RNA-directed DNA methylation
RNA-directed DNA methylation is a biological process in which non-coding RNA molecules direct the addition of DNA methylation to specific DNA sequences. The RdDM pathway is unique to plants, although other mechanisms of RNA-directed chromatin modification have also been described in fungi and animals. To date, the RdDM pathway is best characterized within angiosperms, and particularly within the model plant Arabidopsis thaliana. However, conserved RdDM pathway components and associated small RNAs have also been found in other groups of plants, such as gymnosperms and ferns. The RdDM pathway closely resembles other sRNA pathways, particularly the highly conserved RNAi pathway found in fungi, plants, and animals. Both the RdDM and RNAi pathways produce sRNAs and involve conserved Argonaute, Dicer and RNA-dependent RNA polymerase proteins.
RdDM has been implicated in a number of regulatory processes in plants. The DNA methylation added by RdDM is generally associated with transcriptional repression of the genetic sequences targeted by the pathway. Since DNA methylation patterns in plants are heritable, these changes can often be stably transmitted to progeny. As a result, one prominent role of RdDM is the stable, transgenerational suppression of transposable element activity. RdDM has also been linked to pathogen defense, abiotic stress responses, and the regulation of several key developmental transitions. Although the RdDM pathway has a number of important functions, RdDM-defective mutants in Arabidopsis thaliana are viable and can reproduce, which has enabled detailed genetic studies of the pathway. However, RdDM mutants can have a range of defects in different plant species, including lethality, altered reproductive phenotypes, TE upregulation and genome instability, and increased pathogen sensitivity. Overall, RdDM is an important pathway in plants that regulates a number of processes by establishing and reinforcing specific DNA methylation patterns, which can lead to transgenerational epigenetic effects on gene expression and phenotype.
Biological functions
RdDM is involved in a number of biological processes in the plant, including stress responses, cell-to-cell communication, and the maintenance of genome stability through TE silencing.Transposable element silencing and genome stability
TEs are pieces of DNA that, when expressed, can move around the genome through a copy-and-paste or cut-and-paste mechanism. New TE insertions can disrupt protein coding or gene regulatory sequences, which can harm or kill the host cell or organism. As a result, most organisms have mechanisms for preventing TE expression. This is particularly key in plant genomes, which are often TE-rich. Some plant species, including important crops like maize and wheat, have genomes consisting of upwards of 80% TEs. RdDM plays a key role in silencing these mobile DNA elements in plants by adding DNA methylation over new TE insertions and constantly reinforcing DNA methylation over existing TEs, inhibiting transposition and maintaining long-term genome stability. Although the RdDM mechanism itself is unique to plants, using DNA methylation to silence TEs is a common strategy among eukaryotes.RdDM primarily targets small TEs and TE fragments near genes, which are usually in open, accessible euchromatic regions of the genome that are permissive for gene expression. In these regions, the 'active' chromatin state has a tendency to spread from expressed genes to nearby repressed regions, like TEs, which can cause these TEs to become activated and transpose. Continuous activity by RdDM opposes the spread of active chromatin, maintaining a silent, repressive heterochromatic state over TEs in these otherwise euchromatic regions. In turn, RdDM activity recruits other pathways that help establish and propagate the silent, heterochromatic state. Because of the self-reinforcing nature of these silencing pathways, excessive RdDM activity can also cause the silent, heterochromatic chromatin state over TEs to spread to nearby genes and repress them, with potentially harmful consequences for the organism. Therefore, RdDM activity must be finely tuned to maintain a balance between repressing TEs and allowing expression of nearby genes.
In addition to maintaining stable silencing of TEs, RdDM can also initiate transcriptional silencing of foreign DNA, including novel TE insertions, virus-derived sequences, and transgenes. When TEs integrate near genes, RdDM-mediated silencing of the TEs often affects gene expression. However, this is not always deleterious, and can sometimes be overcome by other processes, or alter gene expression in ways beneficial to the plant. Over evolutionary time, beneficial TEs can become an important part of the mechanism by which a gene is regulated. In one example, the gene ROS1 lies adjacent to a small helitron TE that is normally methylated by RdDM. While DNA methylation is normally associated with transcriptional repression, this is not the case at the ROS1 locus. Instead, methylation of the helitron TE promotes ROS1 expression, so ROS1 expression is lost in mutants of the RdDM pathway that cannot methylate the TE. Interestingly, ROS1 encodes a DNA glycosylase that functions to remove DNA methylation from the genome. The link between ROS1 expression and RdDM activity at this TE ensures that DNA methylation and demethylation activities remain in balance, helping to maintain DNA methylation homeostasis genome-wide. Thus, RdDM-mediated regulation of TEs can lead to beneficial regulatory outcomes.
Some TEs have evolved mechanisms to suppress or escape RdDM-based silencing in order to facilitate their own proliferation, leading to an evolutionary arms race between TEs and their host genomes. In one example, a TE-derived sequence was found to produce sRNAs that trigger post-transcriptional repression of a component of the RdDM pathway, inhibiting RdDM. This sequence may have helped the original TE escape RdDM-based silencing and insert itself into the host genome.
Studying how RdDM targets and represses different types of TEs has led to many major insights into how the RdDM mechanism works. The retrotransposon EVADÉ was one of the first TEs specifically shown to be repressed by RdDM-derived sRNAs. Later work used EVD to trace the mechanism by which a novel TE insertion became silenced, revealing an important mechanistic link between post-transcriptional gene silencing and RdDM. Studies of other retrotransposons, including ONSEN, which is regulated by both RdDM and heat stress, and Athila family TEs, among many others, have also provided valuable insights into RdDM-mediated TE silencing.
Development and reproduction
A number of epigenetic changes required for normal development and reproduction in flowering plants involve RdDM. In a well-studied example, RdDM is required for repression of the FWA gene, which allows for proper timing of flowering in Arabidopsis. The FWA promoter contains tandem repeats that are usually methylated by RdDM, leading to transcriptional repression. Loss of this methylation re-activates FWA expression, causing a late-flowering phenotype. The loss of DNA methylation and associated late-flowering phenotype can be stably transmitted to progeny. Since the demethylated fwa allele leads to a stable, heritable change in the expression of FWA without any change to the DNA sequence, it is a classic example of an epiallele.Mutations in the RdDM pathway can strongly affect gamete formation and seed viability, particularly in plant species with high TE content like maize and Brassica rapa, highlighting the importance of this pathway in plant reproduction. During gamete formation, it has been hypothesized, and in some cases shown, that RdDM helps reinforce TE silencing in the germ cells. In both pollen and ovules, a support cell undergoes epigenetic reprogramming, losing DNA methylation and other epigenetic marks at a number of loci, including TEs. This causes TE re-activation and encourages the production of RdDM-derived sRNAs against these TEs in the support cells. The sRNAs are then thought to move from the support cell to the germ cell in order to reinforce TE silencing in the next generation. This phenomenon has been observed in pollen, but has yet to be shown definitively in the ovule. This role for sRNAs in plants resembles the role of piRNAs in germline development in Drosophila and some other animals. A similar phenomenon may also occur in roots to preserve TE silencing in important stem cell populations.
The RdDM pathway is also involved in regulating imprinted expression at some genes. This unusual parent-of-origin-specific expression pattern occurs at several loci in the endosperm during seed development in flowering plants. A few factors involved in the RdDM pathway are themselves imprinted in diverse species, including A. thaliana, A. lyrata, C. rubella, and maize. RdDM also plays a role in mediating the gene dosage effects seen in seeds derived from interploid crosses, though the mechanism for this remains largely unknown.
There is also evidence that RdDM plays a role in several other aspects of plant development, including seed dormancy, fruit ripening, and other pathways involved in flowering. However, most of these data are correlative, and further study is necessary to understand the role of RdDM in these processes.