Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome

  1. Craig S. Pikaard2,3,7,11
  1. 1Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA;
  2. 2Department of Biology,
  3. 3Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, USA;
  4. 4School of Informatics and Computing, Indiana University, Bloomington, Indiana 47405, USA;
  5. 5Salk Institute, La Jolla, California 92037, USA;
  6. 6Howard Hughes Medical Institute, Salk Institute, La Jolla, California 92037, USA,
  7. 7Howard Hughes Medical Institute, Indiana University, Bloomington, Indiana 47405, USA
    1. 8 These authors contributed equally to this work.

    • Present addresses: 9The University of Western Australia, Perth 6009, Australia;

    • 10 University of Pennsylvania, Philadelphia, PA 19104, USA.

    Abstract

    Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%–60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.

    Keywords

    Footnotes

    • Received June 2, 2012.
    • Accepted July 2, 2012.

    Freely available online through the Genes & Development Open Access option.

    Related Article

    | Table of Contents
    OPEN ACCESS ARTICLE

    Life Science Alliance