RESUMO
RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.
Assuntos
Cromatina/enzimologia , Dano ao DNA , DNA/metabolismo , Anemia de Fanconi/enzimologia , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Proteína de Replicação A/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/efeitos da radiação , DNA/genética , Anemia de Fanconi/genética , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Mitomicina/farmacologia , Mutação , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Interferência de RNA , Rad51 Recombinase/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Reparo de DNA por Recombinação/efeitos da radiação , Proteína de Replicação A/genética , Transfecção , Ubiquitina-Proteína Ligases/genética , Proteína com ValosinaRESUMO
The RNA field is undergoing a renaissance, with a deluge of proteins being identified to bind RNA. Two reports now introduce proteome-wide approaches that identify the peptides that are crosslinked to RNA (Castello et al., 2016; He et al., 2016).
Assuntos
Cromatina/química , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Nucleares/química , Proteoma/química , RNA não Traduzido/química , Proteínas de Ligação a RNA/química , Animais , Sítios de Ligação , Cromatina/metabolismo , Cromatina/efeitos da radiação , Expressão Gênica , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos da radiação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Mapeamento de Peptídeos/métodos , Processos Fotoquímicos , Ligação Proteica , Domínios Proteicos , Proteoma/genética , Proteoma/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Raios UltravioletaRESUMO
Interactions between noncoding RNAs and chromatin proteins play important roles in gene regulation, but the molecular details of most of these interactions are unknown. Using protein-RNA photocrosslinking and mass spectrometry on embryonic stem cell nuclei, we identified and mapped, at peptide resolution, the RNA-binding regions in â¼800 known and previously unknown RNA-binding proteins, many of which are transcriptional regulators and chromatin modifiers. In addition to known RNA-binding motifs, we detected several protein domains previously unknown to function in RNA recognition, as well as non-annotated and/or disordered regions, suggesting that many functional protein-RNA contacts remain unexplored. We identified RNA-binding regions in several chromatin regulators, including TET2, and validated their ability to bind RNA. Thus, proteomic identification of RNA-binding regions (RBR-ID) is a powerful tool to map protein-RNA interactions and will allow rational design of mutants to dissect their function at a mechanistic level.
Assuntos
Cromatina/química , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Nucleares/química , Proteoma/química , RNA não Traduzido/química , Proteínas de Ligação a RNA/química , Animais , Sítios de Ligação , Cromatina/metabolismo , Cromatina/efeitos da radiação , Expressão Gênica , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos da radiação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Mapeamento de Peptídeos/métodos , Processos Fotoquímicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Raios UltravioletaRESUMO
Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage.
Assuntos
Cromatina/efeitos da radiação , Reparo do DNA , Imunofluorescência/métodos , Histonas/genética , Osteoblastos/efeitos da radiação , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Raios UltravioletaRESUMO
The NAD+-dependent deacetylase and mono-ADP-ribosyl transferase SIRT6 stabilizes the genome by promoting DNA double strand break repair, thereby acting as a tumor suppressor. However, whether SIRT6 regulates nucleotide excision repair (NER) remains unknown. Here, we showed that SIRT6 was recruited to sites of UV-induced DNA damage and stimulated the repair of UV-induced DNA damage. Mechanistic studies further indicated that SIRT6 interacted with DDB2, the major sensor initiating global genome NER (GG-NER), and that the interaction was enhanced upon UV irradiation. SIRT6 deacetylated DDB2 at two lysine residues, K35 and K77, upon UV stress and then promoted DDB2 ubiquitination and segregation from chromatin, thereby facilitating downstream signaling. In addition, we characterized several SIRT6 mutations derived from melanoma patients. These SIRT6 mutants ablated the stimulatory effect of SIRT6 on NER and destabilized the genome due to (i) partial loss of enzymatic activity (P27S or H50Y), (ii) a nonsense mutation (R150*) or (iii) high turnover rates (G134W). Overall, we demonstrate that SIRT6 promotes NER by deacetylating DDB2, thereby preventing the onset of melanomagenesis.
Assuntos
Carcinogênese/genética , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Melanoma/genética , Sirtuínas/genética , Carcinogênese/efeitos da radiação , Cromatina/genética , Cromatina/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Células HeLa , Humanos , Melanoma/patologia , Mutação/efeitos da radiação , Ubiquitinação/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
The excision of mutagenic DNA adducts by the nucleotide excision repair (NER) pathway is essential for genome stability, which is key to avoiding genetic diseases, premature aging, cancer and neurologic disorders. Due to the need to process an extraordinarily high damage density embedded in the nucleosome landscape of chromatin, NER activity provides a unique functional caliper to understand how histone modifiers modulate DNA damage responses. At least three distinct lysine methyltransferases (KMTs) targeting histones have been shown to facilitate the detection of ultraviolet (UV) light-induced DNA lesions in the difficult to access DNA wrapped around histones in nucleosomes. By methylating core histones, these KMTs generate docking sites for DNA damage recognition factors before the chromatin structure is ultimately relaxed and the offending lesions are effectively excised. In view of their function in priming nucleosomes for DNA repair, mutations of genes coding for these KMTs are expected to cause the accumulation of DNA damage promoting cancer and other chronic diseases. Research on the question of how KMTs modulate DNA repair might pave the way to the development of pharmacologic agents for novel therapeutic strategies.
Assuntos
Cromatina/genética , Dano ao DNA/genética , Histona Metiltransferases/genética , Histonas/genética , Cromatina/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Instabilidade Genômica/genética , Instabilidade Genômica/efeitos da radiação , Histona Metiltransferases/química , Metilação/efeitos da radiação , Nucleossomos/genética , Nucleossomos/efeitos da radiação , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos da radiação , Raios UltravioletaRESUMO
Multifractionated irradiation is the mainstay of radiation treatment in cancer therapy. Yet, little is known about the cellular DNA repair processes that take place between radiation fractions, even though understanding the molecular mechanisms promoting cancer cell recovery and survival could improve patient outcome and identify new avenues for targeted intervention. To address this knowledge gap, we systematically characterized how cells respond differentially to multifractionated and single-dose radiotherapy, using a combination of genetics-based and functional approaches. We found that both cancer cells and normal fibroblasts exhibited enhanced survival after multifractionated irradiation compared with an equivalent single dose of irradiation, and this effect was entirely dependent on 53BP1-mediated NHEJ. Furthermore, we identified RIF1 as the critical effector of 53BP1. Inhibiting 53BP1 recruitment to damaged chromatin completely abolished the survival advantage after multifractionated irradiation and could not be reversed by suppressing excessive end resection. Analysis of the TCGA database revealed lower expression of 53BP1 pathway genes in prostate cancer, suggesting that multifractionated radiotherapy might be a favorable option for radio-oncologic treatment in this tumor type. We propose that elucidation of DNA repair mechanisms elicited by different irradiation dosing regimens could improve radiotherapy selection for the individual patient and maximize the efficacy of radiotherapy.
Assuntos
Sobrevivência Celular/genética , Neoplasias da Próstata/radioterapia , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Sobrevivência Celular/efeitos da radiação , Cromatina/efeitos da radiação , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Fibroblastos/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HeLa , Humanos , Masculino , Camundongos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos da radiaçãoRESUMO
Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are predominantly repaired by non-homologous end joining (NHEJ). IR-induced DNA damage activates autophagy, an intracellular degradation process that delivers cytoplasmic components to the lysosome. We identified the deubiquitinase USP14 as a novel autophagy substrate and a regulator of IR-induced DNA damage response (DDR) signaling. Inhibition of autophagy increased levels and DSB recruitment of USP14. USP14 antagonized RNF168-dependent ubiquitin signaling and downstream 53BP1 chromatin recruitment. Here we show that autophagy-deficient cells are defective in NHEJ, as indicated by decreased IR-induced foci (IRIF) formation by pS2056-, pT2609-DNA-PKcs, pS1778-53BP1, RIF1 and a reporter assay activation. Moreover, chromatin recruitment of key NHEJ proteins, including, Ku70, Ku80, DNA-PKcs and XLF was diminished in autophagy-deficient cells. USP14 inhibition rescued the activity of NHEJ-DDR proteins in autophagy-deficient cells. Mass spectrometric analysis identified USP14 interaction with core NHEJ proteins, including Ku70, which was validated by co-immunoprecipitation. An in vitro assay revealed that USP14 targeted Ku70 for deubiquitination. AKT, which mediates Ser432-USP14 phosphorylation, was required for IRIF formation by USP14. Similar to USP14 block, AKT inhibition rescued the activity of NHEJ-DDR proteins in autophagy- and PTEN-deficient cells. These findings reveal a novel negative PTEN/Akt-dependent regulation of NHEJ by USP14.
Assuntos
Reparo do DNA por Junção de Extremidades/efeitos da radiação , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ubiquitina Tiolesterase/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Autofagia/efeitos da radiação , Cromatina/genética , Cromatina/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Células HEK293 , Humanos , Autoantígeno Ku/genética , PTEN Fosfo-Hidrolase/deficiência , Radiação Ionizante , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genéticaRESUMO
Protein-protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein-protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein-protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein-protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein-protein interactions at DNA damage sites.
Assuntos
Osteoblastos/efeitos da radiação , Poli(ADP-Ribose) Polimerase-1/genética , Mapeamento de Interação de Proteínas/métodos , Fatores de Transcrição/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Cromatina/efeitos da radiação , Dano ao DNA , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lasers , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Imagem Óptica , Osteoblastos/citologia , Osteoblastos/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Transdução de Sinais , Análise de Célula Única , Fatores de Transcrição/metabolismo , Raios Ultravioleta , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Proteína Vermelha FluorescenteRESUMO
Plants are exposed to the damaging effect of sunlight that induces DNA photolesions. In order to maintain genome integrity, specific DNA repair pathways are mobilized. Upon removal of UV-induced DNA lesions, the accurate re-establishment of epigenome landscape is expected to be a prominent step of these DNA repair pathways. However, it remains poorly documented whether DNA methylation is accurately maintained at photodamaged sites and how photodamage repair pathways contribute to the maintenance of genome/methylome integrities. Using genome wide approaches, we report that UV-C irradiation leads to CHH DNA methylation changes. We identified that the specific DNA repair pathways involved in the repair of UV-induced DNA lesions, Direct Repair (DR), Global Genome Repair (GGR) and small RNA-mediated GGR prevent the excessive alterations of DNA methylation landscape. Moreover, we identified that UV-C irradiation induced chromocenter reorganization and that photodamage repair factors control this dynamics. The methylome changes rely on misregulation of maintenance, de novo and active DNA demethylation pathways highlighting that molecular processes related to genome and methylome integrities are closely interconnected. Importantly, we identified that photolesions are sources of DNA methylation changes in repressive chromatin. This study unveils that DNA repair factors, together with small RNA, act to accurately maintain both genome and methylome integrities at photodamaged silent genomic regions, strengthening the idea that plants have evolved sophisticated interplays between DNA methylation dynamics and DNA repair.
Assuntos
Dano ao DNA/genética , Metilação de DNA/genética , Reparo do DNA/genética , Epigenoma/genética , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Cromatina/genética , Cromatina/efeitos da radiação , Dano ao DNA/efeitos da radiação , Metilação de DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Epigenoma/efeitos da radiação , Genoma de Planta/genética , Genoma de Planta/efeitos da radiação , Raios UltravioletaRESUMO
Chromatin immunoprecipitation (ChIP) is the most widely used approach for identification of genome-associated proteins and their modifications. We have previously introduced a microplate-based ChIP platform, Matrix ChIP, where the entire ChIP procedure is done on the same plate without sample transfers. Compared to conventional ChIP protocols, the Matrix ChIP assay is faster and has increased throughput. However, even with microplate ChIP assays, sample preparation and chromatin fragmentation (which is required to map genomic locations) remains a major bottleneck. We have developed a novel technology (termed 'PIXUL') utilizing an array of ultrasound transducers for simultaneous shearing of samples in standard 96-well microplates. We integrated PIXUL with Matrix ChIP ('PIXUL-ChIP'), that allows for fast, reproducible, low-cost and high-throughput sample preparation and ChIP analysis of 96 samples (cell culture or tissues) in one day. Further, we demonstrated that chromatin prepared using PIXUL can be used in an existing ChIP-seq workflow. Thus, the high-throughput capacity of PIXUL-ChIP provides the means to carry out ChIP-qPCR or ChIP-seq experiments involving dozens of samples. Given the complexity of epigenetic processes, the use of PIXUL-ChIP will advance our understanding of these processes in health and disease, as well as facilitate screening of epigenetic drugs.
Assuntos
Imunoprecipitação da Cromatina/métodos , Epigênese Genética , Animais , Linhagem Celular , Cromatina/efeitos da radiação , DNA/efeitos da radiação , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , RNA Polimerase II/análise , Ondas UltrassônicasRESUMO
Multiple pathways counteract DNA replication stress to prevent genomic instability and tumorigenesis. The recently identified human SDE2 is a genome surveillance protein regulated by PCNA, a DNA clamp and processivity factor at replication forks. Here, we show that SDE2 cleavage after its ubiquitin-like domain generates Lys-SDE2Ct, the C-terminal SDE2 fragment bearing an N-terminal Lys residue. Lys-SDE2Ct constitutes a short-lived physiological substrate of the Arg/N-end rule proteolytic pathway, in which UBR1 and UBR2 ubiquitin ligases mediate the degradation. The Arg/N-end rule and VCP/p97UFD1-NPL4 segregase cooperate to promote phosphorylation-dependent, chromatin-associated Lys-SDE2Ct degradation upon UVC damage. Conversely, cells expressing the degradation-refractory K78V mutant, Val-SDE2Ct, fail to induce RPA phosphorylation and single-stranded DNA formation, leading to defects in PCNA-dependent DNA damage bypass and stalled fork recovery. Together, our study elucidates a previously unappreciated axis connecting the Arg/N-end rule and the p97-mediated proteolysis with the replication stress response, working together to preserve replication fork integrity.
Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Genoma , Proteína de Replicação A/genética , Ubiquitina-Proteína Ligases/genética , Animais , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Cromatina/efeitos da radiação , DNA/metabolismo , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Osteoblastos , Fosforilação/efeitos da radiação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteólise/efeitos da radiação , Proteína de Replicação A/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Raios Ultravioleta , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
Ultraviolet (UV) light-induced mutations are unevenly distributed across skin cancer genomes, but the molecular mechanisms responsible for this heterogeneity are not fully understood. Here, we assessed how nucleosome structure impacts the positions of UV-induced mutations in human melanomas. Analysis of mutation positions from cutaneous melanomas within strongly positioned nucleosomes revealed a striking ~10 base pair (bp) oscillation in mutation density with peaks occurring at dinucleotides facing away from the histone octamer. Additionally, higher mutation density at the nucleosome dyad generated an overarching "translational curvature" across the 147 bp of DNA that constitutes the nucleosome core particle. This periodicity and curvature cannot be explained by sequence biases in nucleosomal DNA. Instead, our genome-wide map of UV-induced cyclobutane pyrimidine dimers (CPDs) indicates that CPD formation is elevated at outward facing dinucleotides, mirroring the oscillation of mutation density within nucleosome-bound DNA. Nucleotide excision repair (NER) activity, as measured by XR-seq, inversely correlated with the curvature of mutation density associated with the translational setting of the nucleosome. While the 10 bp periodicity of mutations is maintained across nucleosomes regardless of chromatin state, histone modifications, and transcription levels, overall mutation density and curvature across the core particle increased with lower transcription levels. Our observations suggest structural conformations of DNA promote CPD formation at specific sites within nucleosomes, and steric hindrance progressively limits lesion repair towards the nucleosome dyad. Both mechanisms create a unique extended mutation signature within strongly positioned nucleosomes across the human genome.
Assuntos
Melanoma/genética , Mutação , Neoplasias Induzidas por Radiação/genética , Nucleossomos/genética , Neoplasias Cutâneas/genética , Cromatina/genética , Cromatina/efeitos da radiação , Reparo do DNA , DNA de Neoplasias/genética , Feminino , Genoma Humano/efeitos da radiação , Código das Histonas/genética , Código das Histonas/efeitos da radiação , Humanos , Masculino , Modelos Genéticos , Nucleossomos/efeitos da radiação , Neoplasias da Próstata/genética , Dímeros de Pirimidina/genética , Raios Ultravioleta/efeitos adversosRESUMO
We propose a comprehensive mathematical model to study the dynamics of ionizing radiation induced Ataxia-telangiectasia mutated (ATM) activation that consists of ATM activation through dual mechanisms: the initiative activation pathway triggered by the DNA damage-induced local chromatin relaxation and the primary activation pathway consisting of a self-activation loop by interplay with chromatin relaxation. The model is expressed as a series of biochemical reactions, governed by a system of differential equations and analyzed by dynamical systems techniques. Radiation induced double strand breaks (DSBs) cause rapid local chromatin relaxation, which is independent of ATM but initiates ATM activation at damage sites. Key to the model description is how chromatin relaxation follows when active ATM phosphorylates KAP-1, which subsequently spreads throughout the chromatin and induces global chromatin relaxation. Additionally, the model describes how oxidative stress activation of ATM triggers a self-activation loop in which PP2A and ATF2 are released so that ATM can undergo autophosphorylation and acetylation for full activation in relaxed chromatin. In contrast, oxidative stress alone can partially activate ATM because phosphorylated ATM remains as a dimer. The model leads to predictions on ATM mediated responses to DSBs, oxidative stress, or both that can be tested by experiments.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/efeitos da radiação , Ataxia Telangiectasia/metabolismo , Cromatina/metabolismo , Cromatina/efeitos da radiação , Modelos Teóricos , Radiação Ionizante , Fator 2 Ativador da Transcrição/metabolismo , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Bleomicina/farmacologia , Proteínas de Ciclo Celular/metabolismo , Cromatina/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Humanos , Estresse Oxidativo , Fosforilação , Transdução de Sinais/fisiologia , Biologia de Sistemas , Proteína 28 com Motivo Tripartido/metabolismoRESUMO
Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable â¼2â min after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.
Assuntos
Cromatina/genética , Proteína Vmw65 do Vírus do Herpes Simples/genética , Transcrição Gênica , Ativação Transcricional/genética , Linhagem Celular Tumoral , Cromatina/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Genes Reporter/genética , Humanos , Cinética , LuzRESUMO
The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.
Assuntos
Epigênese Genética/genética , Epigênese Genética/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Luz , Optogenética/métodos , Transcrição Gênica/efeitos da radiação , Animais , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Cromatina/genética , Cromatina/efeitos da radiação , Criptocromos/metabolismo , Regulação da Expressão Gênica/genética , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/efeitos da radiação , Fatores de Tempo , Transcrição Gênica/genética , Técnicas do Sistema de Duplo-Híbrido , VigíliaRESUMO
DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Dano ao DNA , Proteínas Nucleares/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Acetilação , Adenosina Trifosfatases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Cromatina/química , Cromatina/efeitos da radiação , Montagem e Desmontagem da Cromatina/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Lisina/química , Lisina/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fosforilação/efeitos da radiação , Fator B de Elongação Transcricional Positiva/metabolismo , Isoformas de Proteínas/metabolismo , Radiação Ionizante , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/química , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Light is a major environmental factor regulating flowering time, thus ensuring reproductive success of higher plants. In contrast to our detailed understanding of light quality and photoperiod mechanisms involved, the molecular basis underlying high light-promoted flowering remains elusive. Here we show that, in Arabidopsis, a chloroplast-derived signal is critical for high light-regulated flowering mediated by the FLOWERING LOCUS C (FLC). We also demonstrate that PTM, a PHD transcription factor involved in chloroplast retrograde signaling, perceives such a signal and mediates transcriptional repression of FLC through recruitment of FVE, a component of the histone deacetylase complex. Thus, our data suggest that chloroplasts function as essential sensors of high light to regulate flowering and adaptive responses by triggering nuclear transcriptional changes at the chromatin level.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Flores/genética , Proteínas de Domínio MADS/genética , Dedos de Zinco PHD/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Cloroplastos/genética , Cloroplastos/metabolismo , Cromatina/genética , Cromatina/efeitos da radiação , Flores/crescimento & desenvolvimento , Flores/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Histona Desacetilases/genética , Luz , Transdução de Sinais/genética , Fatores de TranscriçãoRESUMO
This study was performed to investigate in vitro effects of deoxynivalenol (DON) on mice sperm quality parameters including viability, motility and DNA damages at various concentrations and exposure times. Mice spermatozoa were exposed to DON at 0, 2.5, 5 and 10 µM for 1, 3 and 6 hr, motility parameters were evaluated by computer-assisted analysis and viability was examined by colorimetric metabolic activity assay and HOS test. DNA damage was examined by acridine orange staining, and sperm damages via lipid peroxidation pathway were determined by malondialdehyde (MDA) content measurement. DON affected sperm parameters in a concentration- and time-dependent manner. In all test groups, the average path velocity and progressive motile spermatozoa were remarkably reduced. In comparison with the controls, after 1, 3 and 6 hr exposure to DON, viability of spermatozoa was reduced 25, 30 and 49% respectively. DON exposure at 10 µM for 6 hr resulted in 15% DNA damage and 2.5-fold more MDA generation, when compared with nonexposed spermatozoa. Our data suggest that DON causes sperm quality parameters decline in concentration- and time-dependent fashion, which attribute to the reduction in sperm metabolic activity and membrane integrity and equally to increase in lipid peroxidation rate and DNA damage.
Assuntos
Cromatina/efeitos da radiação , Dano ao DNA/efeitos da radiação , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Tricotecenos/toxicidade , Animais , Fusarium/química , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Estresse Oxidativo/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Análise do Sêmen , Espermatozoides/metabolismoRESUMO
One of the most sensitive, time-consuming, and variable steps of chromatin immunoprecipitation (ChIP) is chromatin sonication. Traditionally, this process can take hours to properly sonicate enough chromatin for multiple ChIP assays. Further, the length of sheared DNA is often inconsistent. In order to faithfully measure chemical and structural changes at the chromatin level, sonication needs to be reliable. Thus, chromatin fragmentation by sonication represents a significant bottleneck to downstream quantitative analysis. To improve the consistency and efficiency of chromatin sonication, we developed and tested a cavitation enhancing reagent based on sonically active nanodroplets. Here, we show that nanodroplets increase sonication efficiency by 16-fold and provide more consistent levels of chromatin fragmentation. Using the previously characterized chromatin in vivo assay (CiA) platform, we generated two distinct chromatin states in order to test nanodroplet-assisted sonication sensitivity in measuring post-translational chromatin marks. By comparing euchromatin to chemically induced heterochromatin at the same CiA:Oct4 locus, we quantitatively measure the capability of our new sonication technique to resolve differences in chromatin structure. We confirm that nanodroplet-assisted sonication results are indistinguishable from those of samples processed with traditional sonication in downstream applications. While the processing time for each sample was reduced from 38.4 to 2.3 min, DNA fragment distribution sizes were significantly more consistent with a coefficient of variation 2.7 times lower for samples sonicated in the presence of nanodroplets. In conclusion, sonication utilizing the nanodroplet cavitation enhancement reagent drastically reduces the amount of processing time and provides consistently fragmented chromatin of high quality for downstream applications.