RTEL1 is required for silencing and epigenome stability.

Transcriptional silencing is an essential mechanism for controlling the expression of genes, transgenes and heterochromatic repeats through specific epigenetic marks on chromatin that are maintained during DNA replication. In Arabidopsis, silenced transgenes and heterochromatic sequences are typically associated with high levels of DNA methylation, while silenced genes are enriched in H3K27me3. Reactivation of these loci is often correlated with decreased levels of these repressive epigenetic marks. Here, we report that the DNA helicase REGULATOR OF TELOMERE ELONGATION 1 (RTEL1) is required for transcriptional silencing. RTEL1 deficiency causes upregulation of many genes enriched in H3K27me3 accompanied by a moderate decrease in this mark, but no loss of DNA methylation at reactivated heterochromatic loci. Instead, heterochromatin exhibits DNA hypermethylation and increased H3K27me3 in rtel1. We further find that loss of RTEL1 suppresses the release of heterochromatin silencing caused by the absence of the MOM1 silencing factor. RTEL1 is conserved among eukaryotes and plays a key role in resolving DNA secondary structures during DNA replication. Inducing such aberrant DNA structures using DNA cross-linking agents also results in a loss of transcriptional silencing. These findings uncover unappreciated roles for RTEL1 in transcriptional silencing and in stabilizing DNA methylation and H3K27me3 patterns.

The histone variant H2A.W and linker histone H1 co-regulate heterochromatin accessibility and DNA methylation.

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.

DNA polymerase epsilon is required for heterochromatin maintenance in Arabidopsis.

BACKGROUND: Chromatin organizes DNA and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent, while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. RESULTS: We isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome-wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, the release of heterochromatin silencing in POL2A-deficient mutants paradoxically occurs in a chromatin context of increased levels of these two repressive epigenetic marks. At the nuclear level, the POL2A defect is associated with fragmentation of heterochromatin. CONCLUSION: These results indicate that POL2A is critical to heterochromatin structure and function, and that unhindered replisome progression is required for the faithful propagation of DNA methylation throughout the cell cycle.

PP7L is essential for MAIL1-mediated transposable element silencing and primary root growth.

The two paralogous Arabidopsis genes MAINTENANCE OF MERISTEMS (MAIN) and MAINTENANCE OF MERISTEMS LIKE1 (MAIL1) encode a conserved retrotransposon-related plant mobile domain and are known to be required for silencing of transposable elements (TE) and for primary root development. Loss of function of either MAIN or MAIL1 leads to release of heterochromatic TEs, reduced condensation of pericentromeric heterochromatin, cell death of meristem cells and growth arrest of the primary root soon after germination. Here, we show that they act in one protein complex that also contains the inactive isoform of PROTEIN PHOSPHATASE 7 (PP7), which is named PROTEIN PHOSPHATASE 7-LIKE (PP7L). PP7L was previously shown to be important for chloroplast biogenesis and efficient chloroplast protein synthesis. We show that loss of PP7L function leads to the same root growth phenotype as loss of MAIL1 or MAIN. In addition, pp7l mutants show similar silencing defects. Double mutant analyses confirmed that the three proteins act in the same molecular pathway. The primary root growth arrest, which is associated with cell death of stem cells and their daughter cells, is a consequence of genome instability. Our data demonstrate so far unrecognized functions of an inactive phosphatase isoform in a protein complex that is essential for silencing of heterochromatic elements and for maintenance of genome stability in dividing cells.

A role for MED14 and UVH6 in heterochromatin transcription upon destabilization of silencing.

Constitutive heterochromatin is associated with repressive epigenetic modifications of histones and DNA which silence transcription. Yet, particular mutations or environmental changes can destabilize heterochromatin-associated silencing without noticeable changes in repressive epigenetic marks. Factors allowing transcription in this nonpermissive chromatin context remain poorly known. Here, we show that the transcription factor IIH component UVH6 and the mediator subunit MED14 are both required for heat stress-induced transcriptional changes and release of heterochromatin transcriptional silencing in Arabidopsis thaliana. We find that MED14, but not UVH6, is required for transcription when heterochromatin silencing is destabilized in the absence of stress through mutating the MOM1 silencing factor. In this case, our results raise the possibility that transcription dependency over MED14 might require intact patterns of repressive epigenetic marks. We also uncover that MED14 regulates DNA methylation in non-CG contexts at a subset of RNA-directed DNA methylation target loci. These findings provide insight into the control of heterochromatin transcription upon silencing destabilization and identify MED14 as a regulator of DNA methylation.

Arabidopsis proteins with a transposon-related domain act in gene silencing.

Transposable elements (TEs) are prevalent in most eukaryotes, and host genomes have devised silencing strategies to rein in TE activity. One of these, transcriptional silencing, is generally associated with DNA methylation and short interfering RNAs. Here we show that the Arabidopsis genes MAIL1 and MAIN define an alternative silencing pathway independent of DNA methylation and short interfering RNAs. Mutants for MAIL1 or MAIN exhibit release of silencing and appear to show impaired condensation of pericentromeric heterochromatin. Phylogenetic analysis suggests not only that MAIL1 and MAIN encode a retrotransposon-related plant mobile domain, but also that host plant mobile domains were captured by DNA transposons during plant evolution. Our results reveal a role for Arabidopsis proteins with a transposon-related domain in gene silencing.

Epigenome confrontation triggers immediate reprogramming of DNA methylation and transposon silencing in Arabidopsis thaliana F1 epihybrids.

Genes and transposons can exist in variable DNA methylation states, with potentially differential transcription. How these epialleles emerge is poorly understood. Here, we show that crossing an Arabidopsis thaliana plant with a hypomethylated genome and a normally methylated WT individual results, already in the F1 generation, in widespread changes in DNA methylation and transcription patterns. Novel nonparental and heritable epialleles arise at many genic loci, including a locus that itself controls DNA methylation patterns, but with most of the changes affecting pericentromeric transposons. Although a subset of transposons show immediate resilencing, a large number display decreased DNA methylation, which is associated with de novo or enhanced transcriptional activation and can translate into transposon mobilization in the progeny. Our findings reveal that the combination of distinct epigenomes can be viewed as an epigenomic shock, which is characterized by a round of epigenetic variation creating novel patterns of gene and TE regulation.

Evolutionary history of double-stranded RNA binding proteins in plants: identification of new cofactors involved in easiRNA biogenesis.

In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway.

Parallel action of AtDRB2 and RdDM in the control of transposable element expression.

BACKGROUND: In plants and animals, a large number of double-stranded RNA binding proteins (DRBs) have been shown to act as non-catalytic cofactors of DICERs and to participate in the biogenesis of small RNAs involved in RNA silencing. We have previously shown that the loss of Arabidopsis thaliana's DRB2 protein results in a significant increase in the population of RNA polymerase IV (p4) dependent siRNAs, which are involved in the RNA-directed DNA methylation (RdDM) process. RESULTS: Surprisingly, despite this observation, we show in this work that DRB2 is part of a high molecular weight complex that does not involve RdDM actors but several chromatin regulator proteins, such as MSI4, PRMT4B and HDA19. We show that DRB2 can bind transposable element (TE) transcripts in vivo but that drb2 mutants do not have a significant variation in TE DNA methylation. CONCLUSION: We propose that DRB2 is part of a repressive epigenetic regulator complex involved in a negative feedback loop, adjusting epigenetic state to transcription level at TE loci, in parallel of the RdDM pathway. Loss of DRB2 would mainly result in an increased production of TE transcripts, readily converted in p4-siRNAs by the RdDM machinery.

DNA methylation in an intron of the IBM1 histone demethylase gene stabilizes chromatin modification patterns.

The stability of epigenetic patterns is critical for genome integrity and gene expression. This highly coordinated process involves interrelated positive and negative regulators that impact distinct epigenetic marks, including DNA methylation and dimethylation at histone H3 lysine 9 (H3K9me2). In Arabidopsis, mutations in the DNA methyltransferase MET1, which maintains CG methylation, result in aberrant patterns of other epigenetic marks, including ectopic non-CG methylation and the relocation of H3K9me2 from heterochromatin into gene-rich chromosome regions. Here, we show that the expression of the H3K9 demethylase IBM1 (increase in BONSAI methylation 1) requires DNA methylation. Surprisingly, the regulatory methylated region is contained in an unusually large intron that is conserved in IBM1 orthologues. The re-establishment of IBM1 expression in met1 mutants restored the wild-type H3K9me2 nuclear patterns, non-CG DNA methylation and transcriptional patterns at selected loci, which included DNA demethylase genes. These results provide a mechanistic explanation for long-standing puzzling observations in met1 mutants and reveal yet another layer of control in the interplay between DNA methylation and histone modification, which stabilizes DNA methylation patterns at genes.

Double-stranded RNA binding proteins DRB2 and DRB4 have an antagonistic impact on polymerase IV-dependent siRNA levels in Arabidopsis.

Biogenesis of the vast majority of plant siRNAs depends on the activity of the plant-specific RNA polymerase IV (PolIV) enzyme. As part of the RNA-dependent DNA methylation (RdDM) process, PolIV-dependent siRNAs (p4-siRNAs) are loaded onto an ARGONAUTE4-containing complex and guide de novo DNA methyltransferases to target loci. Here we show that the double-stranded RNA binding proteins DRB2 and DRB4 are required for proper accumulation of p4-siRNAs. In flowers, loss of DRB2 results in increased accumulation of p4-siRNAs but not ta-siRNAs, inverted repeat (IR)-derived siRNAs, or miRNA. Loss of DRB2 does not impair uniparental expression of p4-dependent siRNAs in developing endosperm, indicating that p4-siRNA increased accumulation is not the result of the activation of the polIV pathway in the male gametophyte. In contrast to drb2, drb4 mutants exhibit reduced p4-siRNA levels, but the extent of this reduction is variable, according to the nature and size of the p4-siRNAs. Loss of DRB4 also leads to a spectacular increase of p4-independent IR-derived 24-nt siRNAs, suggesting a reallocation of factors from p4-dependent to p4-independent siRNA pathways in drb4. Opposite effects of drb2 and drb4 mutations on the accumulation of p4-siRNAs were also observed in vegetative tissues. Moreover, transgenic plants overexpressing DRB2 mimicked drb4 mutants at the morphological and molecular levels, confirming the antagonistic roles of DRB2 and DRB4.

Arabidopsis eIF2alpha kinase GCN2 is essential for growth in stress conditions and is activated by wounding.

BACKGROUND: Phosphorylation of eIF2alpha provides a key mechanism for down-regulating protein synthesis in response to nutrient starvation or stresses in mammalian and yeast cells. However, this process has not been well characterized in plants RESULTS: We show here that in response to amino acid and purine starvations, UV, cold shock and wounding, the Arabidopsis GCN2 kinase (AtGCN2) is activated and phosphorylates eIF2alpha. We show that AtGCN2 is essential for plant growth in stress situations and that its activity results in a strong reduction in global protein synthesis. CONCLUSION: Our results suggest that a general amino acid control response is conserved between yeast and plants but that the plant enzyme evolved to fulfill a more general function as an upstream sensor and regulator of diverse stress-response pathways. The activation of AtGCN2 following wounding or exposure to methyl jasmonate, the ethylene precursor 1-Aminocyclopropane-1-carboxylic acid (ACC) and salicylic acid, further suggests that this enzyme could play a role in plant defense against insect herbivores.

SINE RNA induces severe developmental defects in Arabidopsis thaliana and interacts with HYL1 (DRB1), a key member of the DCL1 complex.

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.

The nanovirus-encoded Clink protein affects plant cell cycle regulation through interaction with the retinoblastoma-related protein.

Nanoviruses, multicomponent single-stranded DNA plant viruses, encode a unique cell cycle link protein, Clink, that interacts with retinoblastoma-related proteins (RBR). We have established transgenic Arabidopsis thaliana lines that conditionally express Clink or a Clink variant deficient in RBR binding. By controlled induction of Clink expression, we demonstrated the capacity of the Clink protein to alter RBR function in vivo. We showed that transcription of both S-phase-specific and G2/M-phase-specific genes was up-regulated depending on the RBR-binding proficiency of Clink. Concomitantly, ploidy levels increased in a substantial fraction of leaf cell nuclei. Also, leaf epidermis cells of transgenic plants producing Clink were smaller and more numerous, indicating additional cell divisions in this tissue. Furthermore, cytogenetic analyses following induction of Clink expression in mature leaves revealed the presence of metaphasic and anaphasic nuclei, clear evidence that Clink-mediated RBR inactivation is sufficient to induce quiescent cells to reenter cell cycle progression and, for at least a fraction of them, to pass through mitosis. Expression of Clink had no effect on genes transcribed by RNA polymerases I and III, suggesting that, in contrast to its mammalian homologue, A. thaliana RBR is not involved in the repression of polymerase I and polymerase III transcription. The results of these in vivo analyses firmly establish Clink as a member of the diverse class of multifunctional cell cycle modulator proteins encoded by small DNA viruses.

Synthesis and processing of tRNA-related SINE transcripts in Arabidopsis thaliana.

Despite the ubiquitous distribution of tRNA-related short interspersed elements (SINEs) in eukaryotic species, very little is known about the synthesis and processing of their RNAs. In this work, we have characterized in detail the different RNA populations resulting from the expression of a tRNA-related SINE S1 founder copy in Arabidopsis thaliana. The main population is composed of poly(A)-ending (pa) SINE RNAs, while two minor populations correspond to full-length (fl) or poly(A) minus [small cytoplasmic (sc)] SINE RNAs. Part of the poly(A) minus RNAs is modified by 3'-terminal addition of C or CA nucleotides. All three RNA populations accumulate in the cytoplasm. Using a mutagenesis approach, we show that the poly(A) region and the 3' end unique region, present at the founder locus, are both important for the maturation and the steady-state accumulation of the different S1 RNA populations. The observation that primary SINE transcripts can be post-transcriptionally processed in vivo into a poly(A)-ending species introduces the possibility that this paRNA is used as a retroposition intermediate.

Direct role of a viroid RNA motif in mediating directional RNA trafficking across a specific cellular boundary.

The plasmodesmata and phloem form a symplasmic network that mediates direct cell-cell communication and transport throughout a plant. Selected endogenous RNAs, viral RNAs, and viroids traffic between specific cells or organs via this network. Whether an RNA itself has structural motifs to potentiate trafficking is not well understood. We have used mutational analysis to identify a motif that the noncoding Potato spindle tuber viroid RNA evolved to potentiate its efficient trafficking from the bundle sheath into mesophyll that is vital to establishing systemic infection in tobacco (Nicotiana tabacum). Surprisingly, this motif is not necessary for trafficking in the reverse direction (i.e., from the mesophyll to bundle sheath). It is not required for trafficking between other cell types either. We also found that the requirement for this motif to mediate bundle sheath-to-mesophyll trafficking is dependent on leaf developmental stages. Our results provide genetic evidence that (1) RNA structural motifs can play a direct role in mediating trafficking across a cellular boundary in a defined direction, (2) the bundle sheath-mesophyll boundary serves as a novel regulatory point for RNA trafficking between the phloem and nonvascular tissues, and (3) the symplasmic network remodels its capacity to traffic RNAs during plant development. These findings may help further studies to elucidate the interactions between RNA motifs and cellular factors that potentiate directional trafficking across specific cellular boundaries.

Viroid-induced RNA silencing of GFP-viroid fusion transgenes does not induce extensive spreading of methylation or transitive silencing.

Viroid infection is associated with the production of short interfering RNAs (siRNAs), a hallmark of post-transcriptional gene silencing (PTGS). However, viroid RNAs autonomously replicating in the nucleus have not been shown to trigger the degradation of homologous RNA in the cytoplasm. To investigate the potential of viroids for the induction of gene silencing, non-infectious fragments of potato spindle tuber viroid (PSTVd) cDNA were transcriptionally fused to the 3' end of the green fluorescent protein (GFP)-coding region. Introduction of such constructs into tobacco plants resulted in stable transgene expression. Upon PSTVd infection, transgene expression was suppressed and partial de novo methylation of the transgene was observed. PSTVd-specific siRNA was detected but none was found corresponding to the gfp gene. Methylation was restricted almost entirely to the PSTVd-specific part of the transgene. Neither a gfp transgene construct lacking viroid-specific elements was silenced nor was de novo methylation detected, when it was introduced into the genetic background of the PSTVd-infected plant lines containing silenced GFP:PSTVd transgenes. The absence of gfp-specific siRNAs and of significant methylation within the gfp-coding region demonstrated that neither silencing nor DNA methylation spread from the initiator region into adjacent 5' regions.

Utilization of the IR hybrid dysgenesis system in Drosophila to test in vivo mobilization of synthetic SINEs sharing 3' homology with the I factor.

The current model of short interspersed nuclear element (SINE) mobility suggests that these non-coding retroposons are able to recruit for their own benefits the enzymatic machinery encoded by autonomous long interspersed nuclear elements (LINEs). The recent characterization of potential SINE-LINE partner pairs that share common 3' end sequences concurs with this model and has led to a potent picture of tRNA-derived SINEs consisting of a tripartite functional structure (Mol. Cell. Biol. 16 (1996) 3756; Mol. Biol. Evol. 16 (1999) 1238; Proc. Natl. Acad. Sci. USA 96 (1999) 2869). This structure consist of a 5' polIII tRNA-related promoter region, a central conserved domain and a variable 3' region with homology to the 3' end of LINEs, believed to be essential to direct recognition by the LINE proteins. To test this model in vivo, we have designed synthetic SINEs possessing this 'canonical' structure, including 3' homology to the 3' UTR of the LINE I factor from Drosophila. These synthetic elements were introduced in a Drosophila reactive strain, and SINE retroposition was assessed following dysgenic crosses that are known to induce high levels of I factor germinal transposition. In the progeny from the dysgenic crosses 3400-4000 flies were analyzed but no retroposed copy of the chimeric SINEs was detected, indicating that what is assumed to be a typical SINE structure is not sufficient per se to allow efficient trans-mobilization of our synthetic SINEs by an actively amplifying partner LINE. Alternatively, the apparent absence of natural fly SINEs may underline intrinsic properties of fly biology that are incompatible with the genesis and/or propagation of SINE-like elements.