A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation.

We used a transgene system to study spreading of RNA-directed DNA methylation (RdDM) during transcriptional gene silencing in Arabidopsis thaliana. Forward and reverse genetics approaches using this system delineated a stepwise pathway for the biogenesis of secondary siRNAs and unidirectional spreading of methylation from an upstream enhancer element into downstream sequences. Trans-acting, hairpin-derived primary siRNAs induce primary RdDM, independently of an enhancer-associated 'nascent' RNA, at the target enhancer region. Primary RdDM is a key step in the pathway because it attracts the secondary siRNA-generating machinery, including RNA polymerase IV, RNA-dependent RNA polymerase2 and Dicer-like3 (DCL3). These factors act in a turnover pathway involving a nascent RNA, which normally accumulates stably in non-silenced plants, to produce cis-acting secondary siRNAs that induce methylation in the downstream region. The identification of DCL3 in a forward genetic screen for silencing-defective mutants demonstrated a strict requirement for 24-nt siRNAs to direct methylation. A similar stepwise process for spreading of DNA methylation may occur in mammalian genomes, which are extensively transcribed in upstream regulatory regions.

RNA-directed DNA methylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing in plants.

RNA-directed DNA methylation, which is one of several RNAi-mediated pathways in the nucleus, has been highly elaborated in the plant kingdom. RNA-directed DNA methylation requires for the most part conventional DNA methyltransferases, histone modifying enzymes and RNAi proteins; however, several novel, plant-specific proteins that are essential for this process have been identified recently. DRD1 (defective in RNA-directed DNA methylation) is a putative SWI2/SNF2-like chromatin remodelling protein; DRD2 and DRD3 (renamed NRPD2a and NRPD1b, respectively) are subunits of Pol IVb, a putative RNA polymerase found only in plants. Interestingly, DRD1 and Pol IVb appear to be required not only for RNA-directed de novo methylation, but also for full erasure of methylation when the RNA trigger is withdrawn. These proteins thus have the potential to facilitate dynamic regulation of DNA methylation. Prominent targets of RNA-directed DNA methylation in the Arabidopsis thaliana genome include retrotransposon long terminal repeats (LTRs), which have bidirectional promoter/enhancer activities, and other types of intergenic transposons and repeats. Intergenic solitary LTRs that are targeted for reversible methylation by the DRD1/Pol IVb pathway can potentially act as switches or rheostats for neighboring plant genes. The resulting alterations in gene expression patterns may promote physiological flexibility and adaptation to the environment.

High frequency, cell type-specific visualization of fluorescent-tagged genomic sites in interphase and mitotic cells of living Arabidopsis plants.

BACKGROUND: Interphase chromosome organization and dynamics can be studied in living cells using fluorescent tagging techniques that exploit bacterial operator/repressor systems and auto-fluorescent proteins. A nuclear-localized Repressor Protein-Fluorescent Protein (RP-FP) fusion protein binds to operator repeats integrated as transgene arrays at defined locations in the genome. Under a fluorescence microscope, the tagged sites appear as bright fluorescent dots in living cells. This technique has been used successfully in plants, but is often hampered by low expression of genes encoding RP-FP fusion proteins, perhaps owing to one or more gene silencing mechanisms that are prevalent in plant cells. RESULTS: We used two approaches to overcome this problem. First, we tested mutations in four factors involved in different types of gene silencing and/or epigenetic modifications for their effects on nuclear fluorescence. Only mutations in DDM1, a chromatin remodelling ATPase involved in repeat-induced heterochromatin formation and DNA methylation, released silencing of the RP-FP fusion protein. This result suggested that the operator repeats can trigger silencing of the adjacent gene encoding the RP-FP fusion protein. In the second approach, we transformed the tagged lines with a second T-DNA encoding the RP-FP fusion protein but lacking operator repeats. This strategy avoided operator repeat-induced gene silencing and increased the number of interphase nuclei displaying fluorescent dots. In a further extension of the technique, we show that green fluorescent-tagged sites can be visualized on moving mitotic chromosomes stained with red fluorescent-labelled histone H2B. CONCLUSIONS: The results illustrate the propensity of operator repeat arrays to form heterochromatin that can silence the neighbouring gene encoding the RP-FP fusion protein. Supplying the RP-FP fusion protein in trans from a second T-DNA largely alleviates this problem. Depending on the promoter used to drive expression of the RP-FP fusion protein gene, the fluorescent tagged sites can be visualized at high frequency in different cell types. The ability to observe fluorescent dots on both interphase and mitotic chromosomes allows tagged sites to be tracked throughout the cell cycle. These improvements enhance the versatility of the fluorescent tagging technique for future studies of chromosome arrangement and dynamics in living plants.

Fluorescent transgenes to study interphase chromosomes in living plants.

Fluorescence tagging of genomic sites through the use of bacterial operator/repressor systems combined with fluorescent proteins permits high-resolution analysis of interphase chromosomes in living cells. This technique has been used to study interphase chromosome arrangement and dynamics in yeast, Drosophila, and mammalian cells, but is only beginning to be exploited in plant systems. In this chapter, we describe methods for producing and identifying Arabidopsis thaliana plants harbouring fluorescence-tagged transgenes. The use of these plants to analyze various aspects of interphase chromosome organization and dynamics in living cells using 3D wide-field fluorescence microscopy is discussed. Potential problems encountered when utilizing this technology in plants are considered.

Use of two-color fluorescence-tagged transgenes to study interphase chromosomes in living plants.

Sixteen distinct sites distributed on all five Arabidopsis (Arabidopsis thaliana) chromosomes have been tagged using different fluorescent proteins and one of two different bacterial operator-repressor systems: (1) a yellow fluorescent protein-Tet repressor fusion protein bound to tet operator sequences, or (2) a green or red fluorescent protein-Lac repressor fusion protein bound to lac operator sequences. Individual homozygous lines and progeny of intercrosses between lines have been used to study various aspects of interphase chromosome organization in root cells of living, untreated seedlings. Features reported here include distances between transgene alleles, distances between transgene inserts on different chromosomes, distances between transgene inserts on the same chromatin fiber, alignment of homologous chromosomes, and chromatin movement. The overall findings are consistent with a random and largely static arrangement of interphase chromosomes in nuclei of root cells. These transgenic lines provide tools for in-depth analyses of interphase chromosome organization, expression, and dynamics in living plants.

RNA-directed DNA methylation in Arabidopsis.

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.

HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by double-stranded RNA.

To analyze relationships between RNA signals, DNA methylation and chromatin modifications, we performed a genetic screen to recover Arabidopsis mutants defective in RNA-directed transcriptional silencing and methylation of a nopaline synthase promoter-neomycinphosphotransferase II (NOSpro- NPTII) target gene. Mutants were identified by screening for recovery of kanamycin resistance in the presence of an unlinked silencing complex encoding NOSpro double-stranded RNA. One mutant, rts1 (RNA-mediated transcriptional silencing), displayed moderate recovery of NPTII gene expression and partial loss of methylation in the target NOSpro, predominantly at symmetrical C(N)Gs. The RTS1 gene was isolated by positional cloning and found to encode a putative histone deacetylase, HDA6. The more substantial decrease in methylation of symmetrical compared with asymmetrical cytosines in rts1 mutants suggests that HDA6 is dispensable for RNA-directed de novo methylation, which results in intermediate methylation of cytosines in all sequence contexts, but is necessary for reinforcing primarily C(N)G methylation induced by RNA. Because CG methylation in centromeric and rDNA repeats was not reduced in rts1 mutants, HDA6 might be specialized for the RNA- directed pathway of genome modification.

Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors.

The Arabidopsis genome encodes four Dicer-like (DCL) proteins, two of which contain putative nuclear localization signals. This suggests one or more nuclear pathways for processing double-stranded (ds) RNA in plants. To study the subcellular location of processing of nuclear-encoded dsRNA involved in transcriptional silencing, we examined short interfering (si) RNA and micro (mi) RNA accumulation in transgenic Arabidopsis expressing nuclear and cytoplasmic variants of P19, a viral protein that suppresses posttranscriptional gene silencing. P19 binds specifically to DCL-generated 21- to 25-nucleotide (nt) dsRNAs with 2-nt 3' overhangs and reportedly suppresses the accumulation of all size classes of siRNA. Nuclear P19 resulted in a significant reduction of 21- to 22-nt siRNAs and a 21-nt miRNA, but had a lesser effect on 24-nt siRNAs. Cytoplasmic P19 did not decrease the quantity but resulted in a 2-nt truncation of siRNAs and miRNA. This suggests that the direct products of DCL cleavage of dsRNA precursors of 21- to 22-nt siRNAs and miRNA are present in the nucleus, where their accumulation is partially repressed, and in the cytoplasm, where both normal sized and truncated forms accumulate. DCL1, which contains two putative nuclear localization signals, is required for miRNA production but not siRNA production. DCL1-green fluorescent protein fusion proteins localize to nuclei in transient expression assays, indicating that DCL1 is a nuclear protein. The results are consistent with a model in which dsRNA precursors of miRNAs and at least some 21- to 22-nt siRNAs are processed in the nucleus, the former by nuclear DCL1 and the latter by an unknown nuclear DCL.

Studies on the effects of a flanking repetitive sequence on the expression of single-copy transgenes in Nicotiana sylvestris and in N. sylvestris-N. tomentosiformis hybrids.

To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N. sylvestris and in N. sylvestris-N. tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N. sylvestris lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methylation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active, unmethylated NOSpro-NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced, methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not become methylated in N. sylvestris-N. tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.