36 research outputs found
Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity
No full textInducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal ‘short-term stress memory’ with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring
Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9
No full textGenome editing using CRISPR/Cas9 is considered the best instrument for genome engineering in plants. This methodology is based on the nuclease activity of Cas9 that is guided to specific genome sequences by single guide RNAs (sgRNAs) thus enabling researchers to engineer simple mutations or large chromosomal deletions. Current methodologies for targeted genome editing in plants using CRISPR/Cas9 are however largely inefficient, mostly due to low Cas9 activity, variable sgRNA efficiency and low heritability of genetic lesions. Here, we describe a newly developed strategy to enhance CRISPR/Cas9 efficiency in Arabidopsis thaliana focusing on the design of novel binary vectors (pUbiCAS9-Red and pEciCAS9-Red), the selection of highly efficient sgRNAs, and the use of direct plant regeneration from induced cell cultures. Our work demonstrates that by combining these three independent developments, heritable targeted chromosomal deletions of large gene clusters and intergenic regulatory sequences can be engineered at a high efficiency. Our results demonstrate that this improved CRISPR/Cas9 methodology can provide a fast, efficient and cost-effective tool to engineer targeted heritable chromosomal deletions, which will be instrumental for future high-throughput functional genomics studies in plants
Epigenetic reprogramming rewires transcription during the alternation of generations in Arabidopsis
No full textRepression of chromomethylase 3 prevents epigenetic collateral damage in arabidopsis
No full textDNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Our results are also consistent with CMT3-induced ectopic methylation of promoters or bodies of genes undergoing transcriptional activation reducing their expression. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants
Regulatory logic of endogenous RNAi in silencing de novo genomic conflicts.
No full textAlthough the biological utilities of endogenous RNAi (endo-RNAi) have been largely elusive, recent studies reveal its critical role in the non-model fruitfly Drosophila simulans to suppress selfish genes, whose unchecked activities can severely impair spermatogenesis. In particular, hairpin RNA (hpRNA) loci generate endo-siRNAs that suppress evolutionary novel, X-linked, meiotic drive loci. The consequences of deleting even a single hpRNA (Nmy) in males are profound, as such individuals are nearly incapable of siring male progeny. Here, comparative genomic analyses of D. simulans and D. melanogaster mutants of the core RNAi factor dcr-2 reveal a substantially expanded network of recently-emerged hpRNA-target interactions in the former species. The de novo hpRNA regulatory network in D. simulans provides insight into molecular strategies that underlie hpRNA emergence and their potential roles in sex chromosome conflict. In particular, our data support the existence of ongoing rapid evolution of Nmy/Dox-related networks, and recurrent targeting of testis HMG-box loci by hpRNAs. Importantly, the impact of the endo-RNAi network on gene expression flips the convention for regulatory networks, since we observe strong derepression of targets of the youngest hpRNAs, but only mild effects on the targets of the oldest hpRNAs. These data suggest that endo-RNAi are especially critical during incipient stages of intrinsic sex chromosome conflicts, and that continual cycles of distortion and resolution may contribute to speciation
MBD2 couples DNA methylation to transposable element silencing during male gametogenesis.
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MBD2 couples DNA methylation to transposable element silencing during male gametogenesis.
No full textDNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood1-5. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2 and MBD4 and show that MBD2 acts as a 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 activation at a small subset of TEs in the vegetative cell of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. TE activation in mbd2 became more significant in the mbd5 mbd6 and adcp1 mutant backgrounds, suggesting that MBD2 acts redundantly with other silencing pathways to repress TEs. Overall, our study identifies MBD2 as a methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis
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Single-nucleus RNA-seq reveals that MBD5, MBD6, and SILENZIO maintain silencing in the vegetative cell of developing pollen
No full textSilencing of transposable elements (TEs) drives 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, a companion cell important for fertilization that undergoes chromatin decompaction. Instead, other silencing mutants (met1, ddm1, mom1, morc) show loss of silencing in all pollen nucleus types and somatic cells. We show that TEs repressed by MBD5/6 gain chromatin 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 particularly important when chromatin compaction is compromised
Correction: Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity
No full textNovel <i>D</i>. <i>simulans</i> hpRNAs related to testis HMG Box (tHMG) loci.
No full text(A) Schematic of tHMG loci in D. melanogaster and D. simulans, and the origins of hpRNA suppressors and targets encompassing multiple novel X-linked tHMG-derived loci. tHMG is locally duplicated in D. melanogaster, but the syntenic location of D. simulans bears a single gene. However, D. simulans X chromosome bears multiple de novo hpRNAs with homology to tHMG, a single copy locus and another genomic cluster containing 13 tandem hpRNAs. (B) Genomic details of the hp-tHMG-solo locus, which is a spliced transcript with defined 5’ and 3’ ends, which generates abundant small RNAs from its duplex arm. Note that the 5’ region of hp-tHMG-solo has been locally duplicated. (C) Genomic details of the hp-tHMG-cluster locus. This region bears 13 nearly identical hpRNAs, although the nature of the primary transcript(s) is not clearly evident, although 6 cluster units show evidence for 5’-seq and 3’-seq (detailed in S6 Fig). RNA-seq data across the tHMG-cluster locus appears depleted in the hpRNA duplex regions. Small RNA tracks show Dcr-2-dependent siRNAs from each cluster member. (D, E) Examples of antisense complementarity between tHMG genes and hp-tHMG-siRNAs, including autosomal tHMG1 and hp-tHMG-solo, and tHMG-Xhet1 and tHMG cluster loci.</p
