RESUMO
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 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
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
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.