Yoshiaki Tarutani

Trans -acting small RNA determines dominance relationships in Brassica self-incompatibility

A diploid organism has two copies of each gene, one inherited from each parent. The expression of two inherited alleles is sometimes biased by the effects known as dominant/recessive relationships, which determine the final phenotype of the organism. To explore the mechanisms underlying these relationships, we have examined the monoallelic expression of S-locus protein 11 genes (SP11), which encode the male determinants of self-incompatibility in Brassica. We previously reported that SP11 expression was monoallelic in some S heterozygotes, and that the promoter regions of recessive SP11 alleles were specifically methylated in the anther tapetum. Here we show that this methylation is controlled by trans-acting small non-coding RNA (sRNA). We identified inverted genomic sequences that were similar to the recessive SP11 promoters in the flanking regions of dominant SP11 alleles. These sequences were specifically expressed in the anther tapetum and processed into 24-nucleotide sRNA, named SP11 methylation inducer (Smi). Introduction of the Smi genomic region into the recessive S homozygotes triggered the methylation of the promoter of recessive SP11 alleles and repressed their transcription. This is an example showing sRNA encoded in the flanking region of a dominant allele acts in trans to induce transcriptional silencing of the recessive allele. Our finding may provide new insights into the widespread monoallelic gene expression systems.

Dominance relationships between self-incompatibility alleles controlled by DNA methylation.

In crucifers, the pollen S-determinant gene, SP11, is sporophytically expressed in the anther tapetum, and the pollen self-incompatibility phenotype is determined by the dominance relationships between the two S-haplotypes it carries. We report here that 5′ promoter sequences of recessive SP11 alleles are specifically methylated in the tapetum before the initiation of SP11 transcription. These results suggest that tissue-specific monoallelic de novo DNA methylation is involved in determining the dominance interactions that determine the cruciferous self-incompatibility phenotype.

Centromere-targeted de novo integrations of an LTR retrotransposon of Arabidopsis lyrata

The plant genome evolves with rapid proliferation of LTR-type retrotransposons, which is associated with their clustered accumulation in gene-poor regions, such as centromeres. Despite their major role for plant genome evolution, no mobile LTR element with targeted integration into gene-poor regions has been identified in plants. Here, we report such targeted integrations de novo. We and others have previously shown that an ATCOPIA93 family retrotransposon in Arabidopsis thaliana is mobilized when the DNA methylation machinery is compromised. Although ATCOPIA93 family elements are low copy number in the wild-type A. thaliana genome, high-copy-number related elements are found in the wild-type Arabidopsis lyrata genome, and they show centromere-specific localization. To understand the mechanisms for the clustered accumulation of the A. lyrata elements directly, we introduced one of them, named Tal1 (Transposon of Arabidopsis lyrata 1), into A. thaliana by transformation. The introduced Tal1 was retrotransposed in A. thaliana, and most of the retrotransposed copies were found in centromeric repeats of A. thaliana, suggesting targeted integration. The targeted integration is especially surprising because the centromeric repeat sequences differ considerably between A. lyrata and A. thaliana. Our results revealed unexpectedly dynamic controls for evolution of the transposon-rich heterochromatic regions.

A Pollen Coat–Inducible Autoinhibited Ca2+-ATPase Expressed in Stigmatic Papilla Cells Is Required for Compatible Pollination in the Brassicaceae

In the Brassicaceae, the pollen coat of compatible pollen grains contains signaling factors that induce Ca2+ export from the stigmatic papilla cells. This Ca2+ export is performed by at least one Ca2+ pump, ACA13, and is required for pollen germination and successful pollination. In the Brassicaceae, intraspecific non-self pollen (compatible pollen) can germinate and grow into stigmatic papilla cells, while self-pollen or interspecific pollen is rejected at this stage. However, the mechanisms underlying this selective acceptance of compatible pollen remain unclear. Here, using a cell-impermeant calcium indicator, we showed that the compatible pollen coat contains signaling molecules that stimulate Ca2+ export from the papilla cells. Transcriptome analyses of stigmas suggested that autoinhibited Ca2+-ATPase13 (ACA13) was induced after both compatible pollination and compatible pollen coat treatment. A complementation test using a yeast Saccharomyces cerevisiae strain lacking major Ca2+ transport systems suggested that ACA13 indeed functions as an autoinhibited Ca2+ transporter. ACA13 transcription increased in papilla cells and in transmitting tracts after pollination. ACA13 protein localized to the plasma membrane and to vesicles near the Golgi body and accumulated at the pollen tube penetration site after pollination. The stigma of a T-DNA insertion line of ACA13 exhibited reduced Ca2+ export, as well as defects in compatible pollen germination and seed production. These findings suggest that stigmatic ACA13 functions in the export of Ca2+ to the compatible pollen tube, which promotes successful fertilization.

Mobilization of a plant transposon by expression of the transposon-encoded anti-silencing factor

Transposable elements (TEs) have a major impact on genome evolution, but they are potentially deleterious, and most of them are silenced by epigenetic mechanisms, such as DNA methylation. Here, we report the characterization of a TE encoding an activity to counteract epigenetic silencing by the host. In Arabidopsis thaliana, we identified a mobile copy of the Mutator‐like element (MULE) with degenerated terminal inverted repeats (TIRs). This TE, named Hiun (Hi), is silent in wild‐type plants, but it transposes when DNA methylation is abolished. When a Hi transgene was introduced into the wild‐type background, it induced excision of the endogenous Hi copy, suggesting that Hi is the autonomously mobile copy. In addition, the transgene induced loss of DNA methylation and transcriptional activation of the endogenous Hi. Most importantly, the trans‐activation of Hi depends on a Hi‐encoded protein different from the conserved transposase. Proteins related to this anti‐silencing factor, which we named VANC, are widespread in the non‐TIR MULEs and may have contributed to the recent success of these TEs in natural Arabidopsis populations.

The histone H3 variant H3.3 regulates gene body DNA methylation in Arabidopsis thaliana

BackgroundGene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation.ResultsWe engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner.ConclusionsWe propose that H3.3 prevents recruitment of H1, inhibiting H1’s promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.

Loss of function mutations in the rice chromomethylase OsCMT3a cause a burst of transposition.

Methylation patterns of plants are unique as, in addition to the methylation at CG dinucleotides that occurs in mammals, methylation also occurs at non-CG sites. Genes are methylated at CG sites, but transposable elements (TEs) are methylated at both CG and non-CG sites. The role of non-CG methylation in transcriptional silencing of TEs is being extensively studied at this time, but only very rare transpositions have been reported when non-CG methylation machineries have been compromised. To understand the role of non-CG methylation in TE suppression and in plant development, we characterized rice mutants with changes in the chromomethylase gene, OsCMT3a. oscmt3a mutants exhibited a dramatic decrease in CHG methylation, changes in the expression of some genes and TEs, and pleiotropic developmental abnormalities. Genome resequencing identified eight TE families mobilized in oscmt3a during normal propagation. These TEs included tissue culture-activated copia retrotransposons Tos17 and Tos19 (Lullaby), a pericentromeric clustered high-copy-number non-autonomous gypsy retrotransposon Dasheng, two copia retrotransposons Osr4 and Osr13, a hAT-tip100 transposon DaiZ, a MITE transposon mPing, and a LINE element LINE1-6_OS. We confirmed the transposition of these TEs by polymerase chain reaction (PCR) and/or Southern blot analysis, and showed that transposition was dependent on the oscmt3a mutation. These results demonstrated that OsCMT3a-mediated non-CG DNA methylation plays a critical role in development and in the suppression of a wide spectrum of TEs. These in planta mobile TEs are important for studying the interaction between TEs and the host genome, and for rice functional genomics.

Genome-Wide Negative Feedback Drives Transgenerational DNA Methylation Dynamics in Arabidopsis

Epigenetic variations of phenotypes, especially those associated with DNA methylation, are often inherited over multiple generations in plants. The active and inactive chromatin states are heritable and can be maintained or even be amplified by positive feedback in a transgenerational manner. However, mechanisms controlling the transgenerational DNA methylation dynamics are largely unknown. As an approach to understand the transgenerational dynamics, we examined long-term effect of impaired DNA methylation in Arabidopsis mutants of the chromatin remodeler gene DDM1 (Decrease in DNA Methylation 1) through whole genome DNA methylation sequencing. The ddm1 mutation induces a drastic decrease in DNA methylation of transposable elements (TEs) and repeats in the initial generation, while also inducing ectopic DNA methylation at hundreds of loci. Unexpectedly, this ectopic methylation can only be seen after repeated self-pollination. The ectopic cytosine methylation is found primarily in the non-CG context and starts from 3’ regions within transcription units and spreads upstream. Remarkably, when chromosomes with reduced DNA methylation were introduced from a ddm1 mutant into a DDM1 wild-type background, the ddm1-derived chromosomes also induced analogous de novo accumulation of DNA methylation in trans. These results lead us to propose a model to explain the transgenerational DNA methylation redistribution by genome-wide negative feedback. The global negative feedback, together with local positive feedback, would ensure robust and balanced differentiation of chromatin states within the genome.

Monoallelic gene expression and its mechanisms

Although the majority of genes are expressed equally from both alleles, some genes are differentially expressed. Monoallelic gene expression, the differential gene expression of the alleles such as genomic imprinting, is reported in several organisms and plays significant roles in proper development and diversity in gene expression and phenotypic variation. Recent studies in flowering plants have greatly increased our understanding of the underlying mechanisms of monoallelic gene expression. They indicate that machineries of gene silencing such as DNA methylation, histone modifications, and noncoding RNAs function in monoallelic gene expression. A combination of genetics and high-throughput technologies expands the scope of study on monoallelic gene expression in flowering plants.

A complex dominance hierarchy is controlled by polymorphism of small RNAs and their targets

In diploid organisms, phenotypic traits are often biased by effects known as Mendelian dominant–recessive interactions between inherited alleles. Phenotypic expression of SP11 alleles, which encodes the male determinants of self-incompatibility in Brassica rapa, is governed by a complex dominance hierarchy1–3. Here, we show that a single polymorphic 24 nucleotide small RNA, named SP11 methylation inducer 2 (Smi2), controls the linear dominance hierarchy of the four SP11 alleles (S44 > S60 > S40 > S29). In all dominant–recessive interactions, small RNA variants derived from the linked region of dominant SP11 alleles exhibited high sequence similarity to the promoter regions of recessive SP11 alleles and acted in trans to epigenetically silence their expression. Together with our previous study4, we propose a new model: sequence similarity between polymorphic small RNAs and their target regulates mono-allelic gene expression, which explains the entire five-phased linear dominance hierarchy of the SP11 phenotypic expression in Brassica.