Constructing 3D interaction maps from 1D epigenomes

Eukaryotic chromosomes are folded into higher-order conformations to coordinate genome functions. In addition to long-range chromatin loops, recent chromosome conformation capture (3C)-based studies have indicated higher levels of chromatin structures including compartments and topologically associating domains (TADs), which may serve as units of genome organization and functions. However, the molecular machinery underlying these hierarchically three-dimensional (3D) chromatin architectures remains poorly understood. Via high-throughput assays, including in situ Hi-C, DamID, ChIP-seq, and RNA-seq, we investigated roles of the Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), a nuclear matrix (NM)-associated protein, in 3D genome organization. Upon the depletion of HNRNPU in mouse hepatocytes, the coverage of lamina-associated domains (LADs) in the genome increases from 53.1% to 68.6%, and a global condensation of chromatin was observed. Furthermore, disruption of HNRNPU leads to compartment switching on 7.5% of the genome, decreases TAD boundary strengths at borders between A (active) and B (inactive) compartments, and reduces chromatin loop intensities. Long-range chromatin interactions between and within compartments or TADs are also significantly remodeled upon HNRNPU depletion. Intriguingly, HNRNPU mainly associates with active chromatin, and 80% of HNRNPU peaks coincide with the binding of CTCF or RAD21. Collectively, we demonstrated that HNRNPU functions as a major factor maintaining 3D chromatin architecture, suggesting important roles of NM-associated proteins in genome organization.

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.

The 3D organization of the genome facilitates gene regulation, replication, and repair, making it a key feature of genomic function and one that remains to be properly understood. Over the past two decades, a variety of chromosome conformation capture (3C) methods have delineated genome folding from megabase-scale compartments and topologically associating domains (TADs) down to kilobase-scale enhancer-promoter interactions. Understanding thefunctional role of each layer of genome organization is a gateway to understanding cell state, development, and disease. Here, we discuss the evolution of 3C-based technologies for mapping 3D genome organization. We focus on genomics methods and provide a historical account of the development from 3C to Hi-C. We also discuss ChIP-based techniques that focus on 3D genome organization mediated by specific proteins, capture-based methods that focus on particular regions or regulatory elements, 3C-orthogonal methods that do not rely on restriction digestion and proximity ligation, and methods for mapping the DNA–RNA and RNA–RNA interactomes. We consider the biological discoveries that have come from these methods, examine the mechanistic contributions of CTCF, cohesin, and loop extrusion to genomic folding, and detail the 3D genome field’s current understanding of nuclear architecture. Finally, we give special consideration to Micro-C as an emerging frontier in chromosome conformation capture and discuss recent Micro-C findings uncovering fine-scale chromatin organization in unprecedented detail.This article is categorized under:Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics

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 .

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.

<p>Hematopoietic stem cell (HSC) development is accompanied by dynamic changes in the transcriptional program. How the corresponding transcriptional programs are related to the epigenetic mechanism is poorly understood.</p> <p>To fill this gap, we first profiled the transcriptomes and epigenomes using RNA-Seq and ChIP-Seq for five key developmental stages of HSC emergence in the mouse embryo. Using epigenetic markers, we identified novel 12,000~17,000 enhancers for each developmental stage. We applied a computational tool to link those enhancers to their target genes. Systematical analysis of enhancer-promoter (EP) pairs using network-based strategy reveals multiple novel key transcription factors for early specification of HSC in the mouse embryo.</p> <p>Second, we compared the 3D genome organization, epigenomes, and transcriptome of fetal and adult HSCs in the mouse. We found that higher-order genome structures are largely conserved between fetal and adult HSCs, including chromosomal compartments and topologically associating domains (TADs). However, chromatin interactions within TADs exhibit substantial differences. We found that promoters within 23% (242/1039) of TADs undergo interaction changes. Transcription factor motif analysis of HSC-specific enhancer-promoter loops suggests a role of KLF1 in mediating condition-specific enhancer looping and regulation of genes involved in cell cycle. Our result provides a comprehensive view of the differences in 3D genome organization, epigenome, and transcriptome between fetal and adult HSCs.</p>

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.

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.

Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding

In eukaryotic cells, 3D genome plays an important role in the regulation of gene spatiotemporal expression, which is essential for the biological and developmental processes in a life cycle. In the past decade, the development of high-throughput technologies greatly enhances our ability to map the 3D genome organization, identifies multiple 3D genome structures, and investigates the functional role of 3D genome organization in gene regulation, which facilitates our understandings of cis-regulatory landscape and biological development. Comparing with the comprehensive analyses of 3D genome in mammals and model plants, the progress in soybean is much less. Future development and application of tools to precisely manipulate 3D genome structure at different levels will significantly strengthen the functional genome study and molecular breeding in soybean. Here, we review the recent progresses in 3D genome study and discuss future directions, which may help to improve soybean 3D functional genome study and molecular breeding.

In clonally propagated crops, extensive divergence between haplotypes complicates transcriptional regulation. However, the contribution of the three dimensional (3D) genome organization to these allelic differences and agronomic traits remains unclear. Here, we generated haplotype-resolved 3D genome landscapes for grapevine cultivars with contrasting berry colors and seed traits, integrating them with genomic, epigenomic, and transcriptomic profiles. We found profound 3D architectural divergence between haplotypes, spanning from large-scale A/B compartments down to a level of topologically associated domain (TAD) boundary variation (18.53%∼23.01%) that approached inter-cultivar differences (18.74~21.62%) (p > 0.05). A key mechanism driving these effects involves large-scale, haplotype-specific transitions between distinct TAD states (Active, Inactive, Heterochromatic), which asymmetrically regulate transcription and alter local DNA methylation patterns. Importantly, these structural rearrangements, including TAD boundary shifts, are strongly associated with underlying structural variants (SVs). Critically, this regulatory cascade impacts key agronomic loci, genes controlling berry color (e.g. VvMYBA) and seedlessness determination (e.g. VvSUS2) were positioned at cultivar-specific TAD boundaries, exhibiting presence/absence variations and differential expression patterns. Our findings support a mechanistic model wherein phased 3D chromatin architecture and heterozygous SVs are strongly associated with the regulation of key agronomic traits, paving the way for accelerating the genetic improvement of clonal crops.

Chromatin insulators demarcate the genome into distinct transcriptional domains and contribute to higher-order genome organization. In Drosophila, Su(Hw), CP190, and Mod(mdg4)67.2 are core protein components of the gypsy insulator complex. Multimerization of these core components contributes to the formation of large structures within the nucleus termed insulator bodies. Post-translational modifications of insulator proteins appear to affect insulator body localization and to be required for full insulator activity, but few factors involved in these processes have been identified. To address this gap in understanding, we performed a high-throughput visual screen for Mod(mdg4)67.2-GFP localization using a ubiquitination-related RNAi library. We identified ubiquitination pathway proteins Effete (Eff) and Cullin 4 (Cul4), as novel regulators of CP190 localization and function. Both Eff and Cul4 physically associate with gypsy insulator proteins and promote gypsy-dependent insulator barrier activity. Moreover, Cul4 extensively colocalizes with CP190 on chromatin and assists in the recruitment of CP190 to gypsy sites. Both Eff and Cul4 affect transcription near topologically associating domain (TAD) borders, with Eff specifically altering the three-dimensional (3D) nuclear positioning of gypsy insulator sites. Our findings reveal a novel role for ubiquitination pathway-related enzymes in chromatin insulator activity, 3D genome organization, and gene expression.

The protozoanTrypanosoma cruzi, the causative agent of Chagas disease, exhibits polycistronic transcription and unidimensional genome compartmentalization ofcore(conserved) anddisruptive(virulence factors from multigenic families) genes. Approximately 50% of its genome is repetitive, mainly virulence factor genes. Genomic sequences, including repeats, motifs of architectural proteins, and noncoding RNA loci are crucial for genome folding. Here, we evaluated the genomic features associated with higher-order chromatin organization inT. cruzithrough extensive computational processing of high-throughput chromosome conformation capture (Hi-C) data, accounting for repetitive regions and improvements in genome annotation. Our study revealed that repetitive DNA (multimapped reads) influences 3D chromatin folding, particularly in determining the boundaries of topologically associated domains (TAD)-like structures. Virulence factor genes, unlikecoregenes, form shorter and more compact TAD-like structures enriched in loops, suggesting a gene expression regulatory mechanism. We found nonprotein-coding RNA loci (e.g., tRNAs) and transcription termination sites preferentially located at the boundaries of the TAD-like structures, while pseudogenes and multigenic family genes located in unstructured genomic regions. Our data indicate 3D clustering of tRNA loci, likely optimizing transcription by RNA polymerase III, and a complex interaction between spliced-leader RNA and 18S rRNA loci. Our findings provide insights into 3D genome organization inT. cruzi, contributing to the understanding of supranucleosome-level chromatin organization and suggesting possible links between 3D architecture and gene expression. We draw an analogy to the art of origami (e.g., papers folded into various shapes) resembling the DNA packed in chromatin fibers assuming distinct folds within the nucleus.ImportanceDespite the knowledge about the linear genome sequence and the identification of numerous virulence factors in the protozoan parasiteTrypanosoma cruzi, there has been a limited understanding of how these genomic features are spatially organized within the nucleus and how this organization impacts gene regulation and pathogenicity. By providing a detailed analysis of the three-dimensional chromatin architecture inT. cruzi, our study contributed to filling this gap. We deciphered part of the origami structure hidden in theT. cruzinucleus, showing the unidimensional genomic features are nonrandomly organized in the nuclear 3D landscape. We revealed the possible role of non-protein-coding RNA loci (e.g., tRNAs, SL-RNA, and 18S RNA) in shaping the genomic architecture. These findings provide insights into an additional epigenetic layer that may influence gene expression.Graphical abstractThe spatial organization of chromatin within the nuclei ofT. cruziand its resemblance to origami art. A. Identification of the 3D nuclear architectures withinT. cruzinuclei: topologically associating domains (TADs) and their boundaries; chromatin loops; and 3D networks. Inter- and intrachromosomal interactions reflect DNA‒DNA contacts on the same (cis) and between different (trans) chromosomes. B. Resemblance between origami art and chromatin folding. Steps “a” to “l” show the process of folding a flat piece of paper from its unidimensional view up to its 3D boat form.

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.