RESUMO
Terminal differentiation of muscle cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both muscle-specific and ubiquitous genes. Two distinct families of transcriptional co-activators, GCN5/PCAF and CREB-binding protein (CBP)/p300, are crucial to this process. While both possess histone acetyl-transferase (HAT) activity, previous studies have failed to identify a requirement for CBP/p300 HAT function in myogenic differentiation. We have addressed this issue directly using a chemical inhibitor of CBP/p300 in addition to a negative transdominant mutant. Our results clearly demonstrate that CBP/p300 HAT activity is critical for myogenic terminal differentiation. Furthermore, this requirement is restricted to a subset of events in the differentiation program: cell fusion and specific gene expression. These data help to define the requirements for enzymatic function of distinct coactivators at different stages of the muscle cell differentiation program.
Assuntos
Acetiltransferases/metabolismo , Músculos/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Relação Dose-Resposta a Droga , Proteína p300 Associada a E1A , Genes Dominantes , Genes Reporter , Histona Acetiltransferases , Imuno-Histoquímica , Camundongos , Microscopia de Fluorescência , Mutação , Miogenina/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , TransfecçãoRESUMO
The myogenic protein MyoD requires two nuclear histone acetyltransferases, CREB-binding protein (CBP)/p300 and PCAF, to transactivate muscle promoters. MyoD is acetylated by PCAF in vitro, which seems to increase its affinity for DNA. We here show that MyoD is constitutively acetylated in muscle cells. In vitro, MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located at the boundary of the DNA binding domain. MyoD acetylation by CBP/p300 (as well as by PCAF) increases its activity on a muscle-specific promoter, as assessed by microinjection experiments. MyoD mutants that cannot be acetylated in vitro are not activated in the functional assay. Our results provide direct evidence that MyoD acetylation functionally activates the protein and show that both PCAF and CBP/p300 are candidate enzymes for MyoD acetylation in vivo.
Assuntos
Proteína MyoD/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores/metabolismo , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Proteína p300 Associada a E1A , Histona Acetiltransferases , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Ativação TranscricionalRESUMO
Although the HIV gp120 binding site of CD4 is well characterized, its interaction site with HLA class II molecules is still controversial. Sixty-seven mutations within the four extracellular domains of CD4 were examined for their effects on cell surface expression and conformational changes and for adhesion of HLA class II-expressing B lymphocytes and HIV gp120 binding to CD4-transfected COS cells. Mutations within the gp120 binding site affected both assays similarly, indicating that the two sites fully overlap. A few additional substitutions of residues mapping on the same face of domains 1 and 2 induced decreased rosette formation. Molecular modeling studies indicated that this is likely to be the consequence of conformational changes induced by the mutations. Thus, CD4 appears to interact with HLA class II molecules mainly through the HIV gp120 binding site and possibly through a second minor interaction site mapping on the same face of the molecule.
