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.

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.

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.

Unraveling the Polysaccharide Biosynthesis Potential of Ganoderma lucidum: A Chromosome-Level Assembly Using Hi-C Sequencing.

Ganoderma lucidum exhibits the ability to synthesize a diverse range of biologically active molecules with significant pharmaceutical potential, including xylomannan and fucogalactan, which have demonstrated antitumor activity. However, there exists considerable intra-species variability in the capacity to produce these metabolites at high concentrations, likely reflecting the high genomic diversity observed from a limited number of strains sequenced to date. We employed high-throughput shotgun sequencing to obtain the complete genome sequence of G. lucidum strain 5.1, which is distinguished by its remarkable xylomannan synthesis capabilities. Through the utilization of semi-automatic reordering based on conformation capture (Hi-C) data, we substantially enhanced the assembly process, resulting in the generation of 12 chromosome-level scaffolds with a cumulative length of 39 Mbp. By employing both de novo and homology-based approaches, we performed comprehensive annotation of the genome, thereby identifying a diverse repertoire of genes likely involved in polysaccharide biosynthesis. The genome sequence generated in this study serves as a valuable resource for elucidating the molecular mechanisms underlying the medicinal potential of Ganoderma species, discovering novel pharmaceutically valuable compounds, and elucidating the ecological mechanisms of the species. Furthermore, the chromosome contact map obtained for the first time for this species extends our understanding of 3D fungal genomics and provides insights into the functional and structural organization within the fungal kingdom.

Extrusion fountains are hallmarks of chromosome organization emerging upon zygotic genome activation.

The first activation of gene expression during development (zygotic genome activation, ZGA) is accompanied by massive changes in chromosome organization. The connection between these two processes remains unknown. Using Hi-C for zebrafish embryos, we found that chromosome folding starts by establishing "fountains", novel elements of chromosome organization, emerging selectively at enhancers upon ZGA. Using polymer simulations, we demonstrate that fountains can emerge as sites of targeted cohesin loading and require two-sided, yet desynchronized, loop extrusion. Specific loss of fountains upon loss of pioneer transcription factors that drive ZGA reveals a causal connection between enhancer activity and fountain formation. Finally, we show that fountains emerge in early Medaka and Xenopus embryos; moreover, we found cohesin-dependent fountain pattern on enhancers of mouse embryonic stem cells. Taken together, fountains are the first enhancer-specific elements of chromosome organization; they constitute starting points of chromosome folding during early development, likely serving as sites of targeted cohesin loading.

Enhancer Function in the 3D Genome.

In this review, we consider various aspects of enhancer functioning in the context of the 3D genome. Particular attention is paid to the mechanisms of enhancer-promoter communication and the significance of the spatial juxtaposition of enhancers and promoters in 3D nuclear space. A model of an activator chromatin compartment is substantiated, which provides the possibility of transferring activating factors from an enhancer to a promoter without establishing direct contact between these elements. The mechanisms of selective activation of individual promoters or promoter classes by enhancers are also discussed.

RNA-DNA interactomes of three prokaryotes uncovered by proximity ligation.

Proximity ligation approaches, which are widely used to study the spatial organization of the genome, also make it possible to reveal patterns of RNA-DNA interactions. Here, we use RedC, an RNA-DNA proximity ligation approach, to assess the distribution of major RNA types along the genomes of E. coli, B. subtilis, and thermophilic archaeon T. adornatum. We find that (i) messenger RNAs preferentially interact with their cognate genes and the genes located downstream in the same operon, which is consistent with polycistronic transcription; (ii) ribosomal RNAs preferentially interact with active protein-coding genes in both bacteria and archaea, indicating co-transcriptional translation; and (iii) 6S noncoding RNA, a negative regulator of bacterial transcription, is depleted from active genes in E. coli and B. subtilis. We conclude that the RedC data provide a rich resource for studying both transcription dynamics and the function of noncoding RNAs in microbial organisms.

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.

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.

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.

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.

Recurrent Translocations in Topoisomerase Inhibitor-Related Leukemia Are Determined by the Features of DNA Breaks Rather Than by the Proximity of the Translocating Genes.

Topoisomerase inhibitors are widely used in cancer chemotherapy. However, one of the potential long-term adverse effects of such therapy is acute leukemia. A key feature of such therapy-induced acute myeloid leukemia (t-AML) is recurrent chromosomal translocations involving AML1 (RUNX1) or MLL (KMT2A) genes. The formation of chromosomal translocation depends on the spatial proximity of translocation partners and the mobility of the DNA ends. It is unclear which of these two factors might be decisive for recurrent t-AML translocations. Here, we used fluorescence in situ hybridization (FISH) and chromosome conformation capture followed by sequencing (4C-seq) to investigate double-strand DNA break formation and the mobility of broken ends upon etoposide treatment, as well as contacts between translocation partner genes. We detected the separation of the parts of the broken AML1 gene, as well as the increased mobility of these separated parts. 4C-seq analysis showed no evident contacts of AML1 and MLL with loci, implicated in recurrent t-AML translocations, either before or after etoposide treatment. We suggest that separation of the break ends and their increased non-targeted mobility-but not spatial predisposition of the rearrangement partners-plays a major role in the formation of these translocations.

Dosage Compensation in Drosophila: Its Canonical and Non-Canonical Mechanisms.

Dosage compensation equalizes gene expression in a single male X chromosome with that in the pairs of autosomes and female X chromosomes. In the fruit fly Drosophila, canonical dosage compensation is implemented by the male-specific lethal (MSL) complex functioning in all male somatic cells. This complex contains acetyl transferase males absent on the first (MOF), which performs H4K16 hyperacetylation specifically in the male X chromosome, thus facilitating transcription of the X-linked genes. However, accumulating evidence points to an existence of additional, non-canonical dosage compensation mechanisms operating in somatic and germline cells. In this review, we discuss current advances in the understanding of both canonical and non-canonical mechanisms of dosage compensation in Drosophila.

Genome-Directed Cell Nucleus Assembly.

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.

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.

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 .

Pluripotency factors determine gene expression repertoire at zygotic genome activation.

Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.

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.

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.

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.