The twisted path of the 3D genome: where does it lead?

A new era in 3D genome studies began with the development of the so-called 'C-methods', used for the analysis of spatial contacts between distant genomic elements. However, the idea that spatial genome organization, partitioning of the genome into structural/functional units, and the functional compartmentalization of the cell nucleus are important for the implementation of key functions of the genome arose much earlier. In this Opinion article, we briefly overview how the concept of spatial genome organization has changed over recent decades, discuss current views on the 3D genome and cell nucleus organization, and compare the experimental evidence for the inter-relation between gene regulation and the 3D genome.

Extensive long-range polycomb interactions and weak compartmentalization are hallmarks of human neuronal 3D genome.

Chromatin architecture regulates gene expression and shapes cellular identity, particularly in neuronal cells. Specifically, polycomb group (PcG) proteins enable establishment and maintenance of neuronal cell type by reorganizing chromatin into repressive domains that limit the expression of fate-determining genes and sustain distinct gene expression patterns in neurons. Here, we map the 3D genome architecture in neuronal and non-neuronal cells isolated from the Wernicke's area of four human brains and comprehensively analyze neuron-specific aspects of chromatin organization. We find that genome segregation into active and inactive compartments is greatly reduced in neurons compared to other brain cells. Furthermore, neuronal Hi-C maps reveal strong long-range interactions, forming a specific network of PcG-mediated contacts in neurons that is nearly absent in other brain cells. These interacting loci contain developmental transcription factors with repressed expression in neurons and other mature brain cells. But only in neurons, they are rich in bivalent promoters occupied by H3K4me3 histone modification together with H3K27me3, which points to a possible functional role of PcG contacts in neurons. Importantly, other layers of chromatin organization also exhibit a distinct structure in neurons, characterized by an increase in short-range interactions and a decrease in long-range ones.

HiConfidence: a novel approach uncovering the biological signal in Hi-C data affected by technical biases.

The chromatin interaction assays, particularly Hi-C, enable detailed studies of genome architecture in multiple organisms and model systems, resulting in a deeper understanding of gene expression regulation mechanisms mediated by epigenetics. However, the analysis and interpretation of Hi-C data remain challenging due to technical biases, limiting direct comparisons of datasets obtained in different experiments and laboratories. As a result, removing biases from Hi-C-generated chromatin contact matrices is a critical data analysis step. Our novel approach, HiConfidence, eliminates biases from the Hi-C data by weighing chromatin contacts according to their consistency between replicates so that low-quality replicates do not substantially influence the result. The algorithm is effective for the analysis of global changes in chromatin structures such as compartments and topologically associating domains. We apply the HiConfidence approach to several Hi-C datasets with significant technical biases, that could not be analyzed effectively using existing methods, and obtain meaningful biological conclusions. In particular, HiConfidence aids in the study of how changes in histone acetylation pattern affect chromatin organization in Drosophila melanogaster S2 cells. The method is freely available at GitHub: https://github.com/victorykobets/HiConfidence.

RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins.

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA-DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA-chromatin interactions. Here, we introduce RedChIP, a technique combining RNA-DNA proximity ligation and chromatin immunoprecipitation for identifying RNA-chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA-DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

The two waves in single-cell 3D genomics.

For decades, biochemical methods for the analysis of genome structure and function provided cell-population-averaged data that allowed general principles and tendencies to be disclosed. Microscopy-based studies, which immanently involve single-cell analysis, did not provide sufficient spatial resolution to investigate the particularly small details of 3D genome folding. Nevertheless, these studies demonstrated that mutual positions of chromosome territories within cell nuclei and individual genomic loci within chromosomal territories can vary significantly in individual cells. The development of new technologies in biochemistry and the advent of super-resolution microscopy in the last decade have made possible the full-scale study of 3D genome organization in individual cells. Maps of the 3D genome build based on C-data and super-resolution microscopy are highly consistent and, therefore, biologically relevant. The internal structures of individual chromosomes, loci, and topologically associating domains (TADs) are resolved as well as cell-cycle dynamics. 3D modeling allows one to investigate the physical mechanisms underlying genome folding. Finally, joint profiling of genome topology and epigenetic features will allow 3D genomics to handle complex cell-to-cell heterogeneity. In this review, we summarize the present state of studies into 3D genome organization in individual cells, analyze the technical problems of single-cell studies, and outline perspectives of 3D genomics.

3D genome alterations and editing in pathology.

The human genome is folded into a multi-level 3D structure that controls many nuclear functions including gene expression. Recently, alterations in 3D genome organization were associated with several genetic diseases and cancer. As a consequence, experimental approaches are now being developed to modify the global 3D genome organization and that of specific loci. Here, we discuss emerging experimental approaches of 3D genome editing that may prove useful in biomedicine.

SETDB1 fuels the lung cancer phenotype by modulating epigenome, 3D genome organization and chromatin mechanical properties.

Imbalance in the finely orchestrated system of chromatin-modifying enzymes is a hallmark of many pathologies such as cancers, since causing the affection of the epigenome and transcriptional reprogramming. Here, we demonstrate that a loss-of-function mutation (LOF) of the major histone lysine methyltransferase SETDB1 possessing oncogenic activity in lung cancer cells leads to broad changes in the overall architecture and mechanical properties of the nucleus through genome-wide redistribution of heterochromatin, which perturbs chromatin spatial compartmentalization. Together with the enforced activation of the epithelial expression program, cytoskeleton remodeling, reduced proliferation rate and restricted cellular migration, this leads to the reversed oncogenic potential of lung adenocarcinoma cells. These results emphasize an essential role of chromatin architecture in the determination of oncogenic programs and illustrate a relationship between gene expression, epigenome, 3D genome and nuclear mechanics.

Comparison of genome architecture at two stages of male germline cell differentiation in Drosophila.

Eukaryotic chromosomes are spatially segregated into topologically associating domains (TADs). Some TADs are attached to the nuclear lamina (NL) through lamina-associated domains (LADs). Here, we identified LADs and TADs at two stages of Drosophila spermatogenesis - in bamΔ86 mutant testes which is the commonly used model of spermatogonia (SpG) and in larval testes mainly filled with spermatocytes (SpCs). We found that initiation of SpC-specific transcription correlates with promoters' detachment from the NL and with local spatial insulation of adjacent regions. However, this insulation does not result in the partitioning of inactive TADs into sub-TADs. We also revealed an increased contact frequency between SpC-specific genes in SpCs implying their de novo gathering into transcription factories. In addition, we uncovered the specific X chromosome organization in the male germline. In SpG and SpCs, a single X chromosome is stronger associated with the NL than autosomes. Nevertheless, active chromatin regions in the X chromosome interact with each other more frequently than in autosomes. Moreover, despite the absence of dosage compensation complex in the male germline, randomly inserted SpG-specific reporter is expressed higher in the X chromosome than in autosomes, thus evidencing that non-canonical dosage compensation operates in SpG.

Hi-C metagenomics facilitate comparative genome analysis of bacteria and yeast from spontaneous beer and cider.

Sequence-based analysis of fermented foods and beverages' microbiomes offers insights into their impact on taste and consumer health. High-throughput metagenomics provide detailed taxonomic and functional community profiling, but bacterial and yeast genome reconstruction and mobile genetic elements tracking are to be improved. We established a pipeline for exploring fermented foods microbiomes using metagenomics coupled with chromosome conformation capture (Hi-C metagenomics). The approach was applied to analyze a collection of spontaneously fermented beers and ciders (n = 12). The Hi-C reads were used to reconstruct the metagenome-assembled genomes (MAGs) of bacteria and yeasts facilitating subsequent comparative genomic analysis, assembly scaffolding and exploration of "plasmid-bacteria" links. For a subset of beverages, yeasts were isolated and characterized phenotypically. The reconstructed Hi-C MAGs primarily belonged to the Lactobacillaceae family in beers, along with Acetobacteraceae and Enterobacteriaceae in ciders, exhibiting improved quality compared to conventional metagenomic MAGs. Comparative genomic analysis of Lactobacillaceae Hi-C MAGs revealed clustering by niche and suggested genetic determinants of survival and probiotic potential. For Pediococcus damnosus, Hi-C-based networks of contigs enabled linking bacteria with plasmids. Analyzing phylogeny and accessory genes in the context of known reference genomes offered insights into the niche specialization of beer lactobacilli. The subspecies-level diversity of cider Tatumella spp. was disentangled using a Hi-C-based graph. We obtained highly complete yeast Hi-C MAGs primarily represented by Brettanomyces and Saccharomyces, with Hi-C-facilitated chromosome-level genome assembly for the former. Utilizing Hi-C metagenomics to unravel the genomic content of individual species can provide a deeper understanding of the ecological interactions within the food microbiome, aid in bioprospecting beneficial microorganisms, improving quality control and improving innovative fermented products.

Ron Hancock - a passionate scientist and a Great Man.

This essay is in memoriam of Ronald Hancock (1933 - 2022).

The role of physicochemical processes in the formation of the 3D genome and compartmentalization of the cell nucleus.

The review analyzes the role of physicochemical processes in the formation of the function-dependent architecture of the cell nucleus, built on the platform of a folded genome. The main attention is paid to various forms of the phase separation process, primarily the processes of liquid-liquid phase separation and polymer-polymer phase separation. The role of these processes in the formation of chromatin compartments and maintenance of three-dimensional genome architecture is discussed in detail. The relationship between genome activity and the creation of functional compartments in the cell nucleus is also analyzed.

Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition.

Chromatin is reprogrammed after fertilization to produce a totipotent zygote with the potential to generate a new organism. The maternal genome inherited from the oocyte and the paternal genome provided by sperm coexist as separate haploid nuclei in the zygote. How these two epigenetically distinct genomes are spatially organized is poorly understood. Existing chromosome conformation capture-based methods are not applicable to oocytes and zygotes owing to a paucity of material. To study three-dimensional chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method. Here we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes. Features of genomic organization including compartments, topologically associating domains (TADs) and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells. At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms. Our results demonstrate that the global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells. An understanding of this zygotic chromatin 'ground state' could potentially provide insights into reprogramming cells to a state of totipotency.

Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells.

Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

A Kaleidoscope of Keratin Gene Expression and the Mosaic of Its Regulatory Mechanisms.

Keratins are a family of intermediate filament-forming proteins highly specific to epithelial cells. A combination of expressed keratin genes is a defining property of the epithelium belonging to a certain type, organ/tissue, cell differentiation potential, and at normal or pathological conditions. In a variety of processes such as differentiation and maturation, as well as during acute or chronic injury and malignant transformation, keratin expression undergoes switching: an initial keratin profile changes accordingly to changed cell functions and location within a tissue as well as other parameters of cellular phenotype and physiology. Tight control of keratin expression implies the presence of complex regulatory landscapes within the keratin gene loci. Here, we highlight patterns of keratin expression in different biological conditions and summarize disparate data on mechanisms controlling keratin expression at the level of genomic regulatory elements, transcription factors (TFs), and chromatin spatial structure.

Trisomies Reorganize Human 3D Genome.

Trisomy is the presence of one extra copy of an entire chromosome or its part in a cell nucleus. In humans, autosomal trisomies are associated with severe developmental abnormalities leading to embryonic lethality, miscarriage or pronounced deviations of various organs and systems at birth. Trisomies are characterized by alterations in gene expression level, not exclusively on the trisomic chromosome, but throughout the genome. Here, we applied the high-throughput chromosome conformation capture technique (Hi-C) to study chromatin 3D structure in human chorion cells carrying either additional chromosome 13 (Patau syndrome) or chromosome 16 and in cultured fibroblasts with extra chromosome 18 (Edwards syndrome). The presence of extra chromosomes results in systematic changes of contact frequencies between small and large chromosomes. Analyzing the behavior of individual chromosomes, we found that a limited number of chromosomes change their contact patterns stochastically in trisomic cells and that it could be associated with lamina-associated domains (LAD) and gene content. For trisomy 13 and 18, but not for trisomy 16, the proportion of compacted loci on a chromosome is correlated with LAD content. We also found that regions of the genome that become more compact in trisomic cells are enriched in housekeeping genes, indicating a possible decrease in chromatin accessibility and transcription level of these genes. These results provide a framework for understanding the mechanisms of pan-genome transcription dysregulation in trisomies in the context of chromatin spatial organization.

Pentad: a tool for distance-dependent analysis of Hi-C interactions within and between chromatin compartments.

BACKGROUND: Understanding the role of various factors in 3D genome organization is essential to determine their impact on shaping large-scale chromatin units such as euchromatin (A) and heterochromatin (B) compartments. At this level, chromatin compaction is extensively modulated when transcription and epigenetic profiles change upon cell differentiation and response to various external impacts. However, detailed analysis of chromatin contact patterns within and between compartments is complicated because of a lack of suitable computational methods. RESULTS: We developed a tool, Pentad, to perform calculation, visualisation and quantitative analysis of the average chromatin compartment from the Hi-C matrices in cis, trans, and specified genomic distances. As we demonstrated by applying Pentad to publicly available Hi-C datasets, it helps to reliably detect redistribution of contact frequency in the chromatin compartments and assess alterations in the compartment strength. CONCLUSIONS: Pentad is a simple tool for the analysis of changes in chromatin compartmentalization in various biological conditions. Pentad is freely available at https://github.com/magnitov/pentad .

Perspectives for the reconstruction of 3D chromatin conformation using single cell Hi-C data.

Construction of chromosomes 3D models based on single cell Hi-C data constitute an important challenge. We present a reconstruction approach, DPDchrom, that incorporates basic knowledge whether the reconstructed conformation should be coil-like or globular and spring relaxation at contact sites. In contrast to previously published protocols, DPDchrom can naturally form globular conformation due to the presence of explicit solvent. Benchmarking of this and several other methods on artificial polymer models reveals similar reconstruction accuracy at high contact density and DPDchrom advantage at low contact density. To compare 3D structures insensitively to spatial orientation and scale, we propose the Modified Jaccard Index. We analyzed two sources of the contact dropout: contact radius change and random contact sampling. We found that the reconstruction accuracy exponentially depends on the number of contacts per genomic bin allowing to estimate the reconstruction accuracy in advance. We applied DPDchrom to model chromosome configurations based on single-cell Hi-C data of mouse oocytes and found that these configurations differ significantly from a random one, that is consistent with other studies.

Non-coding RNAs in chromatin folding and nuclear organization.

One of the most intriguing questions facing modern biology concerns how the genome directs the construction of cells, tissues, and whole organisms. It is tempting to suggest that the part of the genome that does not encode proteins contains architectural plans. We are still far from understanding how these plans work at the level of building tissues and the body as a whole. However, the results of recent studies demonstrate that at the cellular level, special non-coding RNAs serve as scaffolds for the construction of various intracellular structures. The term "architectural RNAs" was proposed to designate this subset of non-coding RNAs. In this review, we discuss the role of architectural RNAs in the construction of the cell nucleus and maintenance of the three-dimensional organization of the genome.

Segregation of α- and ß-Globin Gene Cluster in Vertebrate Evolution: Chance or Necessity?

The review is devoted to the patterns of evolution of α- and ß-globin gene domains. A hypothesis is presented according to which segregation of the ancestral cluster of α/ß-globin genes in Amniota occurred due to the performance by α-globins and ß-globins of non-canonical functions not related to oxygen transport.

Domain Model of Eukaryotic Genome Organization: From DNA Loops Fixed on the Nuclear Matrix to TADs.

The article reviews the development of ideas on the domain organization of eukaryotic genome, with special attention on the studies of DNA loops anchored to the nuclear matrix and their role in the emergence of the modern model of eukaryotic genome spatial organization. Critical analysis of results demonstrating that topologically associated chromatin domains are structural-functional blocks of the genome supports the notion that these blocks are fundamentally different from domains whose existence was proposed by the domain hypothesis of eukaryotic genome organization formulated in the 1980s. Based on the discussed evidence, it is concluded that the model postulating that eukaryotic genome is built from uniformly organized structural-functional blocks has proven to be untenable.