RESUMO
Defining the contributions and interactions of paternal and maternal genomes during embryo development is critical to understand the fundamental processes involved in hybrid vigor, hybrid sterility, and reproductive isolation. To determine the parental contributions and their regulation during Arabidopsis embryogenesis, we combined deep-sequencing-based RNA profiling and genetic analyses. At the 2-4 cell stage there is a strong, genome-wide dominance of maternal transcripts, although transcripts are contributed by both parental genomes. At the globular stage the relative paternal contribution is higher, largely due to a gradual activation of the paternal genome. We identified two antagonistic maternal pathways that control these parental contributions. Paternal alleles are initially downregulated by the chromatin siRNA pathway, linked to DNA and histone methylation, whereas transcriptional activation requires maternal activity of the histone chaperone complex CAF1. Our results define maternal epigenetic pathways controlling the parental contributions in plant embryos, which are distinct from those regulating genomic imprinting.
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Arabidopsis/embriologia , Arabidopsis/genética , Epigenômica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Arabidopsis/metabolismo , Perfilação da Expressão Gênica , Genoma de Planta , Histona-Lisina N-Metiltransferase/metabolismo , Óvulo Vegetal/metabolismo , Fatores de Processamento de RNA , RNA Interferente Pequeno/metabolismo , Sementes/genética , Ativação TranscricionalRESUMO
DNA methylation is an important epigenetic mark required for proper gene expression and silencing of transposable elements. DNA methylation patterns can be modified by environmental factors such as pathogen infection, in which modification of DNA methylation can be associated with plant resistance. To counter the plant defense pathways, pathogens produce effector molecules, several of which act as proteasome inhibitors. Here, we investigated the effect of proteasome inhibition by the bacterial virulence factor syringolin A (SylA) on genome-wide DNA methylation. We show that SylA treatment results in an increase of DNA methylation at centromeric and pericentromeric regions of Arabidopsis chromosomes. We identify several CHH differentially methylated regions (DMRs) that are enriched in the proximity of transcriptional start sites. SylA treatment does not result in significant changes in small RNA composition. However, significant changes in genome transcriptional activity can be observed, including a strong upregulation of resistance genes that are located on chromosomal arms. We hypothesize that DNA methylation changes could be linked to the upregulation of some atypical members of the de novo DNA methylation pathway, namely AGO3, AGO9, and DRM1. Our data suggests that modification of genome-wide DNA methylation resulting from an inhibition of the proteasome by bacterial effectors could be part of an epi-genomic arms race against pathogens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Epigenoma , Arabidopsis/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genéticaRESUMO
Rapid plant genome evolution is crucial to adapt to environmental changes. Chromosomal rearrangements and gene copy number variation (CNV) are two important tools for genome evolution and sources for the creation of new genes. However, their emergence takes many generations. In this study, we show that in Arabidopsis thaliana, a significant loss of ribosomal RNA (rRNA) genes with a past history of a mutation for the chromatin assembly factor 1 (CAF1) complex causes rapid changes in the genome structure. Using long-read sequencing and microscopic approaches, we have identified up to 15 independent large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. Our data suggest that these TDDOs appeared within a few generations, leading to the duplication of hundreds of genes. By subsequently focusing on a line only containing 20% of rRNA gene copies (20rDNA line), we investigated the impact of TDDOs on 3D genome organization, gene expression, and cytosine methylation. We found that duplicated genes often accumulate more transcripts. Among them, several are involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and discuss their potential implications for the evolution of plant genomes.
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Arabidopsis/genética , Resistência à Doença/genética , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Genes de RNAr , Expressão Gênica , Genes de Plantas , Genoma de Planta , Instabilidade GenômicaRESUMO
Chromosomes are folded, spatially organized, and regulated by epigenetic marks. How chromosomal architecture is connected to the epigenome is not well understood. We show that chromosomal architecture of Arabidopsis is tightly linked to the epigenetic state. Furthermore, we show how physical constraints, such as nuclear size, correlate with the folding principles of chromatin. We also describe a nuclear structure, termed KNOT, in which genomic regions of all five Arabidopsis chromosomes interact. These KNOT ENGAGED ELEMENT (KEE) regions represent heterochromatic islands within euchromatin. Similar to PIWI-interacting RNA clusters, such as flamenco in Drosophila, KEEs represent preferred landing sites for transposable elements, which may be part of a transposon defense mechanism in the Arabidopsis nucleus.
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Arabidopsis/genética , Cromatina/metabolismo , Cromossomos de Plantas/metabolismo , Drosophila/genética , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/ultraestrutura , Cromatina/química , Cromatina/ultraestrutura , Cromossomos de Plantas/química , Cromossomos de Plantas/ultraestrutura , DNA de Plantas/química , Drosophila/metabolismo , Epigenômica/métodos , Hibridização in Situ Fluorescente , Conformação de Ácido Nucleico , Análise de Componente Principal , Análise de Sequência de DNARESUMO
Arabidopsis encodes 10 ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21-22 nucleotide (nt) long small RNAs (sRNAs) to mediate post-transcriptional gene silencing (PTGS) or 24 nt sRNAs to promote RNA-directed DNA methylation. Using full-locus constructs, we characterized the expression, biochemical properties and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression patterns differ drastically, with AGO2 being expressed in both male and female gametes whereas AGO3 accumulates in aerial vascular terminations and specifically in chalazal seed integuments. Accordingly, AGO3 downregulation alters gene expression in siliques. Similar to AGO2, AGO3 binds sRNAs with a strong 5' adenosine bias, but unlike Arabidopsis AGO2, it binds 24 nt sRNAs most efficiently. AGO3 immunoprecipitation experiments in siliques revealed that these sRNAs mostly correspond to genes and intergenic regions in a manner reflecting their respective accumulation from their loci of origin. AGO3 localizes to the cytoplasm and co-fractionates with polysomes to possibly mediate PTGS via translation inhibition.
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Proteínas de Arabidopsis/fisiologia , Proteínas Argonautas/fisiologia , Flores/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Flores/fisiologia , Duplicação GênicaRESUMO
In recent years, the study of plant three-dimensional nuclear architecture received increasing attention. Enabled by technological advances, our knowledge on nuclear architecture has greatly increased and we can now access large data sets describing its manifold aspects. The principles of nuclear organization in plants do not significantly differ from those in animals. Plant nuclear organization comprises various scales, ranging from gene loops to topologically associating domains to nuclear compartmentalization. However, whether plant three-dimensional chromosomal features also exert similar functions as in animals is less clear. This review discusses recent advances in the fields of three-dimensional chromosome folding and nuclear compartmentalization and describes a novel silencing mechanism, which is closely linked to nuclear architecture.
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Arabidopsis , Núcleo Celular , Animais , Arabidopsis/genética , Núcleo Celular/genética , CromatinaRESUMO
BACKGROUND: Cassava is an important food crop in tropical and sub-tropical regions worldwide. In Africa, cassava production is widely affected by cassava mosaic disease (CMD), which is caused by the African cassava mosaic geminivirus that is transmitted by whiteflies. Cassava breeders often use a single locus, CMD2, for introducing CMD resistance into susceptible cultivars. The CMD2 locus has been genetically mapped to a 10-Mbp region, but its organization and genes as well as their functions are unknown. RESULTS: We report haplotype-resolved de novo assemblies and annotations of the genomes for the African cassava cultivar TME (tropical Manihot esculenta), which is the origin of CMD2, and the CMD-susceptible cultivar 60444. The assemblies provide phased haplotype information for over 80% of the genomes. Haplotype comparison identified novel features previously hidden in collapsed and fragmented cassava genomes, including thousands of allelic variants, inter-haplotype diversity in coding regions, and patterns of diversification through allele-specific expression. Reconstruction of the CMD2 locus revealed a highly complex region with nearly identical gene sets but limited microsynteny between the two cultivars. CONCLUSIONS: The genome maps of the CMD2 locus in both 60444 and TME3, together with the newly annotated genes, will help the identification of the causal genetic basis of CMD2 resistance to geminiviruses. Our de novo cassava genome assemblies will also facilitate genetic mapping approaches to narrow the large CMD2 region to a few candidate genes for better informed strategies to develop robust geminivirus resistance in susceptible cassava cultivars.
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Resistência à Doença/genética , Haplótipos/genética , Manihot/genética , Doenças das Plantas/genética , Mapeamento Cromossômico/métodos , Suscetibilidade a Doenças , Geminiviridae , Predisposição Genética para Doença , Anotação de Sequência MolecularRESUMO
BACKGROUND: The study of nuclear architecture using Chromosome Conformation Capture (3C) technologies is a novel frontier in biology. With further reduction in sequencing costs, the potential of Hi-C in describing nuclear architecture as a phenotype is only about to unfold. To use Hi-C for phenotypic comparisons among different cell types, conditions, or genetic backgrounds, Hi-C data processing needs to be more accessible to biologists. RESULTS: HiCdat provides a simple graphical user interface for data pre-processing and a collection of higher-level data analysis tools implemented in R. Data pre-processing also supports a wide range of additional data types required for in-depth analysis of the Hi-C data (e.g. RNA-Seq, ChIP-Seq, and BS-Seq). CONCLUSIONS: HiCdat is easy-to-use and provides solutions starting from aligned reads up to in-depth analyses. Importantly, HiCdat is focussed on the analysis of larger structural features of chromosomes, their correlation to genomic and epigenomic features, and on comparative studies. It uses simple input and output formats and can therefore easily be integrated into existing workflows or combined with alternative tools.
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Genômica/métodos , RNA/genética , Cromossomos , Humanos , Software , Estatística como AssuntoRESUMO
Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function; it restricts enhancer-promoter communication and contributes to enhancer specificity.
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Research on the three-dimensional (3D) structure of the genome and its distribution within the nuclear space has made a big leap in the last two decades. Work in the animal field has led to significant advances in our general understanding on eukaryotic genome organization. This did not only bring along insights into how the 3D genome interacts with the epigenetic landscape and the transcriptional machinery but also how 3D genome architecture is relevant for fundamental developmental processes, such as cell differentiation. In parallel, the 3D organization of plant genomes have been extensively studied, which resulted in both congruent and novel findings, contributing to a more complete view on how eukaryotic genomes are organized in multiple dimensions. Plant genomes are remarkably diverse in size, composition, and ploidy. Furthermore, as intrinsically sessile organisms without the possibility to relocate to more favorable environments, plants have evolved an elaborate epigenetic repertoire to rapidly respond to environmental challenges. The diversity in genome organization and the complex epigenetic programs make plants ideal study subjects to acquire a better understanding on universal features and inherent constraints of genome organization. Furthermore, considering a wide range of species allows us to study the evolutionary crosstalk between the various levels of genome architecture. In this article, we aim at summarizing important findings on 3D genome architecture obtained in various plant species. These findings cover many aspects of 3D genome organization on a wide range of levels, from gene loops to topologically associated domains and to global 3D chromosome configurations. We present an overview on plant 3D genome organizational features that resemble those in animals and highlight facets that have only been observed in plants to date.
Assuntos
Cromatina , Cromossomos , Humanos , Animais , Cromatina/genética , Genoma , Diferenciação CelularRESUMO
While the pivotal role of linker histone H1 in shaping nucleosome organization is well established, its functional interplays with chromatin factors along the epigenome are just starting to emerge. Here we show that, in Arabidopsis, as in mammals, H1 occupies Polycomb Repressive Complex 2 (PRC2) target genes where it favors chromatin condensation and H3K27me3 deposition. We further show that, contrasting with its conserved function in PRC2 activation at genes, H1 selectively prevents H3K27me3 accumulation at telomeres and large pericentromeric interstitial telomeric repeat (ITR) domains by restricting DNA accessibility to Telomere Repeat Binding (TRB) proteins, a group of H1-related Myb factors mediating PRC2 cis recruitment. This study provides a mechanistic framework by which H1 avoids the formation of gigantic H3K27me3-rich domains at telomeric sequences and contributes to safeguard nucleus architecture.
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Proteínas de Arabidopsis , Arabidopsis , Animais , Histonas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cromatina , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/genética , Telômero/metabolismo , Mamíferos/metabolismoRESUMO
The ability to decipher the three-dimensional chromosome folding in many eukaryotes is a major asset in molecular biology. It is not only required to study the biological relevance of chromosome folding in cellular processes but also for the de novo assembly of genomes of nonmodel species. With lowering DNA sequencing costs, the latter has recently become interesting to many scientists, ranging from molecular biologists that aim to establish new model organisms, to evolutionary biologists and ecologists, interested in genome evolution and diversity. Hi-C is regarded as the method of choice to characterize three-dimensional genome folding and, thus, also has been integrated as a standard method in assembly pipelines. However, Hi-C is a demanding molecular biology technique, and its application can be considerably challenged by the tissue used. Hi-C relies on efficient and pure nuclei isolation, which is, especially in many plant species, inhibited by the tough nature of plant tissues and cell walls. The Hi-C protocol presented here has been optimized for such tissues and has been shown to generate Hi-C samples of sufficient quality in various plant and animal tissues.
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Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Núcleo Celular/genética , Cromossomos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodosRESUMO
The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. After entering interphase, the positions of the scattered centromeres are then stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWN proteins have little effect on chromatin organization involved in the control of gene expression, indicating a robustness of chromatin organization regardless of the type of centromere distribution.
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Centrômero , Membrana Nuclear , Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA , Complexos Multiproteicos , Membrana Nuclear/metabolismoRESUMO
Research on plant three-dimensional (3D) genome architecture made rapid progress over the past 5 years. Numerous Hi-C interaction data sets were generated in a wide range of plant species, allowing for a comprehensive overview on 3D chromosome folding principles in the plant kingdom. Plants lack important genes reported to be vital for chromosome folding in animals. However, similar 3D structures such as topologically associating domains and chromatin loops were identified. Recent studies in Arabidopsis thaliana revealed how chromosomal regions are positioned within the nucleus by determining their association with both, the nuclear periphery and the nucleolus. Additionally, many plant species exhibit high-frequency interactions among KNOT entangled elements, which are associated with safeguarding the genome from invasive DNA elements. Many of the recently published Hi-C data sets were generated to aid de novo genome assembly and remain to date little explored. These data sets represent a valuable resource for future comparative studies, which may lead to a more profound understanding of the evolution of 3D chromosome organization in plants.
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Cromossomos de Plantas/genética , Magnoliopsida/genética , Oryza/genética , AnimaisRESUMO
BACKGROUND: The three-dimensional (3D) organization of chromosomes is linked to epigenetic regulation and transcriptional activity. However, only few functional features of 3D chromatin architecture have been described to date. The KNOT is a 3D chromatin structure in Arabidopsis, comprising 10 interacting genomic regions termed KNOT ENGAGED ELEMENTs (KEEs). KEEs are enriched in transposable elements and associated small RNAs, suggesting a function in transposon biology. RESULTS: Here, we report the KNOT's involvement in regulating invasive DNA elements. Transgenes can specifically interact with the KNOT, leading to perturbations of 3D nuclear organization, which correlates with the transgene's expression: high KNOT interaction frequencies are associated with transgene silencing. KNOT-linked silencing (KLS) cannot readily be connected to canonical silencing mechanisms, such as RNA-directed DNA methylation and post-transcriptional gene silencing, as both cytosine methylation and small RNA abundance do not correlate with KLS. Furthermore, KLS exhibits paramutation-like behavior, as silenced transgenes can lead to the silencing of active transgenes in trans. CONCLUSION: Transgene silencing can be connected to a specific feature of Arabidopsis 3D nuclear organization, namely the KNOT. KLS likely acts either independent of or prior to canonical silencing mechanisms, such that its characterization not only contributes to our understanding of chromosome folding but also provides valuable insights into how genomes are defended against invasive DNA elements.
Assuntos
Arabidopsis/genética , Inativação Gênica , Genoma de Planta , Conformação de Ácido Nucleico , Transgenes , Elementos de DNA TransponíveisRESUMO
BACKGROUND: Chromatin provides a tunable platform for gene expression control. Besides the well-studied core nucleosome, H1 linker histones are abundant chromatin components with intrinsic potential to influence chromatin function. Well studied in animals, little is known about the evolution of H1 function in other eukaryotic lineages for instance plants. Notably, in the model plant Arabidopsis, while H1 is known to influence heterochromatin and DNA methylation, its contribution to transcription, molecular, and cytological chromatin organization remains elusive. RESULTS: We provide a multi-scale functional study of Arabidopsis linker histones. We show that H1-deficient plants are viable yet show phenotypes in seed dormancy, flowering time, lateral root, and stomata formation-complemented by either or both of the major variants. H1 depletion also impairs pluripotent callus formation. Fine-scale chromatin analyses combined with transcriptome and nucleosome profiling reveal distinct roles of H1 on hetero- and euchromatin: H1 is necessary to form heterochromatic domains yet dispensable for silencing of most transposable elements; H1 depletion affects nucleosome density distribution and mobility in euchromatin, spatial arrangement of nanodomains, histone acetylation, and methylation. These drastic changes affect moderately the transcription but reveal a subset of H1-sensitive genes. CONCLUSIONS: H1 variants have a profound impact on the molecular and spatial (nuclear) chromatin organization in Arabidopsis with distinct roles in euchromatin and heterochromatin and a dual causality on gene expression. Phenotypical analyses further suggest the novel possibility that H1-mediated chromatin organization may contribute to the epigenetic control of developmental and cellular transitions.
Assuntos
Arabidopsis/genética , Cromatina/química , Histonas/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Epigênese Genética , Eucromatina/química , Regulação da Expressão Gênica de Plantas , Heterocromatina/química , Histonas/genética , Histonas/metabolismo , Mutação , NucleossomosRESUMO
[This corrects the article DOI: 10.1038/s41438-018-0071-9.].
RESUMO
The introduction of chromosome conformation capture (3C) technologies boosted the field of 3D-genome research and significantly enhanced the available toolset to study chromosomal architecture. 3C technologies not only offer increased resolution compared to the previously dominant cytological approaches but also allow the simultaneous study of genome-wide 3D chromatin contacts, thereby enabling a candidate-free perspective on 3D-genome architecture. Since its introduction in 2002, 3C technologies evolved rapidly and now constitute a collection of tools, each with their strengths and pitfalls with respect to specific research questions. This chapter aims at guiding 3C novices through the labyrinth of potential applications of the various family members, hopefully providing a valuable basis for choosing the appropriate strategy for different research questions.
Assuntos
Cromatina/genética , Cromossomos/química , Imageamento Tridimensional/métodos , Animais , Cromatina/química , Mapeamento Cromossômico , Humanos , Conformação de Ácido NucleicoRESUMO
Brassica rapa comprises several important cultivated vegetables and oil crops. Current reference genome assemblies of Brassica rapa are quite fragmented and not highly contiguous, thereby limiting extensive genetic and genomic analyses. Here, we report an improved assembly of the B. rapa genome (v3.0) using single-molecule sequencing, optical mapping, and chromosome conformation capture technologies (Hi-C). Relative to the previous reference genomes, our assembly features a contig N50 size of 1.45 Mb, representing a ~30-fold improvement. We also identified a new event that occurred in the B. rapa genome ~1.2 million years ago, when a long terminal repeat retrotransposon (LTR-RT) expanded. Further analysis refined the relationship of genome blocks and accurately located the centromeres in the B. rapa genome. The B. rapa genome v3.0 will serve as an important community resource for future genetic and genomic studies in B. rapa. This resource will facilitate breeding efforts in B. rapa, as well as comparative genomic analysis with other Brassica species.
RESUMO
The study of nuclear architecture promises novel insights into genome function and regulation. Hereby, quantitative methods based on chromosome conformation capture (3C) revolutionized the field, as they allow accurate and unbiased characterization of 3D genome organization of genomic regions of interest. The choice of the appropriate 3C derivate is crucial to acquire results suited for a specific research question. Circular 3C (4C) is the method of choice to study the genome-wide 3D architecture of a specific genomic region of interest. Here, I present a robust 4C protocol, established in Arabidopsis thaliana, which can be employed by any experienced molecular biologist and is applicable in various other plant species.