Entering the Next Dimension: Plant Genomes in 3D

Review

Entering the Next Dimension: Plant Genomes in 3D Mariana Sotelo-Silveira,1 Ricardo A. Chávez Montes,2 Jose R. Sotelo-Silveira,3,4 Nayelli Marsch-Martínez,5 and Stefan de Folter2,* After linear sequences of genomes and epigenomic landscape data, the 3D organization of chromatin in the nucleus is the next level to be explored. Different organisms present a general hierarchical organization, with chromosome territories at the top. Chromatin interaction maps, obtained by chromosome conformation capture (3C)-based methodologies, for eight plant species reveal commonalities, but also differences, among them and with animals. The smallest structures, found in high-resolution maps of the Arabidopsis genome, are single genes. Epigenetic marks (histone modification and DNA methylation), transcriptional activity, and chromatin interaction appear to be correlated, and whether structure is the cause or consequence of the function of interacting regions is being actively investigated.

Highlights Chromosome conformation capture (3C)-based methods reveal the 3D genome of eukaryotic organisms. Plant chromosomes are hierarchically organized in chromosome territories as the largest structure and subsequent smaller domains. Hi-C maps of different plant species have uncovered different chromatin interactions. Intrachromosomal interactions are more frequent than interchromosomal interactions.

Chromatin Organization: A New Level of Regulation? Plants can adopt alternative phenotypes in response to the environmental conditions. This phenomenon, called phenotypic plasticity [1,2], is based on the capacity of cells to modify their genetic programs. The plasticity of the cell nuclear program is well illustrated by constant changes in gene activity, guiding the process of plant development [2]. Although the molecular mechanisms underlying this plasticity are not well understood, fine regulation of the biological processes during the development of an organism is crucial. Biological processes are controlled by gene regulatory networks, which are composed of transcription factors (see Glossary) and their targets, but also hormones, microRNAs, peptides, and chromatin-modifying proteins [3–5]. Many factors have been described that influence the transcription output, such as promoter organization, protein–DNA interactions, and protein–protein interactions, that are fine-tuned by the interaction affinities of the respective complexes [6–9]. A new dimension that is being incorporated into these multilevel regulatory networks in multicellular eukaryotes is the physical architecture of chromatin at the subchromosomal level (Figure 1 and Box 1). DNA organization into chromatin allows the long chromosomal fibers to fit into the nucleus, but can restrict access of regulatory proteins to DNA. Chromatin remodeling complexes regulate access to DNA by modifying chromatin structure, type of histone, and nucleosome positioning, thus controlling gene expression [10–16]. Although chromatin accessibility has been correlated to gene expression, not all gene expression changes are associated with changes in chromatin accessibility [7,17]. In eukaryotes, chromatin refers to the linear nucleotide sequence that composes the genome, associated with proteins [12,18–21] (Box 1). The traditional distinction among different chromatin states at specific nuclear regions is based primarily on gene content and gene expression levels. However, gene transcription rate is not uniform within a particular chromatin state, suggesting that 598

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Chromatin contacts frequently correlate with specific epigenetic states. 1

Departamento de Biología Vegetal, Laboratorio de Bioquímica, Facultad de Agronomía, Garzón 809, 12900 Montevideo, Uruguay 2 Unidad de Genómica Avanzada[295_TD$IF], Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Km. 9.6 Libramiento Norte, Carretera IrapuatoLeón, [296_TD$IF]36824 Irapuato, Guanajuato, Mexico 3 Department of Genomics, Instituto de Investigaciones Biológicas Clemente Estable, [298_TD$IF]Av. Italia 3318, 11600 Montevideo, Uruguay 4 Sección Biología Celular, Dept. Cell and Molecular Biology, Facultad de Ciencias, Universidad de la Republica, Igua 4225, Montevideo, Uruguay 5 Departamento de Biotecnología y Bioquímica, Unidad Irapuato, CINVESTAV-IPN, Km. 9.6 Libramiento Norte, Carretera Irapuato-León, [296_TD$IF]36824 Irapuato, Guanajuato, Mexico *Correspondence: [email protected] (S. de Folter).

classification of the latter is a more nuanced subject [22–24]. In Arabidopsis (e.g., Arabidopsis thaliana) and other plants, different chromatin states correlate with different genomic elements (Box 1) [19,23,25–27]. Beyond the linear genome organization and the epigenome, the 3D genome organization within the nucleus in plants is starting to be understood [28,29]. Animals, yeast, and plants present general chromatin packaging similarities in terms of spatial organization of the genome inside the nucleus (Figure 1A). Chromosome folding involves genomic regions that interact and form structural units that include large chromosomal domains and nuclear bodies with different functions (e.g., repressive nuclear structures) [22,28–34]. There are also differences among the organisms studied, related to genome size and gene density [28,35–38]. Development of chromosome conformation capture (3C)-based technologies (see Figure I in Box 2) has opened the possibility of reconstructing the 3D organization of nuclear architectures. These technologies have uncovered a variety of structural interactions between chromosomes [39–42], and have identified the assembly of intranuclear local microenvironments, where regulatory interactions that impact gene transcription are favored [43–47]. This has added a new level of complexity to our understanding of the regulation of genome function. In this review, we focus on the recent developments in the field of plant chromatin structures and their relation to gene expression.

Chromosome Interactions in Plant Nuclei Understanding nuclear architecture depends on prior knowledge of the linear genome [20,32,45,48]. Due to the quality, size (130 Mb), and low repeat content of its genome, Arabidopsis was, initially, the main organism used for plant chromosome architecture studies, based on high-throughput sequencing and other molecular tools [19,22,49,50] (Figure 1B). However, Hi-C studies in rice (Oryza sativa) and barley (Hordeum vulgare), and more recently also in maize (Zea mays), tomato (Solanum lycopersicum), sorghum (Sorghum bicolor), foxtail millet (Setaria italica), and cotton (Gossypium spp.) are broadening our knowledge about the 3D organization of plant genomes [25–27,51,52]. Particularly, one of the latest analyses employed the same type of cells, mesophyll protoplasts (which are relatively uniform and mainly differentiated), of five different species, which allowed the comparison of the 3D genome organization of these species. The latest studies also include the simultaneous characterization of chromatin accessibility, DNA methylation, histone modification, and gene expression, trying to link these characteristics to the 3D organization information [25,27]. Chromosome Territories The eukaryotic nuclear 3D space involves a hierarchy of structures of different sizes [30]. The chromosome territory (CT) is at the top (Figure 1A). Interphase chromosomes can be visualized as being organized into discrete territories that are distributed within the nucleus and relative to other chromosomes [2,43,53–55]. Microscopy and fluorescent in situ hybridization (FISH) have revealed different configurations of chromosome organization in the nucleus of different plants, depending on the way chromosome arms, centromere, and telomeres fold and contact each other: Rabl, Bouquet, and Rosette (reviewed in [2,22,28]). FISH in Arabidopsis revealed that centromeres are located at the nuclear periphery, euchromatic chromosome arm regions form mainly distinct CTs with some loops outside their CT, and telomeres cluster at [29_TD$IF]nucleolus organizer regions (NORs) [53,56,57]. During the past decade, the pioneering 3C method [58], and subsequent 3C-derived methods (4C [59,60], 5C [61,62], Hi-C [63], and ChIA-PET [64]) were developed (see Figure I in Box 2).

Glossary A/B compartment: chromosomes are divided into two sets of loci, named A/B compartments. A compartments are enriched in generich, transcriptionally active chromatin regions and B compartments in gene-poor, transcriptionally silent chromatin regions. Chromatin: a negatively charged, long polymer composed of genomic DNA, histones, and various proteins. The chromatin structure is highly dynamic. The primary function of chromatin is to compact DNA to fit inside the nucleus. Chromosome conformation capture (3C): a method that allows detection of long-range interactions between specific pairs of loci (one versus one). Chromosome territory (CT): the chromosome occupies a defined space inside the nucleus that only intermingles with its immediate neighbors. Cohesin: a protein complex with three core subunits that form a ringshaped structure that can entrap DNA in its lumen. It shapes the genome by looping together CTCF sites along the chromosome, and has an essential role in TAD formation. CTCF: CCCTC-binding protein (CTCF) has, among others, the role of regulating the 3D genome architecture, helping to establish TAD borders. Enhancer: enhancers are noncoding DNA sequences that can be bound by multiple transcription factors (TFs) to activate the expression of genes located up to several Mb away. Fluorescent in situ hybridization (FISH): a technique used to detect the position of a DNA sequence on chromosomes. Gene loops: dynamic structures that juxtapose the 30 ends of genes with their promoters. Hi-C: a 3C-derived method that allows unbiased identification of chromatin interactions across the entire genome (all versus all) Insulator: cis regulatory elements that have a role in structural organization of domains inside the nucleus, due to their ability to block enhancer–promoter interactions and

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These methods provided information of chromosome organization at higher resolution and allowed comprehensive analyses of complete genome 3D folding. 4C and Hi-C experiments in Arabidopsis revealed that intrachromosomal interactions occur preferentially within chromosome arms, implying that they form CTs separated by the centromeres [18,65] (Figure 1B). Telomeres interact with each other, except for those close to the NORs on the short arms of chromosomes 2 and 4, frequently associated with centromeric regions [18]. Pericentromeric regions, seen as compacted in chromocenters, interacted weakly with the rest of the genome [65]. The strongest interactions were observed between pericentromeric heterochromatin and between telomeres [66]. Recently, a rice genome Hi-C map at 50 kb resolution revealed strong signals along a diagonal, indicating that interactions between sequences are close to each other in the linear genome [51]. Moreover, centromeres of different chromosomes were separated, while telomeres clustered together in the nuclear space [51]. Interestingly, the authors also tested the effect of cold conditions in rice chromatin packing, and found that cold caused a general chromosome decondensation. Decondensation of heterochromatin regions and changes in chromatin state have also been observed in Drosophila melanogaster [67] and plants (reviewed in [68]) subjected to stress. Also recently, Hi-C was used to order and orient scaffolds of the barley genome sequence [52] and to study its 3D chromatin nuclear organization. As in other plant genomes, the linear distance between loci determined the Hi-C link frequency. However, the contact frequency increased at distances above 200 Mb, observed as an antidiagonal in the Hi-C map of all chromosomes, indicating that regions in different chromosome arms laid adjacent in the nucleus. Regions near telomeres also presented a high contact frequency, suggesting a Rabl configuration, where both chromosome arms interact and all centromeres are located to one side and all telomeres to the other side of the nucleus [52]. One of the most recent Hi-C studies, performed in five plant species with genome sizes that range from 0.4 to 2.4 Gb [25], found frequent interactions between adjacent loci. Chromosome territories were inferred from the strong intra- and interchromosomal interactions mainly between euchromatin arms in maize, tomato, sorghum, foxtail millet, and rice. The largest genome in this study was the maize genome. Its Hi-C map presented intense interaction signals on the antidiagonal lines, indicating interaction between the two chromosome arms. Moreover, the arms of different chromosomes presented frequent interactions. Also, the centromeric regions of different chromosomes interacted. These interactions were not observed in the other four middle-sized genomes that were compared, but resembled the interactions reported for the large barley genome [25,52]. 4C and Hi-C data provides relevant information about chromatin [30_TD$IF]interactions with itself [63], but not on its location in the nuclear space. In most vertebrates, a radial arrangement, where chromosome gene-poor regions are located at the nuclear periphery and gene-rich regions to the interior, is observed [54,55]. A recent study using restriction enzyme (RE)-mediated ChIP and the Arabidopsis nucleoprotein NUP showed that chromatin regions with stronger contact with the nuclear periphery were mostly repressed domains, for example pericentromeric regions. However, telomeres and interactive heterochromatic islands (IHIs) or KNOT engaged elements (KEEs) (explained below) were preferentially found in the nuclear interior. This arrangement pattern was conserved in different tissues [33,69,70].

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the spreading of heterochromatin silencing effects. KNOT: a nuclear structure in Arabidopsis, consisting of an entanglement of 10 chromosomal regions. These regions represent heterochromatic islands within euchromatin. KNOT engaged elements (KEE) or interactive heterochromatic islands (IHI): KEEs or IHIs are regions enriched in strong interchromosomal interactions of loci far apart in the DNA sequence, observed in Arabidopsis Hi-C maps. Lamina-associated domain (LAD): genomic regions that are in close contact with the nuclear lamina. Most of the genes in LADs are very lowly expressed. Nucleolus organizer regions (NORs): chromosomal regions important for the formation of the nucleolus. Polycomb: proteins that are conserved chromatin factors regulating key developmental genes. Polycomb group proteins bind to discrete genomic elements for the silencing of neighboring genes. Positive strips: local chromatinpacking features that show more frequent interactions with neighboring chromatin. It can be seen as contrasting lines in a 2 kb resolution Hi-C map of the Arabidopsis genome. TCP: family of plant-specific transcription factors. TCP binding sequence motifs were shown to be enriched in TAD borders in rice, which points to a new structural role of this family of proteins. TFIIIC: transcription factor with insulator activity associated with RNA Pol III. Topologically associating domain (TAD): genomic domains enriched in chromatin interactions within them. TADs are megabase-sized and have specific positions in the genome. Transcription factor: protein that can bind DNA cis regulatory elements, thereby regulating gene expression. Transcription factories: specialized sites in the 3D genome where transcription occurs. Each factory contains several stationary RNA polymerase II molecules (4–30 in animals) surrounding a protein-rich core involved in transcription. However, they have not been observed in Arabidopsis.

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Box 1. Chromatin Structure The basic unit of chromatin is the nucleosome, which consists of a histone protein octamer, composed of two protein complexes of each of the four core histones (H2A, H2B, H3, and H4) wrapped by 147 bp of DNA [19,49]. Both plants and animals present separate chromosomal regions with distinct levels of condensation, which were described by cytologists using various chromatin dyes and DNA-binding fluorochromes, and are named euchromatic (decondensed and light staining) and heterochromatic (compact and dense staining) regions [2]. The traditional distinction is that euchromatin is gene-rich and transcriptionally active, while heterochromatin is gene-poor and transcriptionally silent [49]. However, the fact that gene transcription rate is never uniform in all genes present in the same chromatin region signifies that chromatin classification is more complex [22,23]. Chromatin structure is highly dynamic [67,68,88] and it has been suggested to have a liquid-like behavior [89]. Chromatin composition can be modified: at the DNA level (e.g., cytosine methylation) or histone level (acetylation, methylation, phosphorylation, ubiquitination, and nucleosome histone replacement with histone variants). These variations, together with nucleosome positioning, have been shown to regulate the traffic of information encoded by the linear genome [12,28]. In Arabidopsis thaliana, nine chromatin states have been described [19,23,97], which are as follows: (i) five chromatin states associated with expressed genes and proximal upstream promoter regions, which occupy half of the genome and are enriched in active marks such as H3K4me2 and H3K4me3, H3 acetylation, H3K36me3, and H2Bub; (ii) two chromatin states, GC-rich and AT-rich, that correspond to heterochromatin; and (iii) polycomb-regulated regions enriched in H3K27me3 marks. The remaining part of the genome is occupied by intergenic regions that delineate the transitions between the above-mentioned chromatin states. The co-occurrence of H3K4me3 (an active chromatin mark) and H3K27me3 (a polycomb, inactive chromatin mark) in the same chromatin fiber evidences that the Arabidopsis linear genome is arranged in 3D space in a topology that combines active and inactive regions [23]. Moreover, the genome can be subdivided into five major functional elements [19]: (i) proximal promoter and transcription start sites (TSS)/50 -UTRs; (ii) genic regions; (iii) distal regulatory intergenic regions; (iv) polycomb chromatin; and (v) silent heterochromatin. The nine chromatin states highly correlate with the five genomic elements mentioned above [19,23].

Interchromosomal Interactions In Arabidopsis, the majority of long-range interactions occur between loci far apart in the primary DNA sequence that form a structure named the KNOT: KEEs [66], or IHIs [38]. Chromatin surrounding these loci contains patches of heterochromatin in euchromatic arms, marked by H3K9me2, with numerous transposable elements (TE)-related repetitive sequences, but flanked by expressed protein-coding genes [38]. These KEEs/IHIs do not appear to be affected in DNA or H3 methylation mutants, but they are enhanced in the atmorc6 mutant [38]. In the rice Hi-C map, the most prominent interchromosomal interactions occurred between regions at the ends of the chromosomes, suggesting that telomeres of different chromosomes tend to cluster [51]. Interestingly, the Hi-C map of the F1 of a cross between two different barley cultivars revealed that the interaction frequency between homologous chromosomes was not higher than between nonhomologous chromosomes, suggesting no preferential interaction between homologous chromosomes [52]. However, in a study comparing tetraploid and diploid cotton species, interaction among the subgenomes in tetraploid species and a correlation between interaction and expression of homoeologous genes in the different subgenomes was observed [27].

Figure 1. Nuclear Chromosome Organization and Chromatin Interactions. (A) Model of nuclear organization (at different resolutions) described for animal models. Chromosomes at interphase are organized into discrete chromosome territories (CTs). This distribution can affect trans interactions and indicate whether a particular locus is surrounded by an active (A compartment) or repressive environment (B compartment). At higher resolution, different types of subnuclear foci are depicted: lamina-associated domain (LAD), characterized by their low gene density and enrichment in CTCF proteins that separate chromatin environments at the nuclear periphery; insulators, which prevent the spread of heterochromatin into flanking chromatin domains; Pc bodies associated with repressive marks; transcription factories, which are active genes that relocate from chromosome territories for gene expression; and topologically associating domains (TADs), genomic domains enriched with chromatin interactions within them. (B) Example of a Hi-C map of chromatin interaction frequencies. Hi-C profiles reveal that the genome is divided into regions that show high levels of cis interactions (intrachromosomal; diagonal line) and less frequent trans interactions (interchromosomal). The Hi-C contact matrix was drawn using Juicebox [100] and the Arabidopsis thaliana Col dataset [38]; the frequency of the interactions is depicted in a white to red color scale. (C) Examples of TADs. The contact matrix was drawn using the 3D Genome Browser [101] and Hi-C data for a region of approximately 3 Mb of the human chromosome 15 at 25 kb resolution [102]; interaction frequencies are depicted in a white to red scale. (D) Linear representation of hypothetical chromatin contacts between genomic elements that can be found inside a TAD. (E) Representation of possible chromatin loops based on the contacts presented in (D).

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Compartments or Structural Domains The next level down the 3D structural hierarchy of many eukaryotic genomes, below CTs, are chromatin compartments of similar status that cluster to conform the CT [30,55]. First described in human cells, A and B compartments are comprised of multichromosomal domains up to 5 Mb that can be in one chromosome or the zone in which two chromosome territories intermingle (Figure 1A). Gene expression levels are correlated to these compartments: A compartments are gene rich, transcriptionally active, and accessible (DNase I hypersensitive), whereas loci found in B compartments are gene poor, transcriptionally silent, and DNase I insensitive [63]. In Arabidopsis, Hi-C maps at megabase-scale revealed that chromosome arms are organized in domains of interacting regions called structural domains (SDs) and analogous to A/B compartments [36,66]. Loose structural domains (LSDs) represent less compacted euchromatin, while closed structural domains (CSDs) are depleted in active and enriched in repressive epigenetic marks in comparison with LSDs [28,66]. These megabase-sized subdomains, which highly correlate with epigenomic marks, have also recently been found in the rice genome [51]. In rice, Hi-C revealed a mostly euchromatic A (LSD) compartment and a B (CSD), mostly heterochromatic compartment, with generally higher levels of CG and CHG methylation, that included centromeres and covered about 60% of the genome. B compartment/CSDs presented weak interchromosomal interactions, indicating less centromere colocalization than in Arabidopsis. A compartment/LSDs, however, presented a higher interaction frequency, suggesting a most likely localization at the outer layer of chromosome territories [51]. Interestingly, in the barley Hi-C study, the authors concluded that the frequency at which different loci interact is mainly determined by their position along the genomic sequence, in contrast to the open and closed chromatin compartments (A/B, LSD/ CSD) observed at megabase resolution of chromatin organization in animals, Arabidopsis, or rice [52]. This may be due to the large and repeat-rich nature of the barley genome [52,71]. In the five genomes (maize, tomato, sorghum, foxtail millet, and rice) comparative study that used the same approach as for mammalian chromosomes [63], very broad global Box 2. Chromosome Conformation Capture (3C) Technologies The fundamental objective of 3C and 3C-derived methods is to observe chromosome organization by identifying the localization and frequency of the physical interactions between chromosomes [58]. In all 3C methods, first a fixative agent (formaldehyde) is used to crosslink the chromatin in order to obtain a snapshot of all protein–protein and protein–DNA interactions. Fixed chromatin is digested with a restriction enzyme, followed by re-ligation of the crosslinked DNA fragments, allowing sequences that were apart in the linear template, but colocalized in space, to be ligated. The actual sequences present in the ligation products can then be identified by PCR, ligation-mediated amplification, or direct sequencing. Through the determination of the relative frequencies with which different sites have become crosslinked, a global view of the interactions between loci is obtained. 3C allows the detection of interactions between a limited number of known sequences, a one to one approach. Nowadays, there are many variations of this method, allowing the detection of genome-wide unbiased interactions ([294_TD$IF]Figure I) [31,58–60,63,80,98,99]. Circular chromosome conformation capture (4C), contrary to 3C, does not require a priori knowledge of candidate contacting regions and it allows identification of all regions contacting a sequence or loci of interest, called a ‘viewpoint’. 4C has a second round of digestion with a restriction enzyme and another ligation step. The resulting templates are DNA circles that contain the viewpoint plus the contacting sequences. Nowadays, these sequences can be analyzed by next-generation sequencing providing contact maps at a few kilobase resolution [59,60]. Hi-C was developed to interrogate ‘all versus all’ interactions, generating a whole-genome contact map (Figure 1B). Biotin-labeled nucleotides are added to the fragment end overhangs and are used to pull down the fragments after shearing the blunt-end ligated fragments. The ligation junctions are sequenced by paired-end sequencing. The resolution of the pairwise matrix depends on the restriction site density and sequencing depth [63]. It is called in situ Hi-C when the genomic DNA is digested and ligated inside the nucleus [102]. Chromosome conformation capture carbon copy (5C) allows the identification of interactions between multiple selected genomic fragments. Ligation products are hybridized with primers containing universal sequences, and analyzed by next-generation sequencing [98]. Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) combines chromatin immunoprecipitaton (ChIP) with 3C-technology. A specific antibody detects the corresponding protein bound to the ligation junctions of interest. After a pull down, the interacting fragments are then amplified and sequenced [64].

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Figure I. Schematic Representation of Chromosome Conformation Capture (3C) and 3C-Derived Methods. These methods help to elucidate nuclear organization by detecting physical interactions between genetic elements located throughout the genome. Abbreviations: IP, immunoprecipitation; RE, restriction enzyme.

compartments were observed: two A compartments at chromosome tips, and a single B compartment in the center of each chromosome [25]. These global compartments were compatible with active and inactive chromatin marks and differential gene and TE density. Using a chromosome block-based analysis approach, smaller, local A/B compartments with their respective euchromatin and heterochromatin characteristics were detected in the five genomes analyzed. Another study that focused on different cotton species also found A/B compartments in their 3D genome architecture [27]. Tetraploid and their diploid progenitors were analyzed, and A/B compartment switching was observed in the subgenomes of tetraploids when compared with the corresponding diploid progenitor genome, that is, some A compartments in the diploid genome became B compartments in the subgenome of the tetraploid species and vice versa.

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Topologically Associating Domains CTs and compartments (A/B or SDs) are found at megabase resolution. The next level down in the hierarchy was revealed by chromosome folding analyses in animals at higher resolution: topologically associating domains (TADs) divide the genome in defined, autonomously regulated regions that control transcriptional specificities in various cell types [9,30,31,40,72– 75] (Figure 1C). TADs are thought to be defined by genetically encoded boundary elements, subdivided into smaller domains and interspaced with short boundaries or longer inter-TAD regions [74] (Figure 1C–E). Chromatin insulation, observed in animals and yeast, is mediated by proteins that bind specific DNA sequences, which are involved in higher-order, genome-wide chromatin organization, helping to regulate gene function [72]. Chromatin insulators were originally defined as DNA regulatory elements that recruit proteins that establish boundaries between adjacent chromatin domains (Figure 1D,E). Insulators were also shown to block communication between enhancers and nearby promoters in an orientation-dependent manner [32,76]. Hi-C studies in Drosophila suggest that eukaryotic genomes are partitioned into physical domains, which can be clustered based on the linear epigenomic profiles, with insulators highly enriched at domain borders [77,78]. Cohesin and a canonical insulator protein, CTCF, are often found at TAD boundaries; also, active chromatin and high transcriptional rates are related to TAD formation [35,74,79]. In general, TADs seem to be stable across cell types and are evolutionarily conserved (at least in mammals), and have been reported in mouse, human, Drosophila, and budding yeast (Saccharomyces cerevisiae) [34,40,72]. CTCF is absent from Arabidopsis and other plants [36], and clear TADs, 3D structural units separated by defined boundaries, were not so obvious in Arabidopsis as in other organisms [28,29,38]. However, Arabidopsis presented a local chromatin-packing feature, observed as positive strips, in the Hi-C map [36]. Positive strips are kilobase-sized intrachromosomal regions that have higher frequencies of interaction than nearby regions. The chromatin involved in these local structures was enriched in H3K27me3 repressive marks and had a lower rate of gene expression [36]. At higher resolution, regions with weaker interactions, enriched in DNase I hypersensitive sites and epigenetic marks indicative of open chromatin, and highly transcribed genes were identified. These ‘insulator-like’ regions are proposed to be similar to ‘TADboundaries’ in animals [36]. Also, regions with the opposite pattern, indicative of transcriptionally tightly packed repressed heterochromatin, reminiscent of TAD-interior-like regions of animals, were observed [36,38]. Interestingly, a recent study in rice revealed the existence of thousands of TADs [51]. This difference with Arabidopsis may suggest a higher variability in chromatin organization in plants compared with metazoans [51]. In rice, TAD boundary regions are positively associated with active gene expression, and, independently of gene expression levels, they are also enriched with a GC-rich motif that can be recognized by TCP proteins. The presence of ‘TAD-boundarylike’ regions in Arabidopsis [36], and the TCP motif in TAD-boundaries in rice [51], opens the door for studies about ‘insulator-like’ mechanisms in plants. In the comparative five plant species study (maize, tomato, sorghum, foxtail millet, and rice), widespread TAD-like domains were also found, with enriched internal cis interactions and mammalian TAD-like characteristic changes at their borders (enriched for active genes, open chromatin, active histone marks and reduced TEs, and DNA methylation) [25]. These domains were associated with different epigenetic signatures, which were clustered in four major domain types: associated with DNA methylation (repressive domain), open chromatin (active Trends in Plant Science, July 2018, Vol. 23, No. 7

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domain), H3K27me3 marks (polycomb domain), and intermediate type without specific features. These domains strongly resembled Drosophila domains, also classified in these four categories [25,78]. Asymmetric features around the plant domain borders were detected, suggesting that active transcription, chromatin, and epigenome change could drive chromatin domain border formation, as found in Drosophila. Finally, the domains overlapped with A/B local compartments, again similarly to Drosophila. When the proportion of domain types among the five species with different genome sizes was compared, differences were observed. For example, large plant genomes (maize, tomato, sorghum), which have many TEs, presented a higher proportion of repressive domains, while rice, with a smaller genome, contained a lower proportion. Foxtail millet presented the highest proportion of polycomb domains, particularly in chromosome 8, and some of them are also present in a syntenic sorghum chromosome, but not present in the maize genome. In general, when different domains were analyzed across the different species, the authors concluded that plant domains are not conserved among species [25], as observed in mammals [72]. In the cotton Hi-C analysis, midrange intrachromosomal interactions and TAD-like regions were also identified [26] in diploid and tetraploid species [27]. Interestingly, the comparison of diploid genomes and their corresponding tetraploid subgenomes indicated that TAD reorganization occurred after allopolyploidization [27]. Chromatin and Gene Body Loops Further down the hierarchy of the structural organization of the genome, chromatin looping is an interaction at a more local scale. This structure allows regulatory elements to communicate with their target region ([301_TD$IF]Figures 1D,E and 2). Chromatin looping is a common intrachromosomal interaction and has influence on gene expression and transcriptional memory [22,24,31,80,81]. In animals, chromatin looping between enhancers and promoters mainly occurs within TADs [40,82,83] (Figure 1E). In Arabidopsis, a Hi-C analysis at single-gene resolution found over 20 000 chromatin loops in euchromatic chromosome arms [37], and few have known transcription regulation roles [37,84,85]. Gene self-loops are common and transcription start sites (TSSs) tend to form loops with downstream regions, whereas transcription termination sites (TTSs) loop with upstream regions. Moreover, many self-loops have been associated with high gene expression [37]. Enhancer–promoter loops activate immediately adjacent genes, although the regulation of distant genes, the ability of one enhancer to regulate more than one gene, or various enhancers regulating one gene as described in animals, have been barely reported in plants [82]. Based on the whole analysis, structural units corresponding to gene bodies are proposed to be an important component of genome folding in Arabidopsis [37]. Interestingly, in a Drosophila high-resolution (250 bp) Hi-C map, together with other methods, small compartments in active regions that were earlier thought to be TAD borders or inter-TAD regions were identified. Inside these, even smaller domains comprised of single genes were found, where an enrichment of gene body interactions led to the name ‘gene mini-domains’ [35]. Interestingly, in the five[302_TD$IF]-genome study, the authors detected many long-range chromatin interactions (loops) in the genomes of maize and tomato (the largest of the study) but not in the smaller genomes. The regions involved were associated with active epigenetic marks and enriched for genes, and the authors indicated that they may possibly correspond to the interaction of local A compartments, separated by local B compartments [25]. In mammals, loops are mostly found at TAD boundaries, but in maize, loops were found outside the domain and were associated with 606

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Figure 2. From Chromatin Contacts to Gene Loops. Representation of

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A–C

B–C

A–B–C

Opon 1

Opon 2

Opon 3

intrachromosomal contacts for a specific locus in a hypothetical Hi-C experiment. Chromatin contacts are represented as dashed purple lines, and the sites of the interactions are named A, B, and C. The green arrow represents a hypothetical gene present in this region. The interactions identified through Hi-C could represent different gene loops: Option 1 represents the A interactions with C gene loop; Option 2 represents the B interactions with C gene loop; and Option 3 represents the possibility of two loops with A–B–C all interacting. Depending on the resolution of the experiment, some local chromatin contacts may be missed. In this example the distance between A and B is below the resolution threshold of the experiment, and therefore an interaction between A and B could not be detected, even if present (as in Option 3). This simple example illustrates that predicting gene loops is not trivial. Furthermore, it is even more difficult to know if the different gene loop possibilities occur in the same or in distinct cell types.

local A compartments [25]. In this study, loops representing interactions between regulatory regions and genes were most likely not detected, due to the resolution of the analysis. In maize, less than 20% of the loops corresponded to interactions between gene regions and non-gene regions. Since non-gene regions presented high chromatin accessibility, the authors propose that they might represent distal regulatory elements [25]. In cotton, Wang and collaborators [26,27] found frequent chromatin interactions between promoters, distal open chromatin regions (possibly enhancers), and regions with active chromatin marks. The authors classified these interactions as promoter–enhancer, promoter–promoter, and enhancer–enhancer interactions, and found that most of the participating genes appeared to be regulated by multiple long-range chromatin interactions.

3D Genome Organization: Consequence and Cause of [30_TD$IF]Its Function and Activity? The relationship between the different layers of nuclear organization and gene regulation has been studied in various systems. Many studies have correlated the transcriptional activity or repression of genes with their position in nuclear domains, such as the nucleolus, nuclear periphery, or heterochromatin clusters. Also, dynamic chromatin changes have been reported to be interconnected with changes in nuclear organization [304_TD$IF][46,67,68,86–89] (Box 1). The mechanisms that determine the spatial position of a locus, and how position affects function (or vice versa, even when the 3D localization of a gene is not always related to its activity), are beginning to be characterized. The influence of 3D chromatin conformation on gene expression has been reported in different systems, in which the relative position of a gene has been correlated to its activation or repression [73,86,90]. Furthermore, abiotic stresses (e.g., heat or cold) also have an impact on 3D chromatin organization [51,67,68].

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There are several examples of mechanisms involving long- and short-range interactions and their functional implications. Among long-range interaction examples, the KNOT has been proposed to be a structure that protects the Arabidopsis genome from the disruptive potential of some TEs [28,66]. The correlation between epigenetic marks and chromatin interactions is also reflected by the recently discovered role for LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), known to interact with H3K27me3 marks, in the formation of long-range interactions in the Arabidopsis genome [91]. Moreover, Microrchidia (MORC) family ATPases localize to nuclear bodies near the Arabidopsis chromocenters, and this architectural feature was also correlated to the establishment of the pattern of long-range interactions that maintain TE silencing [65]. An interesting example of long-range interactions that affect gene expression is the mechanism behind the differential expression of the maize anthocyanin production b1 epialleles. This mechanism involves the formation of chromatin loops between the gene body and an enhancer located 100 kb upstream [92]. In the B-I epiallele, chromatin in the enhancer region is open, inducing the formation of a multiloop structure between the b1 TSS and additional upstream regions, resulting in high b1 expression. By contrast, in the B0 epiallele, the enhancer chromatin is compacted, the multiloop structure does not form, and b1 is lowly expressed [92]. Another mechanism of chromatin interaction affecting gene expression involves the localization of a certain region in a specific location of the nucleus. An example is the repositioning of the chlorophyll a/b-binding protein CAB gene locus to the nuclear periphery in response to red and far-red light for transcriptional activation [93]. There are also interesting examples in plants of short-range chromatin interactions that are correlated to gene expression [22]. One of the best studied involves the polycomb-mediated epigenetic silencing of FLOWERING LOCUS C (FLC) of Arabidopsis. This MADS box protein represses the expression of genes that switch the meristem to a floral fate. Gradual FLC silencing in cold conditions allows flowering, and involves histone modifications, small RNAs, and long noncoding RNAs (lncRNAs) [86]. Among other events, a chromatin loop is formed that connects the 5and 3ends of FLC extending beyond the polyA site (2.7 kb in size). During vernalization, which leads to FLC silencing, the gene [305_TD$IF]loop is disrupted [84] and FLC loci are physically clustered [94], while H3K27me3 marks are increased at the nucleation site, a mechanism reminiscent of polycomb bodies in mammalian cells [84–86]. A second example, which also involves a developmental switch, is the chromatin loop in the promoter region of the PINOID (PID) auxin response gene. PID affects polar auxin transport during organ development. In response to auxin, the chromatin loop opens and gradually closes to fine-tune PID transcription. This oscillating chromatin 3D structure is physically mediated by an lncRNA (called APOLO) and histone interacting proteins [85]. Some organisms present chromatin structures known as transcription factories, where clustering of active RNA Pol II promotes transcription of colocalized genes [306_TD$IF][30,32,53,95] (Figure 1A). In Arabidopsis, however, highly transcribed genes have a weak tendency to cluster in 3D space at long distances [37,38]. The interaction between two chromatin regions bound by RNA Pol II occurs mainly within genes at short distances (<6 kb) [37,95]. Recently, the study that found ‘gene mini-domains’ in Drosophila also found a high correlation between the transcriptional status and the interaction of genomic regions in Drosophila, Arabidopsis, and other organisms [35]. The authors could predict an Arabidopsis Hi-C contact map based 608

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on the transcriptional state of a region, and propose that transcriptional states of individual genes play a basic role in, and unify, domain and compartment and formation in different organisms [35]. The global role of transcription factors in the formation and function of the different chromatin 3D structures in plants is also beginning to be explored [22,29]. In Arabidopsis, a recent study sought to integrate Hi-C, methylation, ChIP-seq, and ChIP-chip data, and found correlations between chromatin interactions, epigenetic marks, and transcription factor binding [50]. In rice, the finding that TCP motifs are enriched in rice TAD boundaries suggests an exciting and unexpected function for these regulators in plant 3D genome organization [51].

Concluding Remarks and Future Perspectives In general, plant genome organization inside the nucleus is governed by principles similar to those of other eukaryotes [30]: (i) CT represents the higher order structure, and (ii) domains with different epigenomic status that are spatially distant, and conform active and repressive environments, have been clearly observed in most plants analyzed to date [18,25–29,36– 38,51,66]. However, there are also interesting differences between the reported chromosome-packing patterns of plants and animals, and also between the eight plant genomes for which Hi-C maps are available: Arabidopsis, rice, barley, maize, tomato, sorghum, foxtail millet, and cotton [22,25–27,29,36,38,51,52,66]. For example, plant TADs and their borders are certainly nonmammalian-type, and the degree of TAD conservation that has been observed in mammals has not been observed in plant species analyzed to date. Moreover, different plants also display different chromosome-packaging characteristics, such as chromosome contact patterns, some of which may be associated with genome size and epigenomic features. For example, among other differences, barley and maize present a Rabl, or close to Rabl, chromosome organization. In barley, instead of being organized in compartments, loci interactions were found to be mainly determined by their linear position in the genome. Chromatin loops are found in some plant species, but not in all. Gene-sized loops have been observed in Arabidopsis, which also seems to be the only plant species so far in which TADs were not prominent. While some of the differences may also be due to the different resolution and data analysis strategies used in different studies, it appears now (from the available data) that plants share some organization features and also present distinct 3D nuclear architectures.

Outstanding Questions Do chromatin conformation patterns change in different plant cell types? Do different chromatin conformation patterns exist for a certain region in the same cell type at the same time? In plants, do TADs only exist in nuclei of species with a large genome size? Do global chromatin conformational patterns contribute to gene expression and influence developmental processes? Can gene expression changes modify chromatin conformation? Following mitosis, are identical or similar chromatin conformation patterns re-established?

Nevertheless, it is clear that there is a relation between chromatin status, structure, and function or activity, all of which are thought to reinforce each other [22,25–30,96]. Further research can clarify these correlations. Recently, a basic role for the transcriptional state at the gene level in the formation of 3D chromatin structures of different organisms, including Arabidopsis, has been proposed [35]. Clearly, there is still a lot to test and learn. More contact maps at higher resolution of a larger number of plant genomes, differing in size and gene densities, will certainly aid in this quest. Further knowledge will be gained from the comparison of contact maps of the same genome in different environmental conditions or developmental stages (genomes in 4D), of single cell types, or even of single cells. The use of synchronized cells will help to follow changes in chromatin contacts during the cell cycle. Contact maps of F1 hybrid, triploid and polyploid zygotes, cells, and plants will provide more information about how different genomes interact. Another exciting prospect is unraveling the function of transcription factors and other DNA binding proteins in the context of the 3D organization of the genome. The modification of their Trends in Plant Science, July 2018, Vol. 23, No. 7

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binding sites, for example TCP binding sites in rice and other plants, with the aid of genome editing technologies, will help to better understand their role in the establishment of chromosome interactions. Moreover, the global participation of RNAs in the formation or maintenance of chromatin structures is also an open exploration field. Finally, combining time-resolved chromosome conformation with other high-throughput methods and high-resolution microscopy will deepen and refine the knowledge obtained through 3C-derived techniques (see Outstanding Questions). Acknowledgements Work in the S.D.F. laboratory is financed by the Mexican National Council of Science and Technology (CONACyT) grants CB-2012/177739 and FC-2015-2/1061, and the NMM laboratory by the CONACyT grant CB-2015/255069. S.D.F. acknowledges the support of the European Union H2020-MSCA-RISE-2015 project ExpoSEED (grant no. 691109). M.S. S. thanks the Programa de Desarrollo de las Ciencias Básicas (PEDECIBA) from Uruguay, and J.S.S. thanks support by CSIC, ANII, and PEDECIBA.

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