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 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...
Epigenetic inheritance in mammals relies in part on robust propagation of DNA methylation patterns throughout development. We show that the protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), also known as NP95 in mouse and ICBP90 in human, is required for maintaining DNA methylation. UHRF1 colocalizes with the maintenance DNA methyltransferase protein DNMT1 throughout S phase. UHRF1 appears to tether DNMT1 to chromatin through its direct interaction with DNMT1. Furthermore UHRF1 contains a methyl DNA binding domain, the SRA (SET and RING associated) domain, that shows strong preferential binding to hemimethylated CG sites, the physiological substrate for DNMT1. These data suggest that UHRF1 may help recruit DNMT1 to hemimethylated DNA to facilitate faithful maintenance of DNA methylation.
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