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Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing in the vegetative cell of developing pollen

ABSTRACT: Silencing of transposable elements (TEs) drove the evolution of numerous redundant mechanisms of transcriptional regulation. Arabidopsis MBD5, MBD6, and SILENZIO act as TE repressors downstream of DNA methylation. Here we show via single-nucleus RNA-seq of developing male gametophytes that these repressors are critical for TE silencing in the pollen vegetative cell, which undergoes epigenetic reprogramming causing chromatin decompaction to support fertilization by sperm cells. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We found that TEs repressed by MBD5/6 gain accessibility in wild-type vegetative nuclei despite remaining silent, suggesting that loss of DNA compaction makes them sensitive to loss of MBD5/6. Consistently, crossing mbd5/6 to histone 1 mutants, which have decondensed chromatin in leaves, reveals derepression of MBD5/6-dependent TEs in leaves. MBD5/6 and SILENZIO thus act as a silencing system especially important when chromatin compaction is compromised.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE202422 | GEO | 2022/10/27

REPOSITORIES: GEO

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MBD2 couples DNA methylation to Transposable Elements silencing during male gametogenesis

Project description:DNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2, and MBD4, and show that MBD2 acts as a transposable element (TE) repressor during male gametogenesis. MBD2 bound chromatin regions containing high levels of CG methylation, and MBD2 was capable of silencing the FWA gene when tethered to its promoter. MBD2 loss caused TE activation in the vegetative cell (VC) of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. Loss of silencing in mbd2 became more significant in the mbd5 mbd6 or adcp1 mutant backgrounds, as well as in plants with chemically induced genome-wide DNA demethylation, suggesting that MBD2 acts redundantly with other silencing pathways to safeguard TEs from activation. Overall, our study identifies MBD2 as a novel methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis.

2024-01-12 | GSE236290 | GEO

Epigenetic reprogramming and small RNA silencing of transposable elements in pollen.

2014-09-04 | GSE61028 | GEO

Epigenetic reprogramming and small RNA silencing of transposable elements in pollen.

Project description:The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that would transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with down-regulation of the heterochromatin remodeler DECREASE IN DNA METHYLATION 1 and of most TE siRNAs. However, 21 nucleotide siRNA from Athila retrotransposons is generated in pollen and accumulates in sperm, indicating that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes. Mature pollen was collected from Columbia reference strain plants by vacuum filtration (Johnson-Brousseau and McCormick, 2004). DNA and RNA were isolated from a ddm1-2 plant in the Columbia reference background. Small RNAs of 19–28 nt were size selected by denaturing 15% PAGE, and cloned as in Brennecke et al. (2007). Additional details regarding the cloning of small RNAs are found in the Supplemental Data. The small RNA libraries were sequenced on Illumina 1G sequencer. The total number of sequences perfectly matching the Arabidopsis genome were as follows: WT inflorescence, 4,158,848 (2,286,133 unique); WT pollen, 1,034,665 (437,984 unique); ddm1 inflorescence, 4,098,772 (1,637,771 unique); and WT sperm, 760,651 (429,972 unique).

2014-09-04 | E-GEOD-61028 | biostudies-arrayexpress

Natural depletion of H1 in sex cells causes DNA demethylation, heterochromatin decondensation and transposon activation

Project description:Transposable elements (TEs) are largely inactive. Interestingly, a subset of TEs are naturally expressed in the vegetative cell (VC) of the male gametophyte, pollen in Arabidopsis. However, the extent and mechanism of such TE activation were unknown. Through RNA-seq, we identified pollen-activated TEs and annotated the transcriptional start sites (TSSs) and transcriptional termination sites (TTSs) using a program called Mikado. We have previously shown that H1 is expressed in sperm but not in VC, which prompted us to ectopically express H1 in VC for studying TE regulation through RNA-seq and bisulfite-seq. Our RNA-seq data shows that H1 expression in VC represses some of pollen-activated TEs. Furthermore, bisulfite-seq data from the pollen nuclei isolated via fluorescence-activated cell sorting (FACS) shows that H1 expression in VC increases DNA methylation of some of H1-repressed TEs around their TSSs, while leaving other H1-repressed TEs unchanged in DNA methylation. Our results indicate that H1 represses TE expression through both DNA methylation-dependent and -independent mechanisms and that natural depletion of H1 in VC allows TEs to be activated.

2019-05-29 | GSE120519 | GEO

Dynamic co-evolution of transposable elements and the piRNA pathway in East African cichlid fishes

Project description:East African cichlid fishes have radiated in an explosive fashion. The (epi)genetic basis for the abundant phenotypic diversity of these fishes remains largely unknown. As transposable elements (TEs) contribute extensively to genome evolution, we reasoned that TEs may have fuelled cichlid radiations. While TE-derived genetic and epigenetic variability has been associated with phenotypic traits, TE expression and epigenetic silencing remain unexplored in cichlids. Here, we profiled TE expression in African cichlids, and describe dynamic expression patterns during embryogenesis and according to sex. Most TE silencing factors are conserved and expressed in cichlids. We describe an expansion of two truncated Piwil1 genes in Lake Malawi/Nyasa cichlids, encoding a Piwi domain with catalytic potential. To further dissect epigenetic silencing of TEs, we focused on small RNA-driven epigenetic silencing. We detect a small RNA population in gonads consistent with an active Piwi-interacting RNA (piRNA) pathway targeting TEs. We uncover fluid genomic origins of piRNAs in closely related cichlid species. This, along with signatures of positive selection in piRNA pathway factors, points towards fast co-evolution of TEs and the piRNA pathway. Our study is the first step to understand the contribution of ongoing TE-host arms races to the cichlid radiations in Africa.

2024-04-02 | PXD047439 | Pride

Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata [RNA-Seq]

Project description:Transposable elements (TEs) are often the primary determinant of genome size differences among eukaryotes. In plants, the proliferation of TEs is countered through epigenetic silencing mechanisms that prevent transposition. Recent studies using the model plant Arabidopsis thaliana have revealed that methylated TE insertions are often associated with reduced expression of nearby genes, and these insertions may be subject to purifying selection due to their effect on nearby genes. Less is known about the genome-wide patterns of epigenetic silencing of TEs in other plant species. Here, we compare the 24-nt siRNA complement from Arabidopsis thaliana and a closely related congener with a two- to three-fold higher TE copy number, A. lyrata. We show that TEs, and particularly siRNA-targeted TEs, are associated with reduced gene expression within both species and also with gene expression differences between orthologs. In addition, A. lyrata TEs are targeted by a lower fraction of uniquely matching siRNAs, which are associated with more effective silencing of TE expression. Overall, our results suggest that the efficacy of RNA-directed DNA methylation silencing is lower in A. lyrata, a finding that may shed light on the causes of differential TE proliferation among species. 4 A. lyrata mRNA-seq samples

2011-02-11 | E-GEOD-24571 | biostudies-arrayexpress

Transcriptional activity and epigenetic regulation of transposable element in the symbiotic fungus Rhizophagus irregularis

Project description:Arbuscular mycorrhizal (AM) fungi form mutualistic relationships with most land plant species. AM fungi have long been considered as ancient asexuals. Long-term clonal evolution would be remarkable for a eukaryotic lineage and suggests the importance of alternative mechanisms to promote genetic variability facilitating adaptation. Here, we assessed the potential of transposable elements (TEs) for generating genomic diversity. The dynamic expression of TEs during Rhizophagus irregularis spore development suggests ongoing TE activity. We find Mutator-like elements located near genes belonging to highly expanded gene families. Characterising the epigenomic status of R. irregularis provides evidence of DNA methylation and small RNA production occurring at TE loci. Our results support a potential role for TEs in shaping the genome, and roles for DNA methylation and small RNA-mediated silencing in regulating TEs. A well-controlled balance between TE activity and repression may therefore contribute to genome evolution in AM fungi.

2022-02-17 | PXD025245 | Pride

TEX15 silences transposable elements in male germ cells

Project description:This SuperSeries is composed of the SubSeries listed below. DNA methylation is a major silencing mechanism of transposable elements (TEs). Here we report that TEX15, a testis-specific protein, is required for TE silencing. TEX15 is both cytoplasmic and nuclear in embryonic germ cells and functions during genome-wide epigenetic reprogramming. Tex15 mutant exhibits DNA hypomethylation in TEs at a level similar to Mili but not Miwi2 mutant. As loss of Tex15 causes TE de-silencing without abolishing piRNA production, our results identify TEX15 as a new essential epigenetic regulator that appears to function independently or downstream of the piRNA biogenesis machineries to silence TEs by DNA methylation in male germ cells.

2020-05-18 | GSE140571 | GEO

Full-length autonomous transposable elements are preferentially targeted by expression-dependent forms of RNA-directed DNA methylation

Project description:The diversity of small RNA-directed DNA methylation (RdDM) mechanisms have been underestimated due to the nearly complete transcriptional silencing of transposable elements (TEs) in the wild-type reference strains of Arabidopsis thaliana. In plants mutant for the SWI/SNF histone remodeler DDM1, TEs are globally activated due to loss of genome wide heterochromatin condensation. Transcriptionally activated TEs go through additional non-canonical forms of RdDM that are dependent on RNA Polymerase II expression. However, the global targets of the non-canonical RdDM pathway have not been explored. In an attempt to identify and contrast the targets of canonical and expression-dependent non-canonical RdDM, we performed MethylC-seq of genome-wide DNA methylation patterns from several RdDM mutants in either the TE-silent or the TE-active (ddm1) contexts. Arabidopsis wildtype and twenty RdDM pathway mutants

2016-07-22 | E-GEOD-79746 | biostudies-arrayexpress

Polymerase IV Plays a Crucial Role in Pollen Development in Capsella

Project description:In Arabidopsis thaliana, DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA (sRNA) transcripts. These transcripts are processed by DICER-LIKE3 into 24-nt small interfering RNAs (siRNAs) that guide RNA-directed DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nt epigenetically activated siRNAs (easiRNAs), which likely silence TEs via post-transcriptional mechanisms. Despite this proposed role of Pol IV, its loss of function in Arabidopsis does not cause a discernable pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV in the Brassicaceae species Capsella rubella, caused post-meiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were 2 depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nt and 24-nt siRNAs; these processes required Pol IV activity. Arrest of Capsella nrpd1 microspores was accompanied by the deregulation of genes targeted by Pol IV-dependent siRNAs. TEs were much closer to genes in Capsella rubella compared to Arabidopsis thaliana, perhaps explaining the essential role of Pol IV in pollen development in Capsella. Our discovery that Pol IV is functionally required in Capsella microspores emphasizes the relevance of investigating different plant models.

2020-01-23 | GSE136847 | GEO

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