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

Genes Dev. 2012 Aug 15;26(16):1825-36. doi: 10.1101/gad.197772.112. Epub 2012 Aug 1.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Arabidopsis Proteins* / genetics
  • Arabidopsis Proteins* / metabolism
  • Arabidopsis* / genetics
  • Arabidopsis* / metabolism
  • Cytosine / metabolism*
  • DNA Methylation*
  • DNA-Directed RNA Polymerases* / genetics
  • DNA-Directed RNA Polymerases* / metabolism
  • Gene Expression Regulation, Plant
  • Genome, Plant*
  • Mutation
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism*


  • Arabidopsis Proteins
  • RNA, Small Interfering
  • Cytosine
  • RNA polymerase IV, Arabidopsis
  • RNA polymerase V, Arabidopsis
  • DNA-Directed RNA Polymerases