Published online 17 May 2011 Nucleic Acids Research, 2011, Vol. 39, No. 16 6919–6931 doi:10.1093/nar/gkr324 Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis Ikhlak Ahmed 1 , Alexis Sarazin1, Chris Bowler1, Vincent Colot1,* and Hadi Quesneville2 1Institut de Biologie de l’Ecole Normale Supe´ rieure (IBENS), Centre National de la Recherche Scientifique (CNRS) UMR8197 - Institut National de la Sante´ et de la Recherche Me´ dicale (INSERM) U1024, 46 rue d’Ulm, 75230 Paris cedex 05 and 2Unite´ de Recherches en Ge´ nomique-Info, Institut National de la Recherche Agronomique (INRA) UR1164, Centre de recherche de Versailles, Route de Saint Cyr, 78026 Versailles cedex, France Received December 22, 2010; Revised March 31, 2011; Accepted April 20, 2011 ABSTRACT Transposable elements (TEs) and their relics play major roles in genome evolution. However, mobil- ization of TEs is usually deleterious and strongly re- pressed. In plants and mammals, this repression is typically associated with DNA methylation, but the relationship between this epigenetic mark and TE sequences has not been investigated systematic- ally. Here, we present an improved annotation of TE sequences and use it to analyze genome-wide DNA methylation maps obtained at single- nucleotide resolution in Arabidopsis. We show that although the majority of TE sequences are methyl- ated, 26% are not. Moreover, a significant fraction of TE sequences densely methylated at CG, CHG and CHH sites (where H = A, T or C) have no or few matching small interfering RNA (siRNAs) and are therefore unlikely to be targeted by the RNA- directed DNA methylation (RdDM) machinery. We provide evidence that these TE sequences acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, we show that although both methylated and unmethylated TE se- quences located in euchromatin tend to be more abundant closer to genes, this trend is least pronounced for methylated, siRNA-targeted TE se- quences located 50 to genes. Based on these and other findings, we propose that spreading of DNA methylation through promoter regions explains at least in part the negative impact of siRNA-targeted TE sequences on neighboring gene expression. INTRODUCTION Transposable elements (TEs) are ubiquitous components of genomes and their differential accumulation is respon- sible for most of the large variations in genome size seen among eukaryotes. However, mobilization of TEs is inher- ently mutagenic and is therefore a rare event. Repression of transposition involves a variety of mechanisms, including DNA methylation in plants and mammals (1,2). Moreover, TEs are among the fastest evolving sequences, leading over time to the accumulation of degenerate, non-mobile relics. In plants, TE sequences are typically methylated at CG, CHG and CHH sites (where H = A, T or C) in a process that requires numerous factors, including small interfering RNAs (siRNAs) to guide methylation of homologous DNA sequences, and so called de novo and maintenance DNA methyltransferases (2,3). The model plant Arabidopsis offers several advantages for the detailed ex- ploration of the relationship between DNA methylation and TE sequences, such as a small, almost fully sequenced genome (4) and a large collection of mutants affected in the establishment, maintenance or removal of DNA methylation (3). However, despite the fact that DNA methylation in Arabidopsis has been studied genome wide using a variety of approaches, including bisulphite treatment of genomic DNA followed by whole genome sequencing (5,6), patterns of DNA methylation associated with Arabidopsis TE sequences have not been investigated systematically so far. We previously described the development of a highly sensitive TE annotation pipeline that doubled the fraction of the Arabidopsis genome detected as TE sequences compared to the initial annotation (7). In the present study, we have reﬁned this pipeline further and have used the resulting set of annotated TE sequences, *To whom correspondence should be addressed. Tel: +33 1 44 32 35 38; Fax: +33 1 44 32 39 35; Email: colot@biologie.ens.fr The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.