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1.
Nature ; 520(7547): 378-82, 2015 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-25624101

RESUMO

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.


Assuntos
Transformação Celular Neoplásica , Interações Hospedeiro-Parasita , Leucócitos/patologia , Peptidilprolil Isomerase/metabolismo , Theileria/enzimologia , Theileria/patogenicidade , Animais , Bovinos , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Resistência a Medicamentos/genética , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/parasitologia , Peptidilprolil Isomerase de Interação com NIMA , Naftoquinonas/farmacologia , Parasitos/efeitos dos fármacos , Parasitos/enzimologia , Parasitos/patogenicidade , Peptidilprolil Isomerase/antagonistas & inibidores , Peptidilprolil Isomerase/genética , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais/efeitos dos fármacos , Theileria/efeitos dos fármacos , Theileria/genética , Fator de Transcrição AP-1/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/embriologia
2.
Nat Genet ; 25(3): 338-42, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10888886

RESUMO

Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA , Histona Desacetilases/metabolismo , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células 3T3 , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Células HeLa , Histona Desacetilase 1 , Histona Desacetilases/genética , Humanos , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta , Proteína do Retinoblastoma/genética , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética
3.
Mol Cell Biol ; 21(19): 6484-94, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11533237

RESUMO

The E2F transcription factor controls the cell cycle-dependent expression of many S-phase-specific genes. Transcriptional repression of these genes in G(0) and at the beginning of G(1) by the retinoblasma protein Rb is crucial for the proper control of cell proliferation. Rb has been proposed to function, at least in part, through the recruitment of histone deacetylases. However, recent results indicate that other chromatin-modifying enzymes are likely to be involved. Here, we show that Rb also interacts with a histone methyltransferase, which specifically methylates K9 of histone H3. The results of coimmunoprecipitation experiments of endogenous or transfected proteins indicate that this histone methyltransferase is the recently described heterochromatin-associated protein Suv39H1. Interestingly, phosphorylation of Rb in vitro as well as in vivo abolished the Rb-Suv39H1 interaction. We also found that Suv39H1 and Rb cooperate to repress E2F activity and that Suv39H1 could be recruited to E2F1 through its interaction with Rb. Taken together, these data indicate that Suv39H1 is involved in transcriptional repression by Rb and suggest an unexpected link between E2F regulation and heterochromatin.


Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Histona-Lisina N-Metiltransferase , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteína do Retinoblastoma/fisiologia , Divisão Celular , Linhagem Celular , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Células HeLa , Heterocromatina/metabolismo , Histona Metiltransferases , Humanos , Células Jurkat , Metiltransferases/fisiologia , Modelos Genéticos , Fosforilação , Regiões Promotoras Genéticas , Proteínas Metiltransferases , Estrutura Terciária de Proteína , Proteínas Repressoras/fisiologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Transcrição Gênica
4.
Biomed Pharmacother ; 60(9): 633-8, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16962735

RESUMO

MicroRNAs (miRNAs) have recently emerged as being essential for development and for the control of cell proliferation/differentiation in various organisms. However, little is known about miRNA function and mode of action at the cellular level. We have designed a miRNA loss-of-function assay, based on chemically modified locked nucleic acids (LNA) antisense oligonucleotides and usable in tissue culture cells. We show that LNA/DNA mixed oligonucleotides form highly stable duplexes with miRNAs in vitro. Ex vivo, the target miRNA becomes undetectable in cells transfected with the antisense oligonucleotide. The effect is dose-dependent, long-lasting, and specific. Moreover, using a reporter assay, we show that antisense LNA/DNA oligonucleotides inhibit short non-coding RNAs at the functional level. Thus LNA/DNA mixmers represent powerful tools for functional analysis of miRNAs.


Assuntos
MicroRNAs/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Células Cultivadas , Humanos , MicroRNAs/antagonistas & inibidores , Oligonucleotídeos
5.
Nucleic Acids Res ; 29(15): 3131-6, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11470869

RESUMO

The product of the retinoblastoma susceptibility gene, the Rb protein, functions partly through transcriptional repression of E2F-regulated genes. Repression by Rb is mediated, at least in part, by a histone deacetylase complex, whose enzymatic activity relies on HDAC1, HDAC2 or HDAC3. Recently, we have shown that the Rb-associated histone deacetylase complex contains RbAp48 protein, which interacts with HDAC1 and HDAC2. RbAp48 could favour the deacetylation of histones since it binds directly to histone H4. In agreement with that, we show that transcriptional repression of E2F activity requires the presence of RbAp48. HDAC3 was thought not to interact with RbAp48. However, we found that it shared with HDAC1 the ability to favour the recruitment of RbAp48 to Rb. This latter effect was unlikely to be due to activation of Rb function, since HDAC3 did not increase Rb-E2F1 interaction. Rather, we found, surprisingly, that HDAC3 could physically interact with RbAp48 both in vitro and in living cells. Taken together, our data suggest a model in which Rb mediates the recruitment to E2F-regulating promoters of a repressive complex containing either HDAC1, HDAC2 or HDAC3 and the histone-binding protein RbAp48.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Proteínas de Transporte/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Genes Reporter/genética , Histona Desacetilases/genética , Humanos , Camundongos , Modelos Genéticos , Proteínas Nucleares/genética , Testes de Precipitina , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Proteína 4 de Ligação ao Retinoblastoma , Transfecção , Células Tumorais Cultivadas
6.
Nucleic Acids Res ; 27(20): 3995-4000, 1999 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-10497263

RESUMO

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a chimera peptide-TFO able to activate transcription from a target gene. The designed hybrid molecule contains a triplex-forming sequence, linked through a phosphoroamidate bond to several minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). We show here that this TFO-peptide chimera (TFO-P) can specifically recognise its DNA target at physiological salt and pH conditions. Bound to the double-stranded target DNA in a promoter region, the TFO-P is able to activate gene expression. Our results suggest that this type of molecule may prove useful in the design of new tools for artificial modulation of gene expression.


Assuntos
Regulação da Expressão Gênica , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Oligonucleotídeos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ativação Transcricional
7.
Oncogene ; 20(24): 3128-33, 2001 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-11420729

RESUMO

The balance between cell differentiation and proliferation is regulated at the transcriptional level. In the cell cycle, the transition from G1 to S phase (G1/S transition) is of paramount importance in this regard. Indeed, it is only before this point that cells can be oriented toward the differentiation pathway: beyond, cells progress into the cycle in an autonomous manner. The G1/S transition is orchestrated by the transcription factor E2F. E2F controls the expression of a group of checkpoint genes whose products are required either for the G1-to-S transition itself or for DNA replication (e.g. DNA polymerase alpha). E2F activity is repressed in growth-arrested cells and in early G1, and is activated at mid-to-late G1. E2F is controlled by the retinoblastoma tumor suppressor protein Rb. Rb represses E2F mainly by recruiting chromatin remodeling factors (histone deacetylases and SWI/SNF complexes), the DNA methyltransferase DNMT1, and a histone methyltransferase. This review will focus on the molecular mechanisms of E2F repression by Rb during the cell cycle and during cell-cycle exit by differentiating cells. A model in which Rb irreversibly represses E2F-regulated genes in differentiated cells by an epigenetic mechanism linked to heterochromatin, and involving histone H3 and promoter DNA methylation, is discussed.


Assuntos
Proteínas de Transporte , Cromatina/genética , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Proteína do Retinoblastoma/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Divisão Celular , Cromatina/metabolismo , Fatores de Transcrição E2F , Humanos , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Oncogene ; 15(14): 1661-9, 1997 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-9349499

RESUMO

TCFs, which are members of the Ets family of transcription factors, are recruited to the Serum Response Element (SRE) in the c-fos promoter by SRF. These Ets proteins, which are substrates for the MAP kinases, are direct targets of the Ras/MAP kinase signal transduction pathway. In this paper, we demonstrate that one of the TCFs, SAP-1a, displays a significant level of autonomous binding to the SRE Ets box. In contrast to previous observations, deletion of the SRF binding domain did not modulate the autonomous binding of SAP-1a. Also, the autonomous binding was not modulated by the phosphorylation of SAP-1a by MAP kinases. The autonomous binding was also detected in live cells: transfected SAP-1a was able to restore the response of a CArG-less SRE in PC12 cells. The response occurred in the absence of SRF recruitment since a mutant of SAP-1a in which the B-box, a domain required for interaction with SRF, had been deleted was still able to transactivate the CArG-less SRE. The transactivation was repressed by a Ras transdominant negative mutant, indicating the involvement of the Ras/MAP kinase pathway. Taken together, these data demonstrate that SAP-1a is capable of binding to the c-fos SRE in the absence of SRF.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Genes fos , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Nucleares/metabolismo , Células PC12 , Fosforilação , Ratos , Proteínas Recombinantes , Sequências Reguladoras de Ácido Nucleico , Fator de Resposta Sérica , Transfecção , Proteínas Elk-4 do Domínio ets
9.
Oncogene ; 19(20): 2430-7, 2000 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-10828885

RESUMO

Transforming viral proteins such as E1A which force quiescent cells into S phase have two essential cellular target proteins, Rb and CBP/p300. Rb regulates the G1/S transition by controlling the transcription factor E2F. CBP/p300 is a transcriptional co-activator with intrinsic histone acetyl-transferase activity. This activity is regulated in a cell cycle dependent manner and shows a peak at the G1/S transition, suggesting a function for CBP/p300 in this crucial step of the cell cycle. Here, we have artificially modulated CBP/p300 levels in individual cells through microinjection of specific antibodies and expression vectors. We show that CBP/p300 is required for cell proliferation and has an essential function during the G1/S transition. Using the same microinjection system and GFP-reporter vectors, we demonstrate that CBP/p300 is essential for the activity of E2F, a transcription factor that controls the G1/S transition. In addition, our results suggest that CBP HAT activity is required both for the G1/S transition and for E2F activity. Thus CBP/p300 seems to be a versatile protein involved in opposing cellular processes, which raises the question of how its multiple activities are regulated.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Transporte , Proteínas de Ciclo Celular/metabolismo , Fase G1 , Fase S , Células 3T3 , Acetiltransferases/genética , Animais , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Histona Acetiltransferases , Camundongos , Mutagênese , Ligação Proteica , Proteína 1 de Ligação ao Retinoblastoma , Deleção de Sequência , Proteína de Ligação a TATA-Box , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Transcrição de p300-CBP
10.
Oncogene ; 18(20): 3143-51, 1999 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-10340386

RESUMO

Thrombospondin 1 (TSP1) is known for its significant anti-angiogenic properties. In a previous study, we have shown that transient or stable overexpression of the transcription factor c-Jun, in rat fibroblasts, leads to repression of TSP1. We now demonstrate that the c-Jun-induced repression of TSP1 does not occur directly and does not require binding of c-Jun to the TSP1 promoter. Instead, repression involves a factor secreted by c-Jun-overexpressing cells. This secreted factor triggers a signal transduction pathway from the membrane to the nucleus, and these signals lead to the binding of the product of the Wilms' tumor suppressor gene, WT1, to the -210 region of the TSP1 promoter. This region binds WT1 and SP1, but not EGR1, although its sequence fits the consensus binding site for this transcription factor. WT1 overexpression in transfected cells inhibits endogenous TSP1 gene expression and TSP1 transcription in experiments using TSP1 promoter-reporter constructs. The WT1 - KTS isoform is more active in repressing TSP1 transcription than WT1 + KTS, while EGR1 is inactive. Enhancement of WT1 binding to DNA in response to c-Jun does not require de novo protein synthesis. The above mechanism for TSP1 repression could apply to other genes, thus coordinating their regulation in the vicinity of a c-Jun-overexpressing cell. We conclude that WT1, which was discovered as a result of its tumor suppressor properties, may also possess oncogenic characteristics in the c-Jun transformation process, and thus repress the anti-angiogenic protein, TSP1.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Proteínas Proto-Oncogênicas c-jun/genética , Trombospondina 1/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Animais , Linhagem Celular , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas WT1
11.
Gene Expr ; 8(1): 33-42, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10543729

RESUMO

The serum response element (SRE) in the c-fos promoter contains an ets box whose integrity is required for full activation of this proto-oncogene by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. Electrophoretic mobility shift assays (EMSA) detect a protein in nuclear extracts that binds to the wild-type SRE, but not to an SRE containing a mutated ets box. Competition studies using unlabeled probes, and supershift experiments using antibodies and in vitro translated core serum response factor (SRF) indicate that the protein in question is not YY1, SAP-1, nor Elk-1 and that it does not exhibit ternary complex factor (TCF) activity, so that it may correspond to an autonomously binding Ets family protein. The complete disappearance of this "Ets-like autonomous binding factor" upon terminal differentiation of both L6alpha2 myoblastic and PC12 pheochromocytoma cells points to a possible role in the proliferation/differentiation process.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Elementos de Resposta , Células 3T3 , Animais , Diferenciação Celular , Divisão Celular , Regulação para Baixo , Camundongos , Fator de Crescimento Neural/metabolismo , Células PC12 , Ligação Proteica , Ratos , Fator de Resposta Sérica , Células Tumorais Cultivadas
12.
J Biol Chem ; 272(49): 31016-21, 1997 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-9388250

RESUMO

The serum response element is one of the major promoter elements of the immediate early response to extracellular signals. The serum response element includes two main binding sites for proteins: the Ets box, which binds p62(TCF), and the CArG box, which binds p67(SRF). These two proteins are direct targets for signal transduction pathways; p62(TCF) is a nuclear end point of the Ras/mitogen-activated protein kinase pathway, and p67(SRF) is targeted by the Rho/Rac small G-proteins. The mechanism by which the signal is further transduced from the transcription factors to the basal transcriptional machinery is poorly understood. Recent data have suggested that the cAMP-responsive element-binding protein (CREB)-binding protein, a transcriptional adaptor involved in the transactivation through a wide variety of enhancer elements, participates in p62(TCF) activity. We here show that the CREB-binding protein also cooperates in the process of transactivation by p67(SRF). Cotransfections of expression vectors for the CREB-binding protein increased the expression, in response to serum, of reporters under the control of the c-fos serum response element. Interestingly, the C-terminal moiety of the CREB-binding protein was not necessary to observe this effect. The cooperation did not require the Ets box in the serum response element, and the CArG box was sufficient, indicating that the CREB-binding protein is able to cooperate with p67(SRF) in the absence of an Ets protein. Co-immunoprecipitation experiments using cell extracts showed that p67(SRF) could be retained with antibodies directed against the CREB-binding protein, suggesting that the two proteins form a multimolecular complex in live cells. The physical interaction between p67(SRF) and the CREB-binding protein was further confirmed by two-hybrid assays in mammalian cells. Our results indicate that the CREB-binding protein cooperates with p67(SRF) and, thus, suggest that the serum response element is regulated by a multimolecular complex, which includes the CREB-binding protein, p67(SRF), and p62(TCF), with multiple interactions between the components of the complex.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células 3T3 , Animais , Sequência de Bases , Proteína de Ligação a CREB , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , Fator de Resposta Sérica
13.
Prog Cell Cycle Res ; 4: 41-7, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10740813

RESUMO

The critical steps of the cell cycle are generally controlled through the transcriptional regulation of specific subsets of genes. Transcriptional regulation has been recently linked to acetylation or deacetylation of core histone tails: acetylated histone tails are generally associated with active chromatin, whereas deacetylated histone tails are associated with silent parts of the genome. A number of transcriptional co-regulators are histone acetyl-transferases or histone deacetylases. Here, we discuss some of the critical cell cycle steps in which these enzymes are involved.


Assuntos
Ciclo Celular/fisiologia , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Acetilação , Acetiltransferases/metabolismo , Animais , Histona Acetiltransferases , Histona Desacetilases/metabolismo , Transcrição Gênica
14.
Semin Cell Dev Biol ; 10(2): 197-203, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10441073

RESUMO

Cell fate is determined by extracellular signals which are transmitted to the nucleus and result in the transcriptional regulation of specific subsets of genes. Transcriptional regulation has been recently linked to enzymatic activities which are able to acetylate or deacetylate core histone tails. A number of transcriptional co-regulators are histone acetyl-transferases or histone deacetylases. Here, we discuss the involvement of these enzymes in critical steps of cell proliferation or cell differentiation control


Assuntos
Acetiltransferases/fisiologia , Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Regulação da Expressão Gênica/fisiologia , Substâncias de Crescimento/fisiologia , Histona Desacetilases/fisiologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais/fisiologia , Acetilação , Animais , Ciclo Celular , Diferenciação Celular , Divisão Celular , Transformação Celular Neoplásica , Cromatina/metabolismo , Fatores de Transcrição E2F , Regulação da Expressão Gênica/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Histona Acetiltransferases , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/fisiologia , Proteína do Retinoblastoma/fisiologia , Proteína 1 de Ligação ao Retinoblastoma , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição DP1 , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Tretinoína/farmacologia
15.
Nucleic Acids Res ; 26(16): 3869-70, 1998 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-9685509

RESUMO

Histone acetyl-transferases (HATs) seem to be key elements in the regulation of transcription. We have designed an enzymatic assay to quantify HAT enzymatic activity. In this assay, the substrate is a peptide corresponding to the 24 first amino acids of histone H4 which is coupled to biotin. After acetylation using [14C]acetyl-CoA, the peptide is purified on streptavidin beads and the associated radioactivity is measured. This assay is sensitive, rapid and convenient.


Assuntos
Acetiltransferases/análise , Proteínas de Saccharomyces cerevisiae , Células 3T3 , Acetilcoenzima A , Acetilação , Acetiltransferases/isolamento & purificação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Biotina , Radioisótopos de Carbono , Histona Acetiltransferases , Histonas/química , Histonas/genética , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Sensibilidade e Especificidade , Estreptavidina , Especificidade por Substrato
16.
J Biol Chem ; 276(23): 20743-9, 2001 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-11278800

RESUMO

CpG methylation is maintained in daughter chromatids by the action of DNA methyltransferase at the replication fork. An opportunity exists for transcription factors at replication forks to bind their cognate sequences and thereby prevent remethylation by DNA methyltransferase. To test this hypothesis, we injected a linearized, methylated, and partially single-stranded reporter plasmid into the nuclei of Xenopus oocytes and followed changes in the transcriptional activity after DNA replication. We find that dependent on Gal4-VP16, the action of DNA methyltransferase, and replication-coupled chromatin assembly DNA replication provides a window of time in which regulatory factors can activate or repress gene activity. Demethylation in the promoter region near the GAL4 binding sites of the newly synthesized DNA did not occur even though the Gal4 binding sites were occupied and transcription was activated. We conclude that "passive" demethylation at the replication fork is not simply dependent on the presence of DNA binding transcriptional activators.


Assuntos
Metilação de DNA , DNA/biossíntese , Transcrição Gênica , Animais , Sequência de Bases , DNA/genética , Primers do DNA , Ligação Proteica , Transativadores/metabolismo , Xenopus
17.
EMBO Rep ; 2(9): 794-9, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11520855

RESUMO

The transcription factor E2F, which is a key element in the control of cell proliferation, is repressed by Rb and other pocket proteins in growth-arrested differentiating cells, as well as in proliferating cells when they progress through early G1. It is not known whether similar mechanisms are operative in the two situations. A body of data suggests that E2F repression by pocket proteins involves class I histone deacetylases (HDACs). It has been hypothesized that these enzymes are recruited to E2F target promoters where they deacetylate histones. Here we have tested this hypothesis directly by using formaldehyde cross-linked chromatin immunoprecipitation (XChIP) assays to evaluate HDAC association in living cells. Our data show that a histone deacetylase, HDAC-1, is stably bound to an E2F target promoter during early G1 in proliferating cells and released at the G1-S transition. In addition, our results reveal an inverse correlation between HDAC-1 recruitment and histone H4 acetylation on specific lysines.


Assuntos
Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Proteínas de Ligação a DNA , Histona Desacetilases/metabolismo , Histonas/metabolismo , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Acetilação , Animais , Northern Blotting , Western Blotting , Divisão Celular , Células Cultivadas , Cromatina/metabolismo , Fatores de Transcrição E2F , Histona Desacetilase 1 , Lisina/química , Camundongos , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Tempo
18.
Nucleic Acids Res ; 25(17): 3459-64, 1997 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-9254704

RESUMO

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a hairpin-TFO able to recruit transcription factors to a target DNA. The designed oligonucleotide contains a triplex-forming sequence, linked through a nucleotide loop to a double-stranded hairpin including the SRE enhancer of the c-fos gene promoter. We show here that this oligonucleotide can specifically recognise its DNA target at physiological salt and pH conditions. The stability of the triplex formed under these conditions is very high: >90% of the triplex remains intact after 24 h of incubation. Bound to the double-stranded target DNA, the oligonucleotide retains its ability to interact specifically with transcription factors, recruiting them to the proximity of the target DNA. Our results suggest that this type of oligonucleotide may prove useful in the design of new tools for artificial modulation of gene expression.


Assuntos
DNA/metabolismo , Oligonucleotídeos/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , DNA Viral/química , DNA Viral/metabolismo , Genes fos , HIV-2/genética , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Proteínas dos Retroviridae/genética , Vírus da Imunodeficiência Símia/genética , Proteínas Virais Reguladoras e Acessórias/genética
19.
Proc Natl Acad Sci U S A ; 97(7): 3084-8, 2000 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-10716704

RESUMO

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.


Assuntos
DNA Viral/química , Mutação , Conformação de Ácido Nucleico , Sequência de Bases , DNA Viral/genética , Proteínas Fúngicas/genética , HIV-1/genética , HIV-2/genética , Plasmídeos
20.
Biochem Biophys Res Commun ; 262(1): 157-62, 1999 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-10448085

RESUMO

The transcriptional coactivator CBP displays an intrinsic histone acetyl transferase (HAT) activity which seems to participate in transcriptional activation through the destabilization of nucleosome structure. CBP is involved in the activity of several transcription factors that are nuclear endpoints of intracellular signal transduction pathways. In some instances, the transcription factors are phosphorylated upon cell activation, which induces their interaction with CBP. CBP itself is a phosphoprotein and can be phosphorylated by cycle-dependent kinases or by MAP kinases. Here we show that CBP phosphorylation by p44 MAP kinase/ERK1 results in the stimulation of its HAT enzymatic activity. The p44 MAP kinase/ERK1 phosphorylation sites are located in the C-terminal part of the protein, outside of the HAT domain. These sites are required for enzymatic stimulation, suggesting that phosphorylation by p44 MAP kinase/ERK1 induces a conformational change of the CBP molecule. Our data suggest that, in some instances, CBP itself might be a target for signal transduction pathways.


Assuntos
Acetiltransferases/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Células 3T3 , Acetiltransferases/química , Trifosfato de Adenosina/metabolismo , Animais , Proteína de Ligação a CREB , Ativação Enzimática , Histona Acetiltransferases , Histonas/metabolismo , Camundongos , Proteína Quinase 3 Ativada por Mitógeno , Modelos Genéticos , Proteínas Nucleares/química , Nucleossomos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Transdução de Sinais , Fatores de Tempo , Transativadores/química
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