By Mehdi Banan, Ph.D.
Science Content/Business Development Analyst

When double stranded RNAs (dsRNAs) are introduced into a variety of organisms (e.g. worms, mice, fruit flies, plants), they trigger a host response that is generally referred to as RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). In the RNAi pathway, dsRNAs are cleaved into 21-25 nt small interfering RNAs (siRNAs) and incorporated into nuclease complexes which then bind and destroy homologous transcripts (1).

In addition to PTGS, siRNAs can also mediate gene silencing at the transcriptional stage (transcriptional gene silencing or TGS). For example, siRNAs targeting plant promoters have been shown to cause DNA methylation at these promoters and transcriptional silencing of the corresponding gene (2). More recently, siRNAs have been associated with methylation of histone proteins at centromeric regions in fission yeast--a phenomenon that may lead to silencing of transposons present at centromeres (3). The mechanism of siRNA-mediated DNA and histone methylation is not well understood, but may involve siRNA-mediated binding and guidance of methyltransferases to specific DNA regions.

A number of plant and fission yeast TGS genes have been identified (4,5). However, more genes are believed to be involved in the process. Now, in the 31 January 2003 issue of Science, Jacobsen and colleagues identify another plant TGS gene called Argonaut4 (Ago4) (6). The exact role of Ago4 in the TGS pathway is not clear but may include the generation of siRNAs that subsequently mediate TGS.

The Screening Procedure

Jacobsen and colleagues used a novel screening procedure to identify the plant TGS genes. The SUPERMAN (SUP) gene is necessary for formation of stamens and carpels in Arabidopsis. Certain recessive alleles of SUP (called clark kent or clk-st) are hypermethylated and hence silenced, an epigenetic state that is manifested by increased numbers of stamens and carpels in the plant. In their approach, the authors screened for mutations that abolish silencing of the hypermethylated SUP alleles (i.e. mutations in genes involved in maintaining the hypermethylation and TGS of SUP). To this end, clk-st seeds were subjected to chemical mutagenesis and screened for development into plants with a wildtype floral phenotype (i.e. where SUP is expressed). In one such revertant, candidate genes were sequenced and a frameshift mutation was detected in the AGO4 gene. The role of AGO4 in the maintenance of SUP silencing was then verified by complementation tests.

AGO4 is involved in DNA and histone methylation

The authors then showed that AGO4 is necessary for maintaining DNA and histone methylation at the SUP locus. Bisulfite sequencing was used to show that AGO4 mutant plants have reduced DNA methylation at the SUP locus. (Treatment of DNA with bisulfite coverts all non-methylated cytosines into thiamines, whereas methylated cytosines remain unchanged. Therefore, upon DNA sequencing, methylated cytosines can be identified.) Moreover, chromatin immunoprecipitation (CHIP) analysis was used to show that AGO4 mutant plants have reduced histone methylation at the SUP locus. (In CHIP, antibodies are used to immunoprecipitate methylated-histones and the associated DNA. The immunoprecipitated DNA is then identified by PCR).

In addition, AGO4 was found to be necessary for increased DNA and histone methylation at three other loci: an intergenic region next to an imprinted gene (MEA-ISR), a retrotransposon sequence (AtSN1), and a DNA transposon Mu1 sequence (AtMu1).

AGO4 and siRNAs

The authors subsequently used Northern blots to determine levels of siRNAs to the above regions (SUP, MEA-ISR, AtSN1, AtMu1) in wildtype and AGO4 mutant plants. It was hypothesized that these siRNAs might be present at low levels in AGO4 mutant plants (because of low levels of DNA and histone methylation at these regions) and at high levels in wildtype plants. As predicted, such a pattern was seen with the siRNAs to the AtSN1 retrotransposon (present at 70 copies per genome). However, siRNAs to the other three regions (SUP, MEA-ISR, Mu1) were not detected in either the wildtype or AGO4 mutant plants, presumably because siRNAs to single copy genes are present at low levels in cells and below the detection sensitivity of Northern blots.

The Model

The above results suggest the following model for the role of AGO4 in TGS:
AGO4, along with DICER and an RNA-dependent RNA polymerase, could initially participate in the generation of siRNAs to certain DNA regions (as shown for AtSN1). Once generated, these siRNAs could bind to DNA and histone methyltransferases, guiding them to homologous DNA regions. This would result in DNA and histone methylation at those regions and silencing of the genes residing at those loci.

These findings by Jacobsen and colleagues however raise a number of questions. For example, the authors found that AGO4 is responsible for DNA and histone methylation at some centromeric loci (e.g. the AtSN1 retrotransposon) but not others (e.g. the Ta3 retrotransposon). Why is that? Are other Argonaut genes responsible for generating siRNAs to the Ta3 region? Moreover, what is the exact role of AGO4 in siRNA generation? Is it to stabilize the siRNAs, or is it to generate the siRNA? Answers to these questions will undoubtedly add to our understanding of the TGS pathway.

References

1. Zamore PD. (2002). Ancient pathways programmed by small RNAs. Science 296:1265-1269.
2. Mette MF, Aufsatz W, van der Winder J, Matzke MA, and Matzke AJM. (2000). Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 19(19):5194-5201.
3. Allshire R. (2002). RNAi and heterochromatin-a hushed up affair. Science 297:1818-1819.
4. Lindroth AM, Cao X, Jackson JP, Zilberman D, McCallum CM, Henikoff S, and Jacobsen SE (2001). Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 292:2077-2080.
5. Jackson JP, Lindroth AM, Cao X, and Jacobsen SE. (2002). Control of CpNpG DNA methylation by the KRYPTONITE histone H3 methyltransferase. Nature 416:556-560.
6. Zilberman D, Cao X, and Jacobsen SE. (2003). ARGONAUTE4 control of locus-specific siRNAs accumulation and DNA and histone methylation. Science 299:716-719.