PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis - PubMed (original) (raw)

Comparative Study

. 2008 Feb 26;105(8):3145-50.

doi: 10.1073/pnas.0709632105. Epub 2008 Feb 19.

Affiliations

Comparative Study

PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis

Rebecca A Mosher et al. Proc Natl Acad Sci U S A. 2008.

Abstract

DNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

PolIVa is required for siRNA accumulation at most endogenous loci. (A) Representative Venn diagram of siRNA-generating loci. Solid line, loci with siRNAs in WT; dotted line, loci with siRNAs at WT or a greater level in nrpd1a; dashed line, loci with siRNAs present at WT or greater level in nrpd1b. (B and C) Pie charts depicting level of siRNA representation at loci requiring at least one PolIV isoform (gray region in A).

Fig. 2.

Fig. 2.

Genetic dependence of PolIV-dependent loci. siRNA Northern blots of mixed floral tissue confirm that all tested PolIV-dependent loci lose siRNAs in nrpd1a. They also require NRPD2, RDR2, and DCL3 for siRNA accumulation but show various degrees of dependence on NRPD1b. The size markers are estimates based on 20- and 30-nt RNA oligos.

Fig. 3.

Fig. 3.

PolIV-dependent loci exhibit differential association with transposable elements but no inherent repetitive character. (A) Over- or underrepresentation of genomic features at PolIV-independent, A type, and A+B type loci. The locus positions were randomized 100 times, and the associated genomic elements were counted to calculate Z scores: Z = (observed − averagerand)/SDrand. (B and C) Histograms of loci with respect to their genomic repetitiveness. Each locus was BLASTed against the Arabidopsis genome, and the resulting matches (E < 0.001) were counted and plotted.

Fig. 4.

Fig. 4.

PolIV-dependent loci are pericentromerically localized independent of transposable element character. Histograms of relative centromeric bias of sRNA-generating loci. 0, centromere; 1, telomere. The rolling average of the fraction of loci in three 1% windows is plotted. The short arms of chromosomes 2 and 4 were removed for clarity (33). Circles, triangles, and stars mark the first, second, and third quartiles, respectively.

Fig. 5.

Fig. 5.

RNA-directed DNA methylation at PolIVa-dependent loci. Bisulfite sequencing of PolIV-dependent loci 00687 (A), 08002 (B), 10102 (C), and 04138 (D). Data from two biological replicates were combined. Asterisks mark changes that were statistically significant (two-tailed t test, P < 0.01) in both replicates. The total number of clones analyzed is in parenthesis.

Fig. 6.

Fig. 6.

Model of PolIV dependency of siRNA-generating loci. PolIV-independent epigenetic marks (blue stars) recruit PolIVa (pink ovals) to generate siRNAs. In the presence of PolIVb, additional epigenetic marks (yellow triangles) can reinforce recruitment of PolIVa. Additionally, RDR2-mediated amplification of siRNAs can occur when direct repeats are present in the transcript, as in LTR retroelement RNAs.

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