Genome Wide Analysis of DNA Methylation

Summary

Principal Investigator: Joseph Ecker
Affiliation: The Salk Institute
Country: USA
Abstract: This proposal aims to identify functional, non-coding elements in eukaryotic genomes by surveying genome-wide DNA methylation. Cytosine DNA methylation controls developmental gene expression in mammals during genomic imprinting and X-chromosome inactivation, and also silences transposons and other repeated DNA sequences. Hypermethylation of specific tumor suppressor genes contributes to cancer, showing the relevance of this work to human health. Here we propose using whole-genome microarrays (WGAs) to find all methylated sequences in a eukaryotic genome; the "methylome". We will test our methods with the model plant Arabidopsis thaliana, demonstrating their usefulness for the human genome. Arabidopsis is ideal for studying DNA methylation, because it has facile genetics, a small genome, and orthologs of every human DNA methyltransferase. In contrast to the lethality of mouse DNA methyltransferase mutants, Arabidopsis can tolerate mutations that virtually eliminate methylation. Our Arabidopsis thaliana arrays tile the complete genome with 25-mer oligonucleotides, allowing us to precisely measure DNA and RNA hybridization. We will use several different reagents to identify methylated DNA sequences, each coupled with high-throughput analysis using WGAs: bisulfite treatment of genomic DNA, restriction digest with methylation-sensitive enzymes, anti-methylcytosine antibodies, and proteins that bind specifically to methylated DNA. By using complementary and independent methods, we hope to detect DNA methylation with up to single nucleotide resolution. Arabidopsis DNA methyltransferase mutants have well-characterized DNA methylation defects at several endogenous loci. We will use mutants in every major Arabidopsis DNA methyltransferase to test and verify DNA methylation detection methods. RNA silencing mutants will determine how much DNA methylation is guided by small intefering RNAs (siRNAs). To further define functional non-coding elements in the genome, we correlate changes in DNA methylation with transcription and with the presence of antisense gene transcripts, and other non-coding RNAs. Once we have identified all methylated loci, we will use novel sequence libraries to classify them as transposons, other repeats, or unique sequences. This analysis is likely to reveal functional non-coding elements that are invisible to other methods. Since methylation is critical in many mammalian gene regulation phenomena, the methods developed in this proposal will clearly move the ENCODE project toward its goal of identifying functional elements in the human genome.
Funding Period: 2004-09-29 - 2007-06-30
more information: NIH RePORT

Top Publications

  1. ncbi Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis
    Xiaoyu Zhang
    Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA
    Cell 126:1189-201. 2006
  2. ncbi Role of RNA polymerase IV in plant small RNA metabolism
    Xiaoyu Zhang
    Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA
    Proc Natl Acad Sci U S A 104:4536-41. 2007
  3. ncbi Genetic analyses of DNA methyltransferases in Arabidopsis thaliana
    X Zhang
    Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California 90095, USA
    Cold Spring Harb Symp Quant Biol 71:439-47. 2006
  4. ncbi Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis
    Xiaoyu Zhang
    Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America
    PLoS Biol 5:e129. 2007
  5. ncbi The Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation
    Xiaoyu Zhang
    Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California 90095, USA
    Nat Struct Mol Biol 14:869-71. 2007
  6. ncbi Mapping the genome landscape using tiling array technology
    Junshi Yazaki
    The Salk Institute for Biological Studies, Plant Molecular and Cellular, Laboratory 10010 North Torrey Pines Road La Jolla, CA 92037, USA
    Curr Opin Plant Biol 10:534-42. 2007
  7. ncbi Highly integrated single-base resolution maps of the epigenome in Arabidopsis
    Ryan Lister
    Plant Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
    Cell 133:523-36. 2008
  8. ncbi Finding the fifth base: genome-wide sequencing of cytosine methylation
    Ryan Lister
    Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA
    Genome Res 19:959-66. 2009

Detail Information

Publications8

  1. ncbi Genome-wide high-resolution mapping and functional analysis of DNA methylation in arabidopsis
    Xiaoyu Zhang
    Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA
    Cell 126:1189-201. 2006
    ....
  2. ncbi Role of RNA polymerase IV in plant small RNA metabolism
    Xiaoyu Zhang
    Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA
    Proc Natl Acad Sci U S A 104:4536-41. 2007
    ..We also describe a class of RNAP IV-independent siRNAs produced from endogenous single-stranded hairpin RNA precursors...
  3. ncbi Genetic analyses of DNA methyltransferases in Arabidopsis thaliana
    X Zhang
    Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California 90095, USA
    Cold Spring Harb Symp Quant Biol 71:439-47. 2006
    ....
  4. ncbi Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis
    Xiaoyu Zhang
    Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America
    PLoS Biol 5:e129. 2007
    ..The results suggest that different mechanisms may underlie the establishment and spreading of H3K27me3 in plants and animals...
  5. ncbi The Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation
    Xiaoyu Zhang
    Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California 90095, USA
    Nat Struct Mol Biol 14:869-71. 2007
    ..The LHP1 chromodomain also binds H3K27me3 with high affinity, suggesting that LHP1 has functions similar to those of Polycomb...
  6. ncbi Mapping the genome landscape using tiling array technology
    Junshi Yazaki
    The Salk Institute for Biological Studies, Plant Molecular and Cellular, Laboratory 10010 North Torrey Pines Road La Jolla, CA 92037, USA
    Curr Opin Plant Biol 10:534-42. 2007
    ..Further capture and integration of additional types of genome-wide data sets will help to illuminate additional hidden features of the dynamic genomic landscape that are regulated by both genetic and epigenetic pathways in plants...
  7. ncbi Highly integrated single-base resolution maps of the epigenome in Arabidopsis
    Ryan Lister
    Plant Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
    Cell 133:523-36. 2008
    ..Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state...
  8. ncbi Finding the fifth base: genome-wide sequencing of cytosine methylation
    Ryan Lister
    Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA
    Genome Res 19:959-66. 2009
    ..Advanced "next-generation" DNA sequencing technologies are now enabling the global mapping of this epigenetic modification at single-base resolution, providing new insights into the regulation and dynamics of DNA methylation in genomes...