RESUMO
Differentiation of skeletal muscle cells and B lymphocytes is regulated by basic helix-loop-helix (bHLH) proteins. Both differentiation programs are inhibited by the adenovirus E1A oncoprotein. Analysis of E1A mutants has implicated two of its cellular-binding proteins, p300 and CBP, in controlling certain aspects of differentiation. We find that p300 can cooperate with tissue-specific bHLH proteins in activating target genes and requires only the bHLH domain of such proteins to stimulate E box-directed transcription. Importantly, the ability of bHLH proteins to activate transcription correlates with the presence of p300/CBP in E box-dependent DNA-binding complexes, because both phenomena require at least two adjacent E-box motifs. Microinjection of p300/CBP antibodies into myoblasts blocks terminal differentiation, cell fusion, and transcriptional activity of myogenic bHLH proteins. These results suggest that the function of p300/CBP is essential for the execution of key aspects of cellular differentiation.
Assuntos
Linfócitos B/citologia , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Alça-Hélice , Músculo Esquelético/citologia , Proteínas Nucleares/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Proteína de Ligação a CREB , Diferenciação Celular , Extratos Celulares , Fusão Celular/fisiologia , DNA/metabolismo , DNA Recombinante , Proteínas de Ligação a DNA/análise , Proteína p300 Associada a E1A , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Fatores de Transcrição MEF2 , Camundongos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas , Músculo Esquelético/crescimento & desenvolvimento , Proteína MyoD/análise , Proteína MyoD/metabolismo , Fatores de Regulação Miogênica , Miogenina/análise , Miogenina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional/fisiologiaRESUMO
The transcription factor ISGF3 transduces interferon (IFN)-alpha signals and activates the transcription of cellular antiviral defence genes. Adenovirus E1A blocks the IFN-alpha response, allowing unhindered viral replication. ISGF3 consists of Stat1, Stat2 and p48. Here we show that p300 and/or CBP (CREB-binding protein), which are transcription adaptors targeted by E1A, interact specifically with Stat2. Binding occurs between the first cysteine-histidine-rich region of p300/CBP and the carboxy-terminal segment of Stat2, a domain essential for ISGF3 function. We find that this domain of Stat2 has transactivation potential, which correlates with its binding to p300/CBP. Moreover, E1A represses Stat2 transactivation and IFN-alpha-activated transcription by inhibiting p300/CBP function. This provides a new mechanism for inhibition of the IFN-alpha-activated antiviral response by E1A, and supports the view that E1A binding to p300/CBP has functional significance for adenovirus replication in its natural host.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interferon-alfa/fisiologia , Proteínas Nucleares/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Adenoviridae/fisiologia , Proteínas E1A de Adenovirus/metabolismo , Sítios de Ligação , Proteína de Ligação a CREB , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Fator Gênico 3 Estimulado por Interferon , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Ligação Proteica , Fator de Transcrição STAT2 , Transativadores/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Replicação ViralRESUMO
p300 and the CREB-binding protein CBP are two large nuclear phosphoproteins that are structurally highly related. Both function, in part, as transcriptional adapters and are targeted by the adenovirus E1A oncoprotein. We show here that p300 and CBP interact with another transforming protein, the simian virus 40 large T antigen (T). This interaction depends on the integrity of a region of T which is critical for its transforming and mitogenic properties and includes its LXCXE Rb-binding motif. T interferes with normal p300 and CBP function on at least two different levels. The presence of T alters the phosphorylation states of both proteins and inhibits their transcriptional activities on certain promoters. Although E1A and T show little sequence similarity, they interact with the same domain of p300 and CBP, suggesting that this region exhibits considerable flexibility in accommodating diverse protein ligands.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Proteínas Nucleares/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Antígenos Transformantes de Poliomavirus/isolamento & purificação , Sequência de Bases , Sítios de Ligação , Western Blotting , Proteína de Ligação a CREB , Divisão Celular , Proteína p300 Associada a E1A , Eletroforese em Gel de Poliacrilamida , Embrião de Mamíferos , Fibroblastos , Glutationa Transferase , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/biossíntese , Proteínas Nucleares/isolamento & purificação , Oligodesoxirribonucleotídeos , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/isolamento & purificação , TransfecçãoRESUMO
The cellular protein p300 is a target of the adenoviral E1A oncoprotein and is thought to participate in preventing the G0/G1 transition in the cell cycle, activating certain enhancers and stimulating differentiation pathways. CBP is a protein that is associated with and coactivates the transcription factor CREB, mediating the induction by cyclic AMP of certain responsive promoters. The sequences of p300 and CBP are highly related. We show here that p300, like CBP2, can stimulate transcription. This activity is directly and specifically inhibited by E1A. We also find that CBP exists in a DNA-bound complex containing a member of the CREB family and that E1A and CBP interact with one another in vivo. In keeping with the idea that E1A functionally targets CBP, cAMP-dependent transcription is repressed by E1A. Thus, p300 and CBP define a family of transcriptional adaptor proteins that are specifically targeted by the E1A oncoprotein.