RESUMO
The SWI/SNF chromatin remodeling complex has been found mutated in a wide range of human cancers, causing alterations in gene expression patterns, proliferation and DNA damage response that have been linked to poor clinical prognosis. Here, we investigated weather knockdown of ARID1B, one of two mutually exclusive subunits within the SWI/SNF complex, can sensitize colorectal cancer cell lines mutated in the other subunit, ARID1A, to ionizing radiation (IR). ARID1A-mutated colorectal cancer (CRC) cell lines are selectively sensitized to IR after siRNA mediated ARID1B depletion, as measured by clonogenic survival. This is characterized by a decrease in the surviving cell fraction to 87.3% ± 2.1%, 86.0% ± 1.1% and 77.2% ± 1.5% per 1 Gy compared with control siRNA exposed cells in the dose range of 0-6 Gy for the LS180, RKO and SW48 lines, respectively (p < 0.0001, F-test). The magnitude of this dose modifying effect was significantly larger in ARID1A mutated than in non-mutated cell lines (Spearman rank correlation rs = 0.88, p = 0.02). Furthermore, initial formation of RAD51 foci at 4 h after IR, as a measure for homologous recombination repair, was significantly reduced in ARID1A-mutant CRC cell lines but not in the majority of wildtype lines nor in fibroblasts. These findings open up perspectives for targeting ARID1B in combination with radiotherapy to improve outcomes of patients with ARID1A-mutant CRC.
Assuntos
Neoplasias Colorretais/terapia , Proteínas de Ligação a DNA/genética , Tolerância a Radiação/genética , Fatores de Transcrição/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Quimiorradioterapia/métodos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos da radiação , Neoplasias Colorretais/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Humanos , Mutação , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Ensaio Tumoral de Célula-Tronco , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
In recent years several approaches have been developed to address the chromatin status and its changes in eukaryotic cells under different conditions-but only few are applicable in living cells. Fluorescence lifetime imaging microscopy (FLIM) is a functional tool that can be used for the inspection of the molecular environment of fluorophores in living cells. Here, we present the use of single organic minor groove DNA binder dyes in FLIM for measuring chromatin changes following modulation of chromatin structure in living cells. Treatment with histone deacetylase inhibitors led to an increased fluorescence lifetime indicating global chromatin decompaction, whereas hyperosmolarity decreased the lifetime of the used dyes, thus reflecting the expected compaction. In addition, we demonstrate that time domain FLIM data based on single photon counting should be optimized using pile-up and counting loss correction, which affect the readout even at moderate average detector count rates in inhomogeneous samples. Using these corrections and utilizing Hoechst 34580 as chromatin compaction probe, we measured a pan nuclear increase in the lifetime following irradiation with X-rays in living NIH/3T3 cells thus providing a method to measure radiation-induced chromatin decompaction.
Assuntos
Benzimidazóis/farmacologia , Montagem e Desmontagem da Cromatina/efeitos da radiação , DNA/metabolismo , Corantes Fluorescentes/farmacologia , Raios X , Animais , Camundongos , Microscopia de Fluorescência , Células NIH 3T3RESUMO
The main function of the skin, to protect against the environment, is supported by the activity of different stem cell populations. The main focus of this study was elucidating the coping mechanisms of stem cells against the stimulation of constant exposure to genotoxic stresses, both endogenous and exogenous, to ensure long-term function. Investigation of various mouse strains, differing in their DNA repair capacity, enables us to clarify fractionated low-dose irradiation (LDR)-induced consequences for different stem cell populations of the murine hair follicle (HF) in their physiological stem cell niche. Using microscopic techniques combined with flow cytometry, we could show that LDR induces accumulation of persisting; pKu70-independent 53BP1-foci ("chromatin-alterations") in heterochromatic regions of the HF stem cells (HFSCs). These remaining chromatin-alterations result in varying stem cell consequences. CD34-positive HFSCs react by ataxia telangiectasia mutated-dependent, premature senescence, which correlates with global chromatin compaction, whereby apoptosis is prevented by the activity of DNA-dependent protein kinase catalytic subunit. However, distinctively highly damaged HFSCs seem to be sorted out of the niche by differentiation, transferring their chromatin-alterations to more proliferative G protein-coupled receptor 5-positive stem cells. Consequentially, the loss of basal HFSCs is compensated by increased proliferation within the stem cell pool. Despite the initial success of these mechanisms in stem cell population maintenance, the combined effect of the chromatin-alterations and the modification in stem cell pool composition may lead to downstream long-term functional loss of tissue or organs. Stem Cells 2018;36:574-588.
Assuntos
Proliferação de Células/efeitos da radiação , Montagem e Desmontagem da Cromatina/efeitos da radiação , Raios gama , Folículo Piloso/metabolismo , Células-Tronco/metabolismo , Animais , Relação Dose-Resposta à Radiação , Folículo Piloso/patologia , Camundongos , Camundongos Transgênicos , Células-Tronco/patologiaRESUMO
Ultraviolet (UV) light induces mutagenic cyclobutane pyrimidine dimers (CPDs) in nucleosomal DNA that is tightly wrapped around histone octamers. How global-genome nucleotide excision repair (GG-NER) processes CPDs despite that this chromatin arrangement is poorly understood. An increased chromatin association of CHD1 (chromodomain helicase DNA-binding 1) upon UV irradiation indicated possible roles of this chromatin remodeler in the UV damage response. Immunoprecipitation of chromatin fragments revealed that CHD1 co-localizes in part with GG-NER factors. Chromatin fractionation showed that the UV-dependent recruitment of CHD1 occurs to UV lesions in histone-assembled nucleosomal DNA and that this CHD1 relocation requires the lesion sensor XPC (xeroderma pigmentosum group C). In situ immunofluorescence analyses further demonstrate that CHD1 facilitates substrate handover from XPC to the downstream TFIIH (transcription factor IIH). Consequently, CHD1 depletion slows down CPD excision and sensitizes cells to UV-induced cytotoxicity. The finding of a CHD1-driven lesion handover between sequentially acting GG-NER factors on nucleosomal histone octamers suggests that chromatin provides a recognition scaffold enabling the detection of a subset of CPDs.
Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Fator de Transcrição TFIIH/metabolismo , Raios Ultravioleta , Xeroderma Pigmentoso/metabolismo , Morte Celular/efeitos da radiação , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos da radiação , Genoma Humano , Células HEK293 , Células HeLa , Humanos , Nucleossomos/efeitos da radiação , Dímeros de Pirimidina/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Multipotent cells have similar basic features of all stem cells but limitation in ability of self-renewal and differentiation compared with pluripotent cells. Here, we have developed an ultra effective, gene- and chemical-free method of generating extra multipotent (xpotent) cells which have differentiation potential more than limited cell types, by the mechanism of ultrasound-directed permeation of environmental transition-guided cellular reprogramming (Entr). Ultrasound stimulus generated a massive number of Entr-mediated xpotent (x/Entr) spheroids from human dermal fibroblasts (HDFs) 6 days after treatment. The emergence of x/Entr was first initiated by the introduction of human embryonic stem cell (ESC) environments into the HDFs to start fast cellular reprogramming including activation of stress-related kinase signaling pathways, subsequent chromatin remodeling, and expression of pluripotent-related genes via transient membrane damage caused by ultrasound-induced cavitation. And then, pluripotent markers were transported into their adjacent HDFs via direct cell-to-cell connections in order to generate xpotent clusters. The features of x/Entr cells were intermediate between pluripotency and multipotency in terms of pluripotency with three germ layer markers, multi-lineage differentiation potential, and no teratoma formation. This physical stimulus-mediated reprogramming strategy was cost-effective, simple, quick, produced significant yields, and was safe, and can therefore provide a new paradigm for clinical application.
Assuntos
Diferenciação Celular , Reprogramação Celular/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Adulto , Técnicas de Cultura de Células , Linhagem Celular , Autorrenovação Celular , Células Cultivadas , Montagem e Desmontagem da Cromatina/efeitos da radiação , Fibroblastos/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/efeitos da radiação , Humanos , Pessoa de Meia-Idade , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/efeitos da radiação , Ondas UltrassônicasRESUMO
Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. The serial steps in NER involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. Transcription-coupled repair (TCR) is a subpathway of NER dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, I report on recent findings that contribute to the elucidation of TCR mechanisms in the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae and human cells. I review general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations.
Assuntos
Reparo do DNA , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Transcrição Gênica , Animais , Evolução Biológica , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Humanos , Mutagênicos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Especificidade da Espécie , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, and loss of autophagy has been linked to increased genome instability. Here, we report that loss of autophagy is coupled to reduced histone H2A ubiquitination after DNA damage. p62/SQSTM1, which accumulates in autophagy-defective cells, directly binds to and inhibits nuclear RNF168, an E3 ligase essential for histone H2A ubiquitination and DNA damage responses. As a result, DNA repair proteins such as BRCA1, RAP80, and Rad51 cannot be recruited to the sites of DNA double-strand breaks (DSBs), which impairs DSB repair. Moreover, nuclear-localized p62 increased the sensitivity of tumor cells to radiation both in vitro and in vivo, and this required its interaction with RNF168. Our findings indicate that autophagy-deficiency-induced p62 accumulation results in inhibition of histone ubiquitination and highlight the complex relationship between autophagy and the DNA damage response.
Assuntos
Autofagia , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Neoplasias Colorretais/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Sequestossoma-1/metabolismo , Ubiquitinação , Autofagia/efeitos da radiação , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Montagem e Desmontagem da Cromatina/efeitos da radiação , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Reparo do DNA/efeitos da radiação , Células HCT116 , Histonas/metabolismo , Humanos , Interferência de RNA , Tolerância a Radiação , Proteína Sequestossoma-1/genética , Transdução de Sinais , Transfecção , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos da radiaçãoRESUMO
Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos da radiação , Cromatina/metabolismo , Cromatina/efeitos da radiação , Histonas/metabolismo , Animais , Linhagem Celular , Dano ao DNA/efeitos da radiação , Reparo do DNA , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Lasers , Camundongos , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Epigenetic mechanisms determine the access of regulatory factors to DNA during events such as transcription and the DNA damage response. However, the global response of histone modifications and chromatin accessibility to UV exposure remains poorly understood. Here, we report that UV exposure results in a genome-wide reduction in chromatin accessibility, while the distribution of the active regulatory mark H3K27ac undergoes massive reorganization. Genomic loci subjected to epigenetic reprogramming upon UV exposure represent target sites for sequence-specific transcription factors. Most of these are distal regulatory regions, highlighting their importance in the cellular response to UV exposure. Furthermore, UV exposure results in an extensive reorganization of super-enhancers, accompanied by expression changes of associated genes, which may in part contribute to the stress response. Taken together, our study provides the first comprehensive resource for genome-wide chromatin changes upon UV irradiation in relation to gene expression and elucidates new aspects of this relationship.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos da radiação , Cromatina/metabolismo , Dano ao DNA , Epigênese Genética/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Cromatina/genética , Cromatina/patologia , Camundongos , Células NIH 3T3RESUMO
Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.
Assuntos
DNA Polimerase Dirigida por DNA/química , Histonas/química , Radical Hidroxila/química , Nucleossomos/metabolismo , Dímeros de Pirimidina/química , Proteínas Recombinantes de Fusão/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Montagem e Desmontagem da Cromatina/efeitos da radiação , Metilação de DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Desaminação , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Radical Hidroxila/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Nucleossomos/química , Nucleossomos/efeitos da radiação , Regiões Promotoras Genéticas , Dímeros de Pirimidina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Raios UltravioletaRESUMO
Nijmegen breakage syndrome (NBS) is a recessive genetic disorder characterized by increased sensitivity to ionizing radiation (IR) and a high frequency of malignancies. NBS1, a product of the mutated gene in NBS, contains several protein interaction domains in the N-terminus and C-terminus. The C-terminus of NBS1 is essential for interactions with MRE11, a homologous recombination repair nuclease, and ATM, a key player in signal transduction after the generation of DNA double-strand breaks (DSBs), which is induced by IR. Moreover, NBS1 regulates chromatin remodeling during DSB repair by histone H2B ubiquitination through binding to RNF20 at the C-terminus. Thus, NBS1 is considered as the first protein to be recruited to DSB sites, wherein it acts as a sensor or mediator of DSB damage responses. In addition to DSB response, we showed that NBS1 initiates Polη-dependent translesion DNA synthesis by recruiting RAD18 through its binding at the NBS1 C-terminus after UV exposure, and it also functions after the generation of interstrand crosslink DNA damage. Thus, NBS1 has multifunctional roles in response to DNA damage from a variety of genotoxic agents, including IR.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , DNA/biossíntese , DNA/genética , Proteínas Nucleares/metabolismo , Radiação , Animais , Proteínas de Ciclo Celular/química , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos da radiação , DNA/química , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/efeitos da radiação , Humanos , Proteínas Nucleares/químicaRESUMO
Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulator of post replication repair (PRR). The depletion of BAF180, a unique subunit of the PBAF chromatin remodeling complex in human cells results in reduced PCNA ubiquitination leading to less efficient fork progression following DNA damage, but little is known about the mechanism. Here, we report that the expression of exogenous BAF180 in cells promotes PCNA ubiquitination during S-phase after UV irradiation and it persists for many hours. No correlation was observed between the protein level of ubiquitin-specific protease 1 (USP1) and ubiquitinated PCNA in BAF180 expressing cells. Analysis of cells expressing BAF180 deletion mutants showed that the bromo-adjacent homology (BAH) domains are responsible for this effect. Surprisingly, a deletion construct encoding only the BAH domain region is able to increase the level of ubiquitinated PCNA, even though it is unable to be assembled into the PBAF complex. These results suggest that the ATPase-dependent chromatin remodeling activity of PBAF is not necessary, but instead the BAH domains are sufficient to promote PCNA ubiquitination.
Assuntos
Proteínas de Arabidopsis/biossíntese , Proteínas Nucleares/biossíntese , Antígeno Nuclear de Célula em Proliferação/biossíntese , Fatores de Transcrição/biossíntese , Proteases Específicas de Ubiquitina/biossíntese , Ubiquitinação/efeitos da radiação , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/genética , Linhagem Celular , Montagem e Desmontagem da Cromatina/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Estrutura Terciária de Proteína , Fatores de Transcrição/metabolismo , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética , Raios UltravioletaRESUMO
The spatial organization of chromatin can be subject to extensive remodeling in plant somatic cells in response to developmental and environmental signals. However, the mechanisms controlling these dynamic changes and their functional impact on nuclear activity are poorly understood. Here, we determined that light perception triggers a switch between two different nuclear architectural schemes during Arabidopsis postembryonic development. Whereas progressive nucleus expansion and heterochromatin rearrangements in cotyledon cells are achieved similarly under light and dark conditions during germination, the later steps that lead to mature nuclear phenotypes are intimately associated with the photomorphogenic transition in an organ-specific manner. The light signaling integrators DE-ETIOLATED 1 and CONSTITUTIVE PHOTOMORPHOGENIC 1 maintain heterochromatin in a decondensed state in etiolated cotyledons. In contrast, under light conditions cryptochrome-mediated photoperception releases nuclear expansion and heterochromatin compaction within conspicuous chromocenters. For all tested loci, chromatin condensation during photomorphogenesis does not detectably rely on DNA methylation-based processes. Notwithstanding, the efficiency of transcriptional gene silencing may be impacted during the transition, as based on the reactivation of transposable element-driven reporter genes. Finally, we report that global engagement of RNA polymerase II in transcription is highly increased under light conditions, suggesting that cotyledon photomorphogenesis involves a transition from globally quiescent to more active transcriptional states. Given these findings, we propose that light-triggered changes in nuclear architecture underlie interplays between heterochromatin reorganization and transcriptional reprogramming associated with the establishment of photosynthesis.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Transdução de Sinal Luminoso , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos da radiação , Cotilédone/crescimento & desenvolvimento , Cotilédone/metabolismo , Cotilédone/efeitos da radiação , Metilação de DNA , Inativação Gênica , Genes de Plantas , Heterocromatina/genética , Heterocromatina/efeitos da radiação , Peptídeos e Proteínas de Sinalização Intracelular , Transdução de Sinal Luminoso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plantas Geneticamente Modificadas , RNA Polimerase II/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Plântula/efeitos da radiação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Natural illumination conditions are highly variable and because of their sessile life style, plants are forced to acclimate to them at the cellular and molecular level. Changes in light intensity or quality induce changes in the reduction/oxidation (redox) state of the photosynthetic electron chain that acts as a trigger for compensatory acclimation responses comprising functional and structural adjustments of photosynthesis and metabolism. Such responses include redox-controlled changes in plant gene expression in the nucleus and organelles. Here we describe a strategy for the identification of early redox-regulated genes (ERGs) in the nucleus of the model organism Arabidopsis thaliana that respond significantly 30 or 60 min after the generation of a reduction signal in the photosynthetic electron transport chain. By comparing the response of wild-type plants with that of the acclimation mutant stn7, we could specifically identify ERGs. The results reveal a significant impact of chloroplast redox signals on distinct nuclear gene groups including genes for the mitochondrial electron transport chain, tetrapyrrole biosynthesis, carbohydrate metabolism, and signaling lipid synthesis. These expression profiles are clearly different from those observed in response to the reduction of photosynthetic electron transport by high light treatments. Thus, the ERGs identified are unique to redox imbalances in photosynthetic electron transport and were then used for analyzing potential redox-responsive cis-elements, trans-factors, and chromosomal regulatory hot spots. The data identify a novel redox-responsive element and indicate extensive redox control at transcriptional and chromosomal levels that point to an unprecedented impact of redox signals on epigenetic processes.
Assuntos
Arabidopsis/genética , Arabidopsis/efeitos da radiação , Núcleo Celular/genética , Luz , Plastídeos/metabolismo , Transdução de Sinais/efeitos da radiação , Aclimatação/efeitos dos fármacos , Aclimatação/genética , Arabidopsis/fisiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos da radiação , Dibromotimoquinona/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Mutação/genética , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Fotossíntese/efeitos dos fármacos , Fotossíntese/genética , Fotossíntese/efeitos da radiação , Plastídeos/efeitos dos fármacos , Plastídeos/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tetrapirróis/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Light is an important environmental cue that affects physiology and development of Neurospora crassa. The light-sensing transcription factor (TF) WCC, which consists of the GATA-family TFs WC1 and WC2, is required for light-dependent transcription. SUB1, another GATA-family TF, is not a photoreceptor but has also been implicated in light-inducible gene expression. To assess regulation and organization of the network of light-inducible genes, we analyzed the roles of WCC and SUB1 in light-induced transcription and nucleosome remodeling. We show that SUB1 co-regulates a fraction of light-inducible genes together with the WCC. WCC induces nucleosome eviction at its binding sites. Chromatin remodeling is facilitated by SUB1 but SUB1 cannot activate light-inducible genes in the absence of WCC. We identified FF7, a TF with a putative O-acetyl transferase domain, as an interaction partner of SUB1 and show their cooperation in regulation of a fraction of light-inducible and a much larger number of non light-inducible genes. Our data suggest that WCC acts as a general switch for light-induced chromatin remodeling and gene expression. SUB1 and FF7 synergistically determine the extent of light-induction of target genes in common with WCC but have in addition a role in transcription regulation beyond light-induced gene expression.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas Fúngicas/genética , Luz , Fatores de Transcrição/biossíntese , Montagem e Desmontagem da Cromatina/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Neurospora crassa/genética , Neurospora crassa/efeitos da radiação , Fatores de Transcrição/genética , Ativação Transcricional/genética , Ativação Transcricional/efeitos da radiaçãoRESUMO
The aim of this study was to investigate sperm DNA damage induced by chemo- and radiotherapy in patients with testicular cancer to provide data on the extent and persistence of nuclear damage that might affect individual reproductive potential. We evaluated pre- and post-antineoplastic treatment sperm DNA integrity, expressed as DNA Fragmentation Index (DFI), in a large caseload of testicular cancer patients by sperm chromatin structure assay. The mean total DFI for all patients at T0 was 18.0 ± 12.5%. Sperm chromatin profile was markedly impaired at T3 (27.7 ± 17.4%) and T6 (23.2 ± 15.3%), improving considerably at T12 and T24 (14.0 ± 8.9% and 14.4 ± 10.3%). After chemotherapy, we found a marked increase in DFI at T3 and T6 and a significant reduction at T12 and T24 in comparison with the baseline. In contrast, DFI increased at T3 and T6 after radiotherapy but the subsequent reduction was far less marked, reaching baseline values at T12 and T24. Finally, post-treatment DNA damage was not age or histotype dependent, but was more marked in the advanced stage of cancer. In this study, we showed that the chromatin profile may be affected in the months immediately following the end of the treatment, improving after 12-24 months. Our results thus indicate that post-treatment DNA damage is influenced both by the type and intensity of the therapy and by the pathological and clinical stage of the disease.
Assuntos
Antineoplásicos/efeitos adversos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos da radiação , Dano ao DNA , Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos da radiação , Neoplasias Testiculares/terapia , Adulto , Fragmentação do DNA , Fertilidade/efeitos dos fármacos , Fertilidade/efeitos da radiação , Humanos , Estudos Longitudinais , Masculino , Estadiamento de Neoplasias , Radioterapia/efeitos adversos , Contagem de Espermatozoides , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos da radiação , Espermatozoides/patologia , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Radiotherapy treats cancer by inducing DNA double-strand breaks (DSB) in tumor cells using ionizing radiation. However, DNA repair in tumor cells often leads to radioresistance and unsuccessful outcome. Inhibition of DNA repair by targeting repair proteins can increase radiosensitivity of tumor cells. The BRG1 chromatin remodeling enzyme assists DSB repair by stimulating γ-H2AX formation and BRG1 binding to acetylated histones at DSBs via bromodomain (BRD) is critical for this activity. Here, we show that ectopic expression of BRG1-BRD inhibited γ-H2AX and DSB repair after irradiation and increased the radiosensitivity in various human cancer cells, including HT29 colon cancer. Dimerization of BRG1-BRD, increasing its chromatin binding affinity, aggravated the defects in γ-H2AX and DSB repair and further enhanced the radiosensitivity. While little affecting the upstream ATM activation, BRG1-BRD in irradiated HT29 cells inhibited the recruitment of 53BP1 to damaged chromatin, the downstream event of γ-H2AX, and compromised the G2-M checkpoint and increased apoptosis. Importantly, in a xenograft mouse model, BRG1-BRD increased the radiosensitivity of HT29 tumors, which was further enhanced by dimerization. These data suggest that BRG1-BRD radiosensitizes tumor cells by a dominant negative activity against BRG1, which disrupts γ-H2AX and its downstream 53BP1 pathways, leading to inefficient DNA repair, G2-M checkpoint defect, and increased apoptosis. This work therefore identifies BRG1-BRD as a novel tumor radiosensitizer and its action mechanism, providing the first example of chromatin remodeler as a target for improving cancer radiotherapy.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/química , DNA Helicases/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Tolerância a Radiação , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Estrutura Terciária de Proteína , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
BACKGROUND: Plants are sessile organisms that deal with their -sometimes adverse- environment in well-regulated ways. Chromatin remodeling involving SWI/SNF2-type ATPases is thought to be an important epigenetic mechanism for the regulation of gene expression in different developmental programs and for integrating these programs with the response to environmental signals. In this study, we report on the role of chromatin remodeling in Arabidopsis with respect to the variability of growth and gene expression in relationship to environmental conditions. RESULTS: Already modest (2-fold) over-expression of the AtCHR23 ATPase gene in Arabidopsis results in overall reduced growth compared to the wild-type. Detailed analyses show that in the root, the reduction of growth is due to reduced cell elongation. The reduced-growth phenotype requires sufficient light and is magnified by applying deliberate abiotic (salt, osmotic) stress. In contrast, the knockout mutation of AtCHR23 does not lead to such visible phenotypic effects. In addition, we show that over-expression of AtCHR23 increases the variability of growth in populations of genetically identical plants. These data indicate that accurate and controlled expression of AtCHR23 contributes to the stability or robustness of growth. Detailed RNAseq analyses demonstrate that upon AtCHR23 over-expression also the variation of gene expression is increased in a subset of genes that associate with environmental stress. The larger variation of gene expression is confirmed in individual plants with the help of independent qRT-PCR analysis. CONCLUSIONS: Over-expression of AtCHR23 gives Arabidopsis a phenotype that is markedly different from the growth arrest phenotype observed upon over-expression of AtCHR12, the paralog of AtCHR23, in response to abiotic stress. This demonstrates functional sub-specialization of highly similar ATPases in Arabidopsis. Over-expression of AtCHR23 increases the variability of growth among genetically identical individuals in a way that is consistent with increased variability of expression of a distinct subset of genes that associate with environmental stress. We propose that ATCHR23-mediated chromatin remodeling is a potential component of a buffer system in plants that protects against environmentally-induced phenotypic and transcriptional variation.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica de Plantas , Adenosina Trifosfatases/genética , Arabidopsis/enzimologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Montagem e Desmontagem da Cromatina/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas/genética , Hipocótilo/anatomia & histologia , Hipocótilo/efeitos da radiação , Luz , Mutação/genética , Fenótipo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/efeitos da radiação , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/efeitos da radiação , Estresse Fisiológico/genética , Estresse Fisiológico/efeitos da radiação , Regulação para Cima/genética , Regulação para Cima/efeitos da radiaçãoRESUMO
Chromatin organization and structure are crucial for transcriptional regulation, DNA replication, and damage repair. Although initially characterized in remodeling cell wall glucans, the ß-1,3-glucanosyltransferase Gas1 was recently discovered to regulate transcriptional silencing in a manner separable from its activity at the cell wall. However, the function of Gas1 in modulating chromatin remains largely unexplored. Our genetic characterization revealed that GAS1 had critical interactions with genes encoding the histone H3 lysine acetyltransferases Gcn5 and Sas3. Specifically, whereas the gas1 gcn5 double mutant was synthetically lethal, deletion of both GAS1 and SAS3 restored silencing in Saccharomyces cerevisiae. The loss of GAS1 also led to broad DNA damage sensitivity with reduced Rad53 phosphorylation and defective cell cycle checkpoint activation following exposure to select genotoxins. Deletion of SAS3 in the gas1 background restored both Rad53 phosphorylation and checkpoint activation following exposure to genotoxins that trigger the DNA replication checkpoint. Our analysis thus uncovers previously unsuspected functions for both Gas1 and Sas3 in DNA damage response and cell cycle regulation.
Assuntos
DNA Fúngico/metabolismo , Histona Acetiltransferases/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Genes Fúngicos , Genes Letais , Histona Acetiltransferases/genética , Glicoproteínas de Membrana/genética , Mutagênicos/farmacologia , Mutação , Fosforilação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.