RESUMO
Chromatin, the eukaryotic template of genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on the N-termini of histones, such as acetylation, methylation, phosphorylation, and ubiquitination. Distinct histone modifications generate synergistic or antagonistic interaction affinities for novhistone proteins, which in turn dictate dynamic transitions between transcriptionally active or silent states of chromatin. Besides transcription, numerous biological processes, including DNA replication, DNA repair, and recombination, are regulated by chromatin-associated factors. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between the structural or regulatory proteins and DNA and its role in regulating diverse cellular processes in vivo by formaldehyde crosslinking of proteins to proteins and proteins to DNA, followed by immunoprecipitation of the fixed material and detection of the associated DNA. Here we illustrate the overall experimental procedure by taking ChIP analysis of the human telomerase reverse transcriptase promoter as an example.
Assuntos
Imunoprecipitação da Cromatina/métodos , Histonas/química , Fatores de Transcrição/química , Animais , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , Humanos , Nucleossomos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ligação Proteica , Telomerase/química , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The catalytic subunit of telomerase reverse transcriptase (TERT) protects dividing cells from replicative senescence in vitro. Here, we show that expression of TERT mRNA is induced in the ipsilateral cortical neurons after occlusion of the middle cerebral artery in adult mice. Transgenic mice that overexpress TERT showed significant resistance to ischemic brain injury. Among excitotoxicity, oxidative stress, and apoptosis comprising of routes of ischemic neuronal death, NMDA receptor-mediated excitotoxicity was reduced in forebrain cell cultures overexpressing TERT. NMDA-induced accumulation of cytosolic free Ca2+ ([Ca2+]c) was reduced in forebrain neurons from TERT transgenic mice, which was attributable to the rapid flow of [Ca2+]c into the mitochondria from the cytosol without change in Ca2+ influx and efflux through the plasma membrane. The present study provides evidence that TERT is inducible in postmitotic neurons after ischemic brain injury and prevents NMDA neurotoxicity through shift of the cytosolic free Ca2+ into the mitochondria, and thus plays a protective role in ameliorating ischemic neuronal cell death.
Assuntos
Lesão Encefálica Crônica/prevenção & controle , Isquemia Encefálica/terapia , N-Metilaspartato , Síndromes Neurotóxicas/prevenção & controle , Telomerase/biossíntese , Animais , Lesão Encefálica Crônica/induzido quimicamente , Lesão Encefálica Crônica/genética , Isquemia Encefálica/genética , Cálcio/metabolismo , Células Cultivadas , Técnicas de Cocultura , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Agonistas de Aminoácidos Excitatórios , Corantes Fluorescentes , Regulação da Expressão Gênica , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/prevenção & controle , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroglia/citologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Síndromes Neurotóxicas/etiologia , Neurotoxinas , RNA Mensageiro/biossíntese , Telomerase/genéticaRESUMO
The dysregulation of Wnt/beta-catenin signaling and subsequent upregulation of beta-catenin response transcription (CRT) occur frequently in colon cancer cells. Non-steroidal anti-inflammatory drugs (NSAIDs) can repress CRT in colorectal cancer, but little is known about the mechanism of action. We show that the NSAID diclofenac inhibits Wnt/beta-catenin signaling without altering the level of beta-catenin protein and reduces the expression of beta-catenin/TCF-dependent genes. Diclofenac induced the degradation of IkappaBalpha, which increased free nuclear factor kappaB (NF-kappaB) in cells. Also, the ectopic expression of p65, which is a component of NF-kappaB, suppressed CRT. Our findings suggest that diclofenac inhibits Wnt/beta-catenin signaling via the activation of NF-kappaB in colon cancer cells.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Neoplasias do Colo/metabolismo , Proteínas do Citoesqueleto/antagonistas & inibidores , Diclofenaco/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , NF-kappa B/metabolismo , Transativadores/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/metabolismo , Neoplasias do Colo/genética , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sinaptotagmina I , Sinaptotagminas , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas , Proteínas Wnt , beta CateninaRESUMO
Telomerase activity is closely correlated with cellular proliferative activity in human tissues. Human cells with high proliferative potential, such as tumor cells or stem cells, exhibit telomerase activity, whereas most normal human somatic cells do not. Telomerase activity is tightly regulated by the expression of its catalytic subunit human telomerase reverse transcriptase (hTERT). Through an expression cloning approach, we identified E2F-1 as a repressor of the hTERT gene in human tumor cells. Ectopic expression of E2F-1 repressed hTERT promoter activity by inhibiting Sp1 activation of the hTERT promoter. In contrast to the repressor function of E2F-1 in human tumor cells, we demonstrated that E2F-1 is an activator of the hTERT gene in normal human somatic cells. Ectopically expressed E2F-1 activated the hTERT promoter through a noncanonical DNA binding site. E2F-1, E2F-2, and E2F-3 (but not E2F-4 and E2F-5) repressed hTERT promoter activity in human tumor cells, whereas they activated it in normal somatic cells. These contrasting effects of E2F transcription factors on the hTERT promoter could underlie the paradoxical biological activities of E2F, which can both promote and inhibit cellular proliferation and tumorigenesis.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Neoplasias/enzimologia , Proteínas Repressoras/fisiologia , Telomerase/genética , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Fator de Transcrição E2F3 , Fator de Transcrição E2F4 , Fator de Transcrição E2F5 , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Modelos Genéticos , Neoplasias/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/fisiologia , Fator de Transcrição Sp1/fisiologia , Telomerase/biossíntese , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
With advances in determining the entire DNA sequence of the human genome, it is now critical to systematically identify the function of a number of genes in the human genome. These biological challenges, especially those in human diseases, should be addressed in human cells in which conventional (e.g. genetic) approaches have been extremely difficult to implement. To overcome this, several approaches have been initiated. This review will focus on the development of a novel "chemical genetic/genomic approach" that uses small molecules to "probe and identify" the function of genes in specific biological processes or pathways in human cells. Due to the close relationship of small molecules with drugs, these systematic and integrative studies will lead to the "medicinal systems biology approach" which is critical to "formulate and modulate" complex biological (disease) networks by small molecules (drugs) in human bio-systems.
Assuntos
Química Farmacêutica/métodos , Genômica/métodos , Biologia Molecular/métodos , Farmacogenética/métodos , Proteômica/métodos , Animais , Química Farmacêutica/tendências , Biologia Computacional , Desenho de Fármacos , Biblioteca Gênica , Genômica/tendências , Humanos , Modelos Biológicos , Modelos Genéticos , Biologia Molecular/tendências , Farmacogenética/tendências , Proteômica/tendências , Análise de SistemasRESUMO
Most somatic cells encounter an inevitable destiny, senescence. Little progress has been made in identifying small molecules that extend the finite lifespan of normal human cells. Here we show that the intrinsic 'senescence clock' can be reset in a reversible manner by selective modulation of the ataxia telangiectasia-mutated (ATM) protein and ATM- and Rad3-related (ATR) protein with a small molecule, CGK733. This compound was identified by a high-throughput phenotypic screen with automated imaging. Employing a magnetic nanoprobe technology, magnetism-based interaction capture (MAGIC), we identified ATM as the molecular target of CGK733 from a genome-wide screen. CGK733 inhibits ATM and ATR kinase activities and blocks their checkpoint signaling pathways with great selectivity. Consistently, siRNA-mediated knockdown of ATM and ATR induced the proliferation of senescent cells, although with lesser efficiency than CGK733. These results might reflect the specific targeting of the kinase activities of ATM and ATR by CGK733 without affecting any other domains required for cell proliferation.
Assuntos
Benzenoacetamidas/farmacologia , Proteínas de Ciclo Celular/metabolismo , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tioureia/análogos & derivados , Proteínas Supressoras de Tumor/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Benzenoacetamidas/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos , Tioureia/química , Tioureia/farmacologiaRESUMO
Normally, the Wnt/beta-catenin pathway controls developmental processes and homeostasis, but abnormal activation of this pathway is a frequent event during the development of cancer. The key mechanism in regulation of the Wnt/beta-catenin pathway is the amino-terminal phosphorylation of beta-catenin, marking it for proteasomal degradation. Here we present small-molecule-based identification of protein kinase C (PKC)-mediated beta-catenin phosphorylation as a novel mechanism regulating the Wnt/beta-catenin pathway. We used a cell-based chemical screen to identify A23187, which inhibits the Wnt/beta-catenin pathway. PKC was activated by A23187 treatment and subsequently phosphorylated N-terminal serine (Ser) residues of beta-catenin, which promoted beta-catenin degradation. Moreover, the depletion of PKCalpha inhibited the phosphorylation and degradation of beta-catenin. Therefore, our findings suggest that the PKC pathway negatively regulates the beta-catenin level outside of the Wnt/beta-catenin pathway.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Calcimicina/farmacologia , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Ionóforos/farmacologia , Camundongos , Fosforilação , Transporte Proteico , Transdução de SinaisRESUMO
Ni/NiO core/shell nanoparticles having high affinity with polyhistidine were synthesized by decomposition of a Ni surfactant complex followed by air oxidation. Ni/NiO nanoparticles showed selective and efficient binding to histidine-tagged proteins and easy separation by using a magnet. These provided a more convenient way to efficient purification of histidine-tagged proteins compared with the conventional Ni-NTA complex-bound resins and microbeads.
Assuntos
Histidina/química , Nanopartículas Metálicas/química , Níquel/química , Proteínas/química , Magnetismo , Ligação ProteicaRESUMO
Technologies to assess the molecular targets of biomolecules in living cells are lacking. We have developed a technology called magnetism-based interaction capture (MAGIC) that identifies molecular targets on the basis of induced movement of superparamagnetic nanoparticles inside living cells. Efficient intracellular uptake of superparamagnetic nanoparticles (coated with a small molecule of interest) was mediated by a transducible fusogenic peptide. These nanoprobes captured the small molecule's labeled target protein and were translocated in a direction specified by the magnetic field. Use of MAGIC in genome-wide expression screening identified multiple protein targets of a drug. MAGIC was also used to monitor signal-dependent modification and multiple interactions of proteins.
Assuntos
Fenômenos Fisiológicos Celulares , Magnetismo , Técnicas de Sonda Molecular , Sondas Moleculares , Nanoestruturas , Proteínas/metabolismo , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Sobrevivência Celular , Endocitose , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , Microscopia Confocal , Inibidor de NF-kappaB alfa , NF-kappa B/metabolismo , Oligopeptídeos/metabolismo , Fosforilação , Ligação Proteica , Pontos Quânticos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Estreptavidina , Tacrolimo/metabolismo , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
Activation of telomerase is crucial for cells to gain immortality. In human cells, telomerase activity is tightly regulated by the expression of its catalytic subunit, human telomerase reverse transcriptase (hTERT). In most normal human somatic cells, hTERT is not expressed, and its suppression acts as an important gatekeeper against tumorigenesis. Here we describe the systematic analyses of hTERT promoter to understand the transcriptional repression mechanism of the hTERT gene in normal human somatic cells. Through the serial deletion analysis of hTERT promoter in normal human fibroblasts, we identified a critical repressive element on the hTERT promoter. The repressive element formed DNA-protein complexes with Sp1 and Sp3 in nuclear extracts. Using formaldehyde cross-linked chromatin immunoprecipitation analysis, we found that Sp1 and Sp3 were associated with the endogenously repressed hTERT promoter in human fibroblasts. Furthermore, Sp1 and Sp3 interacted with histone deacetylase (HDAC) in these cells. Overexpression of dominant-negative mutants of Sp1 and Sp3, which contained mainly the HDAC2-binding domain, relieved the HDAC-mediated repression of the hTERT promoter. Taken together, these results suggest that Sp1 and Sp3 associate with the hTERT promoter, recruiting HDAC for the localized deacetylation of nucleosomal histones and transcriptional silencing of the hTERT gene in normal human somatic cells.
Assuntos
Histona Desacetilases/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Telomerase/genética , Transcrição Gênica , Sequência de Bases , Células Cultivadas , Cromatina/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Proteínas de Ligação a DNA , Fibroblastos/citologia , Fibroblastos/fisiologia , Inativação Gênica , Genes Reporter , Humanos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Elementos de Resposta/genética , Telomerase/metabolismoRESUMO
Activation of telomerase is crucial for cells to gain immortality. Most normal human somatic cells have a limited proliferative life span, and expression of the rate-limiting telomerase catalytic subunit, known as human telomerase reverse transcriptase (hTERT), has been believed to be tightly repressed. This model of hTERT regulation is challenged by the recent identification of the induction of hTERT in normal cycling human fibroblasts during their transit through S phase. Here we show the small-molecule-based identification of the assembly and disassembly of E2F-pocket protein-histone deacetylase (HDAC) complex as a key mechanistic basis for the repression and activation of hTERT in normal human cells. A cell-based chemical screen was used to identify a small molecule, CGK1026, that derepresses hTERT expression. CGK1026 inhibits the recruitment of HDAC into E2F-pocket protein complexes assembled on the hTERT promoter. Chromatin immunoprecipitation analysis reveals dynamic alterations in hTERT promoter occupancy by E2F and pocket proteins according to the cell cycle-dependent regulation of hTERT. Dominant-negative or protein-knockout strategies to disrupt the assembly of E2F-pocket protein-HDAC complex derepress hTERT and telomerase activity. Taken together with the results on the regulatory function of these complexes in cellular senescence and tumorigenesis, our findings suggest that dynamic assembly of E2F-pocket protein-HDAC complex plays a central role in the regulation of hTERT in a variety of proliferative conditions (e.g., normal cycling, senescent, and tumor cells).
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/metabolismo , Telomerase/metabolismo , Fatores de Transcrição/metabolismo , Divisão Celular/fisiologia , Linhagem Celular , Senescência Celular/fisiologia , Fatores de Transcrição E2F , Indução Enzimática/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Teste de Complementação Genética , Humanos , Regiões Promotoras Genéticas , Telomerase/genéticaRESUMO
Interferon regulatory factor 3 (IRF3) is activated in response to various environmental stresses including viral infection and DNA-damaging agents. However, the biological function of IRF3 in cell growth is not well understood. We demonstrated that IRF3 markedly inhibited growth and colony formation of cells. IRF3 blocked DNA synthesis and induced apoptosis. Based on this negative control of cell growth by IRF3, we examined whether functional loss of IRF3 may contribute to oncogenic transformation. IRF3 activity was specifically inhibited by expression of its dominant negative mutant. This mutant lacks a portion of the DNA binding domain like IRF3a, an alternative splice form of IRF3 in the cells. This dominant negative inhibition blocked expression of specific IRF3 target genes. Mutant IRF3 efficiently transformed NIH3T3 cells, as demonstrated by anchorage-independent growth in soft agar and tumorigenicity in nude mice. These results imply that IRF3 may function as a tumor suppressor and suggest a possible role for the relative levels of IRF3 and its dominant negative mutant in tumorigenesis.