Understanding 3D Genome Organization and Its Effect on Transcriptional Gene Regulation Under Environmental Stress in Plant: A Chromatin Perspective

Abstract

The genome of a eukaryotic organism is comprised of a supra-molecular complex of chromatin fibers and intricately folded three-dimensional (3D) structures. Chromosomal interactions and topological changes in response to the developmental and/or environmental stimuli affect gene expression. Chromatin architecture plays important roles in DNA replication, gene expression, and genome integrity. Higher-order chromatin organizations like chromosome territories (CTs), A/B compartments, topologically associating domains (TADs), and chromatin loops vary among cells, tissues, and species depending on the developmental stage and/or environmental conditions (4D genomics). Every chromosome occupies a separate territory in the interphase nucleus and forms the top layer of hierarchical structure (CTs) in most of the eukaryotes. While the A and B compartments are associated with active (euchromatic) and inactive (heterochromatic) chromatin, respectively, having well-defined genomic/epigenomic features, TADs are the structural units of chromatin. Chromatin architecture like TADs as well as the local interactions between promoter and regulatory elements correlates with the chromatin activity, which alters during environmental stresses due to relocalization of the architectural proteins. Moreover, chromatin looping brings the gene and regulatory elements in close proximity for interactions. The intricate relationship between nucleotide sequence and chromatin architecture requires a more comprehensive understanding to unravel the genome organization and genetic plasticity. During the last decade, advances in chromatin conformation capture techniques for unravelling 3D genome organizations have improved our understanding of genome biology. However, the recent advances, such as Hi-C and ChIA-PET, have substantially increased the resolution, throughput as well our interest in analysing genome organizations. The present review provides an overview of the historical and contemporary perspectives of chromosome conformation capture technologies, their applications in functional genomics, and the constraints in predicting 3D genome organization. We also discuss the future perspectives of understanding high-order chromatin organizations in deciphering transcriptional regulation of gene expression under environmental stress (4D genomics). These might help design the climate-smart crop to meet the ever-growing demands of food, feed, and fodder.

Keywords: 4D genomics; A/B compartment; ChIA-PET; Hi-C; chromatin loop; chromosome territories; single-cell 3D genomics; topologically associating domain.

FIGURE 1

Schematic representation of plant chromatin organization in the nucleus. (A) Hierarchical chromatin organization can be studied mainly at four levels: chromosome territory, chromatin compartments, topologically associating domain (TAD), and chromatin loops. (i) Chromosomes occupy specific territories in the nucleus. In different territories, chromosomes show different morphology, such as Rabl, Rosette, and Bouquet configuration. In Rabl configuration, telomeres and centromeres of chromosomes cluster at two different poles in the nucleus, particularly in plants with larger genomes. In the Rosette configuration, the nucleolus is surrounded by telomeres, while heterochromatin and centromeres are clustered together but euchromatin oozes out freely in the nucleus to form a rosette-like configuration, observed in plants with smaller genome like Arabidopsis. Bouquet configuration is a transient chromatin configuration observed during meiosis in different organisms, including plants, wherein telomeres of the chromosomes are co-localized on a specific site of nuclear periphery, while the rest of the chromatin remains dispersed in the nuclear space. Nucleolus-associated domains (NADs) are chromatin regions that interact with the nucleolus, while the lamina-associated domains (LADs) are associated with the lamina of the nuclear envelope. Chromosome territories are further divided into (ii) A and B compartments, which correspond to euchromatic and heterochromatic regions, respectively. While the A compartment is constituted of high gene density, activating epigenetic modifications, and active transcriptional activity, the B compartment possesses lesser genes, low transcriptional activity, repressive epigenetic modifications, and higher transposon density. (iii) Topologically associated domains (TADs) are relatively independent local units/regions where chromatins interact with each other at a higher frequency than with the surrounding regions. (iv) Number of factors/modifications/readers is involved in the formation of chromatin loops that connects regulatory elements to their target loci in plants. (B) Lower level chromatin interactions (chromatin loops) establish regulatory networks between the distant elements through their physical proximity. The regulatory function of chromatin loops comes due to the formation of (i) heterochromatin/repressive loop by histone modifiers−H3K27me3−polycomb protein−lncRNAs, while (ii) silencing chromatin loop is formed by H3K9me2-reader (ADCP1)−ncRNAs. (iii) Different regions (5′–3′ gene looping) of the same gene, (iv) an enhancer and promoter (enhance–promoter loop) of a gene, (v) the different co-regulated genes (gene-gene loops), (vi) non-coding genomic regions (intergenic loop), and (vii) transcriptional hub/loop formed by H3K4me3 modifiers, RNA Pol-II and eRNAs.

FIGURE 2

An overview of 3D genomics techniques. The techniques can be broadly divided into two categories: one is based on cytological/microscopic examination/imaging; another is based on sequencing. (A) The microscopy-based techniques include WFM (wide-field microscopy), CFM (confocal fluorescence microscopy), ChromEMT (Chrom-electron microscopy tomography), Hi-M (multiplexed, sequential imaging approach) simultaneously reveals 3D chromatin organization and transcriptional activity, SRM (super-resolution microscopy), which include SIM (structured illumination microscopy), SEDM (stimulated emission depletion microscopy), PALM (photoactivated localization microscopy), STORM (stochastic optical reconstruction microscopy), EMI (electron microscopy imaging), and LSFM (light-sheet fluorescence microscopy). (B) While 3D-EMISH (three-dimensional electron microscopy with in situ hybridization) utilizes the advantages of both microscopy (electron microscopy) and labeling (in situ hybridization), the labeling-based techniques include 3D-FISH [such as fluorescence in situ hybridization, MB-FISH (molecular beacon-FISH), Oligo-FISH (oligonucleotides probe-based FISH), GISH (genomic in situ hybridization)], staining with chemical dyes like DAB−DRAQ5 system, immune-staining, and FP-tagging (fluorescent protein-tagging) including tagging with zinc-finger protein, lacO-LacI-GFP system, CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats-nuclease-deficient Cas9), TALE (transcription activator-like effectors with a quantum dot labeling technique). The sequencing-based techniques can be ligation-free or those which require proximity ligation (3C, chromosome conformation capture). (C) While ligation-free techniques include GAM (genome architecture mapping) that combines micro-cutting and sequencing, SPRITE (split-pool recognition of interactions by tag extension), ChIA-Drop (chromatin interaction analysis via droplet-based and barcode-linked sequencing), DamC (DNA adenine methyltransferase-based chromosomal contacts), (D) ligation-based techniques include the advancements in 3C (chromosome conformation capture), like 4C, 5C (chromosome conformation capture carbon copy), methyl-3C (combination of DNA methylation detection and 3C technology), Dip-C (combination of single-cell 3C and transposon-based whole-genome amplification method), T2C (targeted Chromatin Capture), Capture 3C (combination of 3C with oligonucleotide capture technology. Further advancements like ChIA-PET (chromatin interaction analysis by paired-end tag sequencing) and Hi-C (high-throughput chromosome conformation capture), and their combination Hi-ChIP (chromatin conformation method that combines Hi-C with ChIA-PET technology) have advanced the 3D genome architectures. Combination of techniques like Cut-C (antibody-mediated cleavage by tethered nuclease with chromosome conformation capture), Capture Hi-C (combination of Hi-C and hybridization-based capture of targeted genomic regions), in situ Hi-C (DNA–DNA proximity ligation performed in intact nuclei), Micro-C (chromatin fragmented into mononucleosomes using micrococcal nuclease), DNase Hi-C (chromatin fragmented by DNase I), DLO Hi-C (digestion-ligation-only Hi-C), BAT Hi-C (bridge linker-Alul-Tn5 Hi-C), BL Hi-C (bridge linker Hi-C), Trac-looping (transposase-mediated analysis of chromatin looping), Methyl Hi-C (a combination of DNA methylation detection technology and Hi-C), OCEAN Hi-C (open chromatin enrichment and network Hi-C), and Single-cell Hi-C (Hi-C in an individual nucleus). The techniques that have been successfully used in plants are presented in the green box (modified from Pei et al., 2021).

FIGURE 3

An overview of the chromatin conformation capture (3C) and its derivative techniques used for 3D genomic studies. The DNA−protein interactions are fixed in vivo using formaldehyde and then chromatins are fragmented by restriction endonuclease treatment. The cross-linked chromatins are processed differentially for one vs one (3C), one vs many (4C), many vs many (5C, ChIA-PET), or all vs all (Hi-C) interaction analysis. The techniques successfully used in plants are presented in green boxes.

FIGURE 4

Modulation in chromatin accessibility under abiotic stresses in plants. (A) Normally, the lamin-like proteins OsNMCP1 regulate drought tolerance through modulating chromatin accessibility via interaction with a chromatin remodeler OsSWI3C in rice. Switch/Sucrose Non-Fermenting (SWI/SNF) complexes interact with OsSWI3C to change the structure of nucleosome, resulting in gene silencing. Under drought stress, OsNMCP1 gets induced and interacts with OsSWI3C, which releases OsSWI3C from the gene-silencing SWI/SNF complexes, resulting in improved chromatin accessibility and higher expression of drought-responsive genes. (B) Topless-like/Topless-like protein (TPL/TPR) and Indeterminate Spikelet 1 (IDS1) interact with Histone Deacetylase (HDAc) to form an IDS1-TPL-HDA1 transcriptional repression complex through histone deacetylation. Under salt stress, acetylation of H3K9 and H3K14 by histone acetyltransferase (General Control Non-repressed Protein 5, GCN5), contributes to salt tolerance by activating salt stress-responsive genes (e.g. SOS1). (C) Under heat stress, SWI1/SNF1 complex interacts with GCN5 and ARP6 to dissociate H2A.Z (and insertion of H2A into the nucleosome), which causes no transcription of heat-responsive genes. On normal weather, the SWI1/SNF1—ARP6 complex plays important role in placing H2A.Z into the nucleosome. (D) Under cold stress, HOS15, in association with DNA Damaged Binding Protein1 (DDB1) and Cullin 4 (CUL4) acts as E3 ubiquitin ligase which degrades HDAc2C causing hyperacetylation of histone H3 on Cold Regulated (COR) chromatin. This makes binding of CBF proteins to COR promoter through High-expression of Osmotically Responsive Gene 15 (HOS15) leading to active expression of COR genes. Moreover, the GCN5 modulates histone acetylation of COR chromatin. At the normal temperature, HOS15 forms a complex with HDAc2C to repress COR expression via hypoacetylation of the COR chromatin.

FIGURE 5

Comparison of 3D chromatin organization of animals and plants. (A) Loop domain in the mammalian genome. CTCT loops are formed at the domain corner, and these domains are located within a compartment. Small adjacent loop domains form larger domains having nested structures. The dynamics of a loop domain is associated with changes in CTCF binding. (B) In Arabidopsis, chromosome arms are partitioned into loose structural domains (LSDs) and compacted structural domains (CSDs) which are comparable to the local A/B compartments rather than the mammalian TAD and the global compartment domain of large-genome plant. (C) Compartment domains in the large-genome plants often overlap with local compartments having active genes located inside the domain associated with the A compartment. Transposable elements and repressed genes are located in the domain with the B compartment.