Etienne Bucher

An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress

Eukaryotic genomes consist to a significant extent of retrotransposons that are suppressed by host epigenetic mechanisms, preventing their uncontrolled propagation. However, it is not clear how this is achieved. Here we show that in Arabidopsis seedlings subjected to heat stress, a copia-type retrotransposon named ONSEN (Japanese ‘hot spring’) not only became transcriptionally active but also synthesized extrachromosomal DNA copies. Heat-induced ONSEN accumulation was stimulated in mutants impaired in the biogenesis of small interfering RNAs (siRNAs); however, there was no evidence of transposition occurring in vegetative tissues. After stress, both ONSEN transcripts and extrachromosomal DNA gradually decayed and were no longer detected after 20–30 days. Surprisingly, a high frequency of new ONSEN insertions was observed in the progeny of stressed plants deficient in siRNAs. Insertion patterns revealed that this transgenerational retrotransposition occurred during flower development and before gametogenesis. Therefore in plants with compromised siRNA biogenesis, memory of stress was maintained throughout development, priming ONSEN to transpose during differentiation of generative organs. Retrotransposition was not observed in the progeny of wild-type plants subjected to stress or in non-stressed mutant controls, pointing to a crucial role of the siRNA pathway in restricting retrotransposition triggered by environmental stress. Finally, we found that natural and experimentally induced variants in ONSEN insertions confer heat responsiveness to nearby genes, and therefore mobility bursts may generate novel, stress-responsive regulatory gene networks.

Selective epigenetic control of retrotransposition in Arabidopsis

Retrotransposons are mobile genetic elements that populate chromosomes, where the host largely controls their activities. In plants and mammals, retrotransposons are transcriptionally silenced by DNA methylation, which in Arabidopsis is propagated at CG dinucleotides by METHYLTRANSFERASE 1 (MET1). In met1 mutants, however, mobilization of retrotransposons is not observed, despite their transcriptional activation. A post-transcriptional mechanism therefore seems to be preventing retrotransposition. Here we show that a copia-type retrotransposon (Évadé, French for ‘fugitive’) evaded suppression of its movement during inbreeding of hybrid epigenomes consisting of met1- and wild-type-derived chromosomes. Évadé (EVD) reinsertions caused a series of developmental mutations that allowed its identification. Genetic testing of host control of the EVD life cycle showed that transcriptional suppression occurred by CG methylation supported by RNA-directed DNA methylation. On transcriptional reactivation, subsequent steps of the EVD cycle were inhibited by plant-specific RNA polymerase IV/V and the histone methyltransferase KRYPTONITE (KYP). Moreover, genome resequencing demonstrated retrotransposition of EVD but no other potentially active retroelements when this combination of epigenetic mechanisms was compromised. Our results demonstrate that epigenetic control of retrotransposons extends beyond transcriptional suppression and can be individualized for particular elements.

Negative-Strand Tospoviruses and Tenuiviruses Carry a Gene for a Suppressor of Gene Silencing at Analogous Genomic Positions

ABSTRACT Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the Bunyaviridae. Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NSS gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NSS-expressing leaf sectors suggests that the tospoviral NSS protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, a nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing.

A structural-maintenance-of-chromosomes hinge domain–containing protein is required for RNA-directed DNA methylation

RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region. To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region. A screen for mutants defective in meristem silencing and enhancer methylation retrieved six dms complementation groups, which included the known factors DRD1 (ref. 3; a SNF2-like chromatin-remodeling protein) and Pol IVb subunits. Additionally, we identified a previously unknown gene DMS3 (At3g49250), encoding a protein similar to the hinge-domain region of structural maintenance of chromosomes (SMC) proteins. This finding implicates a putative chromosome architectural protein that can potentially link nucleic acids in facilitating an RNAi-mediated epigenetic modification involving secondary siRNAs and spreading of DNA methylation.

Stress-Induced Activation of Heterochromatic Transcription

Constitutive heterochromatin comprising the centromeric and telomeric parts of chromosomes includes DNA marked by high levels of methylation associated with histones modified by repressive marks. These epigenetic modifications silence transcription and ensure stable inheritance of this inert state. Although environmental cues can alter epigenetic marks and lead to modulation of the transcription of genes located in euchromatic parts of the chromosomes, there is no evidence that external stimuli can globally destabilize silencing of constitutive heterochromatin. We have found that heterochromatin-associated silencing in Arabidopsis plants subjected to a particular temperature regime is released in a genome-wide manner. This occurs without alteration of repressive epigenetic modifications and does not involve common epigenetic mechanisms. Such induced release of silencing is mostly transient, and rapid restoration of the silent state occurs without the involvement of factors known to be required for silencing initiation. Thus, our results reveal new regulatory aspects of transcriptional repression in constitutive heterochromatin and open up possibilities to identify the molecular mechanisms involved.

A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation

We used a transgene system to study spreading of RNA‐directed DNA methylation (RdDM) during transcriptional gene silencing in Arabidopsis thaliana. Forward and reverse genetics approaches using this system delineated a stepwise pathway for the biogenesis of secondary siRNAs and unidirectional spreading of methylation from an upstream enhancer element into downstream sequences. Trans‐acting, hairpin‐derived primary siRNAs induce primary RdDM, independently of an enhancer‐associated ‘nascent’ RNA, at the target enhancer region. Primary RdDM is a key step in the pathway because it attracts the secondary siRNA‐generating machinery, including RNA polymerase IV, RNA‐dependent RNA polymerase2 and Dicer‐like3 (DCL3). These factors act in a turnover pathway involving a nascent RNA, which normally accumulates stably in non‐silenced plants, to produce cis‐acting secondary siRNAs that induce methylation in the downstream region. The identification of DCL3 in a forward genetic screen for silencing‐defective mutants demonstrated a strict requirement for 24‐nt siRNAs to direct methylation. A similar stepwise process for spreading of DNA methylation may occur in mammalian genomes, which are extensively transcribed in upstream regulatory regions.

Loss of DNA methylation affects the recombination landscape in Arabidopsis

During sexual reproduction, one-half of the genetic material is deposited in gametes, and a complete set of chromosomes is restored upon fertilization. Reduction of the genetic information before gametogenesis occurs in meiosis, when cross-overs (COs) between homologous chromosomes secure an exchange of their genetic information. COs are not evenly distributed along chromosomes and are suppressed in chromosomal regions encompassing compact, hypermethylated centromeric and pericentromeric DNA. Therefore, it was postulated that DNA hypermethylation is inhibitory to COs. Here, when analyzing meiotic recombination in mutant plants with hypomethylated DNA, we observed unexpected and counterintuitive effects of DNA methylation losses on CO distribution. Recombination was further promoted in the hypomethylated chromosome arms while it was inhibited in heterochromatic regions encompassing pericentromeric DNA. Importantly, the total number of COs was not affected, implying that loss of DNA methylation led to a global redistribution of COs along chromosomes. To determine by which mechanisms altered levels of DNA methylation influence recombination—whether directly in cis or indirectly in trans by changing expression of genes encoding recombination components—we analyzed CO distribution in wild-type lines with randomly scattered and well-mapped hypomethylated chromosomal segments. The results of these experiments, supported by expression profiling data, suggest that DNA methylation affects meiotic recombination in cis. Because DNA methylation exhibits significant variation even within a single species, our results imply that it may influence the evolution of plant genomes through the control of meiotic recombination.

RNA-directed DNA methylation and plant development require an IWR1-type transcription factor

RNA‐directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II‐related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV‐dependent secondary short interfering RNAs, Pol V‐mediated RdDM, Pol V‐dependent synthesis of intergenic non‐coding RNA and expression of many Pol II‐driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant.

Antiviral immune responses in gene-targeted mice expressing the immunoglobulin heavy chain of virus-neutralizing antibodies

Two gene-targeted immunoglobulin heavy chain transgenic mouse strains, TgH(KL25) and TgH(VI10), expressing neutralizing specificities for lymphocytic choriomeningitis virus and vesicular stomatitis virus, respectively, have been generated. Three days after lymphocytic choriomeningitis virus infection, TgH(KL25) mice showed a thymus-independent neutralizing IgM response followed by thymus-dependent (TD) IgG. In contrast, WT mice mounted only a TD IgG response around day 80. These observations indicated that not only structural properties of the virus but also immunological parameters such as the frequency of B cells were indicative for the induction of thymus-independent versus TD Ig responses. Naïve vesicular stomatitis virusspecific Ig heavy chain transgenic mice displayed greatly elevated natural antibody titers. However, despite these high naïve titers, de novo activation of naïve CD4+ T and B cells was not blocked. Therefore, B cells giving rise to natural antibodies do not participate in virus-induced antibody responses.

Epigenetic control of transposon transcription and mobility in Arabidopsis

The mobility of genetic elements called transposable elements (TEs) was discovered half a century ago by Barbara McClintock. Although she had recognized them as chromosomal controlling elements, for much of the consequent time TEs were primarily considered as parasites of the host genome. However the recent explosion of discoveries in the fields of genomics and epigenetics have unambiguously shown the importance of TEs in genome function and evolution. Bursts of endogenous TEs have been reported in plants with epigenetic misregulation, revealing the molecular mechanisms underlying their control. We review here the different steps in TE invasion of the host genome involving epigenetic control and environmental stress responses. As TEs propagate in plant genomes and attract epigenetic marks, their neo-insertions can lead to the formation of new, heritable epigenetic variants (epialleles) of genes in their vicinity and impact on host gene regulatory networks. The epigenetic interplay between TE and genes thus plays a crucial role in the TE-host co-evolution.