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1.
Nucleic Acids Res ; 48(4): 1652-1668, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31930303

RESUMO

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 Ultravioleta
2.
PLoS Genet ; 15(11): e1008476, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738755

RESUMO

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 , /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 , Genoma de Planta/genética , Genoma de Planta/efeitos da radiação , Raios Ultravioleta
3.
J Exp Clin Cancer Res ; 38(1): 203, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101118

RESUMO

BACKGROUND: The VRK1 chromatin kinase regulates the organization of locally altered chromatin induced by DNA damage. The combination of ionizing radiation with inhibitors of DNA damage responses increases the accumulation of DNA damage in cancer cells, which facilitates their antitumor effect, a process regulated by VRK1. METHODS: Tumor cell lines with different genetic backgrounds were treated with olaparib to determine their effect on the activation of DNA repair pathways induced by ionizing radiation. The effect of combining olaparib with depletion of the chromatin kinase VRK1 was studied in the context of double-strand breaks repair pathway after treatment with ionizing radiation. The initiation and progression of DDR were studied by specific histone acetylation, as a marker of local chromatin relaxation, and formation of γH2AX and 53BP1 foci. RESULTS: In this work, we have studied the effect that VRK1 by itself or in collaboration with olaparib, an inhibitor of PARP, has on the DNA oxidative damage induced by irradiation in order to identify its potential as a new drug target. The combination of olaparib and ionizing radiation increases DNA damage permitting a significant reduction of their respective doses to achieve a similar amount of DNA damage detected by γH2AX and 53BP1 foci. Different treatment combinations of olaparib and ionizing radiation permitted to reach the maximum level of DNA damage at lower doses of both treatments. Furthermore, we have studied the effect that depletion of the VRK1 chromatin kinase, a regulator of DDR, has on this response. VRK1 knockdown impaired all steps in the DDR induced by these treatments, which were detected by a reduction of sequential markers such as H4K16 ac, γH2AX, NBS1 and 53BP1. Moreover, this effect of VRK1 is independent of TP53 or ATM, two genes frequently mutated in cancer. CONCLUSION: The protective DNA damage response induced by ionizing radiation is impaired by the combination of olaparib with depletion of VRK1, and can be used to reduce doses of radiation and their associated toxicity. Proteins implicated in DNA damage responses are suitable targets for development of new therapeutic strategies and their combination can be an alternative form of synthetic lethality.


Assuntos
Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Células A549 , Acetilação/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/radioterapia , Fosforilação/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Interferência de RNA , Radiação Ionizante , Mutações Sintéticas Letais/genética
4.
Nucleic Acids Res ; 47(12): e69, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-30927002

RESUMO

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 Ultrassônicas
5.
Andrologia ; 51(5): e13238, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30706512

RESUMO

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 Espermática/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/metabolismo
6.
Nucleic Acids Res ; 47(8): 3996-4010, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30698750

RESUMO

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/metabolismo
7.
Cell Death Dis ; 9(12): 1142, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30442925

RESUMO

Radiation-induced bystander effect (RIBE) is a poorly understood phenomenon wherein non-targeted cells exhibit effects of radiation. We have reported that cell-free chromatin (cfCh) particles that are released from dying cells can integrate into genomes of surrounding healthy cells to induce DNA damage and inflammation. This raised the possibility that RIBE might be induced by cfCh released from irradiated dying cells. When conditioned media from BrdU-labeled irradiated cells were passed through filters of pore size 0.22 µm and incubated with unexposed cells, BrdU-labeled cfCh particles could be seen to readily enter their nuclei to activate H2AX, active Caspase-3, NFκB, and IL-6. A direct relationship was observed with respect to activation of RIBE biomarkers and radiation dose in the range of 0.1-0 Gy. We confirmed by FISH and cytogenetic analysis that cfCh had stably integrated into chromosomes of bystander cells and had led to extensive chromosomal instability. The above RIBE effects could be abrogated when conditioned media were pre-treated with agents that inactivate cfCh, namely, anti-histone antibody complexed nanoparticles (CNPs), DNase I and a novel DNA degrading agent Resveratrol-copper (R-Cu). Lower hemi-body irradiation with γ-rays (0.1-50 Gy) led to activation of H2AX, active Caspase-3, NFκB, and IL-6 in brain cells in a dose-dependent manner. Activation of these RIBE biomarkers could be abrogated by concurrent treatment with CNPs, DNase I and R-Cu indicating that activation of RIBE was not due to radiation scatter to the brain. RIBE activation was seen even when mini-beam radiation was delivered to the umbilical region of mice wherein radiation scatter to brain was negligible and could be abrogated by cfCh inactivating agents. These results indicate that cfCh released from radiation-induced dying cells are activators of RIBE and that it can be prevented by treatment with appropriate cfCh inactivating agents.


Assuntos
Cromatina/genética , Inflamação/tratamento farmacológico , Lesões por Radiação/tratamento farmacológico , Resveratrol/farmacologia , Animais , Efeito Espectador/efeitos dos fármacos , Efeito Espectador/efeitos da radiação , Caspase 3/genética , Sistema Livre de Células/efeitos dos fármacos , Sistema Livre de Células/efeitos da radiação , Cromatina/efeitos dos fármacos , Cromatina/efeitos da radiação , Cobre/farmacologia , Meios de Cultivo Condicionados/farmacologia , Dano ao DNA/efeitos da radiação , Desoxirribonuclease I/genética , Modelos Animais de Doenças , Raios gama/efeitos adversos , Histonas/genética , Humanos , Inflamação/genética , Inflamação/patologia , Interleucina-6/genética , Camundongos , NF-kappa B/genética , Lesões por Radiação/genética , Lesões por Radiação/patologia
8.
PLoS Genet ; 14(11): e1007823, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30485262

RESUMO

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 adversos
9.
Mutat Res Genet Toxicol Environ Mutagen ; 836(Pt A): 109-116, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30389153

RESUMO

Ataxia telangiectasia is a rare autosomal recessive genome instability syndrome caused by mutations in the Ataxia Telangiectasia Mutated gene and characterized by a very high sensitivity to agents inducing double strand breaks such as ionizing radiation. In cells derived from ataxia telangiectasia patients a prominent enhancement of chromosomal aberrations is revealed as a consequence of this radiosensitivity characteristic, arising from defective DNA repair for a small fraction of breaks localized in the less accessible heterochromatin. Moreover, the signaling mediated by ataxia telangiectasia protein kinase also modifies chromatin structure. Even if there is a lot of knowledge concerning biochemical aspects of repair of double strand breaks, no conclusive results on radiosensitivity of structurally- and functionally-different chromatin are available, particularly in ataxia telangiectasia cells. Thus, a wild-type cell line and two ataxia telangiectasia patient derived ones could represent a suitable model to study the possible relationship between chromatin conformation and sensitivity to ionizing radiation. In this context, the effects of both cytosine arabinoside, an inhibitor of DNA repair synthesis, and trichostatin A, a histone deacetylase inhibitor, were tested in normal and ataxia telangiectasia lymphoblastoid cell lines carrying different mutation in the Ataxia Telangiectasia Mutated gene. The response to both inhibitors was investigated analyzing two endpoints, namely, chromosomal aberrations and the removal of DNA lesions by Comet assay, after exposure to X-rays. Results obtained suggest that the modulation of chromatin structure by trichostatin A leading to a more open conformation, decreases radiation-induced chromosomal aberrations in ataxia telangiectasia cells. The reduction in chromosomal instability can be attributed to an enhancement in DNA repair occurring in the presence of the histone deacetylase inhibitor, as its abolishment by the known inhibitor of DNA repair synthesis cytosine arabinoside clearly demonstrates. Data obtained could indicate a pivotal role of chromatin conformation in the radiosensitivity of ataxia telangiectasia cells.


Assuntos
Ataxia Telangiectasia/tratamento farmacológico , Cromatina/química , Reparo do DNA , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Linfócitos/patologia , Radiação Ionizante , Ataxia Telangiectasia/enzimologia , Ataxia Telangiectasia/patologia , Cromatina/efeitos dos fármacos , Cromatina/efeitos da radiação , Ensaio Cometa , Quebras de DNA de Cadeia Dupla , Replicação do DNA , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação
10.
Methods Mol Biol ; 1832: 243-253, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30073531

RESUMO

In eukaryotic cell nuclei, all DNA transactions, including DNA damage repair, take place on a chromatin substrate, the integrity of which is central to gene expression programs and cell identity. However, substantial chromatin rearrangements accompany the repair response, culminating in the deposition of new histones. How the original epigenetic information conveyed by chromatin may be preserved in this context is a burning question. Elucidating the fate of parental histones, which characterize the pre-damage chromatin state, is a key step forward in deciphering the mechanisms that safeguard epigenome stability. Here, we present an in vivo approach for tracking parental histone H3 variant dynamics in real time after UVC laser-induced damage in human cells.


Assuntos
Cromatina/efeitos da radiação , Dano ao DNA , Histonas/metabolismo , Imageamento Tridimensional/métodos , Raios Ultravioleta , Linhagem Celular Tumoral , Humanos , Isoformas de Proteínas/metabolismo , Coloração e Rotulagem
11.
Radiother Oncol ; 129(3): 600-610, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30049456

RESUMO

BACKGROUND AND PURPOSE: High linear-energy-transfer (LET) irradiation (IR) is characterized by unique depth-dose distribution and advantageous biologic effectiveness compared to low-LET-IR, offering promising alternatives for radio-resistant tumors in clinical oncology. While low-LET-IR induces single DNA lesions such as double-strand breaks (DSBs), localized energy deposition along high-LET particle trajectories induces clustered DNA lesions that are more challenging to repair. During DNA damage response (DDR) 53BP1 and ATM are required for Kap1-dependent chromatin relaxation, thereby sustaining heterochromatic DSB repair. Here, spatiotemporal dynamics of chromatin restructuring were visualized during DDR after high-LET and low-LET-IR. MATERIAL AND METHODS: Human fibroblasts were irradiated with high-LET carbon/calcium ions or low-LET photons. At 0.1 h, 0.5 h, 5 h and 24 h post-IR fluorophore- and gold-labeled repair factors (53BP1, pATM, pKAP-1, pKu70) were visualized by immunofluorescence and transmission electron microscopy, to monitor formation and repair of DNA damage in chromatin ultrastructure. To track chromatin remodeling at damage sites, decondensed regions (DCR) were delineated based on local chromatin concentration densities. RESULTS: Low-LET-IR induced single DNA lesions throughout the nucleus, but nearly all DSBs were efficiently rejoined without visible chromatin decompaction. High-LET-IR induced clustered DNA damage and triggered profound changes in chromatin structure along particle trajectories. In DCR multiple heterochromatic DSBs exhibited delayed repair despite cooperative activity of 53BP1, pATM, pKap-1. These closely localized DSBs may disturb efficient repair and subsequent chromatin restoration, thereby affecting large-scale genome organization. CONCLUSION: Clustered damage concentrated in particle trajectories causes persistent rearrangements in chromatin architecture, which may affect structural and functional organization of cell nuclei.


Assuntos
Cromatina/efeitos da radiação , Dano ao DNA , Animais , Células Cultivadas , Cromatina/ultraestrutura , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Autoantígeno Ku/análise , Transferência Linear de Energia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/análise
12.
J Cell Biochem ; 119(10): 8146-8162, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29923310

RESUMO

We studied how deficiency in lamins A/C and lamina-associated polypeptide 2α (Lap2α) affects DNA repair after irradiation. A-type lamins and Lap2α were not recruited to local DNA lesions and did not accumulate to γ-irradiation-induced foci (IRIF), as it is generally observed for well-known marker of DNA lesions, 53BP1 protein. At micro-irradiated chromatin of lmna double knockout (dn) and Lap2α dn cells, 53BP1 protein levels were reduced, compared to locally irradiated wild-type counterpart. Decreased levels of 53BP1 we also observed in whole populations of lmna dn and Lap2α dn cells, irradiated by UV light. We also studied distribution pattern of 53BP1 protein in a genome outside micro-irradiated region. In Lap2α deficient cells, identical fluorescence of mCherry-tagged 53BP1 protein was found at both microirradiated region and surrounding chromatin. However, a well-known marker of double strand breaks, γH2AX, was highly abundant in the lesion-surrounding genome of Lap2α deficient cells. Described changes, induced by irradiation in Lap2α dn cells, were not accompanied by cell cycle changes. In Lap2α dn cells, we additionally performed analysis by FLIM (Fluorescence Lifetime Imaging Microscopy) that showed different dynamic behavior of mCherry-tagged 53BP1 protein pools when it was compared with wild-type (wt) fibroblasts. This analysis revealed three different fractions of mCherry-53BP1 protein. Two of them showed identical exponential decay times (τ1 and τ3), but the decay rate of τ2 and amplitudes of fluorescence decays (A1-A3) were statistically different in wt and Lap2α dn fibroblasts. Moreover, γ-irradiation weakened an interaction between A-type lamins and Lap2α. Together, our results demonstrate how depletion of Lap2α affects DNA damage response (DDR) and how chromatin compactness is changed in Lap2α deficient cells exposed to radiation.


Assuntos
Cromatina/efeitos da radiação , Reparo do DNA , Proteínas de Ligação a DNA/genética , Fibroblastos/efeitos da radiação , Lamina Tipo A/genética , Proteínas de Membrana/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Transformada , Cromatina/química , Cromatina/ultraestrutura , Dano ao DNA , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Raios gama , Regulação da Expressão Gênica , Genes Reporter , Histonas/genética , Histonas/metabolismo , Lamina Tipo A/deficiência , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/deficiência , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta
13.
Biochemistry ; 57(19): 2756-2761, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29658277

RESUMO

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.


Assuntos
Imunoprecipitação da Cromatina/métodos , Fragmentação do DNA/efeitos da radiação , DNA/genética , Sonicação/métodos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cromatina/efeitos da radiação , DNA/química , DNA/efeitos da radiação , Eucromatina/efeitos da radiação , Heterocromatina/efeitos da radiação , Camundongos , Nanopartículas/química , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/genética
15.
J Cell Sci ; 130(24): 4213-4224, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29122982

RESUMO

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 ∼2 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 Genética , Ativação Transcricional/genética , Linhagem Celular Tumoral , Cromatina/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Genes Reporter/genética , Humanos , Cinética , Luz
16.
Carcinogenesis ; 38(10): 976-985, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28981631

RESUMO

Subunit 2 of DNA damage-binding protein complex (DDB2) is an early sensor of nucleotide excision repair (NER) pathway for eliminating DNA damage induced by UV radiation (UVR) and cisplatin treatments of mammalian cells. DDB2 is modified by ubiquitin and poly(ADP-ribose) (PAR) in response to UVR, and these modifications play a crucial role in regulating NER. Here, using immuno-analysis of irradiated cell extracts, we have identified multiple post-irradiation modifications of DDB2 protein. Interestingly, although the DNA lesions induced by both UVR and cisplatin are corrected by NER, only the UV irradiation, but not the cisplatin treatment, induces any discernable DDB2 modifications. We, for the first time, show that the appearance of UVR-induced DDB2 modifications depend on the binding of DDB2 to the damaged chromatin and the participation of functionally active 26S proteasome. The in vitro and in vivo analysis revealed that SUMO-1 conjugations comprise a significant portion of these UVR-induced DDB2 modifications. Mapping of SUMO-modified sites demonstrated that UVR-induced SUMOylation occurs on Lys-309 residue of DDB2 protein. Mutation of Lys-309 to Arg-309 diminished the DDB2 SUMOylation observable both in vitro and in vivo. Moreover, K309R mutated DDB2 lost its function of recruiting XPC to the DNA damage sites, as well as the ability to repair cyclobutane pyrimidine dimers following cellular UV irradiation. Taken together, our results indicate that DDB2 is modified by SUMOylation upon UV irradiation, and this post-translational modification plays an important role in the initial recognition and processing of UVR-induced DNA damage occurring within the context of chromatin.


Assuntos
Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Sumoilação/efeitos da radiação , Cromatina/metabolismo , Cromatina/efeitos da radiação , Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Lisina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Sumoilação/efeitos dos fármacos , Raios Ultravioleta
17.
BMC Genomics ; 18(1): 778, 2017 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-29025389

RESUMO

BACKGROUND: The role of histone modifications in the DNA damage response has been extensively studied in non-plant systems, including mammals and yeast. However, there is a lack of detailed evidence showing how chromatin dynamics, either an individual mark or combined chromatin states, participate in regulating differentially expressed genes in the plant DNA damage response. RESULTS: In this study, we used RNA-seq and ChIP-seq to show that differentially expressed genes (DEGs), in response to ionizing radiation (IR), might be involved in different pathways responsible for the DNA damage response. Moreover, chromatin structures associated with promoters, exons and intergenic regions are significantly affected by IR. Most importantly, either an individual mark or a certain chromatin state was found to be highly correlated with the expression of up-regulated genes. In contrast, only the chromatin states, as opposed to any individual marks tested, are related to the expression of the down-regulated genes. CONCLUSIONS: Our findings demonstrate that IR-related DEGs are modulated by distinct epigenetic mechanisms. Either chromatin states or distinct histone dynamics may act sequentially or in combination in regulating up-regulated genes, but the complex chromatin structure is mainly responsible for the expression of down-regulated genes. Thus, this study provides new insights into how up- and down-regulated genes are epigenetically regulated at the chromatin levels, thereby helping us to understand distinct epigenetic mechanisms that function in the plant DNA damage response.


Assuntos
Cromatina/genética , Cromatina/efeitos da radiação , Radioisótopos de Cobalto/farmacologia , Raios gama , Oryza/genética , Oryza/efeitos da radiação , Transcriptoma/efeitos da radiação , Dano ao DNA , Éxons/genética , Histonas/metabolismo , Análise de Sequência de RNA , Transcrição Genética/efeitos da radiação
18.
Radiat Res ; 188(6): 690-703, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28792846

RESUMO

Monte Carlo based simulation has proven useful in investigating the effect of proton-induced DNA damage and the processes through which this damage occurs. Clustering of ionizations within a small volume can be related to DNA damage through the principles of nanodosimetry. For simulation, it is standard to construct a small volume of water and determine spatial clusters. More recently, realistic DNA geometries have been used, tracking energy depositions within DNA backbone volumes. Traditionally a chromatin fiber is built within the simulation and identically replicated throughout a cell nucleus, representing the cell in interphase. However, the in vivo geometry of the chromatin fiber is still unknown within the literature, with many proposed models. In this work, the Geant4-DNA toolkit was used to build three chromatin models: the solenoid, zig-zag and cross-linked geometries. All fibers were built to the same chromatin density of 4.2 nucleosomes/11 nm. The fibers were then irradiated with protons (LET 5-80 keV/µm) or alpha particles (LET 63-226 keV/µm). Nanodosimetric parameters were scored for each fiber after each LET and used as a comparator among the models. Statistically significant differences were observed in the double-strand break backbone size distributions among the models, although nonsignificant differences were noted among the nanodosimetric parameters. From the data presented in this article, we conclude that selection of the solenoid, zig-zag or cross-linked chromatin model does not significantly affect the calculated nanodosimetric parameters. This allows for a simulation-based cell model to make use of any of these chromatin models for the scoring of direct ion-induced DNA damage.


Assuntos
Partículas alfa , Cromatina/efeitos da radiação , Simulação por Computador , Dano ao DNA , Modelos Biológicos , Nanotecnologia/métodos , Nucleossomos/efeitos da radiação , Prótons , Radiometria/métodos , Algoritmos , Cromatina/ultraestrutura , Histonas , Transferência Linear de Energia , Nucleossomos/ultraestrutura , Eficiência Biológica Relativa
19.
Nat Cell Biol ; 19(9): 1061-1070, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28759028

RESUMO

Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments in senescent cells. The activation of cGAS, in turn, triggers the production of SASP factors via stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS-STING pathway in vitro and we show cGAS-dependent regulation of senescence following irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS-STING pathway as a crucial regulator of senescence and the SASP.


Assuntos
Senescência Celular , Cromatina/enzimologia , Citosol/enzimologia , Imunidade Inata , Nucleotidiltransferases/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Senescência Celular/efeitos da radiação , Cromatina/imunologia , Cromatina/efeitos da radiação , Citosol/imunologia , Citosol/efeitos da radiação , Ativação Enzimática , Feminino , Genótipo , Imunidade Inata/efeitos da radiação , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Estresse Oxidativo , Comunicação Parácrina , Fenótipo , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção
20.
Mol Cell ; 66(5): 622-634.e8, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28575658

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 Valosina
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