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Simple Summary: Genomic DNA is folded into chromatin interaction patterns contributing to logical control of gene expression in mammalian cells, but how these highly ordered structures form is not yet fully understood. To assess to what extent functional gene activity is relative to the positioning of genes within the 3D nuclear landscape, we analyzed genome-wide gene expression and chromatin conformation capture data together in five distinct types of cells. We observed that 3D chromatin repositioning frequently occurs during cell differentiation, and these chromatin relocations are significantly associated with changes in gene expression levels. A set of genomic loci with extraordinarily high gene density participates in the establishment of common subcompartment A1 across the genome in all these five cells. By contrast, regulatory genomic segments enriched in cell type-specific genes are engaged in the formation of variable A1. Both subsets of subcompartment A1 bearing the strongest euchromatin signals harbor topological domains with frequent intradomain interactions to facilitate gene regulation. Thus, our study links the gene transcriptional levels with their subcompartment positioning, suggesting a key role of both constitutive and regulatory transcriptional activity in the 3D genome organization. Three-dimensional genome organization has been increasingly recognized as an important determinant of the precise regulation of gene expression in mammalian cells, yet the relationship between gene transcriptional activity and spatial subcompartment positioning is still not fully comprehended. Here, we first utilized genome-wide Hi-C data to infer eight types of subcompartment (labeled A1, A2, A3, A4, B1, B2, B3, and B4) in mouse embryonic stem cells and four primary differentiated cell types, including thymocytes, macrophages, neural progenitor cells, and cortical neurons. Transitions of subcompartments may confer gene expression changes in different cell types. Intriguingly, we identified two subsets of subcompartments defined by higher gene density and characterized by strongly looped contact domains, named common A1 and variable A1, respectively. We revealed that common A1, which includes highly expressed genes and abundant housekeeping genes, shows a ~2-fold higher gene density than the variable A1, where cell type-specific genes are significantly enriched. Thus, our study supports a model in which both types of genomic loci with constitutive and regulatory high transcriptional activity can drive the subcompartment A1 formation. Special chromatin subcompartment arrangement and intradomain interactions may, in turn, contribute to maintaining proper levels of gene expression, especially for regulatory non-housekeeping genes. [ABSTRACT FROM AUTHOR]

Gene regulation in eukaryotes is profoundly shaped by the 3D organization of chromatin within the cell nucleus. Distal regulatory interactions between enhancers and their target genes are widespread and many causal loci underlying heritable agricultural or clinical traits have been mapped to distal cis-regulatory elements. Dissecting the sequence features that mediate such distal interactions is key to understanding their underlying biology. Deep Learning (DL) models coupled with genome-wide 3C-based sequencing data have emerged as powerful tools to infer the DNA sequence grammar underlying such distal interactions. In this review we show that most DL models have remarkably high prediction accuracy, which indicates that DNA sequence features are important determinants of chromatin looping. However, DL model training has so far been limited to a small set of human cell lines, raising questions about the generalization of these predictions to other tissue-types and species. Furthermore, we find that the model architecture seems less relevant for model performance than the training strategy and the data preparation step. Transfer learning, coupled with functionally curated interactions, appear to be the most promising approach to learn cell-type specific and possibly species- specific sequence features in future applications.

Three-dimensional genome organization has been increasingly recognized as an important determinant of the precise regulation of gene expression in mammalian cells, yet the relationship between gene transcriptional activity and spatial subcompartment positioning is still not fully comprehended. Here, we first utilized genome-wide Hi-C data to infer eight types of subcompartment (labeled A1, A2, A3, A4, B1, B2, B3, and B4) in mouse embryonic stem cells and four primary differentiated cell types, including thymocytes, macrophages, neural progenitor cells, and cortical neurons. Transitions of subcompartments may confer gene expression changes in different cell types. Intriguingly, we identified two subsets of subcompartments defined by higher gene density and characterized by strongly looped contact domains, named common A1 and variable A1, respectively. We revealed that common A1, which includes highly expressed genes and abundant housekeeping genes, shows a ~2-fold higher gene density than the variable A1, where cell type-specific genes are significantly enriched. Thus, our study supports a model in which both types of genomic loci with constitutive and regulatory high transcriptional activity can drive the subcompartment A1 formation. Special chromatin subcompartment arrangement and intradomain interactions may, in turn, contribute to maintaining proper levels of gene expression, especially for regulatory non-housekeeping genes.

Simple Summary: Speckles and other nuclear bodies, the nucleolus and perinucleolar zone, transcription/replication factories and the lamina-associated compartment, serve as a structural basis for various genomic functions. In turn, genome activity and specific chromatin 3D organization directly impact the integrity of intranuclear assemblies, initiating/facilitating their formation and dictating their composition. Thus, the large-scale nucleus structure and genome activity mutually influence each other. The cell nucleus is frequently considered a compartment in which the genome is placed to protect it from external forces. Here, we discuss the evidence demonstrating that the cell nucleus should be considered, rather, as structure built around the folded genome. Decondensing chromosomes provide a scaffold for the assembly of the nuclear envelope after mitosis, whereas genome activity directs the assembly of various nuclear compartments, including nucleolus, speckles and transcription factories. The cell nucleus is frequently considered a cage in which the genome is placed to protect it from various external factors. Inside the nucleus, many functional compartments have been identified that are directly or indirectly involved in implementing genomic DNA's genetic functions. For many years, it was assumed that these compartments are assembled on a proteinaceous scaffold (nuclear matrix), which provides a structural milieu for nuclear compartmentalization and genome folding while simultaneously offering some rigidity to the cell nucleus. The results of research in recent years have made it possible to consider the cell nucleus from a different angle. From the "box" in which the genome is placed, the nucleus has become a kind of mobile exoskeleton, which is formed around the packaged genome, under the influence of transcription and other processes directly related to the genome activity. In this review, we summarize the main arguments in favor of this point of view by analyzing the mechanisms that mediate cell nucleus assembly and support its resistance to mechanical stresses. [ABSTRACT FROM AUTHOR]

The cell nucleus is frequently considered a cage in which the genome is placed to protect it from various external factors. Inside the nucleus, many functional compartments have been identified that are directly or indirectly involved in implementing genomic DNA’s genetic functions. For many years, it was assumed that these compartments are assembled on a proteinaceous scaffold (nuclear matrix), which provides a structural milieu for nuclear compartmentalization and genome folding while simultaneously offering some rigidity to the cell nucleus. The results of research in recent years have made it possible to consider the cell nucleus from a different angle. From the “box” in which the genome is placed, the nucleus has become a kind of mobile exoskeleton, which is formed around the packaged genome, under the influence of transcription and other processes directly related to the genome activity. In this review, we summarize the main arguments in favor of this point of view by analyzing the mechanisms that mediate cell nucleus assembly and support its resistance to mechanical stresses.

How chromosomes are organized within the tridimensional space of the nucleus and how can this organization affect genome function have been long-standing questions on the path to understanding genome activity and its link to disease. In the last decade, high-throughput chromosome conformation capture techniques, such as Hi-C, have facilitated the discovery of new principles of genome folding. Chromosomes are folded in multiple high-order structures, with local contacts between enhancers and promoters, intermediate-level contacts forming Topologically Associating Domains (TADs) and higher-order chromatin structures sequestering chromatin into active and repressive compartments. However, despite the increasing evidence that genome organization can influence its function, we are still far from understanding the underlying mechanisms. Deciphering these mechanisms represents a major challenge for the future, which large, international initiatives, such as 4DN, HCA and LifeTime, aim to collaboratively tackle by using a conjunction of state-of-the-art population-based and single-cell approaches. • Chromatin folds in multiple, partly independent hierarchical layers. • Different layers follow distinct folding rules, providing robustness to the system. • Aberrant folding can lead to developmental disorders, disease and cancer. • Large consortia are deployed to systematically study 3D genome folding & its function. [ABSTRACT FROM AUTHOR]

• C-TALE is a new many-versus-all C-method based on in-solution hybridization. • Probes for C-TALE hybridization are prepared from BAC DNA. • C-TALE enables analysis of DNA folding of megabase-scale genomic regions. • C-TALE is well suited for studying individual chromatin loops. • High-resolution contact maps are achievable in C-TALE at a low sequencing depth. Studies performed using Hi-C and other high-throughput whole-genome C-methods have demonstrated that 3D organization of eukaryotic genomes is functionally relevant. Unfortunately, ultra-deep sequencing of Hi-C libraries necessary to detect loop structures in large vertebrate genomes remains rather expensive. However, many studies are in fact aimed at determining the fine-scale 3D structure of comparatively small genomic regions up to several Mb in length. Such studies typically focus on the spatial structure of domains of coregulated genes, molecular mechanisms of loop formation, and interrogation of functional significance of GWAS-revealed polymorphisms. Therefore, a handful of molecular techniques based on Hi-C have been developed to address such issues. These techniques commonly rely on in-solution hybridization of Hi-C/3C-seq libraries with pools of biotinylated baits covering the region of interest, followed by deep sequencing of the enriched library. Here, we describe a new protocol of this kind, C-TALE (Chromatin TArget Ligation Enrichment). Preparation of hybridization probes from bacterial artificial chromosomes and an additional round of enrichment make C-TALE a cost-effective alternative to existing many-versus-all C-methods. [ABSTRACT FROM AUTHOR]

Changes of nuclear localization of lineage‐specific genes from a transcriptionally inert to permissive environment are a crucial step in establishing the identity of a cell. Noncoding RNA transcription‐mediated genome folding and activation of target gene expression have been found in a variety of cell types. Noncoding RNA ThymoD (thymocyte differentiation factor) transcription at superenhancers is essential for mouse T‐cell lineage commitment. The cessation of ThymoD transcription abolishes transcription‐mediated demethylation, recruiting looping factors such as the cohesin complex, CCCTC‐binding factor (CTCF), ultimately leading to the phenotype of severe combined immunodeficiency and T‐cell leukemia/lymphoma. In this review, we describe the functional role of RNA polymerase II‐mediated transcription at enhancers and in genome folding. We also highlight the involvement of faulty activation or suppression of enhancer transcription and enhancer‐promoter interaction in cancer development. [ABSTRACT FROM AUTHOR]

Eukaryotic chromosomes exhibit a hierarchical organization that spans a spectrum of length scales, ranging from sub-regions known as loops, which typically comprise hundreds of base pairs, to much larger chromosome territories that can encompass a few mega base pairs. Chromosome conformation capture experiments that involve high-throughput sequencing methods combined with microscopy techniques have enabled a new understanding of inter- and intra-chromosomal interactions with unprecedented details. This information also provides mechanistic insights on the relationship between genome architecture and gene expression. In this article, we review the recent findings on three-dimensional interactions among chromosomes at the compartment, topologically associating domain, and loop levels and the impact of these interactions on the transcription process. We also discuss current understanding of various biophysical processes involved in multi-layer structural organization of chromosomes. Then, we discuss the relationships between gene expression and genome structure from perturbative genome-wide association studies. Furthermore, for a better understanding of how chromosome architecture and function are linked, we emphasize the role of epigenetic modifications in the regulation of gene expression. Such an understanding of the relationship between genome architecture and gene expression can provide a new perspective on the range of potential future discoveries and therapeutic research., (© 2023 IOP Publishing Ltd.)

Genome function, replication, integrity, and propagation rely on the dynamic structural organization of chromosomes during the cell cycle. Genome folding in interphase provides regulatory segmentation for appropriate transcriptional control, facilitates ordered genome replication, and contributes to genome integrity by limiting illegitimate recombination. Here, we review recent high-resolution chromosome conformation capture and functional studies that have informed models of the spatial and regulatory compartmentalization of mammalian genomes, and discuss mechanistic models for how CTCF and cohesin control the functional architecture of mammalian chromosomes. [ABSTRACT FROM AUTHOR]

Translating the vast amounts of genomic and epigenomic information accumulated on the linear genome into threedimensional models of nuclear organization is a current major challenge. In response to this challenge, recent technological innovations based on chromosome conformation capture methods in combination with increasingly powerful functional approaches have revealed exciting insights into key aspects of genome regulation. These findings have led to an emerging model where the genome is folded and compartmentalized into highly conserved topological domains that are further divided into functional subdomains containing physical loops that bring cis-regulatory elements to close proximity. Targeted functional experiments, largely based on designable DNA-binding proteins, have begun to define the major architectural proteins required to establish and maintain appropriate genome regulation. Here, we focus on the accessible and wellcharacterized system of pluripotent cells to review the functional role of chromatin organization in regulating pluripotency, differentiation and reprogramming. [ABSTRACT FROM AUTHOR]

Abstract: Spatial conformation of DNA chains during interphase in eukaryotic cell nucleus is relatively dense, yet unknotted and exhibits self-similar fractal properties. In this respect it resembles the space-filling curves of Hilbert, but differs in the experimentally accessible contact probability of distant loci. Here we construct space-filling curves with fractal domain boundaries of dimension close to that of the embedding space and show how these match the statistical properties and the contact probability of the DNA conformation. The present mathematical model should shed light on the statistical ensemble of unknotted dense polymers and ease the modeling of genome folding and related biological processes. [Copyright &y& Elsevier]

The article focuses on a research regarding the creation of DNA flexibility maps of human beings, fruit fly and mouse. It informs that the maps have revealed that human chromosomes have flexible DNA regions also called SPIKEs than the chromosomes of mouse and fruit fly. It also mentions that researchers believe that SPIKEs may be responsible for genome-folding mechanisms in human beings.

Gene regulation in eukaryotes is profoundly shaped by the 3D organization of chromatin within the cell nucleus. Distal regulatory interactions between enhancers and their target genes are widespread and many causal loci underlying heritable agricultural or clinical traits have been mapped to distal cis-regulatory elements. Dissecting the sequence features that mediate such distal interactions is key to understanding their underlying biology. Deep Learning (DL) models coupled with genome-wide 3C-based sequencing data have emerged as powerful tools to infer the DNA sequence grammar underlying such distal interactions. In this review we show that most DL models have remarkably high prediction accuracy, which indicates that DNA sequence features are important determinants of chromatin looping. However, DL model training has so far been limited to a small set of human cell lines, raising questions about the generalization of these predictions to other tissue-types and species. Furthermore, we find that the model architecture seems less relevant for model performance than the training strategy and the data preparation step. Transfer learning, coupled with functionally curated interactions, appear to be the most promising approach to learn cell-type specific and possibly species- specific sequence features in future applications., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo. [Display omitted] • Super-resolution imaging reveals the structural organization of chromatin loops • Increased looping results into chromatin blending • Transcription regulates cohesin distribution and mobility on chromatin • Transcription-mediated supercoiling controls loop formation The mechanisms underlying chromatin loop formation by cohesin-mediated extrusion are still partially unknown. Using super-resolution imaging, Neguembor et al. visualize how loops are organized at the nanoscale level in intact nuclei and show that transcriptionally generated supercoiling regulates loop formation in vivo. [ABSTRACT FROM AUTHOR]

Genetics is a major determinant of susceptibility to autoimmune disorders. Here, we examined whether genome organization provides resilience or susceptibility to sequence variations, and how this would contribute to the molecular etiology of an autoimmune disease. We generated high-resolution maps of linear and 3D genome organization in thymocytes of NOD mice, a model of type 1 diabetes (T1D), and the diabetes-resistant C57BL/6 mice. Multi-enhancer interactions formed at genomic regions harboring genes with prominent roles in T cell development in both strains. However, diabetes risk-conferring loci coalesced enhancers and promoters in NOD, but not C57BL/6 thymocytes. 3D genome mapping of NODxC57BL/6 F1 thymocytes revealed that genomic misfolding in NOD mice is mediated in cis. Moreover, immune cells infiltrating the pancreas of humans with T1D exhibited increased expression of genes located on misfolded loci in mice. Thus, genetic variation leads to altered 3D chromatin architecture and associated changes in gene expression that may underlie autoimmune pathology. • T cell identity genes are hyperconnected in 3D in C57BL/6 and NOD strains • Diabetes-associated regions are hyperconnected in 3D only in NOD strain • The 3D regulatory landscape in NOD mice is mediated in cis • KRAB-ZFP genes are highly expressed in the pancreas of humans with T1D Fasolino and Goldman et al. generate high-resolution maps of linear and 3D genome organization in thymocytes of NOD mice, a model of type 1 diabetes, and reveal that diabetes risk-conferring loci coalesce enhancers and promoters of genes associated with T cell function, altering gene expression. Thus, genetic variation leads to altered chromatin architecture that may underlie autoimmune pathology, with implications for human disease. [ABSTRACT FROM AUTHOR]

A complex cascade of highly regulated processes of cell fate determination, differentiation, proliferation and transdifferentiation dictate the patterning, morphogenesis and growth of the vertebrate skeleton, perturbation of which results in malformation. In humans over 450 different dysplasias involving the skeletal system constitute a significant fraction of documented Mendelian disorders. The combination of clinical, phenotypic characterization of rare human skeletal dysmorphologies, the discovery of causative mutations and functional validation in animal models has contributed enormously to the understanding of molecular control of skeletal development. These studies revealed a myriad of genes and pathways, such as WNT, Hedgehog (HH), planar cell polarity and primary cilia, as key regulators for skeletal patterning, growth and homeostasis. The generation of mouse models recapitulating human congenital skeletal dysplasia has provided mechanistic insights into the diverse pathologies caused by single gene mutations, integrated action of developmental pathways such as WNT and HH and the role of stress responses. Technological developments in whole genome and exome sequencing have accelerated the discovery of disease-causing mutations and are changing approaches for diagnosis. The discovery that non-coding variants and disorganization of the 3D genome are associated with limb patterning disorders has revealed an additional level of complexity in the regulatory framework of skeletal development and disease mechanisms. This chapter focuses on a selection of human skeletal pathologies which illustrate how new findings about the coding and noncoding genome, combined with functional modeling, are contributing to deeper understanding of skeletal development, mechanisms of disease, with therapeutic potential for chondrodysplasias., (© 2019 Elsevier Inc. All rights reserved.)

Translating the vast amounts of genomic and epigenomic information accumulated on the linear genome into three-dimensional models of nuclear organization is a current major challenge. In response to this challenge, recent technological innovations based on chromosome conformation capture methods in combination with increasingly powerful functional approaches have revealed exciting insights into key aspects of genome regulation. These findings have led to an emerging model where the genome is folded and compartmentalized into highly conserved topological domains that are further divided into functional subdomains containing physical loops that bring cis-regulatory elements to close proximity. Targeted functional experiments, largely based on designable DNA-binding proteins, have begun to define the major architectural proteins required to establish and maintain appropriate genome regulation. Here, we focus on the accessible and well-characterized system of pluripotent cells to review the functional role of chromatin organization in regulating pluripotency, differentiation and reprogramming., (© The Author 2015. Published by Oxford University Press.)