The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation.

In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV's largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.

Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation.

Plant multisubunit RNA polymerase V (Pol V) transcription recruits Argonaute-small interfering RNA (siRNA) complexes that specify sites of RNA-directed DNA methylation (RdDM) for gene silencing. Pol V's largest subunit, NRPE1, evolved from the largest subunit of Pol II but has a distinctive C-terminal domain (CTD). We show that the Pol V CTD is dispensable for catalytic activity in vitro yet essential in vivo. One CTD subdomain (DeCL) is required for Pol V function at virtually all loci. Other CTD subdomains have locus-specific effects. In a yeast two-hybrid screen, the 3'→ 5' exoribonuclease RRP6L1 was identified as an interactor with the DeCL and glutamine-serine (QS)-rich subdomains located downstream of an Argonaute-binding subdomain. Experimental evidence indicates that RRP6L1 trims the 3' ends of Pol V transcripts sliced by Argonaute 4 (AGO4), suggesting a model whereby the CTD enables the spatial and temporal coordination of AGO4 and RRP6L1 RNA processing activities.

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair.

The AtRAD21.1 and AtRAD21.3 Arabidopsis cohesins play a synergistic role in somatic DNA double strand break damage repair.

BACKGROUND: The RAD21 cohesin plays, besides its well-recognised role in chromatid cohesion, a role in DNA double strand break (dsb) repair. In Arabidopsis there are three RAD21 paralog genes (AtRAD21.1, AtRAD21.2 and AtRAD21.3), yet only AtRAD21.1 has been shown to be required for DNA dsb damage repair. Further investigation of the role of cohesins in DNA dsb repair was carried out and is here reported. RESULTS: We show for the first time that not only AtRAD21.1 but also AtRAD21.3 play a role in somatic DNA dsb repair. Comet data shows that the lack of either cohesins induces a similar high basal level of DNA dsb in the nuclei and a slower DNA dsb repair kinetics in both cohesin mutants. The observed AtRAD21.3 transcriptional response to DNA dsb induction reinforces further the role of this cohesin in DNA dsb repair. The importance of AtRAD21.3 in DNA dsb damage repair, after exposure to DNA dsb damage inducing agents, is notorious and recognisably evident at the phenotypical level, particularly when the AtRAD21.1 gene is also disrupted. CONCLUSIONS: Our data demonstrates that both Arabidopsis cohesin (AtRAD21.1 and AtRAD21.3) play a role in somatic DNA dsb repair. Furthermore, the phenotypical data from the atrad21.1 atrad21.3 double mutant indicates that these two cohesins function synergistically in DNA dsb repair. The implications of this data are discussed.

The cytological and molecular role of domains rearranged methyltransferase3 in RNA-dependent DNA methylation of Arabidopsis thaliana.

BACKGROUND: Plants have evolved a unique epigenetic process to target DNA cytosine methylation: RNA-directed DNA methylation (RdDM). During RdDM, small RNAs (smRNAs) guide methylation of homologous DNA loci. In Arabidopsis thaliana, the de novo DNA methyltransferase that ultimately methylates cytosines guided by smRNAs in all sequence contexts is Domains Rearranged Methyltransferase 2 (DRM2). Recent reports have shown that DRM2 requires the catalytic mutated paralog DRM3 to exert its function through a still largely unknown process. To shed light on how DRM3 affects RdDM, we have further characterized its role at the molecular and cytological levels. FINDINGS: Although DRM3 is not required for RdDM loci transcriptional silencing, it specifically affects loci's DNA methylation. Interestingly, DRM3 and DRM2 regulate the DNA methylation in a subset of loci differently.Fluorescence In Situ Hybridization and immunolocalization analyses showed that DRM3 is not required for the large-scale nuclear organization of heterochromatin during interphase, with the notable exception of the 45S ribosomal RNA loci. DRM3 localizes exclusively to the nucleus and is enriched in a round-shaped domain located in the nucleolar periphery, in which it colocalizes with components of the RdDM pathway. CONCLUSIONS: Our analyses reinforce the previously proposed chaperone role of DRM3 in RdDM. Overall, our work further demonstrates that DRM3 most likely functions exclusively with DRM2 in RdDM and not with other A. thaliana DNA methyltransferases. However, DRM3's regulation of DNA methylation is likely target- or chromatin context-dependent. DRM3 hypothetically acts in RdDM either upstream of DRM2, or in a parallel step.

DNA topoisomerase 1α promotes transcriptional silencing of transposable elements through DNA methylation and histone lysine 9 dimethylation in Arabidopsis.

RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.

Connecting the dots of RNA-directed DNA methylation in Arabidopsis thaliana.

Noncoding RNAs are the rising stars of genome regulation and are crucial to an organism's metabolism, development, and defense. One of their most notable functions is its ability to direct epigenetic modifications through small RNA molecules to specific genomic regions, ensuring transcriptional regulation, proper genome organization, and maintenance of genome integrity. Here, we review the current knowledge of the spatial organization of the Arabidopsis thaliana RNA-directed DNA methylation pathway within the cell nucleus, which, while known to be essential for the proper establishment of epigenetic modifications, remains poorly understood. We will also discuss possible future cytological approaches that have the potential of unveiling functional insights into how small RNA-directed epigenetics is regulated through the spatiotemporal regulation of its major components within the cell.

Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states.

Eukaryotes can have thousands of 45S ribosomal RNA (rRNA) genes, many of which are silenced during development. Using fluorescence-activated sorting techniques, we show that active rRNA genes in Arabidopsis thaliana are present within sorted nucleoli, whereas silenced rRNA genes are excluded. DNA methyltransferase (met1), histone deacetylase (hda6), or chromatin assembly (caf1) mutants that disrupt silencing abrogate this nucleoplasmic-nucleolar partitioning. Bisulfite sequencing data indicate that active nucleolar rRNA genes are nearly completely demethylated at promoter CGs, whereas silenced genes are nearly fully methylated. Collectively, the data reveal that rRNA genes occupy distinct but changeable nuclear territories according to their epigenetic state.

Intersection of small RNA pathways in Arabidopsis thaliana sub-nuclear domains.

In Arabidopsis thaliana, functionally diverse small RNA (smRNA) pathways bring about decreased RNA accumulation of target genes via several different mechanisms. Cytological experiments have suggested that the processing of microRNAs (miRNAs) and heterochromatic small interfering RNAs (hc-siRNAs) occurs within a specific nuclear domain that can present Cajal Body (CB) characteristics. It is unclear whether single or multiple smRNA-related domains are found within the same CB and how specialization of the smRNA pathways is determined within this specific sub-compartment. To ascertain whether nuclear smRNA centers are spatially related, we localized key proteins required for siRNA or miRNA biogenesis by immunofluorescence analysis. The intranuclear distribution of the proteins revealed that hc-siRNA, miRNA and trans-acting siRNA (ta-siRNA) pathway proteins accumulate and colocalize within a sub-nuclear structure in the nucleolar periphery. Furthermore, colocalization of miRNA- and siRNA-pathway members with CB markers, and reduced wild-type localization patterns in CB mutants indicates that proper nuclear localization of these proteins requires CB integrity. We hypothesize that these nuclear domains could be important for RNA silencing and may partially explain the functional redundancies and interactions among components of the same protein family. The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place.

A histone acetyltransferase regulates active DNA demethylation in Arabidopsis.

Active DNA demethylation is an important part of epigenetic regulation in plants and animals. How active DNA demethylation is regulated and its relationship with histone modification patterns are unclear. Here, we report the discovery of IDM1, a regulator of DNA demethylation in Arabidopsis. IDM1 is required for preventing DNA hypermethylation of highly homologous multicopy genes and other repetitive sequences that are normally targeted for active DNA demethylation by Repressor of Silencing 1 and related 5-methylcytosine DNA glycosylases. IDM1 binds methylated DNA at chromatin sites lacking histone H3K4 di- or trimethylation and acetylates H3 to create a chromatin environment permissible for 5-methylcytosine DNA glycosylases to function. Our study reveals how some genes are indicated by multiple epigenetic marks for active DNA demethylation and protection from silencing.

A DNA 3' phosphatase functions in active DNA demethylation in Arabidopsis.

DNA methylation is an important epigenetic mark established by the combined actions of methylation and demethylation reactions. Plants use a base excision repair pathway for active DNA demethylation. After 5-methylcytosine removal, the Arabidopsis DNA glycosylase/lyase ROS1 incises the DNA backbone and part of the product has a single-nucleotide gap flanked by 3'- and 5'-phosphate termini. Here we show that the DNA phosphatase ZDP removes the blocking 3' phosphate, allowing subsequent DNA polymerization and ligation steps needed to complete the repair reactions. ZDP and ROS1 interact in vitro and colocalize in vivo in nucleoplasmic foci. Extracts from zdp mutant plants are unable to complete DNA demethylation in vitro, and the mutations cause DNA hypermethylation and transcriptional silencing of a reporter gene. Genome-wide methylation analysis in zdp mutant plants identified hundreds of hypermethylated endogenous loci. Our results show that ZDP functions downstream of ROS1 in one branch of the active DNA demethylation pathway.

A subgroup of SGS3-like proteins act redundantly in RNA-directed DNA methylation.

Plant specific SGS3-like proteins are composed of various combinations of an RNA-binding XS domain, a zinc-finger zf-XS domain, a coil-coil domain and a domain of unknown function called XH. In addition to being involved in de novo 2 (IDN2) and SGS3, the Arabidopsis genome encodes 12 uncharacterized SGS3-like proteins. Here, we show that a group of SGS3-like proteins act redundantly in RNA-directed DNA methylation (RdDM) pathway in Arabidopsis. Transcriptome co-expression analyses reveal significantly correlated expression of two SGS3-like proteins, factor of DNA methylation 1 (FDM1) and FDM2 with known genes required for RdDM. The fdm1 and fdm2 double mutations but not the fdm1 or fdm2 single mutations significantly impair DNA methylation at RdDM loci, release transcriptional gene silencing and dramatically reduce the abundance of siRNAs originated from high copy number repeats or transposons. Like IDN2 and SGS3, FDM1 binds dsRNAs with 5' overhangs. Double mutant analyses also reveal that IDN2 and three uncharacterized SGS3-like proteins FDM3, FDM4 and FDM5 have overlapping function with FDM1 in RdDM. Five FDM proteins and IDN2 define a group of SGS3-like proteins that possess all four-signature motifs in Arabidopsis. Thus, our results demonstrate that this group of SGS3-like proteins is an important component of RdDM. This study further enhances our understanding of the SGS3 gene family and the RdDM pathway.

Posttranscriptional gene silencing in nuclei.

In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybean FAD2-1A desaturase intron is sufficient to silence FAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. Silencing FAD2-1 using intronic or 3'-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA-specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants.

Mechanisms of HDA6-mediated rRNA gene silencing: suppression of intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed cytosine methylation.

The Arabidopsis histone deacetylase HDA6 is required to silence transgenes, transposons, and ribosomal RNA (rRNA) genes subjected to nucleolar dominance in genetic hybrids. In nonhybrid Arabidopsis thaliana, we show that a class of 45S rRNA gene variants that is normally inactivated during development fails to be silenced in hda6 mutants. In these mutants, symmetric cytosine methylation at CG and CHG motifs is reduced, and spurious RNA polymerase II (Pol II) transcription occurs throughout the intergenic spacers. The resulting sense and antisense spacer transcripts facilitate a massive overproduction of siRNAs that, in turn, direct de novo cytosine methylation of corresponding gene sequences. However, the resulting de novo DNA methylation fails to suppress Pol I or Pol II transcription in the absence of HDA6 activity; instead, euchromatic histone modifications typical of active genes accumulate. Collectively, the data reveal a futile cycle of unregulated transcription, siRNA production, and siRNA-directed DNA methylation in the absence of HDA6-mediated histone deacetylation. We propose that spurious Pol II transcription throughout the intergenic spacers in hda6 mutants, combined with losses of histone deacetylase activity and/or maintenance DNA methylation, eliminates repressive chromatin modifications needed for developmental rRNA gene dosage control.

JMJ14, a JmjC domain protein, is required for RNA silencing and cell-to-cell movement of an RNA silencing signal in Arabidopsis.

JMJ14 is a histone H3 Lys4 (H3K4) trimethyl demethylase that affects mobile RNA silencing in an Arabidopsis transgene system. It also influences CHH DNA methylation, abundance of endogenous transposon transcripts, and flowering time. JMJ14 acts at a point in RNA silencing pathways that is downstream from RNA-dependent RNA polymerase 2 (RDR2) and Argonaute 4 (AGO4). Our results illustrate a link between RNA silencing and demethylation of histone H3 trimethylysine. We propose that JMJ14 acts downstream from the Argonaute effector complex to demethylate histone H3K4 at the target of RNA silencing.

An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation.

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

Extra views on RNA-dependent DNA methylation and MBD6-dependent heterochromatin formation in nucleolar dominance.

Nucleolar dominance is a widespread epigenetic phenomenon, describing the preferential silencing of ribosomal RNA (rRNA) genes inherited from one progenitor of an interspecific hybrid, independent of maternal or paternal effects. In the allotetraploid hybrid plant species Arabidopsis suecica, A. thaliana-derived rRNA genes are silenced whereas the A. arenosa-derived rRNA genes are transcribed. We reported previously on an RNAi-based screen of DNA methyltransferases, methylcytosine binding proteins and RNA-dependent DNA methylation pathway proteins that identified specific activities required for the establishment or enforcement of nucleolar dominance. Here we present additional molecular and cell biological evidence that siRNA-directed cytosine methylation and the methylcytosine binding protein MBD6 bring about large-scale chromosomal effects on rRNA gene loci subjected to nucleolar dominance in A. suecica.

A conserved transcriptional regulator is required for RNA-directed DNA methylation and plant development.

RNA-directed DNA methylation (RdDM) is a conserved mechanism for epigenetic silencing of transposons and other repetitive elements. We report that the rdm4 (RNA-directed DNA Methylation4) mutation not only impairs RdDM, but also causes pleiotropic developmental defects in Arabidopsis. Both RNA polymerase II (Pol II)- and Pol V-dependent transcripts are affected in the rdm4 mutant. RDM4 encodes a novel protein that is conserved from yeast to humans and interacts with Pol II and Pol V in plants. Our results suggest that RDM4 functions in epigenetic regulation and plant development by serving as a transcriptional regulator for RNA Pol V and Pol II, respectively.

RNA polymerase V functions in Arabidopsis interphase heterochromatin organization independently of the 24-nt siRNA-directed DNA methylation pathway.

In Arabidopsis, pericentromeric repeats, retroelements, and silenced rRNA genes are assembled into heterochromatin within nuclear structures known as chromocenters. The mechanisms governing higher-order heterochromatin organization are poorly understood but 24-nt small interfering RNAs (siRNAs) are known to play key roles in heterochromatin formation. Nuclear RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), and DICER-LIKE 3 (DCL3) are required for biogenesis of 24-nt siRNAs that associate with ARGONAUTE 4 (AGO4). Nuclear RNA polymerase V (Pol V) collaborates with DRD1 (DEFICIENT IN RNA-DEPENDENT DNA METHYLATION 1) to generate transcripts at heterochromatic loci that are hypothesized to bind to siRNA-AGO4 complexes and subsequently recruit the de-novo DNA methylation and/or histone modifying machinery. Here, we report that decondensation of the major pericentromeric repeats and depletion of the heterochromatic mark histone H3 lysine 9 dimethylation at chromocenters occurs specifically in pol V and drd1 mutants. Disruption of pericentromeric repeats condensation is coincident with transcriptional reactivation of specific classes of pericentromeric 180-bp repeats. We further demonstrate that Pol V functions independently of Pol IV, RDR2, and DCL3-mediated siRNA production to affect interphase heterochromatin organization, possibly by involving RNAs that recruit structural or chromatin-modifying proteins.