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Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning


Cytosine DNA methylation is important in regulating gene expression and in silencing transposons and other repetitive sequences1,2. Recent genomic studies in Arabidopsis thaliana 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 levels3,4,5. 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 bisulphite treatment of genomic DNA with ultra-high-throughput sequencing using the Illumina 1G Genome Analyser and Solexa sequencing technology6. This approach, termed BS-Seq, unlike previous microarray-based methods, allows one to sensitively measure cytosine methylation on a genome-wide scale within specific sequence contexts. Here we describe methylation on previously inaccessible components of the genome and analyse 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 that of mouse.

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Figure 1: Methylation of different fractions of the Arabidopsis genome.
Figure 2: Sequence preferences for methylation in CG, CHG and CHH contexts.
Figure 3: Methylation shows periodic patterns.
Figure 4: BS-Seq profiling of methylation mutants in Arabidopsis and mouse.

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  1. Henderson, I. R. & Jacobsen, S. E. Epigenetic inheritance in plants. Nature 447, 418–424 (2007)

    Article  ADS  CAS  Google Scholar 

  2. Goll, M. G. & Bestor, T. H. Eukaryotic cytosine methyltransferases. Annu. Rev. Biochem. 74, 481–514 (2005)

    Article  CAS  Google Scholar 

  3. Zhang, X. et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126, 1189–1201 (2006)

    Article  CAS  Google Scholar 

  4. Zilberman, D., Gehring, M., Tran, R. K., Ballinger, T. & Henikoff, S. Genome-wide analysis of Arabidopsis thaliana DNA methylation uncovers an interdependence between methylation and transcription. Nature Genet. 39, 61–69 (2007)

    Article  CAS  Google Scholar 

  5. Vaughn, M. W. et al. Epigenetic natural variation in Arabidopsis thaliana. PLoS Biol. 5, e174 (2007)

    Article  Google Scholar 

  6. Bentley, D. R. Whole-genome re-sequencing. Curr. Opin. Genet. Dev. 16, 545–552 (2006)

    Article  CAS  Google Scholar 

  7. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl Acad. Sci. USA 89, 1827–1831 (1992)

    Article  ADS  CAS  Google Scholar 

  8. Ngernprasirtsiri, J., Kobayashi, H. & Akazawa, T. DNA methylation as a mechanism of transcriptional regulation in nonphotosynthetic plastids in plant cells. Proc. Natl Acad. Sci. USA 85, 4750–4754 (1988)

    Article  ADS  CAS  Google Scholar 

  9. Tran, R. K. et al. DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes. Curr. Biol. 15, 154–159 (2005)

    Article  CAS  Google Scholar 

  10. Gruenbaum, Y., Naveh-Many, T., Cedar, H. & Razin, A. Sequence specificity of methylation in higher plant DNA. Nature 292, 860–862 (1981)

    Article  ADS  CAS  Google Scholar 

  11. Meyer, P., Niedenhof, I. & ten Lohuis, M. Evidence for cytosine methylation of non-symmetrical sequences in transgenic Petunia hybrida. EMBO J. 13, 2084–2088 (1994)

    Article  CAS  Google Scholar 

  12. Cao, X. & Jacobsen, S. E. Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc. Natl Acad. Sci. USA 99 (Suppl 4). 16491–16498 (2002)

    Article  ADS  CAS  Google Scholar 

  13. Dieguez, M. J., Vaucheret, H., Paszkowski, J. & Mittelsten Scheid, O. Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance. Mol. Gen. Genet. 259, 207–215 (1998)

    Article  CAS  Google Scholar 

  14. Ramsahoye, B. H. et al. Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc. Natl Acad. Sci. USA 97, 5237–5242 (2000)

    Article  ADS  CAS  Google Scholar 

  15. Jia, D., Jurkowska, R. Z., Zhang, X., Jeltsch, A. & Cheng, X. Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature 449, 248–251 (2007)

    Article  ADS  CAS  Google Scholar 

  16. Cao, X. et al. Conserved plant genes with similarity to mammalian de novo DNA methyltransferases. Proc. Natl Acad. Sci. USA 97, 4979–4984 (2000)

    Article  ADS  CAS  Google Scholar 

  17. Bers, E. P., Singh, N. P., Pardonen, V. A., Lutova, L. A. & Zalensky, A. O. Nucleosomal structure and histone H1 subfractional composition of pea (Pisum sativum) root nodules, radicles and callus chromatin. Plant Mol. Biol. 20, 1089–1096 (1992)

    Article  CAS  Google Scholar 

  18. Vershinin, A. V. & Heslop-Harrison, J. S. Comparative analysis of the nucleosomal structure of rye, wheat and their relatives. Plant Mol. Biol. 36, 149–161 (1998)

    Article  CAS  Google Scholar 

  19. Fulnecek, J., Matyasek, R., Kovarik, A. & Bezdek, M. Mapping of 5-methylcytosine residues in Nicotiana tabacum 5S rRNA genes by genomic sequencing. Mol. Gen. Genet. 259, 133–141 (1998)

    Article  CAS  Google Scholar 

  20. Fan, Y. et al. Histone H1 depletion in mammals alters global chromatin structure but causes specific changes in gene regulation. Cell 123, 1199–1212 (2005)

    Article  CAS  Google Scholar 

  21. Zhang, X. & Jacobsen, S. E. Genetic analyses of DNA methyltransferases in Arabidopsis thaliana. Cold Spring Harb. Symp. Quant. Biol. 71, 439–447 (2006)

    Article  CAS  Google Scholar 

  22. Henderson, I. R. et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nature Genet. 38, 721–725 (2006)

    Article  CAS  Google Scholar 

  23. Bostick, M. et al. UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science 317, 1760–1764 (2007)

    Article  ADS  CAS  Google Scholar 

  24. Sharif, J. et al. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450, 908–912 (2007)

    Article  ADS  CAS  Google Scholar 

  25. Meissner, A. et al. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 33, 5868–5877 (2005)

    Article  CAS  Google Scholar 

  26. Rajagopalan, R., Vaucheret, H., Trejo, J. & Bartel, D. P. A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev. 20, 3407–3425 (2006)

    Article  CAS  Google Scholar 

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We thank Y. Bernatavichute for nuclear DNA isolation protocols, A. Clarke for providing embryonic stem cell DNA, A. Girard and G. Hannon for providing mouse germ cell DNA, J. Hetzel for technical assistance, and C. F. Li for assistance with rDNA annotation. S.F. is a Howard Hughes Medical Institute Fellow of the Life Science Research Foundation. X.Z. was supported by a fellowship from the Jonsson Cancer Center Foundation. S.E.J. is an investigator of the Howard Hughes Medical Institute. This work was supported in part by grants from the NSF Plant Genome Research Program and the NIH, and some aspects of the work were performed in the UCLA DNA Microarray Facility.

Author Contributions S.J.C. developed computational methods for mapping and base-calling. S.F. designed and created DNA libraries and performed all molecular biology experiments. S.F., Z.C., B.M. and S.F.N. sequenced the libraries. M.P., S.J.C., S.F. and S.E.J. analysed 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.

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Correspondence to Matteo Pellegrini or Steven E. Jacobsen.

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C. Haudenschild is an employee of Illumina Inc., the manufacturer of the high throughput sequencing system used in this work.

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Cokus, S., Feng, S., Zhang, X. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 (2008).

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