Genome-Wide Association of Histone H3 Lysine Nine Methylation with CHG DNA Methylation in Arabidopsis thaliana Yana V. Bernatavichute1,2, Xiaoyu Zhang3, Shawn Cokus2, Matteo Pellegrini2*, Steven E. Jacobsen1,2,4* 1 Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America, 2 Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America, 3 Department of Plant Biology, University of Georgia, Athens, Georgia, United States of America, 4 Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, California, United States of America Abstract Methylation of histone H3 lysine 9 (H3K9) is a hallmark of transcriptional silencing in many organisms. In Arabidopsis thaliana, dimethylation of H3K9 (H3K9m2) is important in the silencing of transposons and in the control of DNA methylation. We constructed a high-resolution genome-wide map of H3K9m2 methylation by using chromatin immunoprecipitation coupled with whole genome Roche Nimblegen microarrays (ChIP-chip). We observed a very high coincidence between H3K9m2 and CHG methylation (where H is either A,T or C) throughout the genome. The coding regions of genes that are associated exclusively with methylation in a CG context did not contain H3K9m2. In addition, we observed two distinct patterns of H3K9m2. Transposons and other repeat elements present in the euchromatic arms contained small islands of H3K9m2 present at relatively low levels. In contrast, pericentromeric/centromeric regions of Arabidopsis chromosomes contained long, rarely interrupted blocks of H3K9m2 present at much higher average levels than seen in the chromosome arms. These results suggest a complex interplay between H3K9m2 and different types of DNA methylation and suggest that distinct mechanisms control H3K9m2 in different compartments of the genome. Citation: Bernatavichute YV, Zhang X, Cokus S, Pellegrini M, Jacobsen SE (2008) Genome-Wide Association of Histone H3 Lysine Nine Methylation with CHG DNA Methylation in Arabidopsis thaliana. PLoS ONE 3(9): e3156. doi:10.1371/journal.pone.0003156 Editor: Brian P. Dilkes, University of California Davis, United States of America Received May 22, 2008; Accepted July 24, 2008; Published September 8, 2008 Copyright: 2008 Bernatavichute 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: Y.V.B. was supported by USPHS National Research Sevice Award GM07104. X.Z. was supported by a fellowship from the Jonsson Cancer Center Foundation. Jacobsen lab research was supported by NIH grant GM60398. S.E.J. is an investigator of the Howard Hughes Medical Institute. Competing Interests: The authors have declared that no competing interests exist. * E-mail: matteop@mcdb.ucla.edu (MP); jacobsen@ucla.edu (SEJ) Introduction Multiple mechanisms control the formation and maintenance of eukaryotic heterochromatin. One important conserved mecha- nism is the presence of particular post-translational modifications of histones, such as methylation. Histones can be mono, di or tri methylated (m, m2, or m3) and each methylation state can be controlled by different histone methyltransferase enzymes and can be associated with different outcomes [1]. For example, mamma- lian histones modified with tri-methylation at lysine 9 of histone H3 (H3K9m3) are found in regions of silenced chromatin [2,3]. H3K9m3 is associated with pericentromeric heterochromatin and crucial for proper mammalian development, as mutants in H3K9m3 histone methyltransferases, Suv39h1 and Suv39h2, display genomic instability and impaired viability [3–5]. In Arabidopsis, di-methylation of histone H3 lysine 9 (H3K9m2) is associated with heterochromatin formation, while H3K9m3 is found throughout euchromatin [6]. Several histone methyltrans- ferases are responsible for the propagation of H3K9m2, including KRYPTONITE/SUVH4 (KYP), SUVH5 and SUVH6 [7–10]. In addition to eliminating H3K9m2, kyp suvh5 suvh6 triple mutants also reduce DNA methylation and lead to the loss of silent epigenetic states of heterochromatin as observed by transposon reactivation [9]. DNA methylation in Arabidopsis is present in three DNA sequence contexts: CG, CHH and CHG (where H = A, T, or C). The initial establishment of methylation (de novo methylation) in all three sequence contexts requires the DOMAINS REARRANGED METHYLASE 2 DNA methyltransferase (DRM2) [11]. DRM2 appears to be guided by 24 nucleotide small interfering RNAs (siRNAs), because the establishment of methylation is blocked by mutations in several RNA silencing genes that control siRNA biogenesis or utilization including ARGONAUTE4 (AGO4), RNA DEPENDENT RNA POLYMERASE2 (RDR2), DICER-LIKE3 (DCL3), RNA POLYMERASE IVa (NRPD1a), RNA POLYMERASE IVb (NRPD1b), and DRD1 [12–19]. The mechanisms involved in the maintenance of DNA methylation are different depending on the sequence context of the cytosine [20,21]. CG methylation is maintained by the DNA methyltransferase MET1, which is a homolog of mammalian Dnmt1 [20,22–27], and by an accessory factor called UHRF1 in mammals or VIM/ORTH in Arabidopsis [28–31]. CHH methylation is maintained by mechanisms very similar to that of de novo methylation, because drm2 and the same suite of RNA silencing mutants cause losses of CHH methylation [20]. However, the DNA methyltransferase CHROMOMETHYLASE3 (CMT3) also plays some role in CHH maintenance and acts redundantly with DRM2 at some loci [11]. CMT3 is also the main enzyme acting to maintain CHG methylation throughout the genome, however, at some loci DRM2 also plays an important role [11,32]. CHG DNA methylation was found to be linked to H3K9m2 when screens for mutations that reduce CHG DNA methylation uncovered mutations in the KYP locus encoding a histone H3 PLoS ONE | www.plosone.org 1 September 2008 | Volume 3 | Issue 9 | e3156