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TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin‐mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter‐TAD loop formation. Based on the reanalysis of Nano‐C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read‐through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter‐TAD loops is restricted though. We speculate that the DNA‐encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer–promoter loop formation and other genomic processes. [ABSTRACT FROM AUTHOR]

Abstract Over the past two decades, scientists have increasingly realized the importance of the three-dimensional (3D) genome organization in regulating cellular activity. Hi-C and related experiments yield 2D contact matrices that can be used to infer 3D models of chromosome structure. Visualizing and analyzing genomes in 3D space remains challenging. Here, we present ARGV, an augmented reality 3D Genome Viewer. ARGV contains more than 350 pre-computed and annotated genome structures inferred from Hi-C and imaging data. It offers interactive and collaborative visualization of genomes in 3D space, using standard mobile phones or tablets. A user study comparing ARGV to existing tools demonstrates its benefits.

Over the past two decades, scientists have increasingly realized the importance of the three-dimensional (3D) genome organization in regulating cellular activity. Hi-C and related experiments yield 2D contact matrices that can be used to infer 3D models of chromosome structure. Visualizing and analyzing genomes in 3D space remains challenging. Here, we present ARGV, an augmented reality 3D Genome Viewer. ARGV contains more than 350 pre-computed and annotated genome structures inferred from Hi-C and imaging data. It offers interactive and collaborative visualization of genomes in 3D space, using standard mobile phones or tablets. A user study comparing ARGV to existing tools demonstrates its benefits. [ABSTRACT FROM AUTHOR]

Within the three‐dimensional (3D) nuclear space, the genome organizes into a series of orderly structures that impose important influences on gene regulation. T lymphocytes, crucial players in adaptive immune responses, undergo intricate transcriptional remodeling upon activation, leading to differentiation into specific effector and memory T cell subsets. Recent evidence suggests that T cell activation is accompanied by dynamic changes in genome architecture at multiple levels, providing a unique biological context to explore the functional relevance and molecular mechanisms of 3D genome organization. Here, we summarize recent advances that link the reorganization of genome architecture to the remodeling of transcriptional programs and conversion of cell fates during T cell activation and differentiation. We further discuss how various chromatin architecture regulators, including CCCTC‐binding factor and several transcription factors, collectively modulate the genome architecture during this process. [ABSTRACT FROM AUTHOR]

Cellular senescence is acknowledged as a key contributor to organismal ageing and late‐life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and ‐B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM‐induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro‐inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent‐like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity. [ABSTRACT FROM AUTHOR]

The 3-dimensional (3D) structure of the genome is of significant importance for many cellular processes. In this paper, we study the problem of reconstructing the 3D structure of chromosomes from Hi-C data of diploid organisms, which poses additional challenges compared to the better-studied haploid setting. With the help of techniques from algebraic geometry, we prove that a small amount of phased data is sufficient to ensure finite identifiability, both for noiseless and noisy data. In the light of these results, we propose a new 3D reconstruction method based on semidefinite programming, paired with numerical algebraic geometry and local optimization. The performance of this method is tested on several simulated datasets under different noise levels and with different amounts of phased data. We also apply it to a real dataset from mouse X chromosomes, and we are then able to recover previously known structural features. [ABSTRACT FROM AUTHOR]

Eukaryotic genomes are hierarchically packaged into cell nucleus, affecting gene regulation. The genome is organized into multiscale structural units, including chromosome territories, compartments, topologically associating domains (TADs), and DNA loops. The identification of these hierarchical structures has benefited from the development of experimental approaches, such as 3C-based methods (Hi-C, ChIA-PET, etc.), imaging tools (2D-FISH, 3D-FISH, Cryo-FISH, etc.) and ligation-free methods (GAM, SPRITE, etc.). In recent two decades, numerous studies have shown that the 3D organization of genome plays essential roles in multiple cellular processes via various mechanisms, such as regulating enhancer activity and promoter-enhancer interactions. However, there are relatively few studies about the 3D genome in livestock species. Therefore, studies for exploring the function of 3D genomes in livestock are urgently needed to provide a more comprehensive understanding of potential relationships between the genome and production traits. In this review, we summarize the recent advances of 3D genomics and its biological functions in human and mouse studies, drawing inspiration to explore the 3D genomics of livestock species. We then mainly focus on the biological functions of 3D genome organization in muscle development and its implications in animal breeding.

Human life expectancy is constantly increasing and aging has become a major risk factor for many diseases, although the underlying gene regulatory mechanisms are still unclear. Using transcriptomic and chromosomal conformation capture (Hi‐C) data from human skin fibroblasts from individuals across different age groups, we identified a tight coupling between the changes in co‐regulation and co‐localization of genes. We obtained transcription factors, cofactors, and chromatin regulators that could drive the cellular aging process by developing a time‐course prize‐collecting Steiner tree algorithm. In particular, by combining RNA‐Seq data from different age groups and protein–protein interaction data we determined the key transcription regulators and gene regulatory changes at different life stage transitions. We then mapped these transcription regulators to the 3D reorganization of chromatin in young and old skin fibroblasts. Collectively, we identified key transcription regulators whose target genes are spatially rearranged and correlate with changes in their expression, thereby providing potential targets for reverting cellular aging. [ABSTRACT FROM AUTHOR]

CD62L+ central memory CD8+ T (TCM) cells provide enhanced protection than naive cells; however, the underlying mechanism, especially the contribution of higher-order genomic organization, remains unclear. Systematic Hi-C analyses reveal that antigen-experienced CD8+ T cells undergo extensive rewiring of chromatin interactions (ChrInt), with TCM cells harboring specific interaction hubs compared with naive CD8+ T cells, as observed at cytotoxic effector genes such as Ifng and Tbx21. TCM cells also acquire de novo CTCF (CCCTC-binding factor) binding sites, which are not only strongly associated with TCM-specific hubs but also linked to increased activities of local gene promoters and enhancers. Specific ablation of CTCF in TCM cells impairs rapid induction of genes in cytotoxic program, energy supplies, transcription, and translation by recall stimulation. Therefore, acquisition of CTCF binding and ChrInt hubs by TCM cells serves as a chromatin architectural basis for their transcriptomic dynamics in primary response and for imprinting the code of “recall readiness” against secondary challenge. [ABSTRACT FROM AUTHOR]

In eukaryotic genomes, hundreds to thousands of potential start sites of DNA replication named origins are dispersed across each of the linear chromosomes. During S-phase, only a subset of origins is selected in a stochastic manner to assemble bidirectional replication forks and initiate DNA synthesis. Despite substantial progress in our understanding of this complex process, a comprehensive 'identity code' that defines origins based on specific nucleotide sequences, DNA structural features, the local chromatin environment, or 3D genome architecture is still missing. In this article, we review the genetic and epigenetic features of replication origins in yeast and metazoan chromosomes and highlight recent insights into how this flexibility in origin usage contributes to nuclear organization, cell growth, differentiation, and genome stability. [ABSTRACT FROM AUTHOR]

Retrocopies are gene duplicates arising from reverse transcription of mature mRNA transcripts and their insertion back into the genome. While long being regarded as processed pseudogenes, more and more functional retrocopies have been discovered. How the stripped-down retrocopies recover expression capability and become functional paralogs continually intrigues evolutionary biologists. Here, we investigated the function and evolution of retrocopies in the context of 3D genome organization. By mapping retrocopy–parent pairs onto sequencing-based and imaging-based chromatin contact maps in human and mouse cell lines and onto Hi-C interaction maps in 5 other mammals, we found that retrocopies and their parental genes show a higher-than-expected interchromosomal colocalization frequency. The spatial interactions between retrocopies and parental genes occur frequently at loci in active subcompartments and near nuclear speckles. Accordingly, colocalized retrocopies are more actively transcribed and translated and are more evolutionarily conserved than noncolocalized ones. The active transcription of colocalized retrocopies may result from their permissive epigenetic environment and shared regulatory elements with parental genes. Population genetic analysis of retroposed gene copy number variants in human populations revealed that retrocopy insertions are not entirely random in regard to interchromosomal interactions and that colocalized retroposed gene copy number variants are more likely to reach high frequencies, suggesting that both insertion bias and natural selection contribute to the colocalization of retrocopy–parent pairs. Further dissection implies that reduced selection efficacy, rather than positive selection, contributes to the elevated allele frequency of colocalized retroposed gene copy number variants. Overall, our results hint a role of interchromosomal colocalization in the "resurrection" of initially neutral retrocopies. [ABSTRACT FROM AUTHOR]

Lamina‐associated domains are large regions of heterochromatin positioned at the nuclear periphery. These domains have been implicated in gene repression, especially in the context of development. In mammals, LAD organization is dependent on nuclear lamins, inner nuclear membrane proteins, and chromatin state. In addition, chromatin readers and modifier proteins have been implicated in this organization, potentially serving as molecular tethers that interact with both nuclear envelope proteins and chromatin. More recent studies have focused on teasing apart the rules that govern dynamic LAD organization and how LAD organization, in turn, relates to gene regulation and overall 3D genome organization. This review highlights recent studies in mammalian cells uncovering factors that instruct the choreography of LAD organization, re‐organization, and dynamics at the nuclear lamina, including LAD dynamics in interphase and through mitotic exit, when LAD organization is re‐established, as well as intra‐LAD subdomain variations. [ABSTRACT FROM AUTHOR]

The human genome is organized in an extremely complexed yet ordered way within the nucleus. Genome organization plays a critical role in the regulation of gene expression. Viruses manipulate the host machinery to influence host genome organization to favor their survival and promote disease development. Epstein‐Barr virus (EBV) is a common human virus, whose infection is associated with various diseases, including infectious mononucleosis, cancer, and autoimmune disorders. This review summarizes our current knowledge of how EBV uses different strategies to control the cellular 3D genome organization to affect cell gene expression to transform normal cells into lymphoblasts. [ABSTRACT FROM AUTHOR]

Deletions, duplications, insertions, inversions, and translocations, collectively called structural variations (SVs), affect more base pairs of the genome than any other sequence variant. The recent technological advancements in genome sequencing have enabled the discovery of tens of thousands of SVs per human genome. These SVs primarily affect non‐coding DNA sequences, but the difficulties in interpreting their impact limit our understanding of human disease etiology. The functional annotation of non‐coding DNA sequences and methodologies to characterize their three‐dimensional (3D) organization in the nucleus have greatly expanded our understanding of the basic mechanisms underlying gene regulation, thereby improving the interpretation of SVs for their pathogenic impact. Here, we discuss the various mechanisms by which SVs can result in altered gene regulation and how these mechanisms can result in rare genetic disorders. Beyond changing gene expression, SVs can produce novel gene‐intergenic fusion transcripts at the SV breakpoints. [ABSTRACT FROM AUTHOR]

ABSTRACTIn eukaryotic genomes, hundreds to thousands of potential start sites of DNA replication named origins are dispersed across each of the linear chromosomes. During S-phase, only a subset of origins is selected in a stochastic manner to assemble bidirectional replication forks and initiate DNA synthesis. Despite substantial progress in our understanding of this complex process, a comprehensive ‘identity code’ that defines origins based on specific nucleotide sequences, DNA structural features, the local chromatin environment, or 3D genome architecture is still missing. In this article, we review the genetic and epigenetic features of replication origins in yeast and metazoan chromosomes and highlight recent insights into how this flexibility in origin usage contributes to nuclear organization, cell growth, differentiation, and genome stability.

Eukaryotic genomes are spatially organized inside the cell nucleus, forming a threedimensional (3D) architecture that allows for spatial separation of nuclear processes and for controlled expression of genes required for cell identity specification and tissue homeostasis. Hence, it is of no surprise that mis-regulation of genome architecture through rearrangements of the linear genome sequence or epigenetic perturbations are often linked to aberrant gene expression programs in tumor cells. Increasing research efforts have shed light into the causes and consequences of alterations of 3D genome organization. In this review, we summarize the current knowledge on how 3D genome architecture is dysregulated in cancer, with a focus on enhancer highjacking events and their contribution to tumorigenesis. Studying the functional effects of genome architecture perturbations on gene expression in cancer offers a unique opportunity for a deeper understanding of tumor biology and sets the basis for the discovery of novel therapeutic targets.

The three-dimensional organization of chromatin plays a crucial role in gene regulation and cellular processes like deoxyribonucleic acid (DNA) transcription, replication and repair. Hi-C and related techniques provide detailed views of spatial proximities within the nucleus. However, data analysis is challenging partially due to a lack of well-defined, underpinning mathematical frameworks. Recently, recognizing and analyzing geometric patterns in Hi-C data has emerged as a powerful approach. This review provides a summary of algorithms for automatic recognition and analysis of geometric patterns in Hi-C data and their correspondence with chromatin structure. We classify existing algorithms on the basis of the data representation and pattern recognition paradigm they make use of. Finally, we outline some of the challenges ahead and promising future directions. [ABSTRACT FROM AUTHOR]

Abstract Background Recent work has demonstrated that three-dimensional genome organization is directly affected by changes in the levels of nuclear cytoskeletal proteins such as β-actin. The mechanisms which translate changes in 3D genome structure into changes in transcription, however, are not fully understood. Here, we use a comprehensive genomic analysis of cells lacking nuclear β-actin to investigate the mechanistic links between compartment organization, enhancer activity, and gene expression. Results Using HiC-Seq, ATAC-Seq, and RNA-Seq, we first demonstrate that transcriptional and chromatin accessibility changes observed upon β-actin loss are highly enriched in compartment-switching regions. Accessibility changes within compartment switching genes, however, are mainly observed in non-promoter regions which potentially represent distal regulatory elements. Our results also show that β-actin loss induces widespread accumulation of the enhancer-specific epigenetic mark H3K27ac. Using the ABC model of enhancer annotation, we then establish that these epigenetic changes have a direct impact on enhancer activity and underlie transcriptional changes observed upon compartment switching. A complementary analysis of fibroblasts undergoing reprogramming into pluripotent stem cells further confirms that this relationship between compartment switching and enhancer-dependent transcriptional change is not specific to β-actin knockout cells but represents a general mechanism linking compartment-level genome organization to gene expression. Conclusions We demonstrate that enhancer-dependent transcriptional regulation plays a crucial role in driving gene expression changes observed upon compartment-switching. Our results also reveal a novel function of nuclear β-actin in regulating enhancer function by influencing H3K27 acetylation levels.

Abstract Background A growing body of evidence has revealed that the mammalian genome is organized into hierarchical layers that are closely correlated with and may even be causally linked with variations in gene expression. Recent studies have characterized chromatin organization in various porcine tissues and cell types and compared them among species and during the early development of pigs. However, how chromatin organization differs among pig breeds is poorly understood. Results In this study, we investigated the 3D genome organization and performed transcriptome characterization of two adipose depots (upper layer of backfat [ULB] and greater omentum [GOM]) in wild boars and Bama pigs; the latter is a typical indigenous pig in China. We found that over 95% of the A/B compartments and topologically associating domains (TADs) are stable between wild boars and Bama pigs. In contrast, more than 70% of promoter-enhancer interactions (PEIs) are dynamic and widespread, involving over a thousand genes. Alterations in chromatin structure are associated with changes in the expression of genes that are involved in widespread biological functions such as basic cellular functions, endocrine function, energy metabolism and the immune response. Approximately 95% and 97% of the genes associated with reorganized A/B compartments and PEIs in the two pig breeds differed between GOM and ULB, respectively. Conclusions We reported 3D genome organization in adipose depots from different pig breeds. In a comparison of Bama pigs and wild boar, large-scale compartments and TADs were mostly conserved, while fine-scale PEIs were extensively reorganized. The chromatin architecture in these two pig breeds was reorganized in an adipose depot-specific manner. These results contribute to determining the regulatory mechanism of phenotypic differences between Bama pigs and wild boar.

The emergence and progression of cancers is accompanied by a dysregulation of transcriptional programs. The three-dimensional (3D) organization of the human genome has emerged as an important multi-level mediator of gene transcription and regulation. In cancer cells, this organization can be restructured, providing a framework for the deregulation of gene activity. The CTCF protein, initially identified as the product from a tumor suppressor gene, is a jack-of-all-trades for the formation of 3D genome organization in normal cells. Here, we summarize how CTCF is involved in the multi-level organization of the human genome and we discuss emerging insights into how perturbed CTCF function and DNA binding causes the activation of oncogenes in cancer cells, mostly through a process of enhancer hijacking. Moreover, we highlight non-canonical functions of CTCF that can be relevant for the emergence of cancers as well. Finally, we provide guidelines for the computational identification of perturbed CTCF binding and reorganized 3D genome structure in cancer cells.

The discovery of the CRISPR-Cas9 endonuclease has enabled facile genome editing in living cells and organisms. Catalytically inactive Cas9 (dCas9) retains the ability to bind DNA in an RNA-guided fashion, and has additionally been explored as a tool for transcriptional modulation, epigenetic editing, and genome imaging. This Review highlights recent progress and challenges in the development of dCas9 for imaging genomic loci. The emergence and maturation of this technology offers the potential to answer mechanistic questions about chromosome dynamics and three-dimensional genome organization in vivo.

The oncogenic process is orchestrated by a complex network of chromatin remodeling elements that shape the cancer epigenome. Histone variant H2A.Z regulates DNA control elements such as promoters and enhancers in different types of cancer; however, the interplay between H2A.Z and the pancreatic cancer epigenome is unknown. This study analyzed the role of H2A.Z in different DNA regulatory elements. We performed Chromatin Immunoprecipitation Sequencing assays (ChiP-seq) with total H2A.Z and acetylated H2A.Z (acH2A.Z) antibodies and analyzed published data from ChIP-seq, RNA-seq, bromouridine labeling-UV and sequencing (BruUV-seq), Hi-C and ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) in the pancreatic cancer cell line PANC-1. The results indicate that total H2A.Z facilitates the recruitment of RNA polymerase II and transcription factors at promoters and enhancers allowing the expression of pro-oncogenic genes. Interestingly, we demonstrated that H2A.Z is enriched in super-enhancers (SEs) contributing to the transcriptional activation of key genes implicated in tumor development. Importantly, we established that H2A.Z contributes to the three-dimensional (3D) genome organization of pancreatic cancer and that it is a component of the Topological Associated Domains (TADs) boundaries in PANC-1 and that total H2A.Z and acH2A.Z are associated with A and B compartments, respectively. H2A.Z participates in the biology and development of pancreatic cancer by generating a pro-oncogenic transcriptome through its posttranslational modifications, interactions with different partners, and regulatory elements, contributing to the oncogenic 3D genome organization. These data allow us to understand the molecular mechanisms that promote an oncogenic transcriptome in pancreatic cancer mediated by H2A.Z. [ABSTRACT FROM AUTHOR]

Chromosomes inside the nucleus are not located in the form of linear molecules. Instead, there is a complex multilevel genome folding that includes nucleosomes packaging, formation of chromatin loops, domains, compartments, and finally, chromosomal territories. Proper spatial organization play an essential role for the correct functioning of the genome, and is therefore dynamically changed during development or disease. Here we discuss how the organization of the cancer cell genome differs from the healthy genome at various levels. A better understanding of how malignization affects genome organization and long-range gene regulation will help to reveal the molecular mechanisms underlying cancer development and evolution. [ABSTRACT FROM AUTHOR]

Over a decade ago the advent of high-throughput chromosome conformation capture (Hi-C) sparked a new era of 3D genomics. Since then the number of methods for mapping the 3D genome has flourished, enabling an ever-increasing understanding of how DNA is packaged in the nucleus and how the spatiotemporal organization of the genome orchestrates its vital functions. More recently, the next generation of spatial genomics technologies has begun to reveal how genome sequence and 3D genome organization vary between cells in their tissue context. We summarize how the toolkit for charting genome topology has evolved over the past decade and discuss how new technological developments are advancing the field of 3D and spatial genomics. For clarification across the field, we propose to use the term '3D genomics' to denote the study of the spatial positioning of chromatin in the nucleus – including chromatin contacts and associations with nuclear landmarks – whereas the term 'spatial genomics' is used to refer to the study of genome sequence and 3D structure variation between cells in their native tissue context. Combining in situ barcode sequencing and ex situ genome sequencing for spatially resolved genomics is an emerging approach that can be leveraged to address fundamental research questions on intercell genome variability and for cancer diagnostics, allowing high-resolution mapping of intratumor heterogeneity. Together, spatial genomics and spatial transcriptomics promise to revolutionize both biology and medicine by providing unprecedented insights into the spatial organization of tissues and how fundamental cellular processes are orchestrated in multicellular organisms. [ABSTRACT FROM AUTHOR]

Non-genetic information can be inherited across generations in a process known as transgenerational epigenetic inheritance (TEI). In Drosophila, hemizygosity of the Fab-7 regulatory element triggers inheritance of the histone mark H3K27me3 at a homologous locus on another chromosome, resulting in heritable epigenetic differences in eye color. Here, by mutating transcription factor binding sites within the Fab-7 element, we demonstrate the importance of the proteins pleiohomeotic and GAGA factor in the establishment and maintenance of TEI. We show that these proteins function by recruiting the polycomb repressive complex 2 and by mediating interchromosomal chromatin contacts between Fab-7 and its homologous locus, respectively. Using an in vivo synthetic biology system to induce them, we then show that chromatin contacts alone can establish TEI, providing a mechanism by which hemizygosity of one locus can establish epigenetic memory at another distant locus in trans through chromatin contacts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Genome organization is intricately tied to regulating genes and associated cell fate decisions. Here, we examine the positioning and functional significance of human genes, grouped by their lineage restriction level, within the 3D organization of the genome. We reveal that genes of different lineage restriction levels have distinct positioning relationships with both domains and loop anchors, and remarkably consistent relationships with boundaries across cell types. While the functional associations of each group of genes are primarily cell type-specific, associations of conserved genes maintain greater stability across 3D genomic features and disease than recently evolved genes. Furthermore, the expression of these genes across various tissues follows an evolutionary progression, such that RNA levels increase from young lineage restricted genes to ancient genes present in most species. Thus, the distinct relationships of gene evolutionary age, function, and positioning within 3D genomic features contribute to tissue-specific gene regulation in development and disease., Competing Interests: Declaration of Interests The authors declare no competing interests.

Developmental gene regulation requires input from enhancers spread over large genomic distances. Our understanding of long-range enhancer-promoter (E-P) communication, characterized as loops, remains incomplete without addressing the role of intervening chromatin. Here, we examine the topology of the entire Sonic hedgehog (Shh) regulatory domain in individual alleles from the mouse embryonic forebrain. Through sequential Oligopaint labeling and super-resolution microscopy, we find that the Shh locus maintains a compact structure that adopts several diverse configurations independent of Shh expression. The most frequent configuration contained distal E-P contacts at the expense of those more proximal to Shh, consistent with an interconnected loop. Genetic perturbations demonstrate that this long-range E-P communication operates by Shh-expression-independent and dependent mechanisms, involving CTCF binding sites and active enhancers, respectively. We propose a model whereby gene regulatory elements secure long-range E-P interactions amid an inherent architectural framework to coordinate spatiotemporal patterns of gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chromatin structural domains, or topologically associated domains (TADs), are a general organizing principle in chromatin biology. RNA polymerase II (RNAPII) mediates multiple chromatin interactive loops, tethering together as RNAPII-associated chromatin interaction domains (RAIDs) to offer a framework for gene regulation. RAID and TAD alterations have been found to be associated with diseases. They can be further dissected as micro-domains (micro-TADs and micro-RAIDs) by clustering single-molecule chromatin-interactive complexes from next-generation three-dimensional (3D) genome techniques, such as ChIA-Drop. Currently, there are few tools available for micro-domain boundary identification. In this work, we developed the MCI-frcnn deep learning method to train a Faster Region-based Convolutional Neural Network (Faster R-CNN) for micro-domain boundary detection. At the training phase in MCI-frcnn, 50 images of RAIDs from Drosophila RNAPII ChIA-Drop data, containing 261 micro-RAIDs with ground truth boundaries, were trained for 7 days. Using this well-trained MCI-frcnn, we detected micro-RAID boundaries for the input new images, with a fast speed (5.26 fps), high recognition accuracy (AUROC = 0.85, mAP = 0.69), and high boundary region quantification (genomic IoU = 76%). We further applied MCI-frcnn to detect human micro-TADs boundaries using human GM12878 SPRITE data and obtained a high region quantification score (mean gIoU = 85%). In all, the MCI-frcnn deep learning method which we developed in this work is a general tool for micro-domain boundary detection.

Chromosomes inside the nucleus are not located in the form of linear molecules. Instead, there is a complex multilevel genome folding that includes nucleosomes packaging, formation of chromatin loops, domains, compartments, and finally, chromosomal territories. Proper spatial organization play an essential role for the correct functioning of the genome, and is therefore dynamically changed during development or disease. Here we discuss how the organization of the cancer cell genome differs from the healthy genome at various levels. A better understanding of how malignization affects genome organization and long-range gene regulation will help to reveal the molecular mechanisms underlying cancer development and evolution.

Condensins are molecular motors that compact DNA via linear translocation. In Caenorhabditis elegans, the X-chromosome harbors a specialized condensin that participates in dosage compensation (DC). Condensin DC is recruited to and spreads from a small number of recruitment elements on the X-chromosome (rex) and is required for the formation of topologically associating domains (TADs). We take advantage of autosomes that are largely devoid of condensin DC and TADs to address how rex sites and condensin DC give rise to the formation of TADs. When an autosome and X-chromosome are physically fused, despite the spreading of condensin DC into the autosome, no TAD was created. Insertion of a strong rex on the X-chromosome results in the TAD boundary formation regardless of sequence orientation. When the same rex is inserted on an autosome, despite condensin DC recruitment, there was no spreading or features of a TAD. On the other hand, when a ‘super rex’ composed of six rex sites or three separate rex sites are inserted on an autosome, recruitment and spreading of condensin DC led to the formation of TADs. Therefore, recruitment to and spreading from rex sites are necessary and sufficient for recapitulating loop-anchored TADs observed on the X-chromosome. Together our data suggest a model in which rex sites are both loading sites and bidirectional barriers for condensin DC, a one-sided loop-extruder with movable inactive anchor.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. [ABSTRACT FROM AUTHOR]

Germ cells are unique in engendering totipotency, yet the mechanisms underlying this capacity remain elusive. Here, we perform comprehensive and in‐depth nucleome analysis of mouse germ‐cell development in vitro, encompassing pluripotent precursors, primordial germ cells (PGCs) before and after epigenetic reprogramming, and spermatogonia/spermatogonial stem cells (SSCs). Although epigenetic reprogramming, including genome‐wide DNA de‐methylation, creates broadly open chromatin with abundant enhancer‐like signatures, the augmented chromatin insulation safeguards transcriptional fidelity. These insulatory constraints are then erased en masse for spermatogonial development. Notably, despite distinguishing epigenetic programming, including global DNA re‐methylation, the PGCs‐to‐spermatogonia/SSCs development entails further euchromatization. This accompanies substantial erasure of lamina‐associated domains, generating spermatogonia/SSCs with a minimal peripheral attachment of chromatin except for pericentromeres—an architecture conserved in primates. Accordingly, faulty nucleome maturation, including persistent insulation and improper euchromatization, leads to impaired spermatogenic potential. Given that PGCs after epigenetic reprogramming serve as oogenic progenitors as well, our findings elucidate a principle for the nucleome programming that creates gametogenic progenitors in both sexes, defining a basis for nuclear totipotency. Synopsis: Multi‐omics profiling of an in vitro system for mouse germ‐cell development delineates key principles of nucleome programming, including progressive euchromatization and dynamic insulation, as a basis for nuclear totipotency. 3D genome matures unidirectionally over epigenetic reprogramming and programming.Epigenetic reprogramming creates broadly open chromatin with enhanced insulation.Spermatogonia show minimal, peripherally‐positioned, and pericentromeric LADs.Nucleome mis‐programming leads to impaired spermatogenic potential. [ABSTRACT FROM AUTHOR]

Abstract Chromatin interactions play important roles in regulating gene expression. However, the availability of genome-wide chromatin interaction data is limited. We develop a computational method, chromatin interaction neural network (ChINN), to predict chromatin interactions between open chromatin regions using only DNA sequences. ChINN predicts CTCF- and RNA polymerase II-associated and Hi-C chromatin interactions. ChINN shows good across-sample performances and captures various sequence features for chromatin interaction prediction. We apply ChINN to 6 chronic lymphocytic leukemia (CLL) patient samples and a published cohort of 84 CLL open chromatin samples. Our results demonstrate extensive heterogeneity in chromatin interactions among CLL patient samples.

Genome-wide association studies (GWAS) have identified a vast number of variants associated with various complex human diseases and traits. However, most of these GWAS variants reside in non-coding regions producing no proteins, making the interpretation of these variants a daunting challenge. Prior evidence indicates that a subset of non-coding variants detected within or near cis-regulatory elements (e.g., promoters, enhancers, silencers, and insulators) might play a key role in disease etiology by regulating gene expression. Advanced sequencing- and imaging-based technologies, together with powerful computational methods, enabling comprehensive characterization of regulatory DNA interactions, have substantially improved our understanding of the three-dimensional (3D) genome architecture. Recent literature witnesses plenty of examples where using chromosome conformation capture (3C)-based technologies successfully links non-coding variants to their target genes and prioritizes relevant tissues or cell types. These examples illustrate the critical capability of 3D genome organization in annotating non-coding GWAS variants. This review discusses how 3D genome organization information contributes to elucidating the potential roles of non-coding GWAS variants in disease etiology.

Genome organization includes contacts both within a single chromosome and between distinct chromosomes. Thus, regulatory organization in the nucleus may include interplay of these two types of chromosomal interactions with genome activity. Emerging advances in omics and single-cell imaging technologies have allowed new insights into chromosomal contacts, including those of homologs and sister chromatids, and their significance to genome function. In this review, we highlight recent studies in this field and discuss their impact on understanding the principles of chromosome organization and associated functional implications in diverse cellular processes. Specifically, we describe the contributions of intra-chromosomal, inter-homolog, and inter-sister chromatid contacts to genome organization and gene expression.

Simple Summary: Gene expression is primarily controlled at the level of transcription, largely due to binding of transcription factors to specific sites on DNA, namely super-enhancers and promoters. Super-enhancers and enhancers can regulate distal target genes via chromatin looping mechanisms. These long-range enhancer–promoter interactions are often mediated by phase-separated condensates. In this review, we summarize recent insights into super-enhancers and phase-separated condensates and their roles in the transcriptional activation of genes involved in development and diseases such as cancer. In addition, we survey a broad array of drugs that target super-enhancers, three-dimensional genome organization, and phase-separated condensates that could be potentially used for the treatment of cancer. 3D chromatin organization plays an important role in transcription regulation and gene expression. The 3D genome is highly maintained by several architectural proteins, such as CTCF, Yin Yang 1, and cohesin complex. This structural organization brings regulatory DNA elements in close proximity to their target promoters. In this review, we discuss the 3D chromatin organization of super-enhancers and their relationship to phase-separated condensates. Super-enhancers are large clusters of DNA elements. They can physically contact with their target promoters by chromatin looping during transcription. Multiple transcription factors can bind to enhancer and promoter sequences and recruit a complex array of transcriptional co-activators and RNA polymerase II to effect transcriptional activation. Phase-separated condensates of transcription factors and transcriptional co-activators have been implicated in assembling the transcription machinery at particular enhancers. Cancer cells can hijack super-enhancers to drive oncogenic transcription to promote cell survival and proliferation. These dysregulated transcriptional programs can cause cancer cells to become highly dependent on transcriptional regulators, such as Mediator and BRD4. Moreover, the expression of oncogenes that are driven by super-enhancers is sensitive to transcriptional perturbation and often occurs in phase-separated condensates, supporting therapeutic rationales of targeting SE components, 3D genome organization, or dysregulated condensates in cancer. [ABSTRACT FROM AUTHOR]

Studies of the regulatory networks and signals controlling erythropoiesis have brought important insights in several research fields of biology and have been a rich source of discoveries with far-reaching implications beyond erythroid cells biology. The aim of this review is to highlight key recent discoveries and show how studies of erythroid cells bring forward novel concepts and refine current models related to genome and 3D chromatin organization, signaling and disease, with broad interest in life sciences. [ABSTRACT FROM AUTHOR]

Abstract We report SPIN, an integrative computational method to reveal genome-wide intranuclear chromosome positioning and nuclear compartmentalization relative to multiple nuclear structures, which are pivotal for modulating genome function. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and putative associations with nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to other 3D genome features and genome function (transcription and replication timing). SPIN provides critical insights into nuclear spatial and functional compartmentalization.

Over a decade has passed since the development of the Hi-C method for genome-wide analysis of 3D genome organization. Hi-C utilizes next-generation sequencing (NGS) technology to generate large-scale chromatin interaction data, which has accumulated across a diverse range of species and cell types, particularly in eukaryotes. There is thus a growing need to streamline the process of Hi-C data analysis to utilize these data sets effectively. Hi-C generates data that are much larger compared to other NGS techniques such as chromatin immunoprecipitation sequencing (ChIP-seq) or RNA-seq, making the data reanalysis process computationally expensive. In an effort to bridge this resource gap, the 4D Nucleome (4DN) Data Portal has reanalyzed approximately 600 Hi-C data sets, allowing users to access and utilize the analyzed data. In this chapter, we provide detailed instructions for the implementation of the common workflow language (CWL)-based Hi-C analysis pipeline adopted by the 4DN Data Portal ecosystem. This reproducible and portable pipeline generates standard Hi-C contact matrices in formats such as .hic or .mcool from FASTQ files. It enables users to output their own Hi-C data in the same format as those registered in the 4DN Data portal, facilitating comparative analysis using data registered in the portal. Our custom-made scripts are available on GitHub at https://github.com/kuzobuta/4dn_cwl_pipeline ., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.

Background: A growing body of evidence has revealed that the mammalian genome is organized into hierarchical layers that are closely correlated with and may even be causally linked with variations in gene expression. Recent studies have characterized chromatin organization in various porcine tissues and cell types and compared them among species and during the early development of pigs. However, how chromatin organization differs among pig breeds is poorly understood. Results: In this study, we investigated the 3D genome organization and performed transcriptome characterization of two adipose depots (upper layer of backfat [ULB] and greater omentum [GOM]) in wild boars and Bama pigs; the latter is a typical indigenous pig in China. We found that over 95% of the A/B compartments and topologically associating domains (TADs) are stable between wild boars and Bama pigs. In contrast, more than 70% of promoter-enhancer interactions (PEIs) are dynamic and widespread, involving over a thousand genes. Alterations in chromatin structure are associated with changes in the expression of genes that are involved in widespread biological functions such as basic cellular functions, endocrine function, energy metabolism and the immune response. Approximately 95% and 97% of the genes associated with reorganized A/B compartments and PEIs in the two pig breeds differed between GOM and ULB, respectively. Conclusions: We reported 3D genome organization in adipose depots from different pig breeds. In a comparison of Bama pigs and wild boar, large-scale compartments and TADs were mostly conserved, while fine-scale PEIs were extensively reorganized. The chromatin architecture in these two pig breeds was reorganized in an adipose depot-specific manner. These results contribute to determining the regulatory mechanism of phenotypic differences between Bama pigs and wild boar. [ABSTRACT FROM AUTHOR]

Interphase chromatin is organized precisely to facilitate accurate gene expression. The structure–function relationship of chromatin is epitomized in sex chromosome dosage compensation (DC), where sex-linked gene expression is balanced between males and females via sex-specific alterations to three-dimensional chromatin structure. Studies in ZW-bearing species suggest that DC is absent or incomplete in most lineages except butterflies and moths, where male (ZZ) Z chromosome (chZ) expression is reduced by half to equal females (ZW). However, whether one chZ is inactivated (as in mammals) or both are partially repressed (as in Caenorhabditis elegans) is unclear. Using Oligopaints in the silkworm, Bombyx mori, we visualize autosomes and chZ in somatic cells from both sexes. We find that B. mori chromosomes are highly compact relative to Drosophila. We show that in B. mori males, both chZs are similar in size and shape and are more compact than autosomes or the female chZ after DC establishment, suggesting both male chZs are partially and equally downregulated. We also find that in the early stages of DC in females, chZ chromatin becomes more accessible and Z-linked expression increases. Concomitant with these changes, the female chZ repositions toward the nuclear center, revealing nonsequencing-based support for Ohno’s hypothesis. These studies visualizing interphase genome organization and chZ structure in Lepidoptera uncover intriguing similarities between DC in B. mori and C. elegans, despite these lineages harboring evolutionarily distinct sex chromosomes (ZW/XY), suggesting a possible role for holocentricity in DC mechanisms. [ABSTRACT FROM AUTHOR]

Cancer risk by environmental exposure is modulated by an individual's genetics and age at exposure. This age-specific period of susceptibility is referred to as the "Window of Susceptibility" (WOS). Rats have a similar WOS for developing breast cancer. A previous study in rat identified an age-specific long-range regulatory interaction for the cancer gene, Pappa , that is associated with breast cancer susceptibility. However, the global role of three-dimensional genome organization and downstream gene expression programs in the WOS is not known. Therefore, we generated Hi-C and RNA-seq data in rat mammary epithelial cells within and outside the WOS. To systematically identify higher-order changes in 3D genome organization, we developed NE-MVNMF that combines network enhancement followed by multitask non-negative matrix factorization. We examined three-dimensional genome organization dynamics at the level of individual loops as well as higher-order domains. Differential chromatin interactions tend to be associated with differentially up-regulated genes with the WOS and recapitulate several human SNP-gene interactions associated with breast cancer susceptibility. Our approach identified genomic blocks of regions with greater overall differences in contact count between the two time points when the cluster assignments change and identified genes and pathways implicated in early carcinogenesis and cancer treatment. Our results suggest that WOS-specific changes in 3D genome organization are linked to transcriptional changes that may influence susceptibility to breast cancer. [ABSTRACT FROM AUTHOR]

Abstract Background The three-dimensional genome organization is critical for gene regulation and can malfunction in diseases like cancer. As a key regulator of genome organization, CCCTC-binding factor (CTCF) has been characterized as a DNA-binding protein with important functions in maintaining the topological structure of chromatin and inducing DNA looping. Among the prolific binding sites in the genome, several events with altered CTCF occupancy have been reported as associated with effects in physiology or disease. However, hitherto there is no comprehensive survey of genome-wide CTCF binding patterns across different human cancers. Results To dissect functions of CTCF binding, we systematically analyze over 700 CTCF ChIP-seq profiles across human tissues and cancers and identify cancer-specific CTCF binding patterns in six cancer types. We show that cancer-specific lost and gained CTCF binding events are associated with altered chromatin interactions, partially with DNA methylation changes, and rarely with sequence mutations. While lost bindings primarily occur near gene promoters, most gained CTCF binding events exhibit enhancer activities and are induced by oncogenic transcription factors. We validate these findings in T cell acute lymphoblastic leukemia cell lines and patient samples and show that oncogenic NOTCH1 induces specific CTCF binding and they cooperatively activate expression of target genes, indicating transcriptional condensation phenomena. Conclusions Specific CTCF binding events occur in human cancers. Cancer-specific CTCF binding can be induced by other transcription factors to regulate oncogenic gene expression. Our results substantiate CTCF binding alteration as a functional epigenomic signature of cancer.

The three-dimensional (3D) genome organization and its role in biological activities have been investigated for over a decade in the field of cell biology. Recent studies using live-imaging and polymer simulation have suggested that the higher-order chromatin structures are dynamic; the stochastic fluctuations of nucleosomes and genomic loci cannot be captured by bulk-based chromosome conformation capture techniques (Hi-C). In this review, we focus on the physical nature of the 3D genome architecture. We first describe how to decode bulk Hi-C data with polymer modeling. We then introduce our recently developed PHi-C method, a computational tool for modeling the fluctuations of the 3D genome organization in the presence of stochastic thermal noise. We also present another new method that analyzes the dynamic rheology property (represented as microrheology spectra) as a measure of the flexibility and rigidity of genomic regions over time. By applying these methods to real Hi-C data, we highlighted a temporal hierarchy embedded in the 3D genome organization; chromatin interaction boundaries are more rigid than the boundary interior, while functional domains emerge as dynamic fluctuations within a particular time interval. Our methods may bridge the gap between live-cell imaging and Hi-C data and elucidate the nature of the dynamic 3D genome organization.

Cancer risk by environmental exposure is modulated by an individual’s genetics and age at exposure. This age-specific period of susceptibility is referred to as the “Window of Susceptibility” (WOS). Rats have a similar WOS for developing breast cancer. A previous study in rat identified an age-specific long-range regulatory interaction for the cancer gene, Pappa, that is associated with breast cancer susceptibility. However, the global role of three-dimensional genome organization and downstream gene expression programs in the WOS is not known. Therefore, we generated Hi-C and RNA-seq data in rat mammary epithelial cells within and outside the WOS. To systematically identify higher-order changes in 3D genome organization, we developed NE-MVNMF that combines network enhancement followed by multitask non-negative matrix factorization. We examined three-dimensional genome organization dynamics at the level of individual loops as well as higher-order domains. Differential chromatin interactions tend to be associated with differentially up-regulated genes with the WOS and recapitulate several human SNP-gene interactions associated with breast cancer susceptibility. Our approach identified genomic blocks of regions with greater overall differences in contact count between the two time points when the cluster assignments change and identified genes and pathways implicated in early carcinogenesis and cancer treatment. Our results suggest that WOS-specific changes in 3D genome organization are linked to transcriptional changes that may influence susceptibility to breast cancer.

Nasopharyngeal cancer (NPC), a cancer derived from epithelial cells in the nasopharynx, is a cancer common in China, Southeast Asia, and Africa. The three-dimensional (3D) genome organization of nasopharyngeal cancer is poorly understood. A major challenge in understanding the 3D genome organization of cancer samples is the lack of a method for the characterization of chromatin interactions in solid cancer needle biopsy samples. Here, we developed Biop-C, a modified in situ Hi-C method using solid cancer needle biopsy samples. We applied Biop-C to characterize three nasopharyngeal cancer solid cancer needle biopsy patient samples. We identified topologically associated domains (TADs), chromatin interaction loops, and frequently interacting regions (FIREs) at key oncogenes in nasopharyngeal cancer from the Biop-C heatmaps. We observed that the genomic features are shared at some important oncogenes, but the patients also display extensive heterogeneity at certain genomic loci. On analyzing the super enhancer landscape in nasopharyngeal cancer cell lines, we found that the super enhancers are associated with FIREs and can be linked to distal genes via chromatin loops in NPC. Taken together, our results demonstrate the utility of our Biop-C method in investigating 3D genome organization in solid cancers.