Genetic analysis of RNA-directed DNA methylation

RNA-directed DNA methylation contributes substantially to epigenetic regulation of the plant genome. Methylation is guided to homologous DNA target sequences by 24 nucleotide `heterochromatic` small RNAs produced by nucleolar-localized components of the RNA interference (RNAi) machinery and a plant-specific RNA polymerase termed Pol IV. Although the exact functions of RdDM in plants are not entirely understood, it is proposed to have roles in transposon silencing as well as plant development and stress adaptation. We have previously identified several key components of the RdDM machinery in two forward genetic screens based on specially designed transgene silencing systems in the model plant Arabidopsis thaliana. In these silencing systems, promoter-directed small RNAs trigger methylation and silencing of target promoters that drive expression of easily-assayable reporter genes. For the present study, we have exploited an enhancer active in meristems (stem cells where developmental decisions are made) to assemble a sensitive, two-component transgene system that permits a genetic dissection of RdDM, secondary siRNA biogenesis, and spreading of DNA methylation in a developmental context. We have already validated the usefulness of this system by identifying a novel component of the RdDM machinery, termed DMS3, in a new forward genetic screen. DMS3 is a structural maintenance of chromosomes hinge-domain- containing protein, and it is the first of its kind to be implicated in an RNAi-mediated, chromatin modification pathway. A related protein in mice, Smchd1, has recently been found by Blewitt, Whitelaw and coworkers to be required for X chromosome inactivation, another epigenetic phenomenon involving a regulatory RNA (Xist RNA) and spreading of DNA methylation. In the proposed work, we will identify and characterize additional mutants recovered from the new forward genetic screen, determine the contributions of the identified gene products to regulation of transcription and genomic methylation patterns in Arabidopsis, and begin to analyze their mode of action. The results will be important for understanding how silent epigenetic states are nucleated by small RNAs and subsequently spread over larger chromosomal domains, and they should be relevant for both plant and mammalian systems.

We have used a genetic approach to discover new factors required for an important `epigenetic` process in plants that limits the spread of chromosomal or genomic parasites called transposons. If left unchecked, transposons can move around and integrate into plant chromosomes at new sites, thus disrupting the integrity of the plant`s DNA. The important epigenetic process that helps to restrict transposon activity is called `RNA-directed DNA methylation` (RdDM). RdDM provides plants with a very specific means, via small `guide` RNAs, to target transposons for inactivation by a chemical modification (cytosine methylation) of the transposon`s own DNA. Although RdDM is an important genome defense mechanism in plants, little was known until recently about the proteins responsible for facilitating various steps in this pathway. Through a genetic screen that we developed to find mutants defective in RdDM in the model plant Arabidopsis thaliana, we were able to find over a dozen factors required for RdDM. Many of these factors are found only in plants and they were functionally uncharacterized before being implicated in RdDM. Our results have thus contributed significantly to identifying new constituents of the RdDM machinery and they set the stage for detailed analyses of the mechanism of RdDM. It is possible that our results will eventually find importance in agricultural biotechnology by suggesting ways to improve the ability of plants to better contain the activity of transposons or to co-opt RdDM for selective control of plant genes.