Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome
- Andrzej T. Wierzbicki1,8,
- Ross Cocklin2,3,8,
- Anoop Mayampurath4,8,
- Ryan Lister5,9,
- M. Jordan Rowley1,
- Brian D. Gregory5,10,
- Joseph R. Ecker5,6,
- Haixu Tang4 and
- Craig S. Pikaard2,3,7,11
- 1Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA;
- 2Department of Biology,
- 3Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, USA;
- 4School of Informatics and Computing, Indiana University, Bloomington, Indiana 47405, USA;
- 5Salk Institute, La Jolla, California 92037, USA;
- 6Howard Hughes Medical Institute, Salk Institute, La Jolla, California 92037, USA,
- 7Howard Hughes Medical Institute, Indiana University, Bloomington, Indiana 47405, USA
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↵8 These authors contributed equally to this work.
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
- DNA-dependent RNA polymerase
- gene silencing
- DNA methylation
- epigenetics
- short interfering RNA
- RNA-directed DNA methylation
Footnotes
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↵11 Corresponding author
E-mail cpikaard{at}indiana.edu
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Supplemental material is available for this article.
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Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.197772.112.
- Received June 2, 2012.
- Accepted July 2, 2012.
- Copyright © 2012 by Cold Spring Harbor Laboratory Press
Freely available online through the Genes & Development Open Access option.