RNA-directed DNA methylation (RdDM)

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Authors
Robert M. Erdmann, Colette Picard
About the Authors 

Robert M. Erdmann
AFFILIATION: Center for Learning Innovation, University of Minnesota Rochester , Rochester, MN
Orcid icon.png 0000-0002-4325-4196

Colette Picard
AFFILIATION: Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles , Los Angeles, CA
Orcid icon.png 0000-0002-2177-2216



Abstract

RNA-directed DNA methylation (RdDM) is a biological process in which small RNA molecules direct the addition of DNA methylation to specific DNA sequences. The RdDM pathway is unique to plants, and the fullest complement of RdDM pathway components characterized to date is found within angiosperms (flowering plants). Other groups of plants, such as gymnosperms and ferns, possess a subset of conserved RdDM pathway components and also express associated small RNAs. RdDM has been implicated in a number of regulatory processes in plants, and several distinct RdDM pathways have been characterized. The RdDM pathway closely resembles other small RNA pathways, particularly the RNAi pathway found in both plants and animals. Both the RdDM and RNAi pathways produce small RNAs and involve conserved Argonaute, Dicer and RNA-dependent RNA polymerase proteins.

Since the DNA methylation added by RdDM is generally a repressive epigenetic mark, RdDM predominantly leads to transcriptional repression of the genetic sequences targeted by the pathway. One prominent role of RdDM is the suppression of transposable element (TE) activity, but RdDM has also been linked to pathogen defense, abiotic stress responses, and the regulation of several key developmental transitions. RdDM pathway mutants in the model plant Arabidopsis thaliana are viable and can reproduce, which has enabled detailed genetic studies of the pathway. However, in other plant species these mutants can have a range of defects including lethality, altered reproductive phenotypes, TE upregulation and genome instability, and increased pathogen sensitivity. Overall, RdDM is an important epigenetic pathway in plants that regulates a number of processes by establishing and reinforcing specific DNA methylation patterns.

History and discovery of RdDM

RdDM was first described in 1994 in tobacco plants. Researchers observed that when viroids were introduced into the plant and integrated into the plant genome, the viroid sequences, but not the host genome, gained DNA methylation.[1] The deposition of methylation over these foreign viroid sequences helped inhibit viroid replication, and therefore was thought to represent a plant pathogen defense mechanism. The evidence suggested that the viroid RNAs produced during viroid replication were being used by the plant as a template to help target DNA methylation to the viroid sequences. This mechanism was therefore named RNA-directed DNA methylation, or RdDM.[1] The role of small RNAs in the pathway was initially suspected due to the similarity between and co-occurrence of RdDM and RNAi, the latter of which was already known to involve small RNAs (sRNAs).[1] Genetic screens later revealed other components of the pathway, including RNA polymerases IV and V, Dicer-like proteins, Argonautes, and others.[2][3] A detailed review of the RdDM pathway and its components can be found in section 5 below.

Biological functions of RdDM

RdDM is involved in a number of biological processes in the plant, including the maintenance of genome stability through TE silencing, response to stress, and cell-to-cell communication. An overview of the biological functions performed by RdDM is shown in Figure 1.

Figure 1. High level overview illustration of a selection of RdDM’s biological functions.

Repression of transposable elements

Transposable elements (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 contain mechanisms for preventing TE expression. This is particularly key in plant genomes, which are often TE-rich. Some plant species, including crops like maize and wheat, have genomes consisting of upwards of 80% TEs.[4][5] RdDM plays a key role in silencing these mobile DNA elements in plants by adding DNA methylation throughout the length of new TE insertions and reinforcing existing DNA methylation at older TEs, leading to stable repression of TEs that can be maintained from one generation to the next.

Some TEs have evolved mechanisms to suppress or escape RdDM-based silencing in order to facilitate their own proliferation, forming 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 the RdDM methyltransferase DRM2.[6] This sequence may have helped the original TE escape RdDM-based silencing and insert itself into the host genome.

RdDM is not the only mechanism that can silence TEs in plants. TEs in and around the pericentromere are usually repressed by other mechanisms (see section 5.4).[7] TEs silenced by the RdDM pathway, however, tend to be in euchromatin and are often near genes, so RdDM activity at these TEs can have the unintended side-effect of also repressing the nearby gene.[7] Therefore, maintaining a balance between repressing TEs and allowing expression of nearby genes is particularly important in these regions. DNA glycosylases, which remove DNA methylation, play a role in maintaining this balance (see section 5.5). Additionally, recent work has revealed a protein complex in Arabidopsis thaliana that may function specifically to help increase the expression of genes near silenced, methylated TEs.[8] Other, similar mechanisms may still remain to be uncovered. It is also important to note that DNA methylation does not always cause silencing: in rare cases, genes can actually require DNA methylation at specific regulatory sequences in order to be expressed.[9][10] Some RdDM-targeted TEs or TE fragments have also been co-opted by the cell to regulate the expression of nearby genes. These are TEs which, when targeted and methylated by RdDM, affect expression of the nearby gene(s) in a way that is beneficial to the host organism. These TEs are then maintained in the genome due to positive selective pressure.

Cell-to-cell communication

The sRNA molecules produced by RdDM and other pathways are able to move between cells via plasmodesmata, and can also move systemically through the plant via the vasculature.[11][12] This was initially demonstrated in plants engineered to express green fluorescent protein (GFP).[13] The GFP protein produced by these plants caused them to glow green under certain light conditions. When tissue from a second plant expressing a sRNA construct complementary to GFP was grafted onto the GFP-expressing plant, the GFP-expressing plant stopped glowing - after grafting, the sRNAs produced by the second plant were moving into the tissues of the GFP-expressing plant and triggering silencing of the GFP locus. They also showed that a subset of these sRNAs was triggering the addition of DNA methylation to the GFP locus via RdDM. Thus, sRNAs generated through RdDM can act as signaling molecules and cause DNA methylation to be added to complementary loci in cells far from where the sRNAs were originally generated. To date, sRNA movement tied to RdDM activity has been shown to play a role in both plant reproduction and pathogen defense, but this is an area of active research, and our understanding of the function of sRNA movement in these contexts is limited.

Development and reproduction

A number of epigenetic changes occur during development and reproduction in flowering plants, some of which involve RdDM. 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,[14][15] 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 (the sperm and egg cells). In both pollen and ovules, a support cell (the vegetative nucleus in pollen, the central cell in ovules) 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. These sRNAs are then thought to move from the support cell to the germ cell in order to reinforce TE silencing in the next generation.[15] This phenomenon has been observed in pollen but has yet to be shown definitively in the ovule.[16][17] This role for sRNAs in plants resembles the role of piRNAs in germline development in Drosophila and some other animals.

The RdDM pathway is also involved in regulating imprinted expression at some genes[18]; this unusual expression pattern occurs during seed development in flowering plants at several loci. Several components of the RdDM pathway are themselves imprinted in the seed.[19] RdDM also plays a role in mediating the gene dosage effects seen in seeds derived from interploid crosses.[20][21] When a tetraploid plant is used as the pollen donor for a diploid ovule, the resulting seed usually aborts due to increased paternal relative to maternal dosage in the endosperm, leading to excessive growth, proliferation, and eventually seed collapse. Seed abortion rates in these crosses are reduced by pollinating the wild-type ovule with pollen from a tetraploid plant that is also defective in RdDM, suggesting that RdDM plays a role in mediating these dosage effects, though the mechanism remains largely unknown.

There is evidence that RdDM may play a role in several other aspects of plant development, including regulating the timing of flowering[22], seed dormancy[23], and fruit ripening.[24] However, most of these data are correlative, and further study is necessary to understand the role of RdDM in these processes.

Stress response

Abiotic stresses

RdDM has been tied to several abiotic stress responses, including heat stress, drought, phosphate starvation, salt stress, and others.[25] In plants exposed to heat stress, several components of the RdDM pathway become upregulated, and plants with defective RdDM machinery have reduced heat tolerance.[26][27] Under low humidity, leaves produce fewer stomata due to RdDM-mediated downregulation of two genes involved in stomatal development.[28] RdDM becomes downregulated in response to salt stress, and this has been shown to trigger the expression of a transcription factor important in salt stress resistance.[29]

Biotic stresses

RdDM was initially discovered as a response to infection by viroids[1], and along with RNAi plays an important role in defending the plant against viroids and viruses. The RdDM and RNAi machinery recognize viral RNAs and process them into sRNAs, which can then be used both by the RNAi and RdDM pathways to help defend against the invading virus and silence any integrated viral DNA.[30] However, little is known about how the RdDM and RNAi machinery distinguish between viral RNAs and RNAs produced by the host plant. Virus-host interactions are another example of an evolutionary arms race, and many plant viruses encode suppressors of both RdDM and RNAi in an attempt to evade the host plant’s defenses.[30][31][32][33] Mutants defective in RdDM and other methylation-deficient mutants are hypersensitive to viral infection.[33][34]

RdDM is also involved in protecting the plant from other biotic stresses, including bacterial and fungal infections, and predation. Loss of RdDM can have opposing effects on resistance for different pathogens. For example, RdDM mutants have increased susceptibility to the bacterium Agrobacterium tumefaciens, but those same mutants have decreased susceptibility to the bacterium Pseudomonas syringae[29][35], highlighting the complexity of the different pathogen defense pathways and their interactions with RdDM. RdDM mutants also have enhanced susceptibility to several fungal pathogens.[36] Overall, while there is strong evidence that RdDM functions in plant immunity, much remains to be discovered about the role of RdDM in plant immune responses.

Stress and RdDM-mediated epigenetic ‘memory’

Due to the self-reinforcing nature of most DNA methylation pathways, including RdDM, any DNA methylation changes caused by stressors have the potential to be maintained and transmitted to future generations. This has been hypothesized, and in some cases shown, to allow stress-induced DNA methylation changes to act as a ‘memory’ of the stressor, to help prime the plant or its progeny to respond more efficiently to the stress if re-exposed.[37][38] For example, silencing of TEs and viruses is maintained across generations by RdDM and other pathways, and the sRNAs produced by these loci help protect against future invasions by similar TE or viral sequences. There is also evidence that DNA methylation changes due to other stressors, such as salt or heat stress, can persist in the progeny of stressed plants even in the absence of the original stressor.[39] In this study, the persistence of the stress-induced DNA methylation changes required DCL2 and DCL3, suggesting that RdDM was involved in maintaining the altered DNA methylation patterns. In another example, resistance to insect attack was transmitted to progeny via DNA methylation changes, and this inheritance was also dependent on functional RdDM.[37] Thus, RdDM has the potential to alter the plant epigenome in a way that can be transmitted to the following generation, helping to modulate future plant stress responses.

Potential biotechnology applications of RdDM

The RdDM machinery can be ‘tricked’ into targeting and silencing endogenous genes in a highly specific manner, which has a number of potential biotechnological and bioengineering applications. Several different methods can be used to trigger RdDM-based DNA methylation and silencing of specific endogenous genes. One method, called virus-induced gene silencing (VIGS), involves inserting part of the promoter sequence of the desired target gene into a virus.[40] The virus will reproduce the chunk of promoter sequence as part of its own RNA, which is otherwise foreign to the plant. Because the viral RNA is foreign, it will be processed into sRNAs (see section 5), some of which will be complementary to the original target gene’s promoter; these sRNAs and will then recruit RdDM to the locus to add DNA methylation, leading to silencing. In one study, researchers used this method with an engineered Cucumber Mosaic Virus to recruit RdDM to silence a gene that affected flower pigmentation in petunia, and another that affected fruit ripening in tomato.[41] In both cases, they showed that DNA methylation was added to the locus as expected. In petunia, both the gain of DNA methylation and changes in flower coloration were heritable, while only partial silencing and heritability were observed in tomato. VIGS has also been used to silence the FLOWERING WAGENINGEN locus in Arabidopsis, which delays flowering.[40] The same study also showed that the inhibitory effect of VIGS on FWA and flowering can become stronger over the course of succeeding generations.[40]

Another method involves introducing a hairpin RNA construct that is complementary to the target locus. Hairpin RNAs contain an inverted repeat, which causes the RNA molecule to form a double-stranded RNA (dsRNA) structure called an RNA hairpin. Since dsRNAs are a precursor of sRNA synthesis by the RdDM and other sRNA machinery (see section 5), the dsRNA hairpin can be processed into sRNAs which are complementary to the target locus, triggering RdDM at that locus. This method has been used in several studies.[9][42]

Taken together, these studies illustrate that DNA methylation resulting from RdDM activity can be targeted to genetic locations of interest and affect gene expression, and can also be maintained and inherited over multiple generations without outside intervention or manipulation. Recent work has even bypassed RdDM altogether by artificially tethering the enzyme responsible for adding DNA methylation (DRM2; see section 5) or other components of the RdDM pathway directly to specific target loci, using either zinc finger nucleases or CRISPR.[43][44] In these experiments, tethering the RdDM machinery to a specific locus led to gain of DNA methylation at the target site that was often heritable for multiple generations, even once the artificial construct was crossed out. For all of these methods, however, more work needs to be done to optimize the procedure, including minimizing off-target effects and increasing DNA methylation efficiency.

Genetically Modified Organisms (GMOs) have played a large role in recent agricultural research and practice, but have proven controversial, and face regulatory barriers to implementation in some jurisdictions. GMOs are defined by the inclusion of “foreign” genetic material into the genome. The treatment of plants with engineered RNAs or viruses intended to trigger RdDM does not change the underlying DNA sequence of the treated plant’s genome; only the epigenetic state of portions of the DNA sequence already present are altered. As a result, these plants are not considered GMOs. This has led to efforts to utilize RdDM and other RNA-mediated effects to induce agriculturally-beneficial traits.[45] This could take the form of treating weeds in order to increase their susceptibility (or reduce their resistance) to an herbicide, or speeding up plant breeding by inhibiting endogenous genes to provide novel/desirable traits without changing the underlying DNA.[46] However, while this is an area of active interest, there are few broadly implemented applications as of now.

Pathways and mechanisms

This section focuses broadly on the various pathways and mechanisms by which RdDM leads to sequence-specific DNA methylation. A simplified schematic is found in Figure 2 and shows the main factors involved in the RdDM pathway. While most of this section focuses on discoveries made in the model plant Arabidopsis thaliana, the findings are likely applicable to other angiosperms. Conservation of RdDM in other plant species is discussed in more detail in section 6.

Broadly, each version of the RdDM pathway can be split up into two processes: the production of sRNAs, and the recruitment of DNA methylation machinery by those sRNAs to specific target loci. These two activities together comprise RdDM.

Figure 2. Simplified schematic of the canonical and non-canonical RdDM pathways, along with the related RNAi pathway.

Canonical RdDM

The canonical RdDM pathway is, as its name suggests, the most well-characterized RdDM pathway to date. This pathway requires two plant-specific RNA polymerases, RNA Polymerase IV (RNAP IV) and RNA Polymerase V (RNAP V), and is generally involved in maintaining existing DNA methylation patterns at target loci.[30] Canonical RdDM makes up the majority of RdDM activity in a cell.[47]

Production of sRNAs involved in canonical RdDM

The plant-specific RNAP IV is recruited to loci with existing DNA methylation, and transcribes them to produce single-stranded RNAs (ssRNAs). These ssRNAs are immediately converted into double-stranded RNAs (dsRNAs) by an RNA-directed RNA polymerase, RDR2. The dsRNA is then cleaved by DCL3 into 24 nucleotide (nt) small RNAs (sRNAs), and these sRNAs direct canonical RdDM to its target loci. While nearly all sRNAs involved in canonical RdDM are produced this way, a small proportion of 24 nt sRNAs are produced through other pathways. For example, some RNA Polymerase II (RNAP II) transcripts that contain an inverted repeat sequence form double-stranded hairpin structures that can be directly cleaved by DCL3 to form 24 nt sRNAs that can act in canonical RdDM.[48]

Silencing of target loci by canonical RdDM

The 24 nt sRNAs produced by DCL3 associate with the Argonaute proteins AGO4 and AGO6.[30] Argonautes are a highly conserved family of proteins that are involved in a number of sRNA-mediated processes, including RNAi and RdDM. Argonautes bind sRNAs, forming a protein-sRNA duplex, which enables them to recognize and bind sequences complementary to their sRNA partner. In RdDM, the AGO-sRNA duplex recognizes and binds an RNA ‘scaffold’ produced by another plant-specific RNA Polymerase V, RNAP V.[49] The RNAP V scaffolds are thought to provide a molecular tether, so that complementary sRNA-AGO complexes that associate with the scaffold are able to direct DNA methylation to cytosines near the complementary sequence. After binding to the scaffold, the sRNA-AGO complex recruits the DNA methyltransferase enzyme DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which methylates nearby DNA.[50]

Existing DNA methylation helps recruit RNAP IV and increases sRNA production at RdDM target loci, which in turn leads to increased RdDM activity and directs further methylation to nearby DNA sequences. Therefore, the canonical RdDM pathway can be thought of as a positive feedback loop or a self-reinforcing pathway.[30] This continual reinforcement helps maintain DNA methylation patterns and is important for long-term repression of TE activity within the genome.

Non-canonical RdDM

Recent work has revealed a number of additional versions of the RdDM pathway, collectively called non-canonical RdDM.[47] Unlike canonical RdDM, the non-canonical pathways are generally involved in establishing initial DNA methylation at new target loci; this DNA methylation then helps recruit RNAP IV to trigger additional methylation and maintenance of existing DNA methylation via canonical RdDM. In general, non-canonical RdDM is much more closely associated with RNAi and other PTGS pathways than canonical RdDM, and often involves the same factors. Non-canonical RdDM targets relatively few loci in comparison to canonical RdDM, consistent with its role in initiation but not maintenance.[47]

Production of sRNAs involved in non-canonical RdDM

Unlike the sRNAs involved in canonical RdDM, which come predominantly from a single source (RNAP IV), sRNAs involved in non-canonical RdDM are produced through a number of different mechanisms. This allows the non-canonical pathways to receive input from many sources, and to trigger de novo DNA methylation at many different types of loci. The sRNAs involved in non-canonical RdDM are 21-22 nt; this is in contrast to the sRNAs involved in canonical RdDM, which are 24 nt. Note that only a fraction of 21-22nt sRNAs are involved in RdDM; sRNAs in this size range can also be involved in various post-transcriptional gene silencing (PTGS) pathways, including RNAi.

One of the primary sources of 21-22 nt sRNAs is RNAP II transcripts. Some transcripts produced by RNAP II, often those produced from TEs or some non-protein-coding transcripts, are targeted by PTGS pathways like miRNAs or RNAi, leading to cleavage of the RNAP II transcript. The resulting fragments are sometimes converted into dsRNA by RDR6, and can then be processed into 21-22nt sRNAs by DCL2 or DCL4.[51] In other cases, dsRNAs resulting from RDR6 activity are processed by DCL3 instead of DCL2/4, and contribute to canonical RdDM.[52] Double-strand break repair has also been shown to produce 21 nt sRNAs in a manner dependent on RDR6 and DCL2/DCL4.[53] There are a range of potential sources of sRNAs for non-canonical RdDM, and these pathways remain an area of active research.[47]

Silencing of target loci by non-canonical RdDM

As with canonical RdDM, once sRNAs have been produced, they can be used as guides to target complementary RNA sequences. Since different pathways can utilize the 21-22 nt sRNAs for targeting, the pathway a particular sRNA participates in is largely dependent on which AGO it associates with. Many sRNAs resulting from the activity of DCL2 and DCL4 on RNAP II transcripts associate with AGO1, leading to RNAi instead of RdDM. However, some of the sRNAs will instead associate with AGO6, which is also involved in canonical RdDM.[54] The rest of the pathway is the same as in canonical RdDM: AGO6 binds a complementary RNAP V scaffold and recruits DRM2 to trigger DNA methylation of nearby loci. There is also a version of the pathway where the sRNAs instead associate with AGO2, which in complex with NERD (Needed for RDR2-independent DNA methylation) also recruits DRM2 and causes DNA methylation.[55]

Differences between canonical and non-canonical RdDM

Some of the key differences between canonical and non-canonical RdDM are the origin (primarily RNAP IV vs. multiple sources) and size (24 nt vs. 21-22 nt) of the sRNAs used to direct DNA methylation. The pathways also generally involve different RDR, DCL and AGO proteins, but both ultimately lead to recruitment of DRM2 and consequent gain of DNA methylation.

Additional factors involved in RdDM

Sections 5.1 and 5.2 present a high-level summary of the major factors involved in canonical and non-canonical RdDM (see Fig. 2). However, a number of other proteins also have roles in this pathway. These include proteins that help recruit the different RdDM factors to target loci, proteins that help ‘unwind’ the DNA to facilitate access, and proteins that affect sRNA production. These are discussed in more detail in this section, and are also summarized in Table 1. However, this is an area of active research, and much remains to be discovered about the factors involved in RdDM.

Table 1: Examples of additional factors involved in RdDM
Factor(s) Factor type Role in RdDM RdDM interactors Description Evidence
SHH1/DTF1 DNA and chromatin binding protein siRNA production, RNAP IV targeting RNAP IV, RDR2, RDM4, CLSY1 Required for RNAPIV-derived sRNA production at a subset of RdDM loci. Binds both unmethylated H3K4 and methylated H3K9 to recruit RNAPIV. [56][57][58]
CLSY1, CLSY2 putative chromatin remodelers siRNA production, RNAP IV targeting RNAP IV, SHH1 Required for SHH1 interaction with and recruitment of RNAPIV to subset of target loci. Mutually exclusive with loci regulated by CLSY3 and CLSY4; together all four regulate nearly all RNAPIV-sRNAs. Requires H3K9me2, likely through interaction with SHH1. [59][60]
CLSY3, CLSY4 putative chromatin remodelers siRNA production, RNAPIV targeting RNAP IV Involved in recruitment of RNAPIV to subset of target loci. Mutually exclusive with loci regulated by CLSY1 and CLSY2; together all four regulate nearly all RNAPIV-sRNAs. [59][60]
HEN1 RNA methylase siRNA production none Stabilizes diced sRNAs by adding methylation to the 3'-OH groups. [61]
SUVH2, SUVH9 methyl-DNA binding proteins RNAP V targeting DDR complex, MORC1, MORC6 Required for proper localization of the DDR complex and RNAPV. May also associate with MORCs. [62]
DDR complex (RDM1, DMS3, DRD1) putative chromatin remodeling complex RNAP V targeting, DRM2 recruitment DRM2, AGO4, RNAP II Helps recruit RNAP V to target sites. Thought to act as a chromatin remodeler, possibly unwinding DNA in front of RNAPV to facilitate binding. RDM1 also binds single-stranded DNA, and is thought to facilitate recruitment of DRM2 to DNA by the AGO-sRNA complex. [63][64]
RDM3/KTF1 transcription factor Recruitment of AGO-sRNA complex to RNAP V scaffold AGO4 Interacts with AGO4 and helps recruit it to the RNA scaffold produced by RNAP V. [65]
SWI/SNF complex chromatin remodeling complex Effector of silencing downstream of RNAP IV, RNAP V IDN2 Is recruited to RNAP V scaffolds by the IDN2-IDP complex, where it affects nucleosome positioning. This is thought to facilitate DNA methylation by DRM2. [66]
IDN2-IDP complex dsRNA-binding protein Interaction between AGO4-sRNA and RNAP V scaffold SWI/SNF complex Thought to help stabilize base pairing between the AGO-sRNA and RNAP V scaffold RNA. May also facilitate recruitment of the SWI/SNF complex to RNAP V scaffolds. [67][66]
NERD GW repeat- and PHD finger-containing protein sRNA accumulation at new 'non-canonical' RdDM sites AGO2 Involved in 'non-canonical' RdDM through its interaction with AGO2. Required for 21 nt sRNA accumulation at some new RdDM targets, including new TEs. [55]
MORC1, MORC6 GHKL ATPases RNAP V targeting? SUVH2, SUVH9, IDN2, DMS3 MORC1 and MORC6 form a heterodimer; may interact with the DDR complex to recruit RNAP V. However, thought to mainly act downstream of DNA methylation to promote silencing. More work needed to uncover precise role in RdDM. [68][43]
DRM1 DNA methyltransferase Depositing DNA methylation none known A homolog of DRM2 that is only expressed during sexual reproduction, specifically in the egg cell and potentially the early embryo. DRM2 is likely the main RdDM methyltransferase in all other tissues. [69]

Factors involved in sRNA production and RNAP IV targeting

Since DNA methylation can affect the expression of both TEs and genes within plant genomes, specificity of RdDM targeting is essential. Regulation of sRNA production is one way that RdDM activity can be regulated, and a number of factors have been shown to correlate with the production of 24-nucleotide sRNAs. These factors act primarily by regulating the recruitment of RNAP IV.

SHH1/DTF1 helps recruit RNAP IV to chromatin rich in unmethylated H3K4 and methylated H3K9 histones via its SAWADEE domain[58], and is required for sRNA production at a subset of RNAP IV loci. The Classy (CLSY) family proteins are putative chromatin remodelers that also help recruit RNAP IV to its target loci.[70][56] Loss of all four CLSY proteins results in a near total loss of 24-nucleotide sRNA production.[59][60] More specific mechanistic details regarding RNAP IV targeting remain to be elucidated.

Factors involved in RNAP V and DRM2 targeting

RdDM targeting can also be regulated by influencing the localization of RNAP V, and several factors are known to influence RNAP V localization and transcription. The DDR complex, composed of RDM1, DMS3 and DRD1, is thought to unwind DNA downstream of RNAP V to facilitate its transcription.[64] The methyl-DNA binding proteins SUVH2 and SUVH9 interact with the DDR complex and are also required for proper RNAP V localization.[62] These factors help establish the positive feedback loop between DNA methylation and RdDM by recruiting the DDR complex and RNAP V to sites with existing DNA methylation.[62]

Additional factors in the RdDM pathway

A number of other proteins have been shown to play key roles in RdDM (Table 1). DRM2 recruitment is also facilitated by the SWI/SNF chromatin remodeling complex, which is recruited to RNAP V scaffolds by the IDN2-IDP complex.[66] RDM3/KTF1 interacts with AGO4, and helps recruit AGO4-sRNA complexes to RNAP V scaffolds.[71] The interaction between the AGO-sRNA complex and the RNAP V scaffold may also be stabilized by the IDN2-IDP complex.[72] MORC1 and MORC6 are thought to act primarily downstream of RdDM and not in the pathway itself, but have emerged several times in screens for defective RdDM, and there is some evidence that they interact with the DDR complex to recruit RNAP V.[43] This list of factors involved in RdDM is not exhaustive, and will continue growing as more work is done to define this pathway.

RdDM and other DNA methylation pathways - localization and interactions

The RdDM pathway is just one of several pathways involved in establishing and maintaining DNA methylation patterns in the plant. These pathways act together at some loci and independently at others, so this section will briefly describe these other pathways and how they interact with RdDM. As noted in previous sections, RdDM itself tends to favor loci that already have some DNA methylation, as well as loci marked with methylated H3K9 and unmethylated H3K4 (both of which are associated with heterochromatin). In particular, RdDM favors small patches of heterochromatin in otherwise euchromatic regions, such as euchromatic TEs and repetitive regions, and many of these target sites are near genes.[7][73][74] In contrast, RdDM is usually less associated with pericentromeric TEs or other regions, which are instead silenced by the activity of two other DNA methyltransferases, CMT2 and CMT3.

CMT2 and CMT3 are plant-specific DNA methyltransferases, and are well-conserved within plants, with homologs in angiosperms, gymnosperms, and others.[75] In Arabidopsis, both CMT2 and CMT3 recognize and bind the heterochromatic mark H3K9me2 and methylate nearby cytosines in the CHH and CHG contexts (with H signifying any base other than cytosine), respectively.[76] The histone methyltransferases SUVH4/KYP, SUVH5, and SUVH6, in turn, recognize CHG and CHH methylation and add H3K9me2 to nearby histones[77], forming a self-reinforcing feedback loop. RdDM is also recruited to H3K9me2-marked chromatin via SHH1, so there is some overlap between CMT2/CMT3 targets and RdDM targets, particularly in euchromatin. However, loss of RdDM tends to have little effect on DNA methylation levels in pericentromeric regions, since methylation in these regions is predominantly maintained by CMT2/CMT3.[7][74] Conversely, in cmt2;cmt3 mutants, many euchromatic TEs remain methylated, presumably due to the persistent activity of RdDM[7][74], suggesting RdDM is the main pathway responsible for maintaining DNA methylation in euchromatin. In mutants defective in both RdDM and CMT2/CMT3, all non-CG methylation in the genome is eliminated[78], suggesting that together RdDM and CMT2/CMT3 account for all non-CG methylation in the genome.

The highly conserved DNA methyltransferase MET1 (a homolog of DNMT1 in mammals) acts to maintain DNA methylation during DNA replication, but only at CG dinucleotides.[79][80] MET1 helps maintain CG methylation genome-wide, including at RdDM sites. In RdDM mutants, non-CG methylation at RdDM target sites is lost, but most of the CG methylation at those sites persists due to maintenance by MET1.[73]

Another important factor involved in maintaining heterochromatic DNA methylation is the chromatin remodeler DDM1. DDM1 primarily helps facilitate access to heterochromatin by CMT2 and CMT3, but recent evidence from rice and maize suggests that it may also act to inhibit RdDM access to heterochromatin.[7][81][82] However, in euchromatin, there is some evidence that DDM1 instead facilitates RdDM-mediated TE silencing.[83]

Balance between methylation and demethylation

Several mechanisms exist to maintain and reinforce DNA methylation patterns in plants. The RdDM pathway itself is self-reinforcing: existing DNA methylation helps recruit RNAP IV, encouraging additional DNA methylation via canonical RdDM.[30] Positive feedback loops like these can cause DNA methylation activity to spread out from the intended methylated target sites into genes or other regulatory elements, which can negatively affect gene expression.[9]

RdDM and other DNA methylation pathways are opposed by passive and active DNA demethylation. DNA methylation can be lost passively with each cell division, since newly-synthesized strands of DNA lack DNA methylation until the maintenance pathways re-establish previous patterns.[84] DNA methylation can also be actively removed in plants by DNA glycosylases, which remove methylated cytosines via the base-excision repair pathway. In Arabidopsis, there are three proteins responsible for removing DNA methylation in vegetative tissues: ROS1, DML2 and DML3.[85] A fourth DNA glycosylase, DME, also removes DNA methylation but is thought to act primarily in reproductive tissues.[86]

DNA glycosylase activity helps prevent the spread of DNA methylation from RdDM targets to active genes.[87][88] Loss of DNA demethylation in ros1;dml2;dml3 triple mutants leads to a widespread increase in DNA methylation levels, whereas overexpression of ROS1 relative to DNA methylation activity leads to progressive loss of DNA methylation at many loci.[9] Therefore, properly balancing the activity of pathways that add and remove DNA methylation is essential to ensure that stable DNA methylation patterns can be maintained over long timescales. While little is currently known about how the activity of these pathways is kept in equilibrium, recent work found that expression of the DNA demethylase ROS1 is directly tied to DNA methylation: higher levels of DNA methylation over a portion of the ROS1 promoter trigger increased ROS1 expression.[9][89] As a result, ROS1 expression is strongly reduced in RdDM mutants, which lack DNA methylation over the ROS1 promoter.[9] This mechanism helps maintain DNA methylation homeostasis by tuning DNA demethylation activity to DNA methylation activity.

Evolutionary Conservation

Origins of RdDM pathway members

While all eukaryotes share three RNA polymerases (RNAPs I, II and III), plants also have two additional polymerases, RNAP IV and RNAP V. Both RNAP IV and V share an evolutionary origin, deriving from RNAP II.[90] In other eukaryotic kingdoms that lack these two specialized RNAPs, RNAP II transcribes the precursors of small RNAs used in silencing pathways – in fact, RNAP II transcripts are also sometimes processed into sRNAs in plants, as described in the non-canonical RdDM pathway (section 3.2). It has been hypothesized that the origin of both RNAP IV and V is rooted in “escape from adaptive conflict”.[91] The idea is that potential tensions between the “traditional” function of RNAP II and the small RNA biogenesis function could be relieved by duplication and subfunctionalization of the resulting multiple RNA polymerases.

Analyses of evolutionary lineage for RNAP IV and RNAP V are complicated to some extent by the fact that each enzyme is actually comprised of around a dozen subunits. In Arabidopsis thaliana, some of these subunits are shared between RNAP IV and RNAP V, and others are unique to each polymerase, while still others are shared between RNAP II, IV, and V. Orthologs of certain RNAP IV and V subunits have been found in all lineages of land plants, including ferns, liverworts, and mosses (Figure 3).[91] This finding argues for a shared origin of RNAP IV and V dating back to early land / vascular plants.

Figure 3. A schematic depicting the evolutionary conservation of selected RNAP IV and V subunit orthologs within the plant kingdom. Subunits beginning with NRPD are RNAP IV subunits, subunits beginning with NRPE are RNAP V subunits, and subunits labeled as NRPD/E are found in both RNAP IV and V. A filled circle for a subunit indicates that an ortholog for that subunit has been identified within the associated lineage.

Much of the work done to elucidate the genes and proteins involved in the RdDM pathway has been done in Arabidopis thaliana, a model angiosperm. However, studies of RNAP IV and V conducted in maize show some key differences with Arabidopsis. Maize RNAP IV and V differ from each other in terms of only one subunit (the largest one). In Arabidopsis, RNAP IV and V differ from each other in terms of three subunits.[92] However, maize utilizes a set of interchangeable catalytic subunits – two in the case of RNAP IV and three in the case of RNAP V – that provide additional specialization of RNAP functionality.[92] While differences exist, overall there is a broad overlap in RdDM functions and components between different angiosperm species.

Outside of RNAP IV and RNAP V, a high proportion of key RdDM component proteins (for example, DCL3 and AGO4) have orthologs found within each class of land plants, which provides support for the hypothesis that some form of the RdDM pathway was found quite early within common ancestors of the plant kingdom (Figure 4).[91] However, RdDM pathway functionality does change to an appreciable extent between different plant species and lineages. For example, while gymnosperms have functional RNAP IV and produce 24 nucleotide small RNAs, the biogenesis of sRNAs within gymnosperms is much more heavily skewed towards 21 nucleotide than 24 nucleotide sRNAs.[93] Such a difference has clear implications for the potential activity of canonical RdDM within the species with lower quantities of relevant sRNA triggers. Similarly, orthologs of DRM2 are found in various angiosperms, but it does not have known orthologs in other plant lineages.[94] It is possible that angiosperms have the fullest known version of the RdDM pathway, with all other plant lineages possessing robust and functional subsets of the pathway. However, since nearly all of the work on RdDM has been done in angiosperms, it is also possible that alternative versions of RdDM in other lineages have simply not yet been uncovered, particularly if these alternative versions include different proteins or proteins without clear homologs in angiosperms.

Figure 4. A schematic depicting the evolutionary conservation of selected RdDM pathway component orthologs within the plant kingdom. A filled circle for a subunit indicates that an ortholog for that subunit has been identified within the associated lineage.

Relationships with sRNA silencing pathways in other kingdoms

All eukaryotic kingdoms host some form of small RNAs, and as such, RdDM and its small RNAs can be compared with a number of other sRNA-based silencing pathways in plants as well as other kingdoms. One such class of RNAs is the Piwi-interacting RNAs (piRNAs). Much like in RdDM, piRNAs primarily function to target and silence transposons. However, piRNAs are only found in animals, are longer than the small RNAs functioning in RdDM (24-32 nucleotides), and mediate their functions through interactions with a different subclass of AGO proteins, the PIWI proteins, which are absent from plants.[95]

Micro-RNAs (miRNAs) are another class of small RNA with silencing properties. miRNAs are found with both plants and animals, but there are notable differences in miRNA biogenesis and function between the two kingdoms. In plants, complementarity between a miRNA and its target mRNA is usually perfect, or nearly so, whereas animal miRNAs usually have only partial complementarity to their targets.[96] While miRNAs are in a similar size range as RdDM sRNAs (~21 nt), miRNAs associate with a distinct set of Argonaute proteins that silence target RNAs by initiating their degradation or blocking their downstream translation into proteins, rather than recruiting DRM2 to add DNA methylation to nearby DNA. Both RdDM and the miRNA pathways involve related proteins from the Argonaute and Dicer families.

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