Toward cis-regulation in soybean: a 3D genome scope.

To investigate three-dimensional (3D) genome organization in prokaryotic and eukaryotic cells, three main strategies are employed, namely nuclear proximity ligation-based methods, imaging tools (such as fluorescence in situ hybridization (FISH) and its derivatives), and computational/visualization methods. Proximity ligation-based methods are based on digestion and re-ligation of physically proximal cross-linked chromatin fragments accompanied by massively parallel DNA sequencing to measure the relative spatial proximity between genomic loci. Imaging tools enable direct visualization and quantification of spatial distances between genomic loci, and advanced implementation of (super-resolution) microscopy helps to significantly improve the resolution of images. Computational methods are used to map global 3D genome structures at various scales driven by experimental data, and visualization methods are used to visualize genome 3D structures in virtual 3D space-based on algorithms. In this review, we focus on the introduction of novel imaging and visualization methods to study 3D genomes. First, we introduce the progress made recently in 3D genome imaging in both fixed cell and live cells based on long-probe labeling, short-probe labeling, RNA FISH, and the CRISPR system. As the fluorescence-capturing capability of a particular microscope is very important for the sensitivity of bioimaging experiments, we also introduce two novel super-resolution microscopy methods, SDOM and low-power super-resolution STED, which have potential for time-lapse super-resolution live-cell imaging of chromatin. Finally, we review some software tools developed recently to visualize proximity ligation-based data. The imaging and visualization methods are complementary to each other, and all three strategies are not mutually exclusive. These methods provide powerful tools to explore the mechanisms of gene regulation and transcription in cell nuclei.

Three-dimensional (3D) genome organization becomes altered during development, aging and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multi-scale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screens, a cellular barcode readout method (BARC-FISH) and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their 3D chromatin folding conformations, alongside perturbation-paired barcode readout in the same single cells. We discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop extrusion and A–B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome.

It is already more than 10 years after the completion of Human Genome Project. However, the mechanism about how genomic information guides the gene expression in a particular space and time remains to be elucidated. Structure determines function is a consensus that we understand the laws of nature. In biology, this consensus is not only applicable to RNA, protein and other small-scale organic molecules; the same should also apply to macromolecules such as the entire DNA composition of the whole genome. Recent studies have shown that three-dimensional (3D) structure of the genome has an important impact on the regulation of gene transcription and other nuclear functions. Therefore, the study of the 3D structure and function of the whole genome (referred to as 3D genomics) has become a new trend of development in genomics. With the consideration of the genome sequence, gene structure and regulatory elements at the same time, 3D genomics studies the 3D structure of the genome in the nucleus, and its impact on gene transcription, replication, repair, regulation and others biological processes. With the progress of large-scale genome sequencing technology, we already have the relevant technologies such as ChIA-PET and Hi-C for the study of 3D genomics. Preliminary results showed the influence of genome 3D structures to genome functions, which provides the basis for further study of genome structure and function in the spatial nuclear space. This paper summarizes the emergence of 3D genomics, lists the issues and the current progress in this new field, and discusses possible applications of 3D genomics to human health and agriculture researches. We anticipate that 3D genomics will provide novel insights into the genome regulatory functions and provide new opportunities of improving human life qualities.

The three-dimensional structure of chromosomes plays an important role in gene expression regulation and also influences the repair of radiation-induced DNA damage. Genomic aberrations that disrupt chromosome spatial domains can lead to diseases including cancer, but how the 3D genome structure responds to DNA damage is poorly understood. Here, we investigate the impact of DNA damage response and repair on 3D genome folding using Hi-C experiments on wild type cells and ataxia telangiectasia mutated (ATM) patient cells. We irradiate fibroblasts, lymphoblasts, and ATM-deficient fibroblasts with 5 Gy X-rays and perform Hi-C at 30 minutes, 24 hours, or 5 days after irradiation. We observe that 3D genome changes after irradiation are cell type-specific, with lymphoblastoid cells generally showing more contact changes than irradiated fibroblasts. However, all tested repair-proficient cell types exhibit an increased segregation of topologically associating domains (TADs). This TAD boundary strengthening after irradiation is not observed in ATM deficient fibroblasts and may indicate the presence of a mechanism to protect 3D genome structure integrity during DNA damage repair.

Ankylosing Spondylitis (AS) is a chronic inflammatory arthritis of the spine exhibiting a strong genetic background. The mechanistic and functional understanding of the AS-associated genomic loci, identified with Genome Wide Association Studies (GWAS), remains challenging. Chromosome conformation capture (3C) and derivatives are recent techniques which are of great help in elucidating the spatial genome organization and of enormous support in uncover a mechanistic explanation for disease-associated genetic variants. The perturbation of three-dimensional (3D) genome hierarchy may lead to a plethora of human diseases, including rheumatological disorders. Here we illustrate the latest approaches and related findings on the field of genome organization, highlighting how the instability of 3D genome conformation may be among the causes of rheumatological disease phenotypes. We suggest a new perspective on the inclusive potential of a 3C approach to inform GWAS results in rheumatic diseases. 3D genome organization may ultimately lead to a more precise and comprehensive functional interpretation of AS association, which is the starting point for emerging and more specific therapies.

The organization of mammalian genomes within the nucleus features a complex, multiscale three-dimensional (3D) architecture. The functional significance of these 3D genome features, however, remains largely elusive due to limited single-cell technologies that can concurrently profile genome organization and transcriptional activities. Here, we report GAGE-seq, a highly scalable, robust single-cell co-assay that simultaneously measures 3D genome structure and transcriptome within the same cell. Employing GAGE-seq on mouse brain cortex and human bone marrow CD34+ cells, we comprehensively characterized the intricate relationships between 3D genome and gene expression. We found that these multiscale 3D genome features collectively inform cell type-specific gene expressions, hence contributing to defining cell identity at the single-cell level. Integration of GAGE-seq data with spatial transcriptomic data revealed in situ variations of the 3D genome in mouse cortex. Moreover, our observations of lineage commitment in normal human hematopoiesis unveiled notable discordant changes between 3D genome organization and gene expression, underscoring a complex, temporal interplay at the single-cell level that is more nuanced than previously appreciated. Together, GAGE-seq provides a powerful, cost-effective approach for interrogating genome structure and gene expression relationships at the single-cell level across diverse biological contexts.

Chromatin conformation capture (3C)1 and high-throughput 3C (Hi-C)2 assays allow the study of three-dimensional (3D) genome structures in cell populations or tissues, based on average proximities of folded DNA. However, differences between cells can be observed only by single-cell measurements that avoid ensemble averaging3-5. To study 3D chromatin organization and dynamics before and after fertilization in flowering plants, we analysed the 3D genomes of rice eggs, sperm cells, unicellular zygotes and shoot mesophyll cells. We show that chromatin architectures of rice eggs and sperm cells are comparable to those of mesophyll cells and are reorganized after fertilization. The rice single-cell 3D genomes display specific features of chromosome compartments and telomere/centromere configuration compared to those in mammalian single cells. Active and silent chromatin domains combine to form multiple foci in the nuclear space. Notably, the 3D genomes of the eggs and unicellular zygotes contain a compact silent centre (CSC) that is absent in sperm cells. CSC appears to be reorganized after fertilization, and may be involved in the regulation of zygotic genome activation (ZGA). Our results reveal specific 3D genome features of plant gametes and the unicellular zygote, and provide a spatial chromatin basis for ZGA and epigenetic regulation in plants.

Three-dimensional (3D) genomics is an emerging discipline that studies the three-dimensional structure of chromatin and the three-dimensional and functions of genomes. It mainly studies the three-dimensional conformation and functional regulation of intranuclear genomes, such as DNA replication, DNA recombination, genome folding, gene expression regulation, transcription factor regulation mechanism, and the maintenance of three-dimensional conformation of genomes. Self-chromosomal conformation capture (3C) technology has been developed, and 3D genomics and related fields have developed rapidly. In addition, chromatin interaction analysis techniques developed by 3C technologies, such as paired-end tag sequencing (ChIA-PET) and whole-genome chromosome conformation capture (Hi-C), enable scientists to further study the relationship between chromatin conformation and gene regulation in different species. Thus, the spatial conformation of plant, animal, and microbial genomes, transcriptional regulation mechanisms, interaction patterns of chromosomes, and the formation mechanism of spatiotemporal specificity of genomes are revealed. With the help of new experimental technologies, the identification of key genes and signal pathways related to life activities and diseases is sustaining the rapid development of life science, agriculture, and medicine. In this paper, the concept and development of 3D genomics and its application in agricultural science, life science, and medicine are introduced, which provides a theoretical basis for the study of biological life processes.

Metastatic cancer cells and healthy fibroblasts must traverse constrictive spaces to reach secondary sites. After passing through multiple constrictions, cancer cells often experience stable changes to their nucleus morphology, 3D genome structure, and migratory phenotype. Here, we investigate whether fibroblasts (BJ-5ta), which are non-cancerous and have an inherent ability to migrate to fulfill roles in wound repair, likewise experience nuclear and 3D genomic changes with constricted migration. We find that BJ-5ta cells do not get progressively better at migrating with sequential attempts but do experience nuclear deformations and 3D genome alterations at the compartment level after constricted migration. Transient compartment shifts spatially rearranged genes associated with preparation for and response to migration. Unlike the stable changes associated with long term phenotype changes in cancer cells, however, the nucleus deformations recovered back to unmigrated levels following proliferation and cell movement. Some compartment changes persist and might influence responses to future stimuli, but most 3D genome changes revert to the unmigrated state after cell proliferation. Our study shows that non-cancerous migratory cells are not more robust against alterations caused by constricted migration but can recover from the ones that do arise more readily than cancer cells.Significance StatementCancer cells experience stable phenotype shifts and 3D genome changes after constricted migration, but it is unknown whether these changes would occur in innately migratory non-cancerous fibroblasts.Fibroblasts (BJ-5ta) show reversible nuclear morphology and 3D genome alterations after constricted migration.Our results reveal the connection between basic nucleus mechanobiology and genome structure in fibroblasts, showing how these cells adapt and respond to the forces of constriction during processes such as wound healing.

Histone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and overlaps well with lamina-associated domains and the B compartment defined by Hi-C. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. Here, we investigated genome-wide H3K9me2 distribution, transcriptome, and 3D genome organization in mouse embryonic stem cells following the inhibition or depletion of H3K9 methyltransferases (MTases): G9a, GLP, SETDB1, SUV39H1, and SUV39H2. We show that H3K9me2 is regulated by all five MTases; however, H3K9me2 and transcription in the A and B compartments are regulated by different MTases. H3K9me2 in the A compartments is primarily regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments is regulated by all five MTases. Furthermore, decreased H3K9me2 correlates with changes to more active compartmental state that accompanied transcriptional activation. Thus, H3K9me2 contributes to inactive compartment setting.

Prostate cancer (PCa) aggressiveness and propensity to metastasize can be influenced by gene expression patterns, genomic aberrations, and overall cellular and nuclear morphology. The 3D folding of chromosomes into loops, domains, and territories as they are packaged into the nucleus plays a key role in all of these processes, affecting proper gene regulation, DNA replication and repair, and nuclear physical properties. However, while nuclear atypia has long been used as a marker of PCa progression, little is known about what changes in 3D genome interactions occur or how these may influence cancer-promoting gene expression profiles or the nuclear malleability necessary for cancer cells to metastasize. Thus, we set out to characterize the role of the 3D genome changes in PCa, analyzing changes in the 3D genome structure of cell lines that model the continuum of disease. Using chromosome conformation capture (Hi-C) we have conducted a systematic comparison of the 3D genome structure of a normal epithelium model cell line (RWPE) to that of adenocarcinoma (LNCaP), bone metastatic (VCaP, PC3, 22RV1, LNCaP-C42B, MDaPCa2a and MDAPCa2b), and atypical metastasis (Brain - DU145) lines. Our study has shown that the open/closed chromatin compartmentalization identity (A vs B compartment) changes with progression. We have characterized a cohort of 316 genes whose compartmentalization changes from the B to the A compartment, suggesting that those genes become poised for transcription. These include androgen receptor, WNT5A and CDK14, as well as genes located in proximity to TMPRSS2, such as BACE2. The positive correlation between switches of compartmentalization identity and transcription activation has been confirmed by microarray. Of these B/A switch genes, 256 are grouped in 48 proximal clusters. We have also identified 84 genes whose compartments switch from the A to B compartment, suggesting a genomic rearrangement that could result in silencing. Such is the case of certain cadherins, annexins and mediators of inflammation. Of these, 64 are grouped in 16 clusters. Finally, we have observed that compartmentalization changes in chromosome 21 are exacerbated with progression and may explain, in part, the genesis of the TMPRSS2-ERG translocation; one of the main drivers of prostate cancer, present in 40% - 80% of prostate cancer patients. Interestingly, changes in gene transcription are also accompanied by changes in the structure, size, and boundaries of the topologically associating domains (TADs) surrounding the loci of interest, suggesting that 3D genome structure plays a role in the activation of alternate pathways for proliferation and survival. Citation Format: Rebeca San Martin, Priyojit Das, Renata Dos Reis Marques, Rachel P. McCord. Rearrangement of the 3D genome structure in prostate cancer is a potential mechanism for disease exacerbation and classifies cancers according to progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2033.

The three-dimensional (3D) folding of the genome plays a crucial role in genome regulation. However, how 3D genome structure varies between individuals and consequently influences genome function and evolution remains poorly understood. One potential source of this variation is transposable elements (TEs), genomic parasites whose location and composition vary between species and individuals. Hosts typically silence TEs through enriching them with repressive epigenetic marks, turning euchromatic TEs into heterochromatin islands, which were shown to spatially interact with pericentromeric heterochromatin (PCH). Because most TE insertions are present in only a few individuals within Drosophila populations, we asked whether polymorphism in the presence/absence of TEs drives varying 3D structures through TE-PCH spatial interactions. We performed deep-coverage Hi-C of two wild-type Drosophila strains and developed a Hi-C analysis framework enabling allelic comparisons of spatial interactions with PCH. Supporting our hypothesis, nearly 40% of strain-specific euchromatic TEs cause their adjacent euchromatic regions to be spatially closer to PCH than TE-free homologous alleles. These interactions are not limited to specific TE families, and, surprisingly, telomere-proximal TEs show a similar propensity as centromere-proximal TEs to enhance PCH interactions. The most defining feature of TEs involved in PCH interactions is H3K9me3 enrichment, revealing a chromatin-based mechanism for TE-mediated 3D genome organization broadly applicable across TE families and genome locations. Importantly, TEs involved in PCH interactions reduce the expression of adjacent genes and are evolutionarily young, indicating stronger selection against them. Our study reveals a previously uncharacterized mechanism by which TEs influence the function and evolution of host genomes by generating polymorphic 3D genome organization.

Studying the three-dimensional (3D) structure of a genome, including chromatin loops and Topologically Associating Domains (TADs), is essential for understanding how the genome is organized, such as gene activation, cell development, protein-protein interaction, etc. Hi-C protocol enables us to study 3D genome structure and organization. Chromatin 3D structure changes dynamically over time, and modeling these continuous changes is crucial for downstream analysis in various domains such as disease diagnosis, vaccine development, etc. The high expense and impracticality of continuous genome sequencing, particularly what evolves between two timestamps, limit the most effective genomic analysis. It is crucial to develop a straightforward and cost-efficient method for constantly generating genomic data between two timestamps in order to address these constraints. In this study, we developed HiCInterpolate, a 4D spatiotemporal interpolation architecture that accepts two timestamp Hi-C contact matrices to interpolate intermediate Hi-C contact matrices at high resolution. HiCInterpolate predicts the intermediate Hi-C contact map using a deep learning-based flow predictor, and a feature encoder and decoder architecture similar to U-Net. In addition, HiCInterpolate supports downstream analysis of multiple 3D genomic features, including A/B compartments, chromatin loops, TADs, and 3D genome structure, through an integrated analysis pipeline. Across multiple evaluation metrics, including PSNR, SSIM, GenomeDISCO, HiCRep, and LPIPS, HiCInterpolate achieved consistently strong performance. Biological validation further demonstrated preservation of key chromatin organization features, such as chromatin loops, A/B compartments, and TADs. Together, these results indicate that HiCInterpolate provides a robust computer vision-based framework for high-resolution interpolation of intermediate Hi-C contact matrices and facilitates biologically meaningful downstream analyses. HiCInterpolate is publicly available at https://github.com/OluwadareLab/HiCInterpolate .

During the cell cycle, the genome must undergo dramatic changes in structure, from a decondensed, yet highly organized interphase structure to a condensed, generic mitotic chromosome and then back again. For faithful cell division, the genome must be replicated and chromosomes and sister chromatids physically segregated from one another. Throughout these processes, there is feedback and tension between the information-storing role and the physical properties of chromosomes. With a combination of recent techniques in fluorescence microscopy, chromosome conformation capture (Hi-C), biophysical experiments, and computational modeling, we can now attribute mechanisms to many long-observed features of chromosome structure changes during cell division. Apparent conflicts that arise when integrating the concepts from these different proposed mechanisms emphasize that orchestrating chromosome organization during cell division requires a complex system of factors rather than a simple pathway. Cell division is both essential for and threatening to proper genome organization. As interphase three-dimensional (3D) genome structure is quite static at a global level, cell division provides an important window of opportunity to make substantial changes in 3D genome organization in daughter cells, allowing for proper differentiation and development. Mistakes in the process of chromosome condensation or rebuilding the structure after mitosis can lead to diseases such as cancer, premature aging, and neurodegeneration. WIREs Syst Biol Med 2017, 9:e1389. doi: 10.1002/wsbm.1389 For further resources related to this article, please visit the WIREs website.

The human genome is tightly packaged into chromatin whose functional output depends on both one-dimensional (1D) local chromatin states and three-dimensional (3D) genome organization. Currently, chromatin modifications and 3D genome organization are measured by distinct assays. An emerging question is whether it is possible to deduce 3D interactions by integrative analysis of 1D epigenomic data and associate 3D contacts to functionality of the interacting loci. Here we present EpiTensor, an algorithm to identify 3D spatial associations within topologically associating domains (TADs) from 1D maps of histone modifications, chromatin accessibility and RNA-seq. We demonstrate that active promoter–promoter, promoter–enhancer and enhancer–enhancer associations identified by EpiTensor are highly concordant with those detected by Hi-C, ChIA-PET and eQTL analyses at 200 bp resolution. Moreover, EpiTensor has identified a set of interaction hotspots, characterized by higher chromatin and transcriptional activity as well as enriched TF and ncRNA binding across diverse cell types, which may be critical for stabilizing the local 3D interactions.