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166-534: 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 (TE) activity. RdDM has also been linked to pathogen defense, abiotic stress responses, and

332-407: A predator species and its prey (Vermeij, 1987), or a parasite and its host . Alternatively, the arms race may be between members of the same species, as in the manipulation/sales resistance model of communication (Dawkins & Krebs, 1979) or as in runaway evolution or Red Queen effects. One example of an evolutionary arms race is in sexual conflict between the sexes, often described with

498-486: A CpG island while only about 6% of enhancer sequences have a CpG island. CpG islands constitute regulatory sequences, since if CpG islands are methylated in the promoter of a gene this can reduce or silence gene transcription. DNA methylation regulates gene transcription through interaction with methyl binding domain (MBD) proteins , such as MeCP2 , MBD1 and MBD2 . These MBD proteins bind most strongly to highly methylated CpG islands . These MBD proteins have both

664-478: 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 ( EVD )

830-482: 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 (also see 'Biotic stresses' and 'Transgene silencing' below). When TEs integrate near genes, RdDM-mediated silencing of the TEs often affects gene expression. However, this

996-422: A basic bacterial gene is dependent on the strength of its promoter and the presence of activators or repressors. In the absence of other regulatory elements, a promoter's sequence-based affinity for RNA polymerases varies, which results in the production of different amounts of transcript. The variable affinity of RNA polymerase for different promoter sequences is related to regions of consensus sequence upstream of

1162-531: A bat-free habitat to ultrasound and found that all of the adventive species reacted to the ultrasound by slowing their flight times, while only one of the endemic species reacted to the ultrasound signal, indicating a loss of hearing over time in the endemic population. However, the degree of loss or regression depends on the amount of evolutionary time and whether or not the moth species has developed secondary uses for hearing. Some bats are known to use clicks at frequencies above or below moths' hearing ranges. This

1328-730: A byproduct of these PTGS pathways, leading to the initial establishment of a silent, heterochromatic state over the new TE or other target locus. Once that initial silent state is established, Pol IV can be recruited to the locus by CLSY and SHH1, and the canonical RdDM pathway takes over the long-term maintenance of silencing. Therefore, the non-canonical RdDM pathways often act as a temporary bridge between initial post-transcriptional silencing of novel elements by RNAi, and long-term transgenerational transcriptional silencing via canonical RdDM. Consistent with this role in initiation of novel silencing, non-canonical RdDM targets relatively few loci in comparison to canonical RdDM. The primary difference between

1494-574: A chromosome different from one where the promoter resides. Proximal enhancers or promoters of neighboring genes can serve as platforms to recruit more distal elements. Up-regulated expression of genes in mammals can be initiated when signals are transmitted to the promoters associated with the genes. Cis-regulatory DNA sequences that are located in DNA regions distant from the promoters of genes can have very large effects on gene expression, with some genes undergoing up to 100-fold increased expression due to such

1660-518: A cis-regulatory sequence. These cis-regulatory sequences include enhancers , silencers , insulators and tethering elements. Among this constellation of sequences, enhancers and their associated transcription factor proteins have a leading role in the regulation of gene expression. Enhancers are sequences of the genome that are major gene-regulatory elements. Enhancers control cell-type-specific gene expression programs, most often by looping through long distances to come in physical proximity with

1826-434: A combination of specific histone modification and DNA methylation patterns. Repressive chromatin modifications, like DNA methylation, help promote DNA compaction and reduce DNA accessibility, while other modifications help open chromatin and increase accessibility. Methylation of the 9th lysine of histone H3 (H3K9), primarily in the form of H3K9 trimethylation ( H3K9me3 ) in animals and H3K9 dimethylation ( H3K9me2 ) in plants,

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1992-454: A defense response, though to date this has only been shown for RNAi, not RdDM. This section focuses on the pathways and mechanisms by which RdDM leads to sequence-specific DNA methylation. The pathways presented here were characterized primarily in the model plant Arabidopsis thaliana , but are likely similar in other angiosperms. Conservation of RdDM in other plant species is discussed in more detail in 'Evolutionary conservation' below. RdDM

2158-506: A eukaryotic cell carries with it an increase in the complexity of transcriptional regulation. Eukaryotes have three RNA polymerases, known as Pol I , Pol II , and Pol III . Each polymerase has specific targets and activities, and is regulated by independent mechanisms. There are a number of additional mechanisms through which polymerase activity can be controlled. These mechanisms can be generally grouped into three main areas: All three of these systems work in concert to integrate signals from

2324-433: A human cell ) generally bind to specific motifs on an enhancer and a small combination of these enhancer-bound transcription factors, when brought close to a promoter by a DNA loop, govern the level of transcription of the target gene. Mediator (coactivator) (a complex usually consisting of about 26 proteins in an interacting structure) communicates regulatory signals from enhancer DNA-bound transcription factors directly to

2490-402: A hypothetical example, the factors A and B might regulate a distinct set of genes from the combination of factors A and C. This combinatorial nature extends to complexes of far more than two proteins, and allows a very small subset (less than 10%) of the genome to control the transcriptional program of the entire cell. Much of the early understanding of transcription came from bacteria, although

2656-500: 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

2822-458: A megabase long called Topological association domains (TADs) containing dozens of genes regulated by hundreds of enhancers distributed within large genomic regions containing only non-coding sequences. The function of TADs is to regroup enhancers and promoters interacting together within a single large functional domain instead of having them spread in different TADs. However, studies of mouse development point out that two adjacent TADs may regulate

2988-405: A megabase of each other or of low occupancy and inside TADs. High occupancy sites are usually conserved and static while intra-TADs sites are dynamic according to the state of the cell therefore TADs themselves are compartmentalized in subdomains that can be called subTADs from few kb up to a TAD long (19). When architectural binding sites are at less than 100 kb from each other, Mediator proteins are

3154-556: A methyl-CpG-binding domain as well as a transcription repression domain. They bind to methylated DNA and guide or direct protein complexes with chromatin remodeling and/or histone modifying activity to methylated CpG islands. MBD proteins generally repress local chromatin such as by catalyzing the introduction of repressive histone marks, or creating an overall repressive chromatin environment through nucleosome remodeling and chromatin reorganization. Transcription factors are proteins that bind to specific DNA sequences in order to regulate

3320-480: A new predator, competitor, etc. This should not seem surprising, as one species may have been in evolutionary struggles for millions of years while the other might never have faced such pressures. This is a common problem in isolated ecosystems such as Australia or the Hawaiian Islands . In Australia, many invasive species , such as cane toads and rabbits , have spread rapidly due to a lack of competition and

3486-413: A phenomenon called transgene silencing. The discovery of transgene silencing in the 1990s spurred a great deal of interest in understanding the mechanisms behind this silencing. Researchers found that transgene silencing was ubiquitous, occurring in multiple species (including Arabidopsis, Tobacco, and Petunia), and was associated with increased DNA methylation over and around the silenced transgene. Around

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3652-470: A physical competition to occupy the site of binding. If the repressor has a higher affinity for its motif than the activator, transcription would be effectively blocked in the presence of the repressor. Tight regulatory control is achieved by the highly dynamic nature of transcription factors. Again, many different mechanisms exist to control whether a transcription factor is active. These mechanisms include control over protein localization or control over whether

3818-448: A plant. Transgene silencing by RdDM and other mechanisms has therefore proved problematic for plant researchers. Efforts to understand how transgenes become silenced have ultimately helped reveal much of what we now know about the RdDM pathway (see 'History and discovery of RdDM'). In one early example, researchers sequentially transformed plants with two different transgenes that shared some of their DNA sequence. They found that transforming

3984-404: A polymerase is successfully bound to a DNA template, it often requires the assistance of other proteins in order to leave the stable promoter complex and begin elongating the nascent RNA strand. This process is called promoter escape, and is another step at which regulatory elements can act to accelerate or slow the transcription process. Similarly, protein and nucleic acid factors can associate with

4150-399: A result they are often recognized as foreign invaders and targeted for silencing by RdDM and PTGS. Since transgene silencing was a reliable marker of RdDM activity, researchers were able to design genetic screens to identify mutants that failed to trigger silencing at transgenes, reasoning that these genes were likely to be involved in the RdDM pathway. These experiments revealed many parts of

4316-515: A set of interchangeable catalytic subunits – two in the case of Pol IV and three in the case of Pol V – that provide additional specialization of polymerase functionality. While differences exist, overall there is a broad overlap in RdDM functions and components between the different angiosperm species studied to date. Outside of Pol IV and Pol V, a large proportion of key RdDM component proteins (for example, DCL3 and AGO4) have orthologs found within each class of land plants, which provides support for

4482-574: A shared origin of Pol IV and V dating back to early land / vascular plants. Much of the work done to elucidate the genes and proteins involved in the RdDM pathway has been performed in Arabidopsis thaliana , a model angiosperm. However, studies of Pol IV and V conducted in maize show some key differences with Arabidopsis. Maize Pol IV and V differ from each other in terms of only one subunit (the largest one). In Arabidopsis, Pol IV and V differ from each other in terms of three subunits. However, maize utilizes

4648-644: A similar effect, suggesting that multiple different factors involved in maintaining heterochromatin likely facilitate RdDM-mediated DNA methylation maintenance. Loss of RdDM leads to strong loss of non-CG methylation at TEs in gene-rich regions in the chromosome arms, but has little effect on DNA methylation levels in the constitutive heterochromatin around the centromere. This suggests that CMT2 and CMT3, which function primarily to maintain CHG and CHH methylation in dense constitutive heterochromatin, do not depend on RdDM activity. Similarly, in cmt2,cmt3 double mutants, many TEs in

4814-482: A similar function in propagating H3K9me2. More recently, a plant-specific protein, Agenet Domain Containing Protein 1 (ADCP1), was also identified that may function analogously to HP1 in maintaining H3K9me2 levels in heterochromatin, facilitating heterochromatin formation. Ultimately, the constant reinforcement of silencing chromatin modifications at heterochromatic loci creates a repressive chromatin state wherein

4980-511: 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. Perhaps the most analogous pathways to RdDM in another eukaryotic kingdom are

5146-547: A small fraction of 21-22nt sRNAs are involved in RdDM, with the majority instead driving a positive feedback loop amplifying the PTGS response. The functional outcome of a specific 21-22 nt sRNA depends on the AGO protein it ultimately associates with: sRNAs that associate with AGO4, AGO6 or AGO9 result in RdDM and DNA methylation, while sRNAs that associate with other AGOs, like AGO1, primarily result in PTGS. By using 21-22 nt sRNAs derived from

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5312-469: A suite of genes the cell wishes to express in response to some external stimuli such as stress. In addition to processes that regulate transcription at the stage of initiation, mRNA synthesis is also controlled by the rate of transcription elongation. RNA polymerase pauses occur frequently and are regulated by transcription factors, such as NusG and NusA, transcription-translation coupling , and mRNA secondary structure. The added complexity of generating

5478-1310: A variety of sources, non-canonical RdDM can flexibly induce de novo DNA methylation and silencing at many different types of loci. One of the primary sources of 21-22 nt sRNAs is Pol II transcripts. Some of these transcripts, particularly those produced from TEs, viruses, or certain non-protein-coding transcripts, are targeted by PTGS pathways like miRNAs or RNAi, leading to cleavage of the transcript. The resulting fragments can be converted into dsRNA by RDR6 and then processed into 21-22 nt sRNAs by DCL2 or DCL4 . Most of these 21-22 nt sRNAs are loaded into AGO1 and feed back into PTGS, amplifying PTGS efficiency. However, some will instead associate with AGO6, leading to RdDM. dsRNAs resulting from RDR6 activity can also sometimes processed by DCL3 instead of DCL2/4 and trigger RdDM. Additionally, some Pol II transcripts contain inverted repeat sequences, which can form double-stranded hairpin-like structures. These can be cleaved by DCL proteins independent of RDRs to produce either 21-22 nt or 24 nt sRNAs that can participate in RdDM. Similarly, miRNA precursors, which also form hairpin structures and are normally cleaved by DCL1 to produce miRNAs, can instead be cleaved by other DCLs to form sRNAs for RdDM. While most non-canonical RdDM occurs via AGO6 or AGO4, there

5644-470: A vital role in maintaining heterochromatin by propagating H3K9 methylation through a positive feedback loop with the H3K9 methyltransferase SUV39H. H3K9 methylation recruits HP1, which recruits SUV39H to deposit more H3K9 methylation. Though HP1 is conserved in plants, its function in this feedback loop is not. Instead, the positive feedback loops between H3K9me2 and the RdDM and CMT2/3 DNA methylation pathways fulfill

5810-496: A way to reinforce DNA methylation and silencing of TEs in developmentally important cell types, like germ cells and stem cells. Silencing TEs and maintaining genome integrity in these cells is particularly important because they give rise to many other cells, all of which will inherit any defects or mutations in the original stem cell or germ cell. sRNA movement is also involved in plant-pathogen interactions: sRNAs can move from infected cells to distal uninfected tissues in order to prime

5976-556: 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

6142-420: A wide variety of mechanisms. In one mechanism, CpG methylation influences binding of most transcription factors to DNA—in some cases negatively and in others positively. In addition, often they are at the end of a signal transduction pathway that functions to change something about the factor, like its subcellular localization or its activity. Post-translational modifications to transcription factors located in

6308-546: Is a highly conserved repressive modification. Lack of H3K4 methylation (H3K4me0) is also associated with repression, along with several other histone modifications and variants . The combination of DNA methylation, H3K9me2, and H3K4me0 is strongly associated with heterochromatin in plants. Since DNA methylation and repressive histone modifications together define heterochromatin, most DNA methylation pathways in plants recognize and interact with repressive histone marks and vice versa, forming positive feedback loops that help maintain

6474-408: Is a key regulator of DNA methylation in dense heterochromatin, but regulates sites mostly independently from RdDM. Interactions between RdDM and the other three maintenance DNA methylation pathways are limited and predominantly indirect. The DNA methyltransferase MET1 robustly maintains CG methylation genome-wide, including at RdDM target sites. In RdDM mutants, non-CG methylation at RdDM target sites

6640-412: Is a particular transcription factor that is important for regulation of methylation of CpG islands. An EGR1 transcription factor binding site is frequently located in enhancer or promoter sequences. There are about 12,000 binding sites for EGR1 in the mammalian genome and about half of EGR1 binding sites are located in promoters and half in enhancers. The binding of EGR1 to its target DNA binding site

6806-786: Is also a version of the pathway where sRNAs instead associate with AGO2, which together with the NERD complex (Needed for RDR2-independent DNA methylation) recruits DRM2 to target loci and triggers DNA methylation. Since the non-canonical pathways are not yet as well characterized as the canonical RdDM pathway, there likely remain additional sources of sRNAs used for RdDM that have not yet been uncovered. A number of factors involved in RdDM are listed below, along with additional details about their function and corresponding references. Several factors primarily involved in PTGS that sometimes participate in RdDM are also listed. Different chromatin states, like active euchromatin or silent heterochromatin, are defined by

RNA-directed DNA methylation - Misplaced Pages Continue

6972-500: Is also altered in response to signals. The three mammalian DNA methyltransferasess (DNMT1, DNMT3A, and DNMT3B) catalyze the addition of methyl groups to cytosines in DNA. While DNMT1 is a “maintenance” methyltransferase, DNMT3A and DNMT3B can carry out new methylations. There are also two splice protein isoforms produced from the DNMT3A gene: DNA methyltransferase proteins DNMT3A1 and DNMT3A2. The splice isoform DNMT3A2 behaves like

7138-449: 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. In this study, the persistence of the stress-induced DNA methylation changes required several RdDM-related proteins, suggesting that RdDM was involved in maintaining the stress-altered DNA methylation patterns. In another example, resistance to insect attack

7304-546: 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. RdDM helps plants respond to a number of abiotic stresses, such as heat stress, drought, phosphate starvation, salt stress, and others. Many TEs become upregulated under abiotic stress conditions, and thus one function of RdDM in stress response

7470-430: Is also involved in protecting the plant from other biotic stresses, including bacterial infections, fungal infections, and predation. Loss of RdDM can have opposing effects on resistance for different pathogens. For example, some RdDM mutants have increased susceptibility to the bacterium Agrobacterium tumefaciens , but those same mutants have decreased susceptibility to the bacterium Pseudomonas syringae , highlighting

7636-581: Is also recognized by the histone methyltransferases SUVH4/KYP, SUVH5, and SUVH6, which bind to non-CG methylation and add H3K9me2 to nearby histones, closing the positive feedback loop. Similarly, CMT3 and CMT2, the two DNA methyltransferases involved in the maintenance of CHG and CHH methylation respectively, both bind and add DNA methylation to H3K9me2-marked heterochromatin, forming their own feedback loop with SUVH4/5/6. These interactions help strongly reinforce silencing at TEs and other heterochromatic regions. A similar feedback loop occurs in animals. HP1 plays

7802-628: Is also strongly reduced in plants with defective RdDM that lose the ability to methylate that TE and others. This general mechanism helps maintain DNA methylation homeostasis by tuning DNA demethylation activity to DNA methylation activity, helping to ensure that DNA methylation patterns can be stably maintained over time. While all eukaryotes share three RNA polymerases (RNA Pol I, II and III), plants have two additional polymerases, Pol IV and Pol V. Both Pol IV and V share an evolutionary origin, deriving from Pol II. In other eukaryotic kingdoms that lack these two specialized RNA polymerases, Pol II transcribes

7968-490: Is also the only pathway capable of adding DNA methylation de novo to previously unmethylated regions in plants. The RdDM pathway can be split up into two main processes: the production of sRNAs, and the recruitment of DNA methylation machinery by those sRNAs to specific target loci in the DNA. These two activities together constitute RdDM, and ultimately lead to DNA methylation being added to cytosines at specific target loci. The canonical RdDM pathway is, as its name suggests,

8134-477: 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 . 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. TEs are pieces of DNA that, when expressed, can move around

8300-442: Is distinct from eukaryotic transcription, whose basal state is to be off and where co-factors required for transcription initiation are highly gene dependent. Sigma factors are specialized bacterial proteins that bind to RNA polymerases and orchestrate transcription initiation. Sigma factors act as mediators of sequence-specific transcription, such that a single sigma factor can be used for transcription of all housekeeping genes or

8466-407: Is enhanced. The configuration of the genome is essential for enhancer-promoter proximity. Cell-fate decisions are mediated upon highly dynamic genomic reorganizations at interphase to modularly switch on or off entire gene regulatory networks through short to long range chromatin rearrangements. Related studies demonstrate that metazoan genomes are partitioned in structural and functional units around

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8632-584: Is first recruited to silent heterochromatin via its interaction with CLASSY (CLSY) proteins and SAWADEE homeodomain homolog 1 (SHH1) (also see 'Interactions between RdDM and other chromatin modifying pathways' below). Pol IV transcribes these regions to produce short single-stranded RNAs (ssRNAs) roughly 30 to 45 nucleotides in length, each of which is the precursor for a single sRNA. These ssRNAs are converted into double-stranded RNAs (dsRNAs) co-transcriptionally by RNA-directed RNA polymerase 2 (RDR2), which physically associates with Pol IV. The dsRNAs are then cleaved by

8798-624: Is followed by 3’ guanine or CpG sites ). 5-methylcytosine (5-mC) is a methylated form of the DNA base cytosine (see Figure). 5-mC is an epigenetic marker found predominantly within CpG sites. About 28 million CpG dinucleotides occur in the human genome. In most tissues of mammals, on average, 70% to 80% of CpG cytosines are methylated (forming 5-methylCpG or 5-mCpG). Methylated cytosines within 5’cytosine-guanine 3’ sequences often occur in groups, called CpG islands . About 60% of promoter sequences have

8964-477: Is insensitive to cytosine methylation in the DNA. While only small amounts of EGR1 transcription factor protein are detectable in cells that are un-stimulated, translation of the EGR1 gene into protein at one hour after stimulation is drastically elevated. Expression of EGR1 transcription factor proteins, in various types of cells, can be stimulated by growth factors, neurotransmitters, hormones, stress and injury. In

9130-420: Is known about how the RdDM and RNAi machinery distinguish between viral RNAs and RNAs produced by the host plant. Mutants defective in RdDM and other methylation-deficient mutants are often hypersensitive to viral infection. 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. RdDM

9296-418: Is known as the allotonic frequency hypothesis. It argues that the auditory systems in moths have driven their bat predators to use higher or lower frequency echolocation to circumvent the moth hearing. Barbastelle bats have evolved to use a quieter mode of echolocation, calling at a reduced volume and further reducing the volume of their clicks as they close in on prey moths. The lower volume of clicks reduces

9462-414: Is lost, but CG methylation is still maintained, suggesting that MET1 activity is independent of RdDM. However, although met1 mutants lose CG methylation as expected, they also lose much of their non-CG methylation, including at RdDM target loci. At these sites, silencing can still be initiated by RdDM in met1 mutants, but it is not maintained or transmitted to progeny, suggesting that MET1 is important for

9628-503: Is mainly cultivated in the North of France and Belgium. The oomycete Phytophthora infestans is responsible for the potato blight, in particular during the European famine in 1840. Zoospores (mobile spores, characteristics of oomycetes) are liberated by zoosporangia provided from a mycelium and brought by rain or wind before infecting tubers and leaves. Black colours appear on the plant because of

9794-440: 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

9960-892: 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,

10126-873: Is particularly true in the dense constitutive heterochromatin surrounding the centromere. In these regions, the chromatin remodeler DDM1 plays a crucial role in DNA methylation maintenance by displacing nucleosomes temporarily to allow methyltransferases and other factors access the DNA. However, since most RdDM targets are small TEs in open, accessible and gene-rich regions (see “TE silencing and genome stability”), few RdDM sites require DDM1. In fact, dense heterochromatin inhibits RdDM. By contrast, CMT2 and CMT3 preferentially function in constitutive heterochromatin and depend strongly on DDM1 to maintain silencing over these regions. Similarly, MET1, which maintains DNA methylation at CG sites after replication, requires DDM1 to access heterochromatin and maintain CG methylation in those regions. Thus, DDM1

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10292-588: Is relatively stable, and can undergo multiple rounds of transcription initiation. After the binding of TFIIB and TFIID, Pol II the rest of the GTFs can assemble. This assembly is marked by the post-translational modification (typically phosphorylation) of the C-terminal domain (CTD) of Pol II through a number of kinases. The CTD is a large, unstructured domain extending from the RbpI subunit of Pol II, and consists of many repeats of

10458-496: Is seen in isolated populations of newts, which have less toxin in their skin. There are geographic hotspots where levels of tetrodotoxin and resistance are extremely high, showing a close interaction between newts and snakes. The whelk predators used their own shell to open the shell of their prey, oftentimes breaking both shells of the predator and prey in the process. This led to the fitness of larger-shelled prey to be higher and then more selected for through generations, however,

10624-545: Is that angiosperms have the “most complete” 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. All eukaryotic kingdoms host some form of small RNAs. One such class of sRNAs

10790-616: Is the Piwi-interacting RNAs (piRNAs) . Much like in RdDM, piRNAs primarily function to target and silence transposons, particularly in the germline. 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 subfamily, which are absent from plants. MicroRNAs (miRNAs) are another class of small RNA with silencing properties. While miRNAs are in

10956-595: Is the only mechanism in plants that can add DNA methylation to cytosines regardless of sequence context. DNA methylation in plants is typically divided into three categories based on the sequence context of the methylated cytosine: CG, CHG, and CHH, where H is any nucleotide except G. These reflect the different sequence contexts targeted by several DNA methylation pathways in plants. These context-specific pathways are primarily involved in maintaining existing DNA methylation patterns. The highly conserved methyltransferase MET1 (homolog of mammalian DNMT1) maintains DNA methylation in

11122-402: Is to help counter this activation. In one example, the retrotransposon ONSEN is upregulated by heat stress, but normally remains suppressed by RdDM-associated sRNAs and can only transpose efficiently in heat-stressed plants that are also deficient in RdDM. More generally, in plants exposed to heat stress, several components of the RdDM pathway become upregulated, and mutations in some components of

11288-411: The histone acetyltransferases (HATs) , histone methyltransferases (HMTs) , and histone deacetylases (HDACs) , among others. These enzymes can add or remove covalent modifications such as methyl groups, acetyl groups, phosphates, and ubiquitin. Histone modifications serve to recruit other proteins which can either increase the compaction of the chromatin and sequester promoter elements, or to increase

11454-469: The FLOWERING WAGENINGEN ( FWA ) locus in Arabidopsis, which resulted in plants that flowered later than normal. The same study also showed that the inhibitory effect of VIGS on FWA and flowering can become stronger over the course of successful generations. Transcriptional regulation In molecular biology and genetics , transcriptional regulation is the means by which a cell regulates

11620-415: The S. pombe pathways and RdDM share many of the same components, like RNA-directed RNA polymerases and sRNAs, and have similar functions in maintaining heterochromatin. Introducing transgenes into organisms has been a widely used tool in plant genetics research for decades. However, researchers often find that their introduced transgenes are not expressed as strongly as expected, or sometimes even at all,

11786-551: The base excision repair pathway . In Arabidopsis, there are four proteins responsible for removing DNA methylation: Repressor of silencing 1 (ROS1), Demeter (DME), Demeter-like 2 (DML2), and Demeter-like 3 (DML3). These DNA glycosylases help prevent the spread of DNA methylation from RdDM targets to active genes. Loss of active DNA demethylation in ros1;dml2;dml3 triple mutants leads to a widespread increase in DNA methylation levels, whereas ectopic expression of ROS1 leads to progressive loss of DNA methylation at many loci, highlighting

11952-581: The cytosol can cause them to translocate to the nucleus where they can interact with their corresponding enhancers. Other transcription factors are already in the nucleus, and are modified to enable the interaction with partner transcription factors. Some post-translational modifications known to regulate the functional state of transcription factors are phosphorylation , acetylation , SUMOylation and ubiquitylation . Transcription factors can be divided in two main categories: activators and repressors . While activators can interact directly or indirectly with

12118-413: The endoribonuclease Dicer-like 3 ( DCL3 ) into 24 nucleotide (nt) sRNAs. Pol IV, RDR2, and DCL3 alone are sufficient for the production of 24 nt sRNAs in vitro , suggesting that while other factors involved in this part of the pathway may help increase efficiency or specificity, they are not required for Pol IV-mediated sRNA production. While nearly all 24 nt sRNAs involved in RdDM are produced through

12284-430: The endosperm during seed development in flowering plants. A few factors involved in the RdDM pathway are themselves imprinted (favoring expression from the paternal allele) 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

12450-414: 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

12616-409: The promoter sequence of the desired target gene into a virus. 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 targeted for PTGS and processed into sRNAs, some of which will be complementary to the original target gene's promoter. A subset of these sRNAs will recruit the RdDM machinery to

12782-625: The AGO-sRNA duplex finds and binds complementary sequences along an RNA ‘scaffold’ produced by the plant-specific RNA polymerase V (Pol V), with the help of interactions with Suppressor of Ty insertion 5-like (SPT5L), the Involved in de novo 2 - IDN2 Paralog (IDN2-IDP) complex, and the Pol V subunit NRPE1. This leads to the recruitment of the DNA methyltransferase enzyme Domains Rearranged Methyltransferase 2 (DRM2), which methylates nearby DNA. The mechanism by which

12948-574: The AGO-sRNA duplex recruits DRM2 is not yet well understood. Recent work has revealed a number of variations of the RdDM pathway, collectively referred to as non-canonical RdDM. Unlike canonical RdDM, the non-canonical pathways are generally involved in establishing initial DNA methylation at new target loci, like novel TE insertions, rather than maintaining existing heterochromatin. Actively expressing elements like new TE insertions are normally strongly targeted by post-transcriptional gene silencing (PTGS/RNAi) pathways. Non-canonical RdDM occurs primarily as

13114-625: The BRCA1 promoter (see Low expression of BRCA1 in breast and ovarian cancers ). Evolutionary arms race In evolutionary biology , an evolutionary arms race is an ongoing struggle between competing sets of co-evolving genes , phenotypic and behavioral traits that develop escalating adaptations and counter-adaptations against each other, resembling the geopolitical concept of an arms race . These are often described as examples of positive feedback . The co-evolving gene sets may be in different species, as in an evolutionary arms race between

13280-399: The CG context, while two conserved plant-specific methyltransferases, Chromomethylase 3 (CMT3) and CMT2, help maintain CHG and CHH methylation, respectively. Unlike these pathways, RdDM leads to the addition of DNA methylation at all cytosines regardless of their sequence context. Like MET1, CMT2 and CMT3, RdDM is primarily involved in maintaining existing DNA methylation patterns. However, RdDM

13446-486: The CTD. These phosphorylation events promote the transcription process and serve as sites of recruitment for mRNA processing machinery. All three of these kinases respond to upstream signals, and failure to phosphorylate the CTD can lead to a stalled polymerase at the promoter. In vertebrates, the majority of gene promoters contain a CpG island with numerous CpG sites . When many of a gene's promoter CpG sites are methylated

13612-432: The DNA and histones ( nucleosomes ) become tightly packed together. This helps silence gene expression by physically inhibiting access to the DNA, preventing RNA Polymerase II , transcription factors and other proteins from initiating transcription. However, this same compaction also prevents factors involved in heterochromatin maintenance from accessing the DNA, which could lead to the silent, compact state being lost. This

13778-455: The GFP fluorescence was lost: after grafting, the sRNAs being produced in the second plant's tissues were moving into the tissues of the first, GFP-expressing plant, and triggering silencing of GFP. The same study showed that a subset of these mobile sRNAs were triggering the addition of DNA methylation to the GFP locus via RdDM. Therefore, sRNAs involved in RdDM can act as signaling molecules and trigger

13944-514: The Pol IV-RDR2- DCL3 pathway, a small proportion are produced through other pathways. For example, some RNA Polymerase II (Pol 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. In the second part of the pathway, the RdDM DNA methylation machinery is guided to DNA sequences complementary to

14110-533: The RNA polymerase II (RNAP II) enzyme bound to the promoter. Enhancers, when active, are generally transcribed from both strands of DNA with RNA polymerases acting in two different directions, producing two eRNAs as illustrated in the Figure. An inactive enhancer may be bound by an inactive transcription factor. Phosphorylation of the transcription factor may activate it and that activated transcription factor may then activate

14276-416: The RNA polymerase to access the template, but generally lack specificity for different promoter sites. A large part of gene regulation occurs through transcription factors that either recruit or inhibit the binding of the general transcription machinery and/or the polymerase. This can be accomplished through close interactions with core promoter elements, or through the long distance enhancer elements. Once

14442-468: The RdDM machinery reduce heat tolerance, suggesting RdDM plays an important role during heat stress. In addition to regulating TEs under stress conditions, RdDM can also regulate genes in order to trigger appropriate stress responses. Under low humidity, leaves produce fewer stomata due to RdDM-mediated downregulation of two genes involved in stomatal development. Similarly, RdDM becomes downregulated in response to salt stress, and this has been shown to trigger

14608-511: The addition of DNA methylation at complementary loci in cells far away from where the sRNAs were originally generated. Since then, studies have shown that sRNAs can move and direct RdDM both from shoot to root and root to shoot, though the silencing effect is more robust when sRNAs move from shoot to root. Movement of sRNAs that drive RdDM activity plays an important role in plant development, including during reproduction and root development. In both cases, sRNA movement seems to function primarily as

14774-610: The addition of DNA methylation to the targeted promoter and silence the gene. This demonstrated that sRNAs could direct DNA methylation to specific loci. Later efforts showed that the sRNAs involved in RdDM were approximately 24-26 nt long, while the sRNAs associated with RNAi were only about 21-22 nt in length. Soon after, the identification of AGO4 and characterization of its role in RdDM led to predictions, later confirmed, that 24 nt sRNAs were associating with AGO4 and directing DNA methylation to complementary loci. Early work on transgene silencing and RdDM also identified SDE4 as required for

14940-400: The amount of RNA being produced through a variety of mechanisms. Bacteria and eukaryotes have very different strategies of accomplishing control over transcription, but some important features remain conserved between the two. Most importantly is the idea of combinatorial control, which is that any given gene is likely controlled by a specific combination of factors to control transcription. In

15106-418: The architectural proteins cooperate with cohesin. For subTADs larger than 100 kb and TAD boundaries, CTCF is the typical insulator found to interact with cohesion. In eukaryotes, ribosomal rRNA and the tRNAs involved in translation are controlled by RNA polymerase I (Pol I) and RNA polymerase III (Pol III) . RNA Polymerase II (Pol II) is responsible for the production of messenger RNA (mRNA) within

15272-740: The brain, when neurons are activated, EGR1 proteins are up-regulated and they bind to (recruit) the pre-existing TET1 enzymes which are highly expressed in neurons. TET enzymes can catalyse demethylation of 5-methylcytosine. When EGR1 transcription factors bring TET1 enzymes to EGR1 binding sites in promoters, the TET enzymes can demethylate the methylated CpG islands at those promoters. Upon demethylation, these promoters can then initiate transcription of their target genes. Hundreds of genes in neurons are differentially expressed after neuron activation through EGR1 recruitment of TET1 to methylated regulatory sequences in their promoters. The methylation of promoters

15438-607: The canonical and non-canonical RdDM pathways lies in the origin and biogenesis of the sRNAs involved. The canonical RdDM pathway involves 24 nt sRNAs, which are specific to that pathway and come predominantly from a single source (the Pol IV-RDR2 complex). In contrast, the non-canonical RdDM pathways involve 21-22 nt sRNAs from a variety of sources, allowing de novo DNA methylation to be initiated at many different types of loci. These 21-22 nt sRNAs are not specific to non-canonical RdDM, and also function in other PTGS pathways. In fact, only

15604-498: The cell and change the transcriptional program accordingly. While in prokaryotic systems the basal transcription state can be thought of as nonrestrictive (that is, “on” in the absence of modifying factors), eukaryotes have a restrictive basal state which requires the recruitment of other factors in order to generate RNA transcripts. This difference is largely due to the compaction of the eukaryotic genome by winding DNA around histones to form higher order structures. This compaction makes

15770-506: The cell. Particularly for Pol II, much of the regulatory checkpoints in the transcription process occur in the assembly and escape of the pre-initiation complex . A gene-specific combination of transcription factors will recruit TFIID and/or TFIIA to the core promoter, followed by the association of TFIIB , creating a stable complex onto which the rest of the General Transcription Factors (GTFs) can assemble. This complex

15936-514: The chromosome arms remain methylated, presumably due to the persistent activity of RdDM, indicating that loss of CMT2/3 has little effect on RdDM activity. This suggests that RdDM and CMT2/3 function mostly independently and at distinct loci: RdDM is the main pathway responsible for maintaining non-CG DNA methylation in euchromatic, gene rich regions, while CMT2 and CMT3 maintain non-CG DNA methylation in constitutive heterochromatin. In mutants defective in both RdDM and CMT2/CMT3, all non-CG methylation in

16102-439: The complexity of the different pathogen defense pathways and their interactions with RdDM. In addition to naturally-occurring foreign nucleic acid stressors like TEs and viruses, artificially introduced DNA sequences, like transgenes , are also targeted for repression by RdDM. Transgenes are widely used in genetics research to study gene function and regulation, and in plant breeding to introduce novel and desirable properties into

16268-560: The constrains on these same interactions. TAD boundaries are often composed by housekeeping genes, tRNAs, other highly expressed sequences and Short Interspersed Elements (SINE). While these genes may take advantage of their border position to be ubiquitously expressed, they are not directly linked with TAD edge formation. The specific molecules identified at boundaries of TADs are called insulators or architectural proteins because they not only block enhancer leaky expression but also ensure an accurate compartmentalization of cis-regulatory inputs to

16434-430: The conversion of DNA to RNA ( transcription ), thereby orchestrating gene activity . A single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus mount a response. Some examples of this include producing

16600-409: The core machinery of transcription through enhancer binding, repressors predominantly recruit co-repressor complexes leading to transcriptional repression by chromatin condensation of enhancer regions. It may also happen that a repressor may function by allosteric competition against a determined activator to repress gene expression: overlapping DNA-binding motifs for both activators and repressors induce

16766-534: 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

16932-412: The effective successful hunting range, but results in a significantly higher number of moths caught than other, louder bat species. Moths have further evolved the ability to discriminate between high and low echolocation click rates, which indicates whether the bat has just detected their presence or is actively pursuing them. This allows them to decide whether or not defensive ultrasonic clicks are worth

17098-468: The elongation complex and modulate the rate at which the polymerase moves along the DNA template. In eukaryotes, genomic DNA is highly compacted in order to be able to fit it into the nucleus. This is accomplished by winding the DNA around protein octamers called histones , which has consequences for the physical accessibility of parts of the genome at any given time. Significant portions are silenced through histone modifications, and thus are inaccessible to

17264-720: The enhancer to which it is bound (see small red star representing phosphorylation of a transcription factor bound to an enhancer in the illustration). An activated enhancer begins transcription of its RNA before activating a promoter to initiate transcription of messenger RNA from its target gene. Transcriptional initiation, termination and regulation are mediated by “DNA looping” which brings together promoters, enhancers, transcription factors and RNA processing factors to accurately regulate gene expression. Chromosome conformation capture (3C) and more recently Hi-C techniques provided evidence that active chromatin regions are “compacted” in nuclear domains or bodies where transcriptional regulation

17430-536: The expression of a gene. The binding sequence for a transcription factor in DNA is usually about 10 or 11 nucleotides long. As summarized in 2009, Vaquerizas et al. indicated there are approximately 1,400 different transcription factors encoded in the human genome by genes that constitute about 6% of all human protein encoding genes. About 94% of transcription factor binding sites (TFBSs) that are associated with signal-responsive genes occur in enhancers while only about 6% of such TFBSs occur in promoters. EGR1 protein

17596-500: The expression of a given gene. There are approximately 1,400 transcription factors in the human genome and they constitute about 6% of all human protein coding genes. The power of transcription factors resides in their ability to activate and/or repress wide repertoires of downstream target genes. The fact that these transcription factors work in a combinatorial fashion means that only a small subset of an organism's genome encodes transcription factors. Transcription factors function through

17762-433: The expression of a transcription factor important in salt stress resistance. RdDM was initially discovered as a response to infection by viroids, 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 pathways to degrade viral RNA (RNAi) and silence viral DNA (RdDM). However, little

17928-435: The extent and complexity of transcriptional regulation is greater in eukaryotes. Bacterial transcription is governed by three main sequence elements: While these means of transcriptional regulation also exist in eukaryotes, the transcriptional landscape is significantly more complicated both by the number of proteins involved as well as by the presence of introns and the packaging of DNA into histones . The transcription of

18094-441: The form of protein repressors and positive control elements can either increase or decrease transcription. Repressors often physically occupy the promoter location, occluding RNA polymerase from binding. Alternatively a repressor and polymerase may bind to the DNA at the same time with a physical interaction between the repressor preventing the opening of the DNA for access to the minus strand for transcription. This strategy of control

18260-686: The gene becomes silenced. Colorectal cancers typically have 3 to 6 driver mutations and 33 to 66 hitchhiker or passenger mutations. However, transcriptional silencing may be of more importance than mutation in causing progression to cancer. For example, in colorectal cancers about 600 to 800 genes are transcriptionally silenced by CpG island methylation (see regulation of transcription in cancer ). Transcriptional repression in cancer can also occur by other epigenetic mechanisms, such as altered expression of microRNAs . In breast cancer, transcriptional repression of BRCA1 may occur more frequently by over-expressed microRNA-182 than by hypermethylation of

18426-418: The gene promoter inaccessible without the assistance of other factors in the nucleus, and thus chromatin structure is a common site of regulation. Similar to the sigma factors in prokaryotes, the general transcription factors (GTFs) are a set of factors in eukaryotes that are required for all transcription events. These factors are responsible for stabilizing binding interactions and opening the DNA helix to allow

18592-462: The genome is eliminated, demonstrating that together RdDM and CMT2/CMT3 account for all non-CG methylation in the genome. Most DNA methylation mechanisms in plants are self-reinforcing (see above), including RdDM: Pol IV and Pol V are both recruited to heterochromatic regions that already have DNA methylation, encouraging additional DNA methylation via canonical RdDM. Positive feedback loops like these can cause DNA methylation activity to spread out from

18758-499: 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

18924-460: 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

19090-417: The genome, and these sRNAs direct the addition of K3K9 methylation to maintain/spread heterochromatin. Meanwhile, CTGS is directed by AGO1-bound sRNAs, similar to PTGS within plants, and results in the inhibition of transcription by Pol II, as well as to Pol II release. Unlike RdDM, TGS and CTGS in S. pombe do not rely on transcription from non-Pol II sources or lead to the addition of DNA methylation. However,

19256-456: The heptad sequence YSPTSPS. TFIIH , the helicase that remains associated with Pol II throughout transcription, also contains a subunit with kinase activity which will phosphorylate the serines 5 in the heptad sequence. Similarly, both CDK8 (a subunit of the massive multiprotein Mediator complex) and CDK9 (a subunit of the p-TEFb elongation factor), have kinase activity towards other residues on

19422-425: The highly toxic frogs, with the frogs that discharge mucus somewhere in between. The snakes would also spend generously more time gaped between the release of the highly toxic frogs than the short gaped time between the release of the frogs that discharge mucus. Therefore, the snakes have a much higher advantage of being able to cope with the different frogs defensive mechanisms, while the frogs could eventually increase

19588-652: The hypothesis that some form of the RdDM pathway evolved early within the plant lineage. However, RdDM pathway functionality does appear to change to an appreciable extent between different plant species and lineages. For example, while gymnosperms have functional Pol IV and produce 24 nt small RNAs, the biogenesis of sRNAs within gymnosperms is much more heavily skewed towards 21 nt than 24 nt sRNAs. This suggests that canonical RdDM may be rarer or less pronounced in gymnosperms than in angiosperms. Similarly, while orthologs of DRM2 are found in various angiosperms, there are no known DRM2 orthologs in other plant lineages. One possibility

19754-411: The importance of balancing DNA methylation and demethylation activity. Interestingly, expression of the DNA demethylase ROS1 is directly tied to RdDM activity: DNA methylation over a TE targeted by RdDM in the ROS1 promoter is required for ROS1 expression, though other factors are also involved in regulating ROS1 . Since ROS1 expression is tied to DNA methylation at a specific TE, ROS1 expression

19920-517: The individual fitness gain. Genetic change accumulation in both populations explains a constant adaptation to have lower fitness costs and avoid extinction in accordance with the Red Queen's hypothesis suggested by Leigh Van Valen in 1973. The Bintje potato is derived from a cross between Munstersen and Fransen potato varieties. It was created in the Netherlands in the early 20th century and now

20086-419: The infection of its cellular system necessary for the multiplication of the oomycete infectious population. The parasite contains virulent-avirulent allelic combinations in several microsatellite loci, likewise the host contains several multiloci resistance genes (or R gene ). That interaction is called gene-for-gene relationship and is, in general, widespread in plant diseases. Expression of genetic patterns in

20252-541: The intended methylated target sites into genes or other regulatory elements, which can negatively affect gene expression. To prevent this spreading, DNA methylation pathways are opposed by passive and active DNA demethylation. DNA methylation can be lost passively with each cell division, because newly synthesized strands of DNA lack DNA methylation until it is re-added by one of the maintenance DNA methylation pathways. DNA methylation can also be actively removed in plants by DNA glycosylases , which remove methylated cytosines via

20418-473: The mRNA that encode enzymes to adapt to a change in a food source, producing the gene products involved in cell cycle specific activities, and producing the gene products responsible for cellular differentiation in multicellular eukaryotes, as studied in evolutionary developmental biology . The regulation of transcription is a vital process in all living organisms. It is orchestrated by transcription factors and other proteins working in concert to finely tune

20584-472: The maintenance, but not initiation, of silencing at a subset of RdDM target loci. This effect is likely indirect: loss of MET1 leads to loss of H3K9me2 at some sites, which inhibits the recruitment of Pol IV and therefore prevents maintenance of DNA methylation via canonical RdDM, although the non-canonical pathways (which do not involve Pol IV) are not affected. Loss of the histone deacetylase HDA6, which facilitates maintenance methylation by MET1 at some loci, has

20750-475: The most well-characterized RdDM pathway to date. Canonical RdDM is preferentially recruited to regions that are already DNA-methylated and heterochromatic, and acts to reinforce existing DNA methylation patterns at these loci, forming a positive feedback loop. Canonical RdDM makes up the majority of RdDM activity in a cell. The first part of the RdDM pathway revolves around the biogenesis of sRNAs. A plant-specific RNA polymerase complex, RNA Polymerase IV (Pol IV),

20916-416: The necessity for the pathogen to have the best virulent alleles to infect the organism and for the host to have the best resistant alleles to survive parasitism. As a consequence, allele frequencies vary through time depending on the size of virulent and resistant populations (fluctuation of genetic selection pressure) and generation time (mutation rate) where some genotypes are preferentially selected thanks to

21082-571: 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

21248-532: The pathway, including RNA Pol IV and V, Dicer-like proteins , Argonautes, and others. The involvement of sRNAs in RdDM was initially suspected due to the similarity between RdDM and RNAi, the latter of which had recently been shown to involve small RNAs. To test whether sRNAs were involved in RdDM, RNA hairpin structures complementary to a specific gene promoter were introduced into Arabidopsis and Tobacco. The hairpin RNAs were processed into sRNAs, which were able to trigger

21414-471: The plant via the vasculature. They therefore have the potential to act as signaling molecules. This has been demonstrated in plants engineered to express green fluorescent protein (GFP). 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,

21580-470: The polymerases or their cofactors. The highest level of transcription regulation occurs through the rearrangement of histones in order to expose or sequester genes, because these processes have the ability to render entire regions of a chromosome inaccessible such as what occurs in imprinting. Histone rearrangement is facilitated by post-translational modifications to the tails of the core histones. A wide variety of modifications can be made by enzymes such as

21746-422: The potency of their toxic knowing the snakes would adapt to that change as well, such as the snakes having venom themselves for the initial attack. The coevolution is still highly asymmetrical because of the advantage the predators have over their prey. When a species has not been subject to an arms race previously, it may be at a severe disadvantage and face extinction well before it could ever hope to adapt to

21912-486: The potential to be maintained and transmitted to future generations. This can allow stress-induced DNA methylation changes to act as a ‘memory’ of the stressor and help prime the plant or its progeny to respond more efficiently to the stress if re-exposed. For example, RdDM-derived sRNAs against TEs or viruses that have already integrated into the genome and been silenced serve as a 'memory' of those prior infections, protecting against future invasions by similar sequences. There

22078-440: The precursors of small RNAs used in silencing pathways – in fact, Pol II transcripts are also sometimes processed into sRNAs in plants. It has been hypothesized that the origin of both Pol IV and Pol V is rooted in “escape from adaptive conflict”. The idea is that potential tensions between the “traditional” function of Pol II and the small RNA biogenesis function could be relieved by duplication of Pol II and subfunctionalization of

22244-424: The predator's population selected for those who were more efficient at opening the larger-shelled prey. This example is an excellent example of asymmetrical arms race because while the prey is evolving a physical trait, the predators are adapting in a much different way. Floodplain death adders eat three types of frogs: one nontoxic, one producing mucus when taken by the predator, and the highly toxic frogs, however,

22410-544: The product of a classical immediate-early gene and, for instance, it is robustly and transiently produced after neuronal activation. Where the DNA methyltransferase isoform DNMT3A2 binds and adds methyl groups to cytosines appears to be determined by histone post translational modifications. On the other hand, neural activation causes degradation of DNMT3A1 accompanied by reduced methylation of at least one evaluated targeted promoter. Transcription factors are proteins that bind to specific DNA sequences in order to regulate

22576-424: The production of most sRNAs involved in RdDM. SDE4 would later be identified as the largest subunit of Pol IV, and renamed NRPD1. A number of studies published in quick succession from multiple research groups, utilizing both forward and reverse genetic approaches, went on to identify and characterize Pol IV and Pol V as highly specialized plant RNA polymerases involved in RdDM. The Pol IV / Pol V naming convention

22742-462: The promoter of a target gene. The loop is stabilized by a dimer of a connector protein (e.g. dimer of CTCF or YY1 ), with one member of the dimer anchored to its binding motif on the enhancer and the other member anchored to its binding motif on the promoter (represented by the red zigzags in the illustration). Several cell function specific transcription factor proteins (in 2018 Lambert et al. indicated there were about 1,600 transcription factors in

22908-453: The promoter or within the first intron of the regulated gene, or distal, in introns of neighboring genes or intergenic regions far away from the locus. Through DNA looping, active enhancers contact the promoter dependently of the core DNA binding motif promoter specificity. Promoter-enhancer dichotomy provides the basis for the functional interaction between transcription factors and transcriptional core machinery to trigger RNA Pol II escape from

23074-411: The promoter. Whereas one could think that there is a 1:1 enhancer-promoter ratio, studies of the human genome predict that an active promoter interacts with 4 to 5 enhancers. Similarly, enhancers can regulate more than one gene without linkage restriction and are said to “skip” neighboring genes to regulate more distant ones. Even though infrequent, transcriptional regulation can involve elements located in

23240-487: The promoters of their target genes. In a study of brain cortical neurons, 24,937 loops were found, bringing enhancers to promoters. Multiple enhancers, each often at tens or hundred of thousands of nucleotides distant from their target genes, loop to their target gene promoters and coordinate with each other to control expression of their common target gene. The schematic illustration in this section shows an enhancer looping around to come into close physical proximity with

23406-610: The protein can bind DNA. An example of this is the protein HSF1 , which remains bound to Hsp70 in the cytosol and is only translocated into the nucleus upon cellular stress such as heat shock. Thus the genes under the control of this transcription factor will remain untranscribed unless the cell is subjected to stress. Enhancers or cis-regulatory modules/elements (CRM/CRE) are non-coding DNA sequences containing multiple activator and repressor binding sites. Enhancers range from 200 bp to 1 kb in length and can be either proximal, 5’ upstream to

23572-568: The protein in such a way as to hamper or prevent binding of the toxin, conferring resistance. In turn, resistance creates a selective pressure that favors newts that produce more toxin. That in its turn imposes a selective pressure favoring snakes with mutations conferring even greater resistance. This evolutionary arms race has resulted in the newts producing levels of toxin far in excess of that needed to kill any other predator. In populations where garter snakes and newts live together, higher levels of tetrodotoxin and resistance to it are observed in

23738-479: 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

23904-579: The repressive chromatin state. The RdDM-associated protein SHH1 recognizes H3K4me0 and H3K9me2 at heterochromatic loci and recruits Pol IV to these loci to trigger additional DNA methylation at these regions. Similarly, SUVH2 and SUVH9 help recruit Pol V to loci with DNA methylation. Thus, both major parts of the canonical RdDM pathway are preferentially recruited to regions that are already in the silent, heterochromatic state marked by DNA methylation, H3K9me2, and H3K4me0. DNA methylation at these same heterochromatic loci

24070-571: The resulting multiple RNA polymerases. Analyses of evolutionary lineage for Pol IV and Pol V are complicated to some extent by the fact that each enzyme is actually composed of at least 12 subunits . In Arabidopsis thaliana , some subunits are shared between Pol IV and Pol V, some are unique to each polymerase, and some are shared between Pol II, IV, and V. Orthologs of certain Pol IV and V subunits have been found in all lineages of land plants, including ferns, liverworts, and mosses. These findings argue for

24236-487: The sRNA directed transcriptional gene silencing (TGS) and co-transcriptional gene silencing (CTGS) pathways in Schizosaccharomyces pombe . In S. pombe , TGS directs methylation of H3K9, leading to heterochromatin formation, and is directed by sRNAs produced from the targeted regions. Similar to canonical RdDM, this pathway is a positive feedback loop: sRNAs are generated preferentially from heterochromatin-rich areas of

24402-436: The sRNAs generated in the first part of the pathway. One strand from each 24 nt double-stranded sRNA is loaded into Argonaute (AGO) proteins AGO4, AGO6, or AGO9. AGO3 may also be able to function in this pathway. Argonautes are a large, highly conserved family of proteins that can bind sRNAs, forming a protein-sRNA duplex that enables them to recognize and bind other RNA sequences complementary to their sRNA partner. Once formed,

24568-514: The same gene cluster. The most relevant study on limb evolution shows that the TAD at the 5’ of the HoxD gene cluster in tetrapod genomes drives its expression in the distal limb bud embryos, giving rise to the hand, while the one located at 3’ side does it in the proximal limb bud, giving rise to the arm. Still, it is not known whether TADs are an adaptive strategy to enhance regulatory interactions or an effect of

24734-406: The same time in 1994, work in tobacco plants had revealed a new pathway involving RNAs that resulted in DNA methylation. Researchers found 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. The deposition of methylation over these foreign viroid sequences helped inhibit viroid replication, and

24900-479: The second transgene into the plants led to the first transgene gaining DNA methylation and becoming inactivated. This provided an early clue that there existed a trans-acting, sequence-based mechanism for transcriptional silencing of foreign DNA, later shown to be RdDM. Due to the heritability of DNA methylation patterns in plants, and the self-reinforcing nature of RdDM and other DNA methylation pathways, any DNA methylation changes caused by environmental stressors have

25066-429: The selective advantage for either species is increased height. An asymmetrical arms race involves contrasting selection pressures, such as the case of cheetahs and gazelles, where cheetahs evolve to be better at hunting and killing while gazelles evolve not to hunt and kill, but rather to evade capture. Selective pressure between two species can include host-parasite coevolution . This antagonistic relationship leads to

25232-425: The silent, heterochromatic state (see 'Interactions between RdDM and other chromatin modifying pathways'). 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

25398-402: The snakes have also found if they wait to consume their toxic prey the potency decreases. In this specific case, the asymmetry enabled the snakes to overcome the chemical defenses of the toxic frogs after their death. The results of the study showed that the snake became accustomed to the differences in the frogs by their hold and release timing, always holding the nontoxic, while always releasing

25564-475: The spacing between histones and allow the association of transcription factors or polymerase on open DNA. For example, H3K27 trimethylation by the polycomb complex PRC2 causes chromosomal compaction and gene silencing. These histone modifications may be created by the cell, or inherited in an epigenetic fashion from a parent. Transcription regulation at about 60% of promoters is controlled by methylation of cytosines within CpG dinucleotides (where 5’ cytosine

25730-541: The target gene to add DNA methylation. 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. 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

25896-403: The targeted promoter. These insulators are DNA-binding proteins like CTCF and TFIIIC that help recruiting structural partners such as cohesins and condensins. The localization and binding of architectural proteins to their corresponding binding sites is regulated by post-translational modifications. DNA binding motifs recognized by architectural proteins are either of high occupancy and at around

26062-432: The term Fisherian runaway . Thierry Lodé emphasized the role of such antagonistic interactions in evolution leading to character displacements and antagonistic coevolution . Arms races may be classified as either symmetrical or asymmetrical. In a symmetrical arms race, selection pressure acts on participants in the same direction. An example of this is trees growing taller as a result of competition for light, where

26228-421: The time and energy expenditure. Rough-skinned newts have skin glands that contain a powerful nerve poison, tetrodotoxin , as an anti-predator adaptation . Throughout much of the newt's range, the common garter snake is resistant to the toxin. While in principle the toxin binds to a tube-shaped protein that acts as a sodium channel in the snake's nerve cells, a mutation in several snake populations configures

26394-408: The transcription start site. The more nucleotides of a promoter that agree with the consensus sequence, the stronger the affinity of the promoter for RNA Polymerase likely is. In the absence of other regulatory elements, the default state of a bacterial transcript is to be in the “on” configuration, resulting in the production of some amount of transcript. This means that transcriptional regulation in

26560-625: The two species is a combination of resistance and virulence characteristics in order to have the best survival rate. Bats have evolved to use echolocation to detect and catch their prey. Moths have in turn evolved to detect the echolocation calls of hunting bats, and evoke evasive flight maneuvers, or reply with their own ultrasonic clicks to confuse the bat's echolocation. The Arctiidae subfamily of Noctuid moths uniquely respond to bat echolocation in three prevailing hypotheses: startle, sonar jamming, and acoustic aposematic defense. All these differences depend on specific environmental settings and

26726-582: The two species respectively. Where the species are separated, the toxin levels and resistance are lower. While isolated garter snakes have lower resistance, they still demonstrate an ability to resist low levels of the toxin, suggesting an ancestral predisposition to tetrodotoxin resistance. The lower levels of resistance in separated populations suggest a fitness cost of both toxin production and resistance. Snakes with high levels of tetrodotoxin resistance crawl more slowly than isolated populations of snakes, making them more vulnerable to predation. The same pattern

26892-459: The type of echolocation call; however, these hypotheses are not mutually exclusive and can be used by the same moth for defense. The different defense mechanisms have been shown to be directly responsive to bat echolocation through sympatry studies. In places with spatial or temporal isolation between bats and their prey, the moth species hearing mechanism tends to regress. Fullard et al. (2004) compared adventive and endemic Noctiid moth species in

27058-485: Was adopted shortly thereafter. Since the mechanism underlying the sequence-specificity of RdDM is well known, RdDM 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 genes. One method, called virus-induced gene silencing (VIGS), involves inserting part of

27224-604: 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. A number of epigenetic changes required for normal development and reproduction in flowering plants involve RdDM. In

27390-452: Was therefore 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. RdDM turned out to be the solution to the transgene mystery: like viroids and viruses, transgenes are foreign sequences, and as

27556-469: Was transmitted to progeny via DNA methylation changes, and this inheritance was also dependent on functional sRNA biogenesis pathways. Thus, RdDM can potentially alter the plant epigenome in response to stress, and helps maintain these changes to modulate future stress responses in the affected plant and its descendants. The sRNA molecules produced by RdDM and other pathways are able to move between cells via plasmodesmata, and can also move systemically through

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