Cytosine DNA methylation is important in regulating gene expression and in silencing transposons and other repetitive sequences 1, 2 . Recent genomic studies in Arabidopsis have revealed that many endogenous genes are methylated either within their promoters or within their transcribed regions, and that gene methylation is highly correlated with transcription levels 3-5 . However, plants have different types of methylation controlled by different genetic pathways, and detailed information on the methylation status of each cytosine in any given genome is lacking. To this end, we generated a map at single base pair resolution of methylated cytosines for Arabidopsis, by combining bisulfite treatment of genomic DNA with ultra-high-throughput sequencing using the Illumina 1G Genome Analyzer and Solexa sequencing technology 6 . This approach, termed BS-Seq, unlike previous microarray-based methods, allows one to sensitively measure cytosine methylation on a genomewide scale within specific sequence contexts. We describe methylation on previously inaccessible components of the genome along with an analysis of the DNA methylation sequence composition and distribution. We also describe the effect of various DNA methylation mutants on genome-wide methylation patterns, and demonstrate that our newly developed library construction and computational methods can be applied to large genomes such as mouse.To generate a DNA methylation map at one nucleotide resolution across the genome, we adapted the Illumina 1G Genome Analyzer using Solexa sequencing technology (Illumina GA) for shotgun sequencing of bisulfite-treated Arabidopsis genomic DNA. Sodium bisulfite converts unmethylated cytosines to uracils, but 5-methylcytosines remain unconverted. Hence, Author Information. Reprints and permissions information is available at www.nature.com/reprints. The authors declare competing financial interests: details accompany the full-text HTML version of the paper at www.nature.com/nature. Correspondence and requests for materials should be addressed to S.E.J. (jacobsen@ucla.edu) or M.P. (matteop@mcdb.ucla.edu). 6 These authors contributed equally to this work. 7 Present address: Department of Plant Biology, University of Georgia, Athens, Georgia 30602, USA. Author Contributions. S.J.C. developed computational methods for mapping and basecalling. S.F. designed and created DNA libraries and performed all molecular biology experiments. S.F., Z.C., B.M., and S.F.N. sequenced libraries. M.P., S.J.C., S.F., and S.E.J. analyzed data. S.E.J. and M.P. designed and directed the study. X.Z., C.D.H., and S.P. assisted in the design of experiments. S.F. and S.J.C. wrote the manuscript. HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript after polymerase chain reaction amplification, unmethylated cytosines appear as thymines and methylated cytosines appear as cytosines 7 . We created genomic DNA libraries after bisulfite conversion and produced ~3.8 billion nucleotides of high quality sequence which successfully mapped to the...

Cytosine methylation is important for transposon silencing and epigenetic regulation of endogenous genes, although the extent to which this DNA modification functions to regulate the genome is still unknown. Here we report the first comprehensive DNA methylation map of an entire genome, at 35 base pair resolution, using the flowering plant Arabidopsis thaliana as a model. We find that pericentromeric heterochromatin, repetitive sequences, and regions producing small interfering RNAs are heavily methylated. Unexpectedly, over one-third of expressed genes contain methylation within transcribed regions, whereas only approximately 5% of genes show methylation within promoter regions. Interestingly, genes methylated in transcribed regions are highly expressed and constitutively active, whereas promoter-methylated genes show a greater degree of tissue-specific expression. Whole-genome tiling-array transcriptional profiling of DNA methyltransferase null mutants identified hundreds of genes and intergenic noncoding RNAs with altered expression levels, many of which may be epigenetically controlled by DNA methylation.

Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes. We found that patterns of methylation are very similar in flowering plants with methylated cytosines detected in all sequence contexts, whereas CG methylation predominates in animals. Vertebrates have methylation throughout the genome except for CpG islands. Gene body methylation is conserved with clear preference for exons in most organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honey bee. Among the eight organisms, the green alga Chlamydomonas has the most unusual pattern of methylation, having non-CG methylation enriched in exons of genes rather than in repeats and transposons. In addition, the Dnmt1 cofactor Uhrf1 has a conserved function in maintaining CG methylation in both transposons and gene bodies in the mouse, Arabidopsis, and zebrafish genomes.BS-Seq | epigenetic profiling | DNA methylation | gene body methylation | UHRF1C ytosine DNA methylation is an epigenetic mark important in many gene regulatory systems, including genomic imprinting, X-chromosome inactivation, silencing of transposons and other repetitive DNA sequences, as well as expression of endogenous genes. Methylation is conserved in most major eukaryotic groups, including many plants, animals, and fungi, although it has been lost from certain model organisms such as the budding yeast Saccharomyces cerevisiae and nematode worm Caenorhabditis elegans (1-3). DNA methylation can be categorized into three types according to the sequence context of the cytosines, namely CG, CHG, and CHH (H = A, C, or T). CG methylation is maintained by conserved Dnmt1 DNA methyltransferase enzymes. CHH methylation, and, to some extent CHG methylation, is generally maintained by the activity of the conserved Dnmt3 methyltransferases, whereas high levels of CHG methylation seen in the model plant Arabidopsis are maintained by the plant-specific methyltransferase CMT3 (2, 3). Generally speaking, DNA methylation is thought to occur "globally" in vertebrates, with CG sites being heavily methylated genome-wide except for those in CpG islands, whereas invertebrates, plants, and fungi have "mosaic" methylation, characterized by interspersed methylated and unmethylated domains (4). These differences are an interesting starting point for studying divergence in methylation pathways and regulatory mechanisms; however, determining precise genomescale methylation patterns has been a challenge for complex genomes until the recent development of high-throughput sequencing technology. In this paper, we generated shotgun bisulfite sequencing data to profile DNA methylation in eight eukaryotic organisms. These organisms display wide variations in methylati...

Trimethylation of histone H3 lysine 27 (H3K27me3) plays critical roles in regulating animal development, and in several cases, H3K27me3 is also required for the proper expression of developmentally important genes in plants. However, the extent to which H3K27me3 regulates plant genes on a genome-wide scale remains unknown. In addition, it is not clear whether the establishment and spreading of H3K27me3 occur through the same mechanisms in plants and animals. We identified regions containing H3K27me3 in the genome of the flowering plant Arabidopsis thaliana using a high-density whole-genome tiling microarray. The results suggest that H3K27me3 is a major silencing mechanism in plants that regulates an unexpectedly large number of genes in Arabidopsis (~4,400), and that the maintenance of H3K27me3 is largely independent of other epigenetic pathways, such as DNA methylation or RNA interference. Unlike in animals, where H3K27m3 occupies large genomic regions, in Arabidopsis, we found that H3K27m3 domains were largely restricted to the transcribed regions of single genes. Furthermore, unlike in animals systems, H3K27m3 domains were not preferentially associated with low–nucleosome density regions. The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals.

Epigenetic reprogramming including demethylation of DNA occurs in mammalian primordial germ cells (PGCs) and in early embryos, and is important for the erasure of imprints and epimutations, and the return to pluripotency [1][2][3][4][5][6][7][8][9] . The extent of this reprogramming and its molecular mechanisms are poorly understood. We previously showed that the cytidine deaminases Aid and Apobec1 can deaminate 5-methylcytosine in vitro and in E coli, and in the mouse are expressed in tissues in which demethylation occurs 10 . Here we profiled DNA methylation throughout the genome by unbiased bisulfite Next Generation Sequencing 11-13 (BS-Seq) in wildtype and Aid deficient PGCs at E13.5. Wildtype PGCs revealed dramatic genome-wide erasure of methylation to a level below that of methylation deficient (Np95-/-) ES cells, with female PGCs being less methylated than male ones. By contrast, Aid deficient PGCs were up to three times more methylated than wildtype ones; this substantial difference occurred throughout the genome, with introns, intergenic regions and transposons being relatively more methylated than exons. Relative hypermethylation in Aid deficient PGCs was confirmed by analysis of individual loci in the genome. Our results reveal that erasure of DNA methylation in the germ line is a global process, hence limiting the potential for transgenerational epigenetic inheritance. Aid deficiency interferes with genome-wide erasure of DNA methylation patterns, suggesting that Aid has a critical function in epigenetic reprogramming and potentially in restricting the inheritance of epimutations in mammals.In plants active demethylation occurs widely (including in imprinted genes) and is carried out by 5meC glycosylases such as Demeter and Demeter like proteins [14][15][16] . This class of enzymes does not appear to exist in mammalian genomes, but instead we found that the cytosine Correspondence and requests for materials should be addressed to S.E. J. (jacobsen@ucla.edu) or W.R. (wolf.reik@bbsrc.ac.uk). * These authors contributed equally to this work Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author contributions C.P. and W.D. isolated tissue samples and PGCs, assessed the purity of the samples, and prepared DNA. C.P. undertook genetic crosses and determined weights of mouse pups. S.F. constructed bisulfite libraries and did Illumina Solexa sequencing, S.J.C., S.A., and M.P. carried out mapping, base-calling, and computational analyses. C.P., W.D., S.F., S.J.C., S.A., M.P., S.E.J and W.R. analysed data. C.P., W.D., S.F., S.E.J and W.R. designed experiments; S.E.J and W.R. designed and directed the study. C.P. and W.R. wrote the manuscript. deaminases Aid and Apobec1 can deaminate 5meC both in vitro and in E coli 10 , suggesting deamination of 5meC followed by T:G base excision repair by glycosylases such as Tdg or Mbd4 as an equivalent pathway for demethylation of DNA. Recently it has been shown that co-expression of Aid and Mbd4 in Zebrafish embryos ca...