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It is known that major nuclear processes occur differently in the nucleolus when compared to the remainder of the nucleus. This variation could be associated with the nature of the nucleolus, which is considered a multi-component liquid environment. Among the numerous nucleolar proteins, Treacle holds a special place due to a wealth of experimental evidence highlighting its significance for transcription as well as DNA repair in the nucleolus. However, it remains unclear how Treacle exerts its multifaceted influence on nucleolar processes from a mechanistic perspective. In this study, we demonstrate that Treacle can undergo phase separation in living cells and characterize the structural basis of its condensation and generation of cooperative Treacle-TOPBP1 liquid assemblies. Our data clearly show that the phase separation of Treacle serves a key role in ribosomal DNA transcription and DNA repair. In these processes, Treacle biomolecular condensates serve as a platform for attracting and retaining transcription machinery (UBF1, Pol I) or repair factors (TOPBP1, ATR, and others) on ribosomal genes. Moreover, Treacle and TOPBP1 can form cooperative nuclear biomolecular condensates in the absence of DNA damage. These damage-independent Treacle-TOPBP1 liquid droplets act as fully functional molecular platforms for DNA damage response activation. Our data indicate that by forming biomolecular condensates, Treacle can facilitate intracellular processes and target them to the required intranuclear locations.

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.

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.

Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA–DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. Analysis of the RNA–DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.

How the nuclear lamina (NL) impacts on global chromatin architecture is poorly understood. Here, we show that NL disruption in Drosophila S2 cells leads to chromatin compaction and repositioning from the nuclear envelope. This increases the chromatin density in a fraction of topologically-associating domains (TADs) enriched in active chromatin and enhances interactions between active and inactive chromatin. Importantly, upon NL disruption the NL-associated TADs become more acetylated at histone H3 and less compact, while background transcription is derepressed. Two-colour FISH confirms that a TAD becomes less compact following its release from the NL. Finally, polymer simulations show that chromatin binding to the NL can per se compact attached TADs. Collectively, our findings demonstrate a dual function of the NL in shaping the 3D genome. Attachment of TADs to the NL makes them more condensed but decreases the overall chromatin density in the nucleus by stretching interphase chromosomes., 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.

Although replication stress response has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Velichko et al. have studied the molecular mechanisms involved in protecting the cell from replication stress induced in rDNA (nucleolus) of the higher eukaryotes., Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress–response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle–TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle–TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on F&ouml, rster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

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.

Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes. 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 modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome nanodomains 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, but increased further mixing of the chromatin compartments and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions 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.

Synthetic lethality occurs when simultaneous perturbations of two genes or molecular processes result in a loss of cell viability. The number of known synthetically lethal interactions is growing steadily. We review here synthetically lethal interactions of ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). These kinases are appropriate for synthetic lethal therapies because their genes are frequently mutated in cancer, and specific inhibitors are currently in clinical trials. Understanding synthetically lethal interactions of a particular gene or gene family can facilitate predicting new synthetically lethal interactions, therapy toxicity, and mechanisms of resistance, as well as defining the spectrum of tumors amenable to these therapeutic approaches.

Chromosomes in many organisms, including Drosophila and mammals, are folded into topologically associating domains (TADs). Increasing evidence suggests that TAD folding is hierarchical, wherein subdomains combine to form larger superdomains, instead of a sequence of nonoverlapping domains. Here, we studied the hierarchical structure of TADs in Drosophila. We show that the boundaries of TADs of different hierarchical levels are characterized by the presence of different portions of active chromatin, but do not vary in the binding of architectural proteins, such as CCCTC binding factor or cohesin. The apparent hierarchy of TADs in Drosophila chromosomes is not likely to have functional importance but rather reflects various options of long-range chromatin folding directed by the distribution of active and inactive chromatin segments and may represent population average.

Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA–DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. We found two microRNAs—MIR3648 and MIR3687 transcribed from the rRNA locus—that are associated with inactive chromatin genome wide. These miRNAs favor bulk heterochromatin over Polycomb-repressed chromatin and interact preferentially with late-replicating genomic regions. Analysis of the RNA–DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.

G-quadruplex (G4) sites in the human genome frequently colocalize with CCCTC-binding factor (CTCF)-bound sites in CpG islands (CGIs). We aimed to clarify the role of G4s in CTCF positioning. Molecular modeling data suggested direct interactions, so we performed in vitro binding assays with quadruplex-forming sequences from CGIs in the human genome. G4s bound CTCF with Kd values similar to that of the control duplex, while respective i-motifs exhibited no affinity for CTCF. Using ChIP-qPCR assays, we showed that G4-stabilizing ligands enhance CTCF occupancy at a G4-prone site in STAT3 gene. In view of the reportedly increased CTCF affinity for hypomethylated DNA, we next questioned whether G4s also facilitate CTCF recruitment to CGIs via protecting CpG sites from methylation. Bioinformatics analysis of previously published data argued against such a possibility. Finally, we questioned whether G4s facilitate CTCF recruitment by affecting chromatin structure. We showed that three architectural chromatin proteins of the high mobility group colocalize with G4s in the genome and recognize parallel-stranded or mixed-topology G4s in vitro. One of such proteins, HMGN3, contributes to the association between G4s and CTCF according to our bioinformatics analysis. These findings support both direct and indirect roles of G4s in CTCF recruitment.

The contribution of nucleoli to the cellular stress response has been discussed for over a decade. Stress-induced inhibition of RNA polymerase I-dependent transcription is hypothesized as a possible effector program in such a response. In this study, we report a new mechanism by which ribosomal DNA transcription can be inhibited in response to cellular stress. Specifically, we demonstrate that mild hypoosmotic stress induces stabilization of R loops in ribosomal genes and thus provokes the nucleoli-specific DNA damage response, which is governed by the ATM- and Rad3-related (ATR) kinase. Activation of ATR in nucleoli strongly depends on Treacle, which is needed for efficient recruitment/retention of TopBP1 in nucleoli. Subsequent ATR-mediated activation of ATM results in repression of nucleolar transcription.

Recently we characterized a class of anti-cancer agents (curaxins) that disturbs DNA/histone interactions within nucleosomes. Here, using a combination of genomic and in vitro approaches, we demonstrate that curaxins strongly affect spatial genome organization and compromise enhancer-promoter communication, which is necessary for the expression of several oncogenes, including MYC. We further show that curaxins selectively inhibit enhancer-regulated transcription of chromatinized templates in cell-free conditions. Genomic studies also suggest that curaxins induce partial depletion of CTCF from its binding sites, which contributes to the observed changes in genome topology. Thus, curaxins can be classified as epigenetic drugs that target the 3D genome organization., 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.

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.

Interphase chromatin in animals is constrained by interactions with the nuclear lamina (NL) – a protein mesh lining nuclear envelope and composed from lamins and lamin-associated proteins. About 30% of chromatin is located near the NL in a living cell forming densely packed lamina-associated domains (LADs). LADs largely overlap with topologically-associating domains (TADs) build up from repressed chromatin, and contain predominately inactive genes, thus playing a role in transcription regulation. Here, we show that the NL disruption in Drosophila leads to en mass chromatin compactisation and redistribution towards the nuclear interior. This results in an increase of contact frequency between active and repressed fractions of the genome accompanied by the transcription upregulation in LADs. A subset of TADs characterized by high level of LADs become less compact, that could be driven by the loss of interactions with the nuclear envelope as revealed by polymer modelling. These results indicate that the NL in Drosophila cells plays a role in the specific spatial positioning of the chromatin inside the nucleus, its compartmentalization and at least partially controls the packaging density and low-level transcription in LADs.

Cellular senescence, a form of cell cycle arrest, is one of the cellular responses to different types of exogenous and endogenous damage. The senescence phenotype can be induced in vitro by oncogene overexpression and/or DNA damage. Recently, we have reported a novel mechanism of cellular senescence induction by mild genotoxic stress. Specifically, we have shown that the formation of a small number of DNA lesions in normal and cancer cells during S phase leads to cellular senescence-like arrest within the same cell cycle. Here, based on this mechanism, we suggest an approach to remotely induce premature senescence in human cell cultures using short-term light irradiation. We used the genetically encoded photosensitizers, tandem KillerRed and miniSOG, targeted to chromatin by fusion to core histone H2B to induce moderate levels of DNA damage by light in S phase cells. We showed that the cells that express the H2B-fused photosensitizers acquire a senescence phenotype upon illumination with the appropriate light source. Furthermore, we demonstrated that both chromatin-targeted tandem KillerRed (produces O2¯) and miniSOG (produces 1O2) induce single-stranded DNA breaks upon light illumination. Interestingly, miniSOG was also able to induce double-stranded DNA breaks.

Although the heat-stress response has been extensively studied for decades, very little is known about its effects on nucleic acids and nucleic acid-associated processes. This is due to the fact that the research has focused on the study of heat shock proteins and factors (HSPs and HSFs), their involvement in the regulation of transcription, protein homeostasis, etc. Recently, there has been some progress in the study of heat stress effects on DNA integrity. In this review, we summarize and discuss well-known and potential mechanisms of formation of various heat stress-induced DNA damage.

The comet assay is one of the most widely used approaches for detecting DNA damage; generally, it provides information on the cell population-averaged level of DNA damage. Here, we present an automatic technique for easy measurement of standard comet characteristics and an annotation of the cell cycle phase of each comet. The approach includes the modified neutral comet assay and a pipeline for CellProfiler software designed to analyze DNA damage-related characteristics and annotate the cell cycle phase of each comet. Using this technique we have performed cell cycle phase-specific analysis of DNA damage induced by the topoisomerase II poison etoposide and have shown that the sensitivity of cells to this drug dramatically differed according to their cell cycle phase. It became evident from our results that the proposed protocol provides important additional information that often remains hidden in a standard comet analysis of an asynchronous cell population. J. Cell. Biochem. 117: 2209-2214, 2016. © 2016 Wiley Periodicals, Inc.

The comet assay is one of the most widely used approaches for detecting DNA damage; generally, it provides information on the cell population-averaged level of DNA damage. Here, we present an automatic technique for easy measurement of standard comet characteristics and an annotation of the cell cycle phase of each comet. The approach includes the modified neutral comet assay and a pipeline for CellProfiler software designed to analyze DNA damage-related characteristics and annotate the cell cycle phase of each comet. Using this technique we have performed cell cycle phase-specific analysis of DNA damage induced by the topoisomerase II poison etoposide and have shown that the sensitivity of cells to this drug dramatically differed according to their cell cycle phase. It became evident from our results that the proposed protocol provides important additional information that often remains hidden in a standard comet analysis of an asynchronous cell population. J. Cell. Biochem. 117: 2209-2214, 2016. © 2016 Wiley Periodicals, Inc.

Cellular senescence, a form of cell cycle arrest, is one of the cellular responses to different types of exogenous and endogenous damage. The senescence phenotype can be induced in vitro by oncogene overexpression and/or DNA damage. Recently, we have reported a novel mechanism of cellular senescence induction by mild genotoxic stress. Specifically, we have shown that the formation of a small number of DNA lesions in normal and cancer cells during S phase leads to cellular senescence-like arrest within the same cell cycle. Here, based on this mechanism, we suggest an approach to remotely induce premature senescence in human cell cultures using short-term light irradiation. We used the genetically encoded photosensitizers, tandem KillerRed and miniSOG, targeted to chromatin by fusion to core histone H2B to induce moderate levels of DNA damage by light in S phase cells. We showed that the cells that express the H2B-fused photosensitizers acquire a senescence phenotype upon illumination with the appropriate light source. Furthermore, we demonstrated that both chromatin-targeted tandem KillerRed (produces O2−) and miniSOG (produces 1O2) induce single-stranded DNA breaks upon light illumination. Interestingly, miniSOG was also able to induce double-stranded DNA breaks.