Author Summary In contrast to alleles, epialleles have identical DNA sequence and differ only in gene expression and chromatin features. Epialleles are heritable and can also contribute to phenotypes. How this variation originates is unclear. In this study, we analyzed two epialleles found in Arabidopsis for the difference between their chromatin features and their potential to change state. We muta- genized plants with the inactive epiallele and recovered mutants with restored gene expression. In several cases, this was connected with different rearrangements down- stream of the epiallele that caused a switch of the epigenetic configuration further upstream. Therefore, sequence alterations, for example by transposon activity or recombination events, may trigger similar heritable changes of chromatin and gene expression in their proximity and could create new epialleles. tion, they also differ in DNA methylation [21]. We screened for a switch between the epialleles, by scoring for restored hygromycin resistance after T-DNA mutagenesis of the diploid S line. We identified two trans-acting factors whose nature indicated an epigenetic ‘double lock’ at the silent epiallele [22]. In contrast to many other silent genes, silencing could only be released by simultaneous interference with methylation of DNA and histones. Six mutations from the same screen were mapped to the resistance gene itself. These cis-mutations provided the opportunity to study the nature and effect of DNA sequence changes on gene expression, chromatin organization, and genetic stability. We describe these new alleles in detail and compare them with the R and S epialleles. We show that different, and non-overlapping, sequence changes downstream of the HPT gene can restore the expression of the upstream promoter, to a similar extent as the mutations interfering with the chromatin factors in trans. Such small sequence alterations that cause epigenetic changes at some distance may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions/excisions, recombination events, or DNA repair processes and may thereby contribute to the origin of new epialleles. Results Epialleles Differ in Chromatin Features and Small RNA Abundance The HPT gene is inserted in an AT-rich intergenic region on Arabidopsis thaliana chromosome 3 [20]. Previous investigations, and published data from genome-wide screens for chromatin features [20,23–24], indicated that the genomic localization itself is unlikely to influence the epigenetic state of the HPT gene, as no prominent epigenetic modifications are present in the neighbor- hood of the insertion. Resistant and sensitive Arabidopsis lines with the different epialleles had been generated from the same progenitor line homozygous for the HPT gene, thereby being supposedly isogenic. Nevertheless, the lack of transcription initiation in the hygromycin-sensitive lines could have been due to a DNA sequence mutation in a regulatory region, for example, a transcription factor binding site. Also, the structure of the insert had not been analyzed in detail. Therefore, active and inactive versions were amplified from genomic DNA of the respective lines. Both epialleles are potentially fully functional and have identical sequences. The 35S promoter (P1) is flanked upstream by a 661 bp fragment derived from the plasmid vector (V1). A rearrangement between two vector molecules prior to, or during, the integration Genetic Rearrangements Modify Nearby Chromatin of the transgene into the plant genome caused a duplication of the adjacent vector sequence (V2) and the 35S promoter (P2), resulting in two tandem repeats (Figure 1A). The polyadenylation signal from the CaMV 35S terminator following the HPT ORF lacks 151 bp compared to the transformation construct and has therefore lost its termination function (DT), causing read through of the P1 transcript into the flanking plant genome sequence (Figure 1A). P2 is followed by a 505 bp non-protein coding fragment (NC) harboring sequences of bovine carrier DNA used to assist PEG-mediated direct gene transfer to mesophyll protoplasts [25], interspersed with 54 nucleotides without homology to known sequences. This heterologous DNA is transcribed by P2, giving rise to a smaller non-coding transcript (P2 transcript) (Figure 1A). Resistant plants produce the longer P1 and the shorter P2 transcripts, while both promoters are inactive in sensitive plants (Figure 1B and Figure S6). Therefore, the isogenic inserts differ only by gene expression, and R and S represent true epialleles. The different expression states were suspected to originate from distinct chromatin configuration, and previous studies had provided evidence for opposing DNA methylation at the epialleles, especially pronounced at the transcription factor binding sites ([20–21], Figure 1C). As DNA methylation and silencing are usually correlated with specific changes of the DNA-associated proteins, we investigated histone modifications and nucleosome occupancy at the epialleles by chromatin immunoprecipitation. This revealed significant differences between the epialleles along the whole transgenic insert. While expressing lines (R) were primarily marked by trimethylation of histone H3 at lysine residue 4 (H3K4me3), typically enriched in euchromatic regions, epialleles in silenced lines (S) have nucleosomes with a modification characteristic of heterochromatin, namely dimethylated lysines at position 9 (H3K9me2) (Figure 1D). These marks, also including low levels of H3 dimethylated at position 27 (H3K27me2), only extend a short distance from the transgene into the flanking plant DNA (Figure S2), indicating limited spreading in transcriptional direction. Beside the specific modifications, we also observed an overall reduced association with H3 in line R compared to S (Figure 1E), probably rendering the promoters more accessible for the transcription machinery. While the epialleles clearly differed in their local chromatin configuration, this did not have any effect on their nuclear localization (Figure S3). Both epialleles were stably inherited over a minimum of eight generations of self-pollination, without any evidence for spontane- ous switches in the germ line. To also study the stability of epialleles in undifferentiated cells, we initiated callus cultures, starting with cotyledons of resistant, sensitive, and non-transgenic plants, and propagated the calli for up to six months under non-selective conditions. We screened callus tissue at several time points for its ability to grow under hygromycin selection for up to 5 weeks. Calli derived from R lines were resistant whereas calli obtained from S or non-transgenic lines died on selection plates. We also determined chromatin modifications and DNA methylation in callus tissue grown on non-selective medium, with results comparable to those of leaf tissue (Figure S4). This demonstrates similar states and stable maintenance of epialleles even upon dedifferentiation. We screened for the involvement of antisense and/or small RNAs in silencing maintenance. Significant promoter activity of the NC region was excluded (Figure S5A), and specific antisense RNA in line S could also not be detected, neither by northern blotting (Figure S5B) nor by RT-PCR (data not shown). Nevertheless, we generated libraries from size-fractionated 19 nt to 26 nt RNAs prepared from flower buds of plants containing either the sensitive or resistant epiallele. Both libraries were sequenced (Table S1) and the reads screened for alignment with PLoS Genetics | www.plosgenetics.org 2 October 2011 | Volume 7 | Issue 10 | e1002331