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Quadrana, L.

Publications and source records attributed to Quadrana, L..

At least 19 recordsLinked to original sources

GH1-HMGA proteins restrict RNA-directed DNA methylation and antagonize linker histone H1 at euchromatic transposable elements

In plant genomes, transposable elements (TEs) are silenced by CG and non-CG DNA methylation, with non-CG methylation maintained by CMT chromomethylases and the RNA-directed DNA methylation (RdDM) pathway. In Arabidopsis, linker histone H1 differentially influences these pathways, as loss of H1 causes non-CG hypermethylation at pericentromeric TEs but hypomethylation at euchromatic TEs in gene-rich regions. The molecular basis of this antagonism has remained unclear. Here, we show that GH1-HMGAs, a family of H1-related High Mobility Group proteins, bind AT-rich sequences at euchromatic TEs and prevent RdDM-dependent non-CG methylation by limiting siRNA accumulation. Furthermore, GH1-HMGA and H1 compete for chromatin association at these TEs, as loss of GH1-HMGA triggers ectopic H1 redistribution, while loss of H1 increases GH1-HMGA enrichment, providing a mechanistic explanation for the non-CG hypomethylation observed in h1 mutants. Collectively, these findings reveal how GH1-HMGA proteins specifically restrain RdDM activity at euchromatic TEs and elucidate how the H1/GH1-HMGA balance spatially partitions distinct DNA methylation pathways across the genome.

genetics↗

Evolution of transposons as controlling elements

Transposons were originally described as controlling elements, yet the molecular basis and evolutionary origin of their transcriptional regulatory activities remain elusive. Here, we show that transcriptional regulatory activity is an intrinsic property of transposases (TPases). We demonstrate that the Arabidopsis AtMu1 TPase is sufficient to induce sequence-specific transcriptional activation of cognate copies, revealing a pre-existing cis-regulatory network. We further demonstrate that long-recognized Spm transposon-encoded transcriptional regulator TnpA evolved from an ancestral TPase, linking transcriptional regulators to TPases. Across eukaryotes, we identify multiple domesticated TPase-derived proteins that recurrently lack catalytic residues for transposition, consistent with a one-step regulatory co-option through loss of catalytic activity. Together, our results reveal TPases as autonomous sequence-specific transcriptional regulators and provide a direct evolutionary route from TPases to transcription factors.

genetics↗

Centromeric origin of Arabidopsis interstitial telomeric repeat domains

The mechanisms underlying the emergence of new DNA repeat classes remain poorly understood. One such example is the formation of interstitial telomeric repeat (ITR) domains commonly viewed as telomere-fossil byproducts of chromosome rearrangements. Here, we report that in Arabidopsis thaliana, ITRs arise from centromeres as centromeric-telomeric mosaic satellites that expand into higher-order structures via in situ amplification. Long-read sequencing of mutation accumulation lines and forward simulations of mutational processes confirmed the propensity of centromeres to form telomeric motifs. During establishment, ITRs adopt an atypical chromatin state that excludes CENH3 incorporation, thereby enabling escape from the functional constraints of centromere identity. Accordingly, a pangenomic survey of natural accessions showed that ITRs vary abruptly and independently of telomere length, notably through transposon-associated block duplications. These findings identify centromeres as major sources of telomeric sequences and an original route for the emergence of new DNA satellite types.

genomics↗

SuB3 a Simple Subcellular Fractionation Protocol for Localization Studies of Nucleic Acids and Proteins

Understanding the subcellular localization of RNA and proteins is critical to dissecting gene regulation in eukaryotic organisms. However, this task is elusive as existing fractionation methods often rely on protoplast isolation or commercial kits, that are labor-intensive, costly, and can introduce stress-induced transcriptomic and proteomic changes. Here, we present a simple, rapid, and cost-effective protocol for the fractionation of nuclear and cytoplasmic components directly from diverse plant tissues, that does not require protoplastization. This Subcellular-fractionation protocol in 3 steps (a.k.a. "bueno, bonito y barato"- spanish for "good, nice and cheap"-), referred to as "SuB3", yields nuclear- and cytoplasmic- enriched subcellular fractions suitable for downstream applications such as RT-PCR, RNA/cDNA sequencing, and Western blotting. The procedure is based on sequential detergent-assisted extraction and centrifugation and enables the simultaneous isolation of RNA and protein from the same biological material. Due to its simplicity, speed, and broad compatibility, this protocol is a valuable tool for plant molecular biology laboratories investigating subcellular dynamics of gene expression.

molecular biology↗

Somatic mobility of transposons is explosive and shaped by distinct integration biases in Arabidopsis thaliana

BackgroundThe movement of transposable elements (TEs) in somatic cells generates genetic and phenotypic diversity across the soma of individual organisms. This process is especially important in plants that display a sessile lifestyle, can regenerate from somatic cells, and continuously establish their germline from somatic stem cells. Yet, the scale and integration biases by which TEs may shape the plant soma remain unknown. ResultsWe adapt high-throughput TE sequencing and introduce a new computational pipeline, "deNOVOEnrich", to identify and analyse somatic transposition events in Arabidopsis thaliana. We detect [~]200,000 new TE insertions across wild-type and epigenetic mutant backgrounds, revealing en masse active transposition in the plant soma. Similar to germline dynamics, somatic integration is non-random and TE-specific. Focusing on the ONSEN, EVADE and AtCOPIA21 families, we find they preferentially integrate into chromosomal arms and genic DNA, and are enriched over H2A.Z, H3K27me3 and H3K4me1 labelled chromatin. However, ONSEN dynamically targets active and open chromatin, while EVADE displays a depletion in these regions, consistent with niche targeting at the local epigenetic level and spatial distribution across the genome ecosystem. We further demonstrate that environmentally-responsive genes such as resistance (R) genes and biosynthetic gene clusters are preferred integration targets, highlighting the adaptive potential of somatic transposition. ConclusionsOur work demonstrates that transposon amplification is an explosive and structured event that generates cellular mosaicism in the soma of A. thaliana. We speculate that this phenomenon is common across plants under permissive conditions, both in experimental and natural settings.

plant biology↗

Entry and exit of Fragilariopsis cylindrus diatoms from the polar night: evidence for chromatin-mediated genome control

Regulation of the epigenome landscape plays a crucial role in adaptation to environmental changes. However, the molecular mechanisms by which epigenome landscapes are established and tuned to adjust the cellular status to environmental cues remain poorly understood, especially in unicellular photosynthetic eukaryotes. Polar microalgae must adapt to extreme variations in light and temperature for survival and therefore represent excellent models to understand the diversity of gene regulatory mechanisms, yet chromatin-based mechanisms have never been explored in these organisms. In this study, we combined genome-wide profiling of histone and DNA modifications with transcriptomic analyses of the marine polar diatom Fragilariopsis cylindrus upon entry and exit of a 3-month-long polar night. We show that, in agreement with its established role in transcription facilitation, histone H2B monobiquitination (H2Bub) is modulated at active genes upon prolonged darkness to light re-exposure. In contrast, histone H3 lysine 27 trimethylation (H3K27me3) and DNA cytosine methylation (CG methylation), two modifications typically associated with the silencing of genes or transposable elements (TEs), remained stable throughout the transitions, reinforcing their role in transcriptional repression, particularly at TEs but also at a few genes. We further demonstrate that H3K27me3 and DNA methylation are physically associated at TEs, probably reflecting a dual locking system enabling F.cylindrus cells to cope with the invasive nature of these genetic elements under extreme environmental conditions. These findings enhance our understanding of genome and epigenome regulation in diatoms in response to environmental variability and open new avenues for exploring the role played by chromatin-level regulation in the unique evolutionary history of polar unicellular eukaryotes.

genomics↗

Transposable elements are prevalent vectors of transgenerational epigenetic inheritance in nature

Loss of DNA methylation over transposable elements (TEs) can affect neighboring genes and be epigenetically inherited in plants, yet the determinants and significance of this additional system of inheritance are unknown. Here, we demonstrate at thousands of TE loci across the Arabidopsis thaliana genome, that experimentally-induced hypomethylation can be transmitted transgenerationally and reveal the role of small RNAs derived from related copies in counteracting this transmission. Using data from >700 strains collected worldwide, we uncover natural hypomethylation at hundreds of the same TE loci, often situated near stress-responsive genes. Like their experimental counterparts, most natural epivariants we tested can be inherited without DNA sequence changes and are therefore bona fide epialleles, although genetic factors modulate their recurrence or persistence. Crucially, we demonstrate that TE-mediated epiallelic variation associated with differential gene expression is generally causal and may be target of selection in specific environments, thus establishing its importance in nature.

genomics↗

Ultra-Sensitive Detection of Transposon Insertions Across Multiple Families by Transposable Element Display Sequencing

BackgroundMobilization of transposable elements (TEs) can generate large effect mutations. However, because new TE insertions are challenging to detect and transposition is typically rare, the actual rate and landscape of new insertions remains unexplored for most TEs. ResultsHere, we introduce a TE display sequencing approach that leverages target amplification of TE extremities to detect non-reference TE insertions with high sensitivity and specificity. By implementing this approach on serial dilutions of genomic DNA from A. thaliana lines carrying different repertoires of new TE insertions, we show that the method can detect TE insertions that are present at frequencies as low as 1:250 000 within a DNA sample. In addition, TE display sequencing can be multiplexed to simultaneously detect insertions for distinct TE families, including both retrotransposons and DNA transposons, increasing its versatility and cost-effectiveness to investigate complex "mobilomes". Importantly, when combined with nanopore sequencing, this approach enables the identification of insertions using long-reads and achieves a turn around time from DNA extraction to insertion identification of less than 24h, significantly reducing the time-to-answer. Analysis of TE insertions in large populations of A. thaliana plants undergoing a transposition burst demonstrate the power of the multiplex TE display sequencing to assess the rates and allele frequencies of heritable insertions, enabling its implementation to study large-scale evolve and resequence experiments. Furthermore, we found that [~]6% of de novo TE insertions show recurrent allele frequency changes consistent with either positive or negative selection. ConclusionsTE display sequencing is an ultra-sensitive, specific, quick, and cost-effective approach to investigate the rate and landscape of new insertions for multiple TEs in large scale population experiments. We provide a step-by-step experimental protocol as well as ready-to-use bioinformatic pipelines, ensuring straightforward implementation of the method.

genomics↗

Transposable element products, functions, and regulatory networks in Arabidopsis

Transposable elements (TEs) are DNA sequences with the ability to propagate themselves within genomes. Their mobilization is catalyzed by self-encoded factors, yet these factors have been poorly investigated. Here, we leveraged extensive long-and short-read transcriptome data, structural predictions, and regulatory networks analyses, to construct a comprehensive atlas of TE transcripts and their encoded products in the model organism Arabidopsis thaliana. We uncovered hundreds of transcriptionally competent TEs, each potentially encoding multiple proteins either through distinct genes, alternative splicing, or post-translational processing. Structural-based protein analyses revealed hitherto unidentified domains, uncovering proteins with multimerization and DNA binding domains forming macromolecular complexes. Furthermore, we demonstrate that TE expression is highly intertwined with the transcriptional network of cellular genes, and identified transcription factors and cis-regulatory elements associated with their coordinated expression during development or in response to environmental cues. This comprehensive functional atlas provides a valuable resource for studying the mechanisms involved in transposition and their consequences for genome and organismal function.

genomics↗

Simultaneous Profiling of Chromatin Accessibility and DNA Methylation in Complete Plant Genomes Using Long-Read Sequencing

Epigenetic regulations, including chromatin accessibility, nucleosome positioning, and DNA methylation intricately shape genome function. However, current chromatin profiling techniques relying on short-read sequencing technologies fail to characterise highly repetitive genomic regions and cannot detect multiple chromatin features simultaneously. Here, we performed Simultaneous Accessibility and DNA Methylation Sequencing (SAM-seq) of purified plant nuclei. Thanks to the use of long-read nanopore sequencing, SAM-seq enables high-resolution profiling of m6A-tagged chromatin accessibility together with endogenous cytosine methylation in plants. Analysis of naked genomic DNA revealed significant sequence preference biases of m6A-MTases, controllable through a normalisation step. By applying SAM-seq to Arabidopsis and maize nuclei we obtained fine-grained accessibility and DNA methylation landscapes genome-wide. We uncovered crosstalk between chromatin accessibility and DNA methylation within nucleosomes of genes, TEs, and centromeric repeats. SAM-seq also detects DNA footprints over cis-regulatory regions. Furthermore, using the single-molecule information provided by SAM-seq we identified extensive cellular heterogeneity at chromatin domains with antagonistic chromatin marks, suggesting that bivalency reflects cell-specific regulations. SAM-seq is a powerful approach to simultaneously study multiple epigenetic features over unique and repetitive sequences, opening new opportunities for the investigation of epigenetic mechanisms.

genomics↗

Retrotransposon-driven environmental regulation of FLC leads to adaptive response to herbicide

The mobilization of retrotransposons yields major-effect mutations. Here, we report an adaptive retrotransposon insertion within the first intron of the Arabidopsis floral-repressor locus FLOWERING LOCUS C (FLC). The insertion-mutation augments the environmental sensitivity of FLC by affecting the balance between coding and non-coding transcript isoforms in response to environmental threads. We show that this balance is modulated epigenetically by DNA methylation and orchestrated by IBM2, a factor involved in the processing of intronic heterochromatin. The stress-sensitive allele of FLC has recently spread across populations subjected to recurrent chemical weeding, and we demonstrate that retrotransposon-driven acceleration of life cycle represents a rapid response to herbicide. Our findings illustrate how retrotransposition can create environmentally-sensitive alleles that facilitate adaptation to anthropogenic disturbances of the environment.

plant biology↗

The molecular basis of phenotypic plasticity evolves in response to environmental predictability

Phenotypic plasticity, the response of a given genotype to its environment of development, is a ubiquitous feature of life, enabling organisms to cope with variation in their environment. Theoretical studies predict that, under stationary environmental variation, the level of plasticity should evolve to match the predictability of selection at the timing of development. However, we still lack critical empirical evidence on the extent to which selection on phenotypic plasticity cascades down from higher phenotypic levels to their underlying molecular basis. Here, we used experimental evolution under controlled environmental fluctuations, to test whether the evolution of phenotypic plasticity in responses to environmental predictability ({rho}2) occurred across biological levels, going from DNA methylation to gene expression to cell morphology. Transcriptomic results indicate clear effects of salinity and{rho} 2xsalinity interaction on gene expression, thus identifying sets of genes involved in plasticity and its evolution. These transcriptomic effects were independent of DNA methylation changes in cis. However we did find{rho} 2-specific responses of DNA methylation to salinity change, albeit weaker than for gene expression. Overall, we found consistent evolution of reduced plasticity in less predictable environments for DNA methylation, gene expression, and cell morphology. Our results provide the first clear empirical signature of plasticity evolution at multiple levels in response to environmental predictability, and highlight the importance of experimental evolution to address predictions from evolutionary theory, as well as investigate the molecular basis of plasticity evolution.

evolutionary biology↗

CRISPR/Cas9 gene editing uncovers the role of CTR1 and ROS1 in melon fruit ripening and epigenetic regulation

Melon (Cucumis melo L.) has emerged as an alternative model to study fruit ripening due to the coexistence of climacteric and non-climacteric varieties. The previous characterization of a major QTL ETHQV8.1 sufficient to trigger climacteric ripening in a non-climacteric background allowed the identification within the QTL interval of a negative regulator of ripening CmCTR1-like (MELO3C024518), and a putative DNA demethylase CmROS1 (MELO3C024516), the orthologue of DML2, a DNA demethylase regulating fruit ripening in tomato. To understand the role of these genes in climacteric ripening, we generated homozygous CRISPR knockout mutants of CmCTR1-like and CmROS1 in a climacteric genetic background. The climacteric behavior was altered in both loss-of-function mutants in two summer seasons with an advanced ethylene production profile compared to the climacteric wild type, suggesting a role of both genes in climacteric ripening in melon. Single cytosine methylome analyses of the CmROS1 knockout mutant revealed DNA methylation changes in the promoter regions of key ripening genes as ACS1, ETR1 and ACO1, and ripening associated-transcription factors as NAC-NOR, RIN and CNR, suggesting the importance of CmROS1-mediated DNA demethylation for triggering fruit ripening in melon.

plant biology↗

Sequence-unrelated long noncoding RNAs converged to modulate the activity of conserved epigenetic machineries across kingdoms

RNA-DNA hybrid (R-loop)-associated long noncoding RNAs (lncRNAs), including the Arabidopsis lncRNA AUXIN-REGULATED PROMOTER LOOP (APOLO), are emerging as important regulators of three-dimensional chromatin conformation and gene transcriptional activity. Here, we showed that in addition to the PRC1-component LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), APOLO interacts with the methylcytosine-binding protein VARIANT IN METHYLATION 1 (VIM1), a conserved homolog of the mammalian DNA methylation regulator UBIQUITIN-LIKE CONTAINING PHD AND RING FINGER DOMAINS 1 (UHRF1). The APOLO-VIM1-LHP1 complex directly regulates the transcription of the auxin biosynthesis gene YUCCA2 by dynamically determining DNA methylation and H3K27me3 deposition over its promoter during the plant thermomorphogenic response. Strikingly, we demonstrated that the lncRNA UHRF1 Protein Associated Transcript (UPAT), a direct interactor of UHRF1 in humans, can be recognized by VIM1 and LHP1 in plant cells, despite the lack of sequence homology between UPAT and APOLO. In addition, we showed that increased levels of APOLO or UPAT hamper VIM1 and LHP1 binding to YUCCA2 promoter. Collectively, our results uncover a new mechanism in which a plant lncRNA coordinates Polycomb action and DNA methylation, and reveal that evolutionary unrelated lncRNAs may exert similar functions across kingdoms.

plant biology↗

Efficient detection of transposable element insertion polymorphisms between genomes using short-read sequencing data

Transposable elements (TEs) are powerful generators of major-effect mutations, most of which are deleterious at the species level and maintained at very low frequencies within populations. As reference genomes can only capture a minor fraction of such variants, methods were developed to detect TE insertion polymorphisms (TIPs) in non-reference genomes from short-read sequencing data, which are becoming increasingly available. We present here a bioinformatic framework combining an improved version of the SPLITREADER and TEPID pipelines to detect non-reference TE presence and reference TE absence variants, respectively. We benchmark our method on ten non-reference Arabidopsis thaliana genomes and demonstrate its high specificity and sensitivity in the detection of TIPs between genomes.

bioinformatics↗

The impact of transposable elements on tomato diversity

Tomatoes come in a multitude of shapes and flavors despite a narrow genetic pool. Here, we leveraged whole-genome resequencing data available for 602 cultivated and wild accessions to determine the contribution of transposable elements (TEs) to tomato diversity. We identified 6,906 TE insertions polymorphisms (TIPs), which result from the mobilization of 337 distinct TE families. Most TIPs are low frequency variants and disproportionately located within or adjacent to genes involved in environmental response. In addition, we show that genic TE insertions tend to have strong transcriptional effects and can notably lead to the generation of multiple transcript isoforms. We also uncovered through genome-wide association studies (GWAS) ~180 TIPs associated with extreme variations in major agronomic traits or secondary metabolites. Importantly, these TIPs tend to affect loci that are distinct from those tagged by SNPs. Collectively, our findings suggest a unique and important role for TE mobilization in tomato diversification, with important implications for future breeding.

plant biology↗

Polycomb Repressive Complex 2 antagonizes linker DNA methylation and chromatin compaction in Arabidopsis thaliana

In plants and mammals, DNA methylation is a hallmark of transposable element (TE) sequences that contributes to their epigenetic silencing. In contrast, histone H3 lysine 27 trimethylation (H3K27me3), which is deposited by the Polycomb Repressive Complex 2 (PRC2), is a hallmark of repressed genes. Nevertheless, there is a growing body of evidence for a functional interplay between these pathways. In particular, many TE sequences acquire H3K27me3 when they lose DNA methylation and it has been proposed that PRC2 can serve as a back-up silencing system for hypomethylated TEs. Here, we describe in the flowering plant Arabidopsis thaliana the gain of H3K27m3 at hundreds of TEs in the mutant ddm1, which is defective in the maintenance of DNA methylation specifically over TE and other repeat sequences. Importantly, we show that this gain essentially depends on CURLY LEAF (CLF), which is one of two otherwise partially redundant H3K27 methyltransferases active in vegetative tissues. Finally, our results challenge the notion that PRC2 can be a compensatory silencing system for hypomethylated TEs, as the complete loss of H3K27me3 in ddm1 clf double mutant plants was not associated with further reactivation of TE expression nor with a burst of transposition. Instead, and surprisingly, ddm1 clf plants exhibited less activated TEs, and a chromatin recompaction as well as hypermethylation of linker DNA compared to ddm1. Thus, we have described an unexpected genetic interaction between DNA methylation and Polycomb silencing pathways, where a mutation in PRC2 does not aggravate the molecular phenotypes linked to TE hypomethylation in ddm1 but instead partially suppresses them. Author summaryEpigenetic marks are covalent modifications of the DNA or its associated proteins (Histones) that impact gene expression in a heritable manner without changing DNA sequence. In plants and mammals, DNA methylation and trimethylation of Lysine 27 of Histone 3 (H3K27me3) are two conserved, major epigenetic systems that mediate the transcriptional silencing of transposons (invasive mobile genetic elements) and of developmental genes respectively. However, in the absence of DNA methylation, H3K27me3 marks can be recruited to transposons, suggesting that the two silencing systems can be compensatory. To test this hypothesis, we analyzed a compound DNA methylation and H3K27me3 mutant of the plant model Arabidopsis thaliana (importantly, mammals harboring equivalent mutations would not be viable). First, this approach allowed us to gain mechanistic insights into the recruitment of H3K27me3 at transposons. Furthermore, we also showed that transposon silencing release in the DNA methylation mutant was not enhanced, contrary to our initial hypothesis, but, surprisingly, partially suppressed by a mutation in H3K27me3 deposition. Thus, our genomic analysis revealed an unexpected and antagonistic genetic interaction between two major silencing pathways whose interplay is at the heart of many biological processes, including cancer.

molecular biology↗

Natural epiallelic variation is associated with quantitative resistance to the pathogen Plasmodiophora brassicae

Clubroot caused by the protist Plasmodiophora brassicae is a major disease affecting cultivated Brassicaceae. Here, we uncover the existence of a natural epigenetic variation that is associated with partial resistance to clubroot in Arabidopsis, by using QTL fine mapping followed by extensive DNA sequence and methylation analyses. We show that at QTL Pb-At5.2, DNA methylation variation is extensive across accessions and strictly correlates with expression variation of the two neighboring genes At5g47260 and At5g47280, which encode NLR-immune receptors. Moreover, these natural variants are stably inherited and are not consistently associated with any nucleotide variation. These findings suggest a direct role for epigenetic variation in quantitative resistance of plants to pathogen attacks.

genetics↗