Genetic Rearrangements Can Modify Chromatin Features at Epialleles Andrea M. Foerster1, Huy Q. Dinh1,2, Laura Sedman1, Bonnie Wohlrab 1 , Ortrun Mittelsten Scheid1* 1 Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna, Austria, 2 Center for Integrative Bioinformatics Vienna, Max F. Perutz Laboratories (MFPL), Vienna, Austria Abstract Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence- identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non- overlapping deletions or rearrangements in the 39 regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles. Citation: Foerster AM, Dinh HQ, Sedman L, Wohlrab B, Mittelsten Scheid O (2011) Genetic Rearrangements Can Modify Chromatin Features at Epialleles. PLoS Genet 7(10): e1002331. doi:10.1371/journal.pgen.1002331 Editor: Tetsuji Kakutani, National Institute of Genetics, Japan Received May 13, 2011; Accepted August 18, 2011; Published October 20, 2011 Copyright: 2011 Foerster et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Austrian Science Fund (FWF P18986-B17) and the EU Network of Excellence ‘‘Epigenome’’ to OMS and from Wiener Wissenschafts-, Forschungs- und Technologie Fonds (WWTF) to A. von Haeseler, co-supervisor of HQD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ortrun.mittelsten_scheid@gmi.oeaw.ac.at Introduction Epialleles are heritable states of different gene expression from sequence-identical genes and have been described in several organisms [1–3]. Like genetically different alleles, epialleles contribute to phenotypic heterogeneity [4–5]. While the muta- genic processes creating DNA sequence allele variations are relatively well understood, little is known about how and when epialleles originate, and it is difficult to investigate this in statu nascendi. In plants, epialleles were described as natural variants [6– 9], mutation-induced [10–12], or associated with tissue-culture [13–15]. Once established, epialleles can acquire stability over many generations; however, they have much higher reversion rates than genetic alleles. Therefore, analyzing the switch from one epigenetic state to the other at well-characterized epialleles can provide insight into their natural origin. Pairs of epialleles are characterized by antithetic histone modifications at the associated nucleosomes, transcriptional activity at the expressed form, and transcriptional gene silencing (TGS) at the other. In some fungi, mammals, and higher plants, the latter is connected with cytosine methylation at the epiallele [e.g. 6,16–17]. Several pairs of epialleles in plants define easily scored phenotypes like morphology [6,10], development [9], pigmentation [7,18], or reporter gene expression [19–20]. Some epialleles, as well as many other epigenetically controlled genes, have been used for mutant screens and have helped identify many different proteins and RNAs whose presence or absence can cause transient or stable changes of epiallele expression, or influence epigenetic regulation in general. There is also a wealth of data on the influence of drug treatments, sequence determinants, and the role of genomic neighborhood, on epigenetic regulation. Arabidopsis thaliana has been the plant model of choice for genetic analysis of switching between epiallelic states, based on the rich genetic and genomic resources available. The experimental system in our study is based on a pair of epialleles in Arabidopsis thaliana containing either an expressed or silent hygromycin phospho- transferase gene (HPT). Active transcription confers resistance to the antibiotic while the inactive epiallele renders the plant sensitive. Gene expression can be selected for on antibiotic- containing medium but does not affect the plants during non- selective growth. The epialleles were found in tetraploid plants obtained by regeneration from protoplasts [20]. While some lines had resistant progeny and expressed the HPT gene, other lines had silenced the HPT and produced only sensitive progeny. The R and S epialleles (determining resistance and sensitivity on hygromycin, respectively) were maintained in their particular expression state after diploidization and for all generations of self-pollination analyzed so far (Figure S1). Beside their differences in transcrip- PLoS Genetics | www.plosgenetics.org 1 October 2011 | Volume 7 | Issue 10 | e1002331