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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 Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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.

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., Competing Interests: The authors declare that they have no conflict of interest.

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.

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.

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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

Catabolic stimuli induce a coordinate expression of the 20S proteasome subunits in skeletal muscles. However, contradictory data have been obtained for the 19S regulatory complex (RC) subunits, which could reflect differential regulation at the transcriptional and/or translational level. To address this point we used a well-established model of muscle atrophy (hindlimb suspension) and determined the mRNA levels for 19S subunits belonging to both the base (non-ATPase S1, ATPases S7 and S8) and the lid (S14) of the 19S RC. Concomitant increased mRNA levels were observed for all studied subunits in rat soleus muscles after 9 days of unloading. In addition, analysis of polysome profiles showed a similar proportion of actively translated mRNA (50%) in unloaded and control soleus muscle. Furthermore, the repressed pool of messenger ribonucleoparticles (mRNPs) was low in both control (14%) and unloaded (15%) animals. Our data show that representative 19S subunits (S7 and S8) were efficiently translated, suggesting a coordinate production of 19S RC subunits. The 19S RC is responsible for the binding of polyubiquitin conjugates that are subsequently degraded inside the 20S proteasome core particle. We observed that soleus muscle atrophy was accompanied by an accumulation of ubiquitin conjugates. Purification of ubiquitin conjugates using the S5a 19S subunit followed by deubiquitination identified telethonin as a 26S proteasome substrate. In conclusion, muscle atrophy induces a concomitant expression of 26S proteasome subunits. Substrates to be degraded include a protein required for maintaining the structural integrity of sarcomeres.