybridized with an HPT probe. (D) DNA rearrangements determined after amplification and sequencing and transcript variation determined by 39RACE and sequencing. Wild type (W); inactive (S) and active (R) epiallele, resistant cis-mutants derived from line S (RD1-6). The dashed part of the NC region indicates the overlap with small RNA reads homologous to P1/P2 (see Figure 2B). doi:10.1371/journal.pgen.1002331.g003 PLoS Genetics | www.plosgenetics.org 5 October 2011 | Volume 7 | Issue 10 | e1002331