RESUMO
BACKGROUND: Many plant secondary metabolites (PSMs) were shown to intercalate into DNA helix or interact with DNA grooves. This may influence histone-DNA interactions changeing chromatin structure and genome functioning. METHODS: Nucleosome stability and linker histone H1.2, H1.4 and H1.5 localizations were studied in HeLa cells after the treatment with 15 PSMs, which are DNA-binders and possess anticancer activity according to published data. Chromatin remodeler CBL0137 was used as a control. Effects of PSMs were studied using fluorescent microscopy, flowcytometry, quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), western-blotting. RESULTS: We showed that 1-hour treatment with CBL0137 strongly inhibited DNA synthesis and caused intensive linker histone depletion consistent with nucleosome destabilization. None of PSMs caused nucleosome destabilization, while most of them demonstrated significant influence on linker histone localizations. In particular, cell treatment with 11 PSMs at non-toxic concentrations induced significant translocation of the histone H1.5 to nucleoli and most of PSMs caused depletion of the histones H1.2 and H1.4 from chromatin fraction. Curcumin, resveratrol, berberine, naringenin, and quercetin caused significant redistribution of all three variants of the studied linker histones showing some overlap of PSM effects on linker histone DNA-binding. We demonstrated that PSMs, which induced the most significant redistribution of the histone H1.5 (berberine, curcumin and naringenin), influence the proportion of cells synthesizing DNA, expressing or non-expressing cyclin B and influence cell cycle distribution. Berberine induction of H1.5 translocations to nucleoli was shown to occur independently on the phases of cell cycle (metaphase was not analyzed). CONCLUSIONS: For the first time we revealed PSM influence on linker histone location in cell nuclei that opens a new direction of PSM research as anticancer agents.
Assuntos
Cromatina , Histonas , Histonas/metabolismo , Humanos , Células HeLa , Cromatina/metabolismo , Nucleossomos/metabolismo , Nucleossomos/efeitos dos fármacos , Metabolismo Secundário , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos/farmacologiaRESUMO
Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing's sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug's mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.
Assuntos
Antineoplásicos , Histona Desmetilases , Nucleossomos , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/metabolismo , Humanos , Nucleossomos/metabolismo , Nucleossomos/efeitos dos fármacos , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Linhagem Celular Tumoral , Histonas/metabolismo , Tranilcipromina/farmacologia , Especificidade por Substrato , CinéticaRESUMO
Methylprednisolone is a glucocorticoid and can negatively influence immune defense mechanisms. During bacterial infections in the dog, neutrophils infiltrate infected tissue and mediate antimicrobial effects with different mechanisms such as phagocytosis and neutrophil extracellular trap (NET) formation. Here, we investigated the influence of methylprednisolone on canine NET formation and neutrophil killing efficiency of Gram positive and Gram negative bacteria. Therefore, canine blood derived neutrophils were treated with different concentrations of methylprednisolone over time. The survival factor of Staphylococcus pseudintermedius, Streptococcus canis or Escherichia coli was determined in presence of stimulated neutrophils. Additionally, free DNA and nucleosomes as NET marker were analyzed in supernatants and neutrophils were assessed for NET formation by immunofluorescence microscopy. Methylprednisolone concentrations of 62.5 and 625 µg/mL enhanced the neutrophil killing of Gram positive bacteria, whereas no significant influence was detected for the Gram negative Escherichia coli. Interestingly, higher amounts of free DNA were detected under methylprednisolone stimulation in a concentration dependency and in the presence of Streptococcus canis and Escherichia coli. The nucleosome release by neutrophils is induced by bacterial infection and differs depending on the concentration of methylprednisolone. Furthermore, immunofluorescence microscopy analysis identified methylprednisolone at a concentration of 62.5 µg/mL as a NET inducer. In summary, methylprednisolone enhances NET-formation and time-dependent and concentration-dependent the bactericidal effect of canine neutrophils on Gram positive bacteria.
Assuntos
Antibacterianos/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Metilprednisolona/farmacologia , Neutrófilos/efeitos dos fármacos , Animais , Cães , Feminino , Masculino , Nucleossomos/efeitos dos fármacos , Fagocitose/efeitos dos fármacosRESUMO
Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed.
Assuntos
Período de Replicação do DNA/genética , Replicação do DNA/efeitos dos fármacos , Raízes de Plantas/genética , Zea mays/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Centrômero/efeitos dos fármacos , Centrômero/genética , Replicação do DNA/genética , Período de Replicação do DNA/efeitos dos fármacos , DNA de Plantas/efeitos dos fármacos , DNA de Plantas/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Endocitose/efeitos dos fármacos , Meristema/efeitos dos fármacos , Meristema/genética , Mitose/efeitos dos fármacos , Mitose/genética , Nucleossomos/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Fase S/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Telomerase (hTERT) reactivation and sustained expression is a key event in the process of cellular transformation. Therefore, the identification of the mechanisms regulating hTERT expression is of great interest for the development of new anticancer therapies. Although the epigenetic state of hTERT gene promoter is important, we still lack a clear understanding of the mechanisms by which epigenetic changes affect hTERT expression. Retinoids are well-known inducers of granulocytic maturation in acute promyelocytic leukemia (APL). We have previously shown that retinoids repressed hTERT expression in the absence of maturation leading to growth arrest and cell death. Exploring the mechanisms of this repression, we showed that transcription factor binding was dependent on the epigenetic status of hTERT promoter. In the present study, we used APL cells lines and publicly available datasets from APL patients to further investigate the integrated epigenetic events that promote hTERT promoter transition from its silent to its active state, and inversely. We showed, in APL patients, that the methylation of the distal domain of hTERT core promoter was altered and correlated with the outcome of the disease. Further studies combining complementary approaches carried out on APL cell lines highlighted the significance of a domain outside the minimal promoter, localized around 5 kb upstream from the transcription start site, in activating hTERT. This domain is characterized by DNA hypomethylation and H3K4Me3 deposition. Our findings suggest a cooperative interplay between hTERT promoter methylation, chromatin accessibility, and histone modifications that force the revisiting of previously proposed concepts regarding hTERT epigenetic regulation. They represent, therefore, a major advance in predicting sensitivity to retinoid-induced hTERT repression and, more generally, in the potential development of therapies targeting hTERT expression in cancers.
Assuntos
Metilação de DNA/genética , Regulação Leucêmica da Expressão Gênica , Código das Histonas/genética , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/genética , Telomerase/genética , Tretinoína/uso terapêutico , Linhagem Celular Tumoral , Cromatina/metabolismo , Análise por Conglomerados , Ilhas de CpG/genética , Epigênese Genética/efeitos dos fármacos , Loci Gênicos , Genoma Humano , Humanos , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/metabolismo , Tretinoína/farmacologiaRESUMO
Leber's hereditary optic neuropathy (LHON) is one of the most common mitochondrial diseases caused by point mutations in mitochondrial DNA (mtDNA). The majority of diagnosed LHON cases are caused by a point mutation at position 11,778 in the mitochondrial genome. LHON mainly affects young men in their 20s and 30s with usually poor visual prognosis. It remains unexplained why men are more likely to develop the disease and why only retinal ganglion cells are affected. In this study, a cell model was used for the first time to investigate the influence of testosterone on the cell death mechanism apoptosis and on an autophagy/mitophagy. Cells with m.11778G > A were found to be significantly more susceptible to nucleosome formation and effector caspase activation that serve as hallmarks of apoptotic cell death. Cells having this mutation expressed higher levels of mitophagic receptors BNIP3 and BNIP3L/Nix in a medium with testosterone. Moreover, cells having the mutation exhibited greater mitochondrial mass, which suggests these cells have a decreased cell survival. The observed decrease in cell survival was supported by the observed increase in apoptotic cell death. Autophagy was analyzed after inhibition with Bafilomycin A1 (Baf A1). The results indicate impairment in autophagy in LHON cells due to lower autophagic flux supported by observed lower levels of autophagosome marker LC3-II. The observed impaired lower autophagic flux in mutant cells correlated with increased levels of BNIP3 and BNIP3L/Nix in mutant cells.
Assuntos
Autofagia/efeitos dos fármacos , Mitofagia/genética , Atrofia Óptica Hereditária de Leber/tratamento farmacológico , Testosterona/farmacologia , Adulto , Apoptose/efeitos dos fármacos , Células Sanguíneas/efeitos dos fármacos , DNA Mitocondrial/genética , Feminino , Genoma Mitocondrial/genética , Humanos , Macrolídeos/farmacologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Nucleossomos/efeitos dos fármacos , Nucleossomos/genética , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/metabolismo , Atrofia Óptica Hereditária de Leber/patologia , Mutação Puntual/genética , Células Ganglionares da Retina/efeitos dos fármacos , Testosterona/metabolismoRESUMO
Nitrogen mustards have long been used in cancer chemotherapy, and their cytotoxicity has traditionally been attributed to the formation of DNA interstrand cross-links and DNA monoalkylation. Recent studies have shown that exposure to nitrogen mustards also induces the formation of DNA-protein cross-links (DPCs) via bridging between N7 of a deoxyguanosine residue in the DNA and the side chain of a Cys residue in the protein. However, the formation of nitrogen mustard-induced DNA-histone cross-links has never been observed. Herein, we demonstrate that treating reconstituted nucleosome core particles (NCPs) with the nitrogen mustard mechlorethamine results in the formation of DNA-histone cross-links in addition to DNA monoalkylation and interstrand cross-link formation. The yields of these three types of DNA lesions in the NCPs decreased in the following order: DNA monoalkylation â« DNA interstrand cross-links > DNA-histone cross-links. Mechanistic studies involving tailless histones and competitive inhibition by a polyamine demonstrated that Lys residues in the N- and C-terminal tails of the histones were the predominant sites involved in DNA-histone cross-link formation. Given that NCPs are the fundamental repeating units of chromatin in eukaryotes, our findings suggest that nitrogen mustard-induced formation of DNA-histone cross-links may occur in living cells and that DPC formation may contribute to the cytotoxicity of nitrogen mustards.
Assuntos
Alquilantes/química , Reagentes de Ligações Cruzadas/química , DNA/efeitos dos fármacos , Histonas/efeitos dos fármacos , Mecloretamina/química , Nucleossomos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , DNA/química , Histonas/química , Masculino , Modelos Químicos , Nucleossomos/química , Salmão , Espermatozoides/químicaRESUMO
Metal cations are associated with many biological processes. The effects of these cations on nucleic acids and chromatin were extensively studied in the early stages of nucleic acid and chromatin research. The results revealed that some monovalent and divalent metal cations, including Mg2+, profoundly affect the conformations and stabilities of nucleic acids, the folding of chromatin fibers, and the extent of chromosome condensation. Apart from these effects, there have only been a few reports on the functions of these cations. In 2007 and 2013, however, Mg2+-implicated novel phenomena were found: Mg2+ facilitates or enables both self-assembly of identical double-stranded (ds) DNA molecules and self-assembly of identical nucleosomes in vitro. These phenomena may be deeply implicated in the heterochromatin domain formation and chromatin-based phase separation. Furthermore, a recent study showed that elevation of the intranuclear Mg2+ concentration causes unusual differentiation of mouse ES (embryonic stem) cells. All of these phenomena seem to be closely related to one another. Mg2+ seems to be a key regulator of chromatin dynamics and chromatin-based biological processes.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Magnésio/farmacologia , Nucleossomos/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Cromatina/química , Cromatina/metabolismo , Humanos , Magnésio/metabolismo , Camundongos , Conformação Molecular/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Nucleossomos/metabolismoRESUMO
Creating access to DNA double-strand break (DSB) sites in the chromatin context is an essential step during the repair process, but much remains to be determined about its regulatory mechanisms. Here, using a novel reporter cassette for simultaneous detection of homologous recombination (HR) and nonhomologous end joining (NHEJ) at the same chromosomal site, we report that the efficiency of HR but not NHEJ negatively correlates with nucleosome density. We demonstrate that PARP1 is required for HR by modulating nucleosome density at damage sites. Mechanistic studies indicate that the ATPase domain of BRG1 and the ZnF domain of SIRT1 interact with poly-ADP ribose (PAR) in response to DNA damage, and are responsible for bringing the two factors to broken DNA ends. At DNA damage sites, BRG1 and SIRT1 physically interact, whereupon SIRT1 deacetylates BRG1 at lysine residues 1029 and 1033, stimulating its ATPase activity to remodel chromatin and promote HR.
Assuntos
DNA Helicases/genética , DNA/genética , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Reparo de DNA por Recombinação , Sirtuína 1/genética , Fatores de Transcrição/genética , Sítios de Ligação , Linhagem Celular , Linhagem Celular Tumoral , Cloroquina/farmacologia , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Fenantrenos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Sirtuína 1/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for normal localization of the histone chaperone, FACT. In yeast, H2B-Ub is deubiquitinated by Ubp8, a subunit of SAGA, and Ubp10. Although they target the same substrate, loss of Ubp8 and Ubp10 cause different phenotypes and alter the transcription of different genes. We show that Ubp10 has poor activity on yeast nucleosomes, but that the addition of FACT stimulates Ubp10 activity on nucleosomes and not on other substrates. Consistent with a role for FACT in deubiquitinating H2B in vivo, a FACT mutant strain shows elevated levels of H2B-Ub. Combination of FACT mutants with deletion of Ubp10, but not Ubp8, confers increased sensitivity to hydroxyurea and activates a cryptic transcription reporter, suggesting that FACT and Ubp10 may coordinate nucleosome assembly during DNA replication and transcription. Our findings reveal unexpected interplay between H2B deubiquitination and nucleosome dynamics.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Alelos , Replicação do DNA/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Hidroxiureia/farmacologia , Mutação/genética , Nucleossomos/efeitos dos fármacos , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transcrição Gênica/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
This work highlights recent studies in epigenetic mechanisms that play a role in alcoholism, which is a complex multifactorial disorder. There is a large body of evidence showing that alcohol can modify gene expression through epigenetic processes, namely DNA methylation and nucleosomal remodeling via histone modifications. In that regard, chronic exposure to ethanol modifies DNA and histone methylation, histone acetylation, and microRNA expression. The alcohol-mediated chromatin remodeling in the brain promotes the transition from use to abuse and addiction. Unravelling the multiplex pattern of molecular modifications induced by ethanol could support the development of new therapies for alcoholism and drug addiction targeting epigenetic processes.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Etanol/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Histonas/genética , Histonas/metabolismo , Humanos , Nucleossomos/efeitos dos fármacos , Nucleossomos/genéticaRESUMO
The rapid development of nanotechnology has led to the use of silver nanoparticles (AgNPs) in biomedical applications, including antibacterial, antiviral, anti-inflammatory, and anticancer therapies. The molecular mechanism of AgNPs-induced cytotoxicity has not been studied thoroughly using a combination of cellular assays and RNA sequencing (RNA-Seq) analysis. In this study, we prepared AgNPs using myricetin, an anti-oxidant polyphenol, and studied their effects on NIH3T3 mouse embryonic fibroblasts as an in vitro model system to explore the potential biomedical applications of AgNPs. AgNPs induced loss of cell viability and cell proliferation in a dose-dependent manner, as evident by increased leakage of lactate dehydrogenase (LDH) from cells. Reactive oxygen species (ROS) were a potential source of cytotoxicity. AgNPs also incrementally increased oxidative stress and the level of malondialdehyde, depleted glutathione and superoxide dismutase, reduced mitochondrial membrane potential and adenosine triphosphate (ATP), and caused DNA damage by increasing the level of 8-hydroxy-2'-deoxyguanosine and the expressions of the p53 and p21 genes in NIH3T3 cells. Thus, activation of oxidative stress may be crucial for NIH3T3 cytotoxicity. Interestingly, gene ontology (GO) term analysis revealed alterations in epigenetics-related biological processes including nucleosome assembly and DNA methylation due to AgNPs exposure. This study is the first demonstration that AgNPs can alter bulk histone gene expression. Therefore, our genome-scale study suggests that the apoptosis observed in NIH3T3 cells treated with AgNPs is mediated by the repression of genes required for cell survival and the aberrant enhancement of nucleosome assembly components to induce apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose/genética , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Endocitose/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Malondialdeído/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/ultraestrutura , Camundongos , Células NIH 3T3 , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Eletricidade Estática , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
In chronic kidney disease (CKD), the number of circulating neutrophils are increased, and this is usually accompanied by an increased basal activation state. However, the possible association between neutrophil extracellular traps (NETs) with vascular complications has not been evaluated. We assessed the relationship between NETs, autophagy and endothelial dysfunction in maintenance hemodialysis (MHD) patients. NET formation, neutrophil elastase (NE) activities, and serum nucleosome levels were measured in MHD (nâ¯=â¯60) and controls (nâ¯=â¯20). Basal NET formation were markedly increased in MHD patient compared to controls. After PMA stimulation, MHD neutrophils showed significantly increased NETs formation response than controls. The degree of NETs was strongly associated with lower flow-mediated dilatation(%) of brachial artery even after adjustment for cardiovascular risk factors and uremic toxins. Moreover, MHD neutrophils showed increased basal autophagy activity. Interestingly, the levels of NETs were markedly augmented after autophagy inhibition, suggesting a protective role of autophagy in excessive NET formation.
Assuntos
Autofagia , Artéria Braquial/fisiopatologia , Endotélio Vascular/fisiopatologia , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Insuficiência Renal Crônica/metabolismo , Vasodilatação/fisiologia , Adulto , Idoso , Estudos de Casos e Controles , Endotélio Vascular/efeitos dos fármacos , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Humanos , Elastase de Leucócito , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Diálise Renal , Insuficiência Renal Crônica/fisiopatologia , Acetato de Tetradecanoilforbol/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
Safeguarding cell function and identity following a genotoxic stress challenge entails a tight coordination of DNA damage signaling and repair with chromatin maintenance. How this coordination is achieved and with what impact on chromatin integrity remains elusive. Here, we address these questions by investigating the mechanisms governing the distribution in mammalian chromatin of the histone variant H2A.X, a central player in damage signaling. We reveal that H2A.X is deposited de novo at sites of DNA damage in a repair-coupled manner, whereas the H2A.Z variant is evicted, thus reshaping the chromatin landscape at repair sites. Our mechanistic studies further identify the histone chaperone FACT (facilitates chromatin transcription) as responsible for the deposition of newly synthesized H2A.X. Functionally, we demonstrate that FACT potentiates H2A.X-dependent signaling of DNA damage. We propose that new H2A.X deposition in chromatin reflects DNA damage experience and may help tailor DNA damage signaling to repair progression.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/genética , Fatores de Elongação da Transcrição/genética , Alfa-Amanitina/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Morfolinas/farmacologia , Células NIH 3T3 , Nucleossomos/química , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Venenos/farmacologia , Pirimidinas/farmacologia , Pironas/farmacologia , Transdução de Sinais , Fatores de Elongação da Transcrição/metabolismoRESUMO
Mitosis encompasses key molecular changes including chromatin condensation, nuclear envelope breakdown, and reduced transcription levels. Immediately after mitosis, the interphase chromatin structure is reestablished and transcription resumes. The reestablishment of the interphase chromatin is probably achieved by "bookmarking," i.e., the retention of at least partial information during mitosis. To gain a deeper understanding of the contribution of histone modifications to the mitotic bookmarking process, we merged proteomics, immunofluorescence, and ChIP-seq approaches. We focused on key histone modifications and employed HeLa-S3 cells as a model system. Generally, in spite of the general hypoacetylation observed during mitosis, we observed a global concordance between the genomic organization of histone modifications in interphase and mitosis, suggesting that the epigenomic landscape may serve as a component of the mitotic bookmarking process. Next, we investigated the nucleosome that enters nucleosome depleted regions (NDRs) during mitosis. We observed that in â¼60% of the NDRs, the entering nucleosome is distinct from the surrounding highly acetylated nucleosomes and appears to have either low levels of acetylation or high levels of phosphorylation in adjacent residues (since adjacent phosphorylation may interfere with the ability to detect acetylation). Inhibition of histone deacetylases (HDACs) by the small molecule TSA reverts this pattern, suggesting that these nucleosomes are specifically deacetylated during mitosis. Altogether, by merging multiple approaches, our study provides evidence to support a model where histone modifications may play a role in mitotic bookmarking and uncovers new insights into the deposition of nucleosomes during mitosis.
Assuntos
Histonas/metabolismo , Mitose , Nucleossomos/genética , Acetilação/efeitos dos fármacos , Imunoprecipitação da Cromatina , Células HeLa , Código das Histonas , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Fosforilação , ProteômicaRESUMO
Hypercapnia, the elevation of CO2 in blood and tissues, commonly occurs in severe acute and chronic respiratory diseases, and is associated with increased risk of mortality. Recent studies have shown that hypercapnia adversely affects innate immunity, host defense, lung edema clearance and cell proliferation. Airway epithelial dysfunction is a feature of advanced lung disease, but the effect of hypercapnia on airway epithelium is unknown. Thus, in the current study we examined the effect of normoxic hypercapnia (20% CO2 for 24 h) vs normocapnia (5% CO2), on global gene expression in differentiated normal human airway epithelial cells. Gene expression was assessed on Affymetrix microarrays, and subjected to gene ontology analysis for biological process and cluster-network representation. We found that hypercapnia downregulated the expression of 183 genes and upregulated 126. Among these, major gene clusters linked to immune responses and nucleosome assembly were largely downregulated, while lipid metabolism genes were largely upregulated. The overwhelming majority of these genes were not previously known to be regulated by CO2. These changes in gene expression indicate the potential for hypercapnia to impact bronchial epithelial cell function in ways that may contribute to poor clinical outcomes in patients with severe acute or advanced chronic lung diseases.
Assuntos
Dióxido de Carbono/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Hipercapnia/complicações , Pneumopatias/patologia , Mucosa Respiratória/efeitos dos fármacos , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Brônquios/patologia , Dióxido de Carbono/sangue , Diferenciação Celular , Células Cultivadas , Doença Crônica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Perfilação da Expressão Gênica , Humanos , Hipercapnia/sangue , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Pneumopatias/etiologia , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , SarcoglicanopatiasRESUMO
Sperm chromatin packaging is a very complex and highly regulated phenomenon. While most of the sperm chromatin is replaced by protamines, some are retained in nucleosomes. It is recently being recognised that these nucleosomes are intentionally retained and could be contributing to the expression of genes in the very early stages of embryogenesis. Endocrine disruption has been previously shown to affect reproductive outcome and sperm DNA methylation. This study aims to decipher the possibility of changes in nucleosome occupancy in sperm chromatin, induced by tamoxifen (selective oestrogen receptor modulator) and cyproterone acetate (androgen antagonist). We used next-generation sequencing approach (MNase-Seq) to identify changes in the nucleosome landscape of the spermatozoa. We demonstrated that endocrine disruption affects nucleosome occupancy at critical regions of the genome and many of them harbour genes relevant for embryogenesis. This study emphasises that environmental factors could affect embryo development by way of modulating male epigenetic factors.
Assuntos
Disruptores Endócrinos/toxicidade , Nucleossomos/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Acetato de Ciproterona , Impressão Genômica , Masculino , Nucleossomos/metabolismo , Ratos Sprague-Dawley , Espermatozoides/metabolismo , Tamoxifeno , Sítio de Iniciação de TranscriçãoRESUMO
The histone lysine methyltransferase nuclear receptor-binding SET domain protein 2 (NSD2, also known as WHSC1/MMSET) is an epigenetic modifier and is thought to play a driving role in oncogenesis. Both NSD2 overexpression and point mutations that increase its catalytic activity are associated with several human cancers. Although NSD2 is an attractive therapeutic target, no potent, selective, and bioactive small molecule inhibitors of NSD2 have been reported to date, possibly due to the challenges of developing high-throughput assays for NSD2. Here, to establish a platform for the discovery and development of selective NSD2 inhibitors, we optimized and implemented multiple assays. We performed quantitative high-throughput screening with full-length WT NSD2 and a nucleosome substrate against a diverse collection of bioactive small molecules comprising 16,251 compounds. We further interrogated 174 inhibitory compounds identified in the primary screen with orthogonal and counter assays and with activity assays based on the clinically relevant NSD2 variants E1099K and T1150A. We selected five confirmed inhibitors for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. We found that all five NSD2 inhibitors bind the catalytic SET domain and one exhibited apparent activity in cells, validating the workflow and providing a template for identifying selective NSD2 inhibitors. In summary, we have established a robust discovery pipeline for identifying potent NSD2 inhibitors from small-molecule libraries.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Nucleossomos/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala/métodos , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Nucleossomos/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The megabase-sized length of chromatin is highly relevant to the state of chromatin in vivo, where it is subject to a highly crowded environment and is organized in topologically associating domains of similar dimension. We developed an in vitro experimental chromatin model system reconstituted from T4 DNA (approximately 166 kbp) and histone octamers and studied the monomolecular compaction of this megabase-sized chromatin fiber under the influence of macromolecular crowding. We used single-molecule fluorescence microscopy and observed compaction in aqueous solutions containing poly(ethylene glycol) in the presence of monovalent (Na+ and K+) and divalent (Mg2+) cations. Both DNA and chromatin demonstrated compaction under comparable conditions in the presence of poly(ethylene glycol) and Na+ or Mg2+ salt. However, the mechanism of the compaction changed from a first-order phase transition for DNA to a continuous folding for megabase-sized chromatin fibers. A more efficient and pronounced chromatin compaction was observed in the presence of Na+ compared to K+. A flow-stretching technique to unfold DNA and chromatin coils was used to gain further insight into the morphology of partially folded chromatin fibers. The results revealed a distribution of partially folded chromatin fibers. This variability is likely the result of the heterogeneous distribution of nucleosomes on the DNA chain. The packaging of DNA in the form of chromatin in the crowded nuclear environment appears essential to ensure gradual conformational changes of DNA.
Assuntos
Cromatina/metabolismo , DNA Viral/metabolismo , Bacteriófago T4 , Cromatina/efeitos dos fármacos , Histonas/metabolismo , Humanos , Magnésio/farmacologia , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Polietilenoglicóis/farmacologia , Sódio/farmacologiaRESUMO
The regulatory mechanisms that ensure an accurate control of gene transcription are central to cellular function, development and disease. Such mechanisms rely largely on noncoding regulatory sequences that allow the establishment and maintenance of cell identity and tissue-specific cellular functions.The study of chromatin structure and nucleosome positioning allowed revealing transcription factor accessible genomic sites with regulatory potential, facilitating the comprehension of tissue-specific cis-regulatory networks. Recently a new technique coupled with high-throughput sequencing named Assay for Transposase Accessible Chromatin (ATAC-seq) emerged as an efficient method to chart open chromatin genome wide. The application of such technique to different cell types allowed unmasking tissue-specific regulatory elements and characterizing cis-regulatory networks. Herein we describe the implementation of the ATAC-seq method to human pancreatic islets, a tissue playing a central role in the control of glucose metabolism.