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.

Comparison of cell response to chromatin and DNA damage.

DNA-targeting drugs are widely used for anti-cancer treatment. Many of these drugs cause different types of DNA damage, i.e. alterations in the chemical structure of DNA molecule. However, molecules binding to DNA may also interfere with DNA packing into chromatin. Interestingly, some molecules do not cause any changes in DNA chemical structure but interfere with DNA binding to histones and nucleosome wrapping. This results in histone loss from chromatin and destabilization of nucleosomes, a phenomenon that we call chromatin damage. Although the cellular response to DNA damage is well-studied, the consequences of chromatin damage are not. Moreover, many drugs used to study DNA damage also cause chromatin damage, therefore there is no clarity on which effects are caused by DNA or chromatin damage. In this study, we aimed to clarify this issue. We treated normal and tumor cells with bleomycin, nuclease mimicking drug which cut predominantly nucleosome-free DNA and therefore causes DNA damage in the form of DNA breaks, and CBL0137, which causes chromatin damage without direct DNA damage. We describe similarities and differences between the consequences of DNA and chromatin damage. Both agents were more toxic for tumor than normal cells, but while DNA damage causes senescence in both normal and tumor cells, chromatin damage does not. Both agents activated p53, but chromatin damage leads to the accumulation of higher levels of unmodified p53, which transcriptional activity was similar to or lower than that of p53 activated by DNA damage. Most importantly, we found that while transcriptional changes caused by DNA damage are limited by p53-dependent activation of a small number of p53 targets, chromatin damage activated many folds more genes in p53 independent manner.

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.

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.

Weak interactions in higher-order chromatin organization.

The detailed principles of the hierarchical folding of eukaryotic chromosomes have been revealed during the last two decades. Along with structures composing three-dimensional (3D) genome organization (chromatin compartments, topologically associating domains, chromatin loops, etc.), the molecular mechanisms that are involved in their establishment and maintenance have been characterized. Generally, protein-protein and protein-DNA interactions underlie the spatial genome organization in eukaryotes. However, it is becoming increasingly evident that weak interactions, which exist in biological systems, also contribute to the 3D genome. Here, we provide a snapshot of our current understanding of the role of the weak interactions in the establishment and maintenance of the 3D genome organization. We discuss how weak biological forces, such as entropic forces operating in crowded solutions, electrostatic interactions of the biomolecules, liquid-liquid phase separation, DNA supercoiling, and RNA environment participate in chromosome segregation into structural and functional units and drive intranuclear functional compartmentalization.

Studying RNA-DNA interactome by Red-C identifies noncoding RNAs associated with various chromatin types and reveals transcription dynamics.

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.

C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries.

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.

Hypoosmotic stress induces R loop formation in nucleoli and ATR/ATM-dependent silencing of nucleolar transcription.

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.

DNA G-Quadruplexes Contribute to CTCF Recruitment.

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.

Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose.

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 Förster 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.

Mechanism of heat stress-induced cellular senescence elucidates the exclusive vulnerability of early S-phase cells to mild genotoxic stress.

Heat stress is one of the best-studied cellular stress factors; however, little is known about its delayed effects. Here, we demonstrate that heat stress induces p21-dependent cellular senescence-like cell cycle arrest. Notably, only early S-phase cells undergo such an arrest in response to heat stress. The encounter of DNA replication forks with topoisomerase I-generated single-stranded DNA breaks resulted in the generation of persistent double-stranded DNA breaks was found to be a primary cause of heat stress-induced cellular senescence in these cells. This investigation of heat stress-induced cellular senescence elucidates the mechanisms underlying the exclusive sensitivity of early S-phase cells to ultra-low doses of agents that induce single-stranded DNA breaks.

Automated Analysis of Cell Cycle Phase-Specific DNA Damage Reveals Phase-Specific Differences in Cell Sensitivity to Etoposide.

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.

Heat shock-induced dissociation of TRF2 from telomeres does not initiate a telomere-dependent DNA damage response.

Telomeric repeat binding factor 2 (TRF2) is a well-studied shelterin complex subunit that plays a major role in the protection of chomosome ends and the prevention of the telomere-associated DNA damage response. We show that heat shock induces the dissociation of TRF2 from telomeres in human primary and cancer cell cultures. TRF2 is not simply degraded in response to heat shock, but redistributed thoughout the nucleoplasm. This TRF2 depletion/redistribution does not initiate the DNA damage response at chomosome termini.

Mechanisms of heat shock response in mammals.

Heat shock (HS) is one of the best-studied exogenous cellular stresses. The cellular response to HS utilizes ancient molecular networks that are based primarily on the action of stress-induced heat shock proteins and HS factors. However, in one way or another, all cellular compartments and metabolic processes are involved in such a response. In this review, we aimed to summarize the experimental data concerning all aspects of the HS response in mammalian cells, such as HS-induced structural and functional alterations of cell membranes, the cytoskeleton and cellular organelles; the associated pathways that result in different modes of cell death and cell cycle arrest; and the effects of HS on transcription, splicing, translation, DNA repair, and replication.

Comparison of cell response to chromatin and DNA damage.

DNA-targeting drugs may damage DNA or chromatin. Many anti-cancer drugs damage both, making it difficult to understand their mechanisms of action. Using molecules causing DNA breaks without altering nucleosome structure (bleomycin) or destabilizing nucleosomes without damaging DNA (curaxin), we investigated the consequences of DNA or chromatin damage in normal and tumor cells. As expected, DNA damage caused p53-dependent growth arrest followed by senescence. Chromatin damage caused higher p53 accumulation than DNA damage; however, growth arrest was p53-independent and did not result in senescence. Chromatin damage activated the transcription of multiple genes, including classical p53 targets, in a p53-independent manner. Although these genes were not highly expressed in basal conditions, they had chromatin organization around the transcription start sites (TSS) characteristic of most highly expressed genes and the highest level of paused RNA polymerase. We hypothesized that nucleosomes around the TSS of these genes were the most sensitive to chromatin damage. Therefore, nucleosome loss upon curaxin treatment would enable transcription without the assistance of sequence-specific transcription factors. We confirmed this hypothesis by showing greater nucleosome loss around the TSS of these genes upon curaxin treatment and activation of a p53-specific reporter in p53-null cells by chromatin-damaging agents but not DNA-damaging agents.

A multi-reference poly-conformational method for in silico design, optimization, and repositioning of pharmaceutical compounds illustrated for selected SARS-CoV-2 ligands.

Background: This work presents a novel computational multi-reference poly-conformational algorithm for design, optimization, and repositioning of pharmaceutical compounds. Methods: The algorithm searches for candidates by comparing similarities between conformers of the same compound and identifies target compounds, whose conformers are collectively close to the conformers of each compound in the reference set. Reference compounds may possess highly variable MoAs, which directly, and simultaneously, shape the properties of target candidate compounds. Results: The algorithm functionality has been case study validated in silico, by scoring ChEMBL drugs against FDA-approved reference compounds that either have the highest predicted binding affinity to our chosen SARS-CoV-2 targets or are confirmed to be inhibiting such targets in-vivo. All our top scoring ChEMBL compounds also turned out to be either high-affinity ligands to the chosen targets (as confirmed in separate studies) or show significant efficacy, in-vivo, against those selected targets. In addition to method case study validation, in silico search for new compounds within two virtual libraries from the Enamine database is presented. The library's virtual compounds have been compared to the same set of reference drugs that we used for case study validation: Olaparib, Tadalafil, Ergotamine and Remdesivir. The large reference set of four potential SARS-CoV-2 compounds has been selected, since no drug has been identified to be 100% effective against the virus so far, possibly because each candidate drug was targeting only one, particular MoA. The goal here was to introduce a new methodology for identifying potential candidate(s) that cover multiple MoA-s presented within a set of reference compounds.

Novel Efficient Multistage Lead Optimization Pipeline Experimentally Validated for DYRK1B Selective Inhibitors.

In addition to general challenges in drug discovery such as the identification of lead compounds in time- and cost-effective ways, specific challenges also exist. Particularly, it is necessary to develop pharmacological inhibitors that effectively discriminate between closely related molecular targets. DYRK1B kinase is considered a valuable target for cancer-specific mono- or combination chemotherapy; however, the inhibition of its closely related DYRK1A kinase is not beneficial. Existing inhibitors target both kinases with essentially the same efficiency, and the unavailability of the DYRK1B crystal structure makes the discovery of DYRK1B-specific inhibitors even more challenging. Here, we propose a novel multi-stage compound discovery pipeline aimed at in silico identification of both potent and selective small molecules from a large set of initial candidates. The method uses structure-based docking and ligand-based quantitative structure-activity relationship modeling. This approach allowed us to identify lead and runner-up small-molecule compounds targeting DYRK1B with high efficiency and specificity.

Transcriptional regulation and spatial organisation of the human AML1/RUNX1 gene.

The transcription factor RUNX1 is a key regulator of haematopoiesis in vertebrates. In humans, the 260-kb long gene coding for this transcription factor is located on chromosome 21. This gene is transcribed from two alternative promoters that are commonly referred to as the distal and the proximal promoters. In model experiments, these two promoters were found to be active in cells of different lineages, although RUNX1 is preferentially expressed in haematopoietic cells. In the present study, we attempted to identify the regulatory elements that could guide tissue-specific expression of the RUNX1 gene. Two such regulatory elements were found within the RUNX1 gene. One of these elements, located within intron 1, is a haematopoietic-specific enhancer. The second regulatory element, located within intron 5.2, contributes to the formation of an active chromatin hub, which integrates the above-mentioned enhancer and the P1 and P2 promoters.

Chromatin loops, illegitimate recombination, and genome evolution.

Chromosomal rearrangements frequently occur at specific places ("hot spots") in the genome. These recombination hot spots are usually separated by 50-100 kb regions of DNA that are rarely involved in rearrangements. It is quite likely that there is a correlation between the above-mentioned distances and the average size of DNA loops fixed at the nuclear matrix. Recent studies have demonstrated that DNA loop anchorage regions can be fairly long and can harbor DNA recombination hot spots. We previously proposed that chromosomal DNA loops may constitute the basic units of genome organization in higher eukaryotes. In this review, we consider recombination between DNA loop anchorage regions as a possible source of genome evolution.

Co-Regulated Genes and Gene Clusters.

There are many co-regulated genes in eukaryotic cells. The coordinated activation or repression of such genes occurs at specific stages of differentiation, or under the influence of external stimuli. As a rule, co-regulated genes are dispersed in the genome. However, there are also gene clusters, which contain paralogous genes that encode proteins with similar functions. In this aspect, they differ significantly from bacterial operons containing functionally linked genes that are not paralogs. In this review, we discuss the reasons for the existence of gene clusters in vertebrate cells and propose that clustering is necessary to ensure the possibility of selective activation of one of several similar genes.