A protein complex required for polymerase V transcripts and RNA- directed DNA methylation in Arabidopsis

Abstract

DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM). Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3), we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 and DMS3, is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V.

Figure 1

Complementation of mutants with epitope tagged DRD1 and DMS3. Analysis of DNA methylation at the MEA-ISR locus by Southern blotting after digestion of genomic DNA with the methylation-sensitive restriction enzyme, MspI. Bands representing methylation (ME) or a lack of methylation (un ME) are indicated. Digestion of genomic DNA extracted from wild-type plants of the Colombia (Col) ecotype serve as a positive control for DNA methylation levels. Panel (A) shows the loss of DNA methylation in the drd1-6 mutant alone and the restoration of DNA methylation after transformation of this mutant with a transgene carrying the indicated Flag and Myc epitope tagged fusion of the DRD1 gene under the control of its endogenous promoter (pDRD1). Panel (B) shows the loss of DNA methylation in the dms3-4 mutant alone and the restoration of DNA methylation after transformation of this mutant with a transgene carrying the indicated Flag epitope tagged fusion of the DMS3 gene under the control of its endogenous promoter (pDMS3). Complementation assays shown were conducted using tissue from T3 homozygous transgenic plant lines. BLRP, Biotin Ligase Recognition Peptide. See also Tables S2 and S3.

Figure 2

Characterization of DDR complex components. (A–D) Streptavidin (SA) pulldown and co-immunopurification assays confirming interactions from Mass Spectrometric analyses. The BLRP tag is biotinylated in vivo allowing interaction with streptavidin. Input lanes confirm expression of the epitope fusions proteins and the endogenous NRPE1 or RDM1 proteins in the parental lines indicated above each lane. F1 represents a cross between the two parental lines. Since these F1 lines only possess a single copy of each transgene, they exhibit lower expression levels as compared to the parental lines. SA pulldown lanes show co-purification of (A) DRD1 with DMS3, (B) DRD1 with RDM1 and (C) DRD1 with NRPE1 and Flag co-immunoprecipitation lanes show (D) DMS3 with RDM1. In (C), protein extracts from Col and nrpe1-12 plants are included to confirm the identity of the co-precipitating band. For each western blot, the antibody used is indicated (upper Left). See also Figure S1.

Figure 3

Gel filtration of co-purifying proteins. The elution profiles of NRPE1, RDM1, 9xMyc-DRD1, and DMS3-3xFlag-BLRP on a Superose6 column were detected using antibodies against endogenous NRPE1, endogenous RDM1 and either the Myc or Flag epitope, respectively. Fraction numbers and sizing standards are indicated. In fractions 62 and 64 nonspecific background bands are marked by an asterisk (*). See also Figure S2.

Figure 4

DNA methylation and IGN transcript defects in a ros1 rdm1 mutant. (A) Southern blot analysis as described in Figure 1 using DNA from wild type Col plants or from the indicated mutant plants. (B and C) Quantitative Reverse-Transcriptase PCR analysis of the abundance of Pol V-dependent transcripts corresponding to the (B) IGN5 and (C) MEA-ISR loci in the indicated genetic backgrounds after normalization to the level of an ACTIN transcript. Error bars represent the standard deviation among at least three biological replicas.