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Abstract Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Here, using DamID-seq, we identified Elys binding sites in Drosophila late embryos and divided them into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. Our findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin.

Abstract 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.

Abstract 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 .

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.

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.

Abstract Cohesin is a key organizer of chromatin folding in eukaryotic cells. The two main activities of this ring-shaped protein complex are the maintenance of sister chromatid cohesion and the establishment of long-range DNA–DNA interactions through the process of loop extrusion. Although the basic principles of both cohesion and loop extrusion have been described, we still do not understand several crucial mechanistic details. One of such unresolved issues is the question of whether a cohesin ring topologically embraces DNA string(s) during loop extrusion. Here, we show that cohesin complexes residing on CTCF-occupied genomic sites in mammalian cells do not interact with DNA topologically. We assessed the stability of cohesin-dependent loops and cohesin association with chromatin in high-ionic-strength conditions in G1-synchronized HeLa cells. We found that increased salt concentration completely displaces cohesin from those genomic regions that correspond to CTCF-defined loop anchors. Unsurprisingly, CTCF-anchored cohesin loops also dissipate in these conditions. Because topologically engaged cohesin is considered to be salt resistant, our data corroborate a non-topological model of loop extrusion. We also propose a model of cohesin activity throughout the interphase, which essentially equates the termination of non-topological loop extrusion with topological loading of cohesin. This theoretical framework enables a parsimonious explanation of various seemingly contradictory experimental findings.

Abstract Chromatin loops represent one of the major levels of hierarchical folding of the genome. Although the situation is evolving, current methods have various difficulties with the accurate mapping of loops even in mammalian Hi-C data, and most of them fail to identify chromatin loops in animal species with substantially different genome architecture. This paper presents the loop and significant contact annotation (LASCA) pipeline, which uses Weibull distribution-based modeling to effectively identify loops and enhancer–promoter interactions in Hi-C data from evolutionarily distant species: from yeast and worms to mammals. Available at: https://github.com/ArtemLuzhin/LASCA_pipeline .

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.

Genomes are partitioned into topologically associating domains (TADs). Here the authors present single-nucleus Hi-C maps in Drosophila at 10 kb resolution, demonstrating the presence of chromatin compartments in individual nuclei, and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which resembles population TAD profiles.

Gut microbiome in critically ill patients shows profound dysbiosis. The most vulnerable is the subgroup of chronically critically ill (CCI) patients – those suffering from long-term dependence on support systems in intensive care units. It is important to investigate their microbiome as a potential reservoir of opportunistic taxa causing co-infections and a morbidity factor. We explored dynamics of microbiome composition in the CCI patients by combining “shotgun” metagenomics with chromosome conformation capture (Hi-C). Stool samples were collected at 2 time points from 2 patients with severe brain injury with different outcomes within a 1–2-week interval. The metagenome-assembled genomes (MAGs) were reconstructed based on the Hi-C data using a novel hicSPAdes method (along with the bin3c method for comparison), as well as independently of the Hi-C using MetaBAT2. The resistomes of the samples were derived using a novel assembly graph-based approach. Links of bacteria to antibiotic resistance genes, plasmids and viruses were analyzed using Hi-C-based networks. The gut community structure was enriched in opportunistic microorganisms. The binning using hicSPAdes was superior to the conventional WGS-based binning as well as to the bin3c in terms of the number, completeness and contamination of the reconstructed MAGs. Using Klebsiella pneumoniae as an example, we showed how chromosome conformation capture can aid comparative genomic analysis of clinically important pathogens. Diverse associations of resistome with antimicrobial therapy from the level of assembly graphs to gene content were discovered. Analysis of Hi-C networks suggested multiple “host-plasmid” and “host-phage” links. Hi-C metagenomics is a promising technique for investigating clinical microbiome samples. It provides a community composition profile with increased details on bacterial gene content and mobile genetic elements compared to conventional metagenomics. The ability of Hi-C binning to encompass the MAG’s plasmid content facilitates metagenomic evaluation of virulence and drug resistance dynamics in clinically relevant opportunistic pathogens. These findings will help to identify the targets for developing cost-effective and rapid tests for assessing microbiome-related health risks.

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. Author summary Reconstruction of 3D configuration of chromosomes based on single cell Hi-C data (contact matrix) constitutes an important way to understand the specific features of genome packaging in individual cells. For this purpose, we develop method DPDchrom based on dissipative particle dynamics. Two sources of the contact dropout, contact radius change and random contact sampling, have different impact to the reconstruction accuracy suggesting unequal value of contacts. We found the exponential dependence of the reconstruction accuracy on the number of contacts per genomic bin. We reconstructed entire nucleus of mouse using single cell Hi-C data and found significant dissimilarity of conformations reconstructed using experimental and shuffled data. These findings open perspectives to study 3D arrangement of cis-regulatory elements orchestrating transcription program.

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.

Curaxins are a recently discovered class of anti-cancer agents that disturbs DNA/histone interactions within. Here the authors provide evidence that curaxins affect the spatial genome organization and compromise enhancer-promoter communication necessary for expression of several oncogenes, including MYC.

The role of the nuclear lamina (NL) in chromatin architecture is still poorly understood. Here, the authors provide evidence that disruption of the NL in Drosophila cells leads to overall chromatin compaction and repositioning from the nuclear envelope, whereas lamina-associated regions become less compacted and transcription within them is increased.

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.

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.

Due to their exceptional simplicity of organization, viruses rely on the resources, molecular mechanisms, macromolecular complexes, regulatory pathways, and functional compartments of the host cell for an effective infection process. The nucleolus plays an important role in the process of interaction between the virus and the infected cell. The interactions of viral proteins and nucleic acids with the nucleolus during the infection process are universal phenomena and have been described for almost all taxonomic groups. During infection, proteins of the nucleolus in association with viral components can be directly used for the processes of replication and transcription of viral nucleic acids and the assembly and transport of viral particles. In the course of a viral infection, the usurpation of the nucleolus functions occurs and the usurpation is accompanied by profound changes in ribosome biogenesis. Recent studies have demonstrated that the nucleolus is a multifunctional and dynamic compartment. In addition to the biogenesis of ribosomes, it is involved in regulating the cell cycle and apoptosis, responding to cellular stress, repairing DNA, and transcribing RNA polymerase II-dependent genes. A viral infection can be accompanied by targeted transport of viral proteins to the nucleolus, massive release of resident proteins of the nucleolus into the nucleoplasm and cytoplasm, the movement of non-nucleolar proteins into the nucleolar compartment, and the temporary localization of viral nucleic acids in the nucleolus. The interaction of viral and nucleolar proteins interferes with canonical and non-canonical functions of the nucleolus and results in a change in the physiology of the host cell: cell cycle arrest, intensification or arrest of ribosome biogenesis, induction or inhibition of apoptosis, and the modification of signaling cascades involved in the stress response. The nucleolus is, therefore, an important target during viral infection. In this review, we discuss the functional impact of viral proteins and nucleic acid interaction with the nucleolus during infection.

Currently, seven molecular subtypes of prostate cancer (PCa) are known, the most common of which being the subtype characterized by the presence of the TMPRSS2–ERG fusion transcript. While there is a considerable amount of work devoted to the influence of this transcript on the prognosis of the disease, data on its role in the progression and prognosis of PCa remain controversial. The present study is devoted to the analysis of the association between the TMPRSS2–ERG transcript and the biochemical recurrence of PCa. The study included two cohorts: the RNA–Seq sample of Russian patients with PCa (n = 72) and the TCGA–PRAD data (n = 203). The results of the analysis of the association between the TMPRSS2–ERG transcript and biochemical recurrence were contradictory. The differential expression analysis (biochemical recurrence cases versus biochemical recurrence-free) and the gene set enrichment analysis revealed a list of genes involved in major cellular pathways. The GNL3, QSOX2, SSPO, and SYS1 genes were selected as predictors of the potential prognostic model (AUC = 1.000 for a cohort of Russian patients with PCa and AUC = 0.779 for a TCGA–PRAD cohort).

Abstract In mammals, genomic DNA that is roughly 2 m long is folded to fit the size of the cell nucleus that has a diameter of about 10 μm. The folding of genomic DNA is mediated via assembly of DNA-protein complex, chromatin. In addition to the reduction of genomic DNA linear dimensions, the assembly of chromatin allows to discriminate and to mark active (transcribed) and repressed (non-transcribed) genes. Consequently, epigenetic regulation of gene expression occurs at the level of DNA packaging in chromatin. Taking into account the increasing attention of scientific community toward epigenetic systems of gene regulation, it is very important to understand how DNA folding in chromatin is related to gene activity. For many years the hierarchical model of DNA folding was the most popular. It was assumed that nucleosome fiber (10-nm fiber) is folded into 30-nm fiber and further on into chromatin loops attached to a nuclear/chromosome scaffold. Recent studies have demonstrated that there is much less regularity in chromatin folding within the cell nucleus. The very existence of 30-nm chromatin fibers in living cells was questioned. On the other hand, it was found that chromosomes are partitioned into self-interacting spatial domains that restrict the area of enhancers action. Thus, TADs can be considered as structural-functional domains of the chromosomes. Here we discuss the modern view of DNA packaging within the cell nucleus in relation to the regulation of gene expression. Special attention is paid to the possible mechanisms of the chromatin fiber self-assembly into TADs. We discuss the model postulating that partitioning of the chromosome into TADs is determined by the distribution of active and inactive chromatin segments along the chromosome. This article was specially invited by the editors and represents work by leading researchers.

Abstract Background In homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation. Results We found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation. Conclusions Our findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing topologically associating domain.

The functioning of a cell at various organizational levels is determined by the interactions between macromolecules that promote cellular organelle formation and orchestrate metabolic pathways via the control of enzymatic activities. Although highly specific and relatively stable protein-protein, protein-DNA, and protein-RNA interactions are traditionally suggested as the drivers for cellular function realization, recent advances in the discovery of weak multivalent interactions have uncovered the role of so-called macromolecule condensates. These structures, which are highly divergent in size, composition, function, and cellular localization are predominantly formed by liquid-liquid phase separation (LLPS): a physical-chemical process where an initially homogenous solution turns into two distinct phases, one of which contains the major portion of the dissolved macromolecules and the other one containing the solvent. In a living cell, LLPS drives the formation of membrane-less organelles such as the nucleolus, nuclear bodies, and viral replication factories and facilitates the assembly of complex macromolecule aggregates possessing regulatory, structural, and enzymatic functions. Here, we discuss the role of LLPS in the spatial organization of eukaryotic chromatin and regulation of gene expression in normal and pathological conditions.

Hyperthermia has been used as an adjuvant treatment for radio- and chemotherapy for decades. In addition to its effects on perfusion and oxygenation of cancer tissues, hyperthermia can enhance the efficacy of DNA-damaging treatments such as radiotherapy and chemotherapy. Although it is believed that the adjuvant effects are based on hyperthermia-induced dysfunction of DNA repair systems, the mechanisms of these dysfunctions remain elusive. Here, we propose that elevated temperatures can induce chromatin trapping (c-trapping) of essential factors, particularly those involved in DNA repair, and thus enhance the sensitization of cancer cells to DNA-damaging therapeutics. Using mass spectrometry-based proteomics, we identified proteins that could potentially undergo c-trapping in response to hyperthermia. Functional analyses of several identified factors involved in DNA repair demonstrated that c-trapping could indeed be a mechanism of hyperthermia-induced transient deficiency of DNA repair systems. Based on our proteomics data, we showed for the first time that hyperthermia could inhibit maturation of Okazaki fragments and activate a corresponding poly(ADP-ribose) polymerase-dependent DNA damage response. Together, our data suggest that chromatin trapping of factors involved in DNA repair and replication contributes to heat-induced radio- and chemosensitization.