RNA-directed DNA methylation in Arabidopsis

Abstract

In plants, double-stranded RNA that is processed to short RNAs approximately 21-24 nt in length can trigger two types of epigenetic gene silencing. Posttranscriptional gene silencing, which is related to RNA interference in animals and quelling in fungi, involves targeted elimination of homologous mRNA in the cytoplasm. RNA-directed DNA methylation involves de novo methylation of almost all cytosine residues within a region of RNA-DNA sequence identity. RNA-directed DNA methylation is presumed to be responsible for the methylation observed in protein coding regions of posttranscriptionally silenced genes. Moreover, a type of transcriptional gene silencing and de novo methylation of homologous promoters in trans can occur if a double-stranded RNA contains promoter sequences. Although RNA-directed DNA methylation has been described so far only in plants, there is increasing evidence that RNA can also target genome modifications in other organisms. To understand how RNA directs methylation to identical DNA sequences and how changes in chromatin configuration contribute to initiating or maintaining DNA methylation induced by RNA, a promoter double-stranded RNA-mediated transcriptional gene silencing system has been established in Arabidopsis. A genetic analysis of this system is helping to unravel the relationships among RNA signals, DNA methylation, and chromatin structure.

Figure 1

Structures and methylation analysis of the silencer NOSpro IR (Upper) and target NOSpro–NPTII gene (Lower). NOSpro sequences are depicted as heavy black arrows. Enzymes and probes used for DNA blot analyses are indicated. Abbreviations: Pv, PvuII; D, DdeI; S, SacII; N, NheI; Hi, HindIII; E, EcoRI; B, BstUI; Ba, BamHI; P, PstI. To assess methylation in the target NOSpro, an E and P double digest was performed (the minus lanes in Figs. 4A and 6 A, C, E, and G) and one of several methylation-sensitive enzymes (B, D, S, N, Ba) was added. Methylation in the NOSpro IR was tested by digesting with Pv and Hi (the minus lanes in Figs. 4B and 6 B, D, F, and H), together with either B, D, S, or N. Filled, half-filled, and open circles, squares, and triangles (CG, CNG, and CNN, respectively) indicate >90%, ≈50%, and <10% cytosine (C) methylation, respectively. Open squares or triangles below each map for the enzymes D, N, and B indicate that the top and bottom DNA strands contain C residues in different sequence contexts (e.g., CG and CNG, or CNG and CNN). The NheI site (underlined) is in the sequence context: 5′-CAGCTACG^mCAA-3′ (top); and 3′-GT^mCGATCGTT-5′ (bottom).

Figure 2

RNA analysis. (A) RNase protection reveals the ≈0.3-kb NOSpro dsRNA transcribed from the silencer NOSpro IR. NT, normal untransformed plants. (B) Total RNA used in A probed with an actin probe from tobacco and an eIF-4A probe from Arabidopsis as loading controls. (C) Detection of NOSpro short RNAs (sense probe) produced by means of dsRNA cleavage. Identical results were obtained with an antisense probe. (D) Nuclear run-on analysis demonstrating transcriptional down-regulation of the NOSpro–NPTII target gene in the presence of the silencing locus, which encodes HPT and NOSpro dsRNA. A constitutively expressed ribulose 1,5-bisphosphate carboxylase (rubisco) gene was used as a control. Positive controls in A and C were prepared from tobacco plants transformed with the 35Spro–NOSproIR construct.

Figure 3

Phenotypic analysis of silencing. NOSpro–NPTII target gene expression is assayed by Kan^R; the silencing locus by Hyg^R. (A) Selfing a plant homozygous for an active target gene produces 100% Kan^R progeny. Selfing a plant homozygous for the target locus and hemizygous for the silencing locus, revealed by 75% Hyg^R, produces only 25% Kan^R progeny. Kan^R seedlings lack the silencer, indicated by 0% (Kan/Hyg)^R. (B) Mottled Kan^R seedling in the first generation after crossing out the silencing locus (Right is an enlargement of the boxed region in Left; white and green patches represent Kan^S and Kan^R regions, respectively). (C Left) Kan^S seedlings before removing the 35Spro with Cre recombinase. (Right) Kan^R seedlings two generations after removing the 35Spro. (D) Ranges of phenotypes on Kan-containing medium (plus signs, different degrees of Kan^R; minus sign, Kan^S) in seedlings after three generations of homozygosity for the ddm1 and met1 mutations, based on 5 plus signs for wild-type levels of Kan^R in seedlings containing the target locus in the unsilenced state.

Figure 4

Methylation analysis. (A) Target NOSpro. (B) Silencer NOSpro IR. (C) NOSpro dsRNA. (D) Centromeric repeats. Methylation of the target and silencing loci were analyzed by using the enzymes and probes described in Fig. 1. For met1, ddm1, and mom1 mutants, methylation was analyzed by using DNA isolated from plants that had been homozygous for the respective mutation for two generations. Methylation of centromeric repeats was analyzed by using HpaII (H: ^mC^mCGG) and MspI (M: ^mCCGG). The unmethylated control for the silencer NOSpro IR consisted of a λ genomic clone containing the silencing locus. Shifts to the smaller fragments indicate no methylation at a particular site. Arrows in A and B indicate position of methylated fragment; in C and D, arrows represent the sizes of the indicated features. n.d., not determined.

Figure 5

Bisulfite sequencing. The ≈300-bp NOSpro sequence is shown with the region of identity to NOSpro dsRNA underlined. Methylation (filled symbols) in 10 cloned PCR fragments from the upper DNA strand is indicated. Symbols are described in the Fig. 1 legend. The positions of restriction enzyme sites used in the DNA blot analyses are indicated (abbreviations are given in the Fig. 1 legend). The four boxed regions represent transcriptional regulatory elements (61), which contain short IRs (arrows). The transcription start site is indicated by the bent arrow at −1. The sequence of the primers used is indicated. Methylation does not infiltrate significantly into NPTII coding sequences.

Figure 6

Methylation analysis in the presence and absence of NOSpro dsRNA. (A, C, E, and G) Target NOSpro. (B, D, F, and H) Silencer NOSpro IR. The enzymes and probes used are described in Fig. 1. The arrows to the left of each blot show the position of the expected unmethylated fragment. Results from two lines containing the Cre-altered silencer are shown. (G) The NOSpro–NPTII bands of interest are flanked by blue dots. The large hybridizing fragment in the minus lanes and the fragment in the D lanes running just below the NOSpro–NPTII band should be disregarded as they are caused by a second NPTII gene (not visible in A, C, and E) used for bacterial selection during cloning (25). The size of the fragment in the minus lanes in B, D, F, and H is shifted after the Cre cross (indicated by red dots) because of removal of the 35Spro and is independent of methylation. Because of an unmethylated DdeI site in the 35Spro (Fig. 1 Upper), the size of the fragment of the unaltered silencer shifts after addition of DdeI (H).