RESUMO
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.
Assuntos
Cromatina , Genoma , Núcleo Celular/genética , Regulação da Expressão GênicaRESUMO
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.
Assuntos
Cromatina/química , Glicóis/farmacologia , Cromatina/efeitos dos fármacos , Elementos Facilitadores Genéticos/efeitos dos fármacos , Genoma Humano , Células HeLa , Humanos , Microscopia , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
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.
Assuntos
Eucariotos , Matriz Nuclear , Cromatina/genética , DNA/genética , Eucariotos/genética , GenomaRESUMO
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.
Assuntos
Cromatina/química , DNA Super-Helicoidal , RNA , Eletricidade EstáticaRESUMO
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.
Assuntos
Cromatina/genética , DNA/genética , Genoma/genética , RNA não Traduzido/genética , Transcrição Gênica , Núcleo Celular/genética , Humanos , RNA Mensageiro/genética , RNA não Traduzido/isolamento & purificação , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Mapeamento Cromossômico/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Biotinilação , Linhagem Celular , Cromatina/química , Cromatina/genética , Cromatina/isolamento & purificação , Cromatina/metabolismo , Mapeamento Cromossômico/economia , Cromossomos Artificiais Bacterianos/genética , DNA/genética , DNA/isolamento & purificação , DNA/metabolismo , Biblioteca Gênica , Genômica/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico/métodosRESUMO
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.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Proteínas de Transporte/genética , Nucléolo Celular/metabolismo , DNA Ribossômico/genética , Proteínas de Ligação a DNA/genética , Inativação Gênica , Proteínas Nucleares/genética , Pressão Osmótica , Estruturas R-Loop , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Sobrevivência Celular , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Replicação do DNA , Dactinomicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Técnicas de Inativação de Genes , Histonas/metabolismo , Humanos , Soluções Hipotônicas/farmacologia , Camundongos , Proteínas Nucleares/fisiologia , Fosfoproteínas/fisiologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Ilhas de CpG , Metilação de DNA , Quadruplex G , Genoma Humano , Fator de Ligação a CCCTC/genética , Cromatina/genética , Humanos , Células K562RESUMO
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.
Assuntos
Proteínas de Bactérias/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/análise , Proteínas Luminescentes/análise , Poli Adenosina Difosfato Ribose/análise , Proteínas de Bactérias/genética , Técnicas Biossensoriais/métodos , Linhagem Celular , Corantes Fluorescentes/metabolismo , Humanos , Proteínas Luminescentes/genética , Fases de Leitura Aberta , Domínios Proteicos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Ubiquitina-Proteína Ligases/análise , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
Assuntos
Senescência Celular , Dano ao DNA , Resposta ao Choque Térmico/genética , Pontos de Checagem da Fase S do Ciclo Celular/genética , Camptotecina/farmacologia , Pontos de Checagem do Ciclo Celular , Células Cultivadas , Quebras de DNA de Cadeia Simples , Reparo do DNA , Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Feminino , Células HeLa , Humanos , Fase S/genética , Inibidores da Topoisomerase I/farmacologiaRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Etoposídeo/farmacologia , Processamento de Imagem Assistida por Computador/métodos , Automação , Western Blotting , Ensaio Cometa , Dano ao DNA , Células HeLa , Humanos , SoftwareRESUMO
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.
Assuntos
Dano ao DNA/fisiologia , Temperatura Alta/efeitos adversos , Telômero/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Humanos , Células MCF-7 , Telômero/genéticaRESUMO
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.
Assuntos
Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Animais , Ciclo Celular/fisiologia , Morte Celular/fisiologia , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Replicação do DNA , HumanosRESUMO
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.
Assuntos
Cromossomos Humanos Par 21/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/biossíntese , Células-Tronco Hematopoéticas/metabolismo , Elementos de Resposta/fisiologia , Transcrição Gênica/fisiologia , Cromossomos Humanos Par 21/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Íntrons/fisiologia , Células Jurkat , Células K562RESUMO
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.
Assuntos
Cromatina/química , Cromatina/genética , Evolução Molecular , Genoma/genética , Recombinação Genética/genética , Animais , DNA/genética , DNA/metabolismo , DNA Topoisomerases Tipo II/metabolismo , HumanosRESUMO
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.
Assuntos
Evolução Molecular , Família Multigênica , Animais , Caderinas/genética , Caderinas/metabolismo , Células Eritroides/metabolismo , Globinas/genética , Globinas/metabolismo , HumanosRESUMO
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 .
Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos/genética , Genoma/genética , Regiões Promotoras Genéticas/genética , Animais , Cromossomos/genética , Genômica , Humanos , Mamíferos/genética , Anotação de Sequência Molecular , Software , Leveduras/genéticaRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Nucléolo Celular/metabolismo , Dano ao DNA , Replicação do DNA , DNA Ribossômico/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Afidicolina/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/genética , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/genética , DNA Ribossômico/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Células HCT116 , Células HeLa , Humanos , Hidroxiureia/farmacologia , Microscopia de Fluorescência , Proteínas Nucleares/genética , Fosfoproteínas/genética , Ligação Proteica , Transporte Proteico , Transdução de SinaisRESUMO
The role of 3D genome organization in the precise regulation of gene expression is well established. Accordingly, the mechanistic connections between 3D genome alterations and disease development are becoming increasingly apparent. This opinion article provides a snapshot of our current understanding of the 3D genome alterations associated with cancers. We discuss potential connections of the 3D genome and cancer transcriptional addiction phenomenon as well as molecular mechanisms of action of 3D genome-disrupting drugs. Finally, we highlight issues and perspectives raised by the discovery of the first pharmaceutical strongly affecting 3D genome organization.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Genoma/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Animais , Cromatina/genética , DNA/genética , Epigenômica/métodos , Humanos , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
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.