RESUMO
The SWI-SNF and SAGA complexes possess ATP-dependent nucleosome remodelling activity and histone acetyltransferase (HAT) activity, respectively. Mutations that eliminate the ATPase activity of the SWI-SNF complex, or the HAT activity of SAGA, abolish proper chromatin remodelling at the PHO8 promoter in vivo. These effects are mechanistically distinct, since the absence of SWI-SNF freezes chromatin in the repressed state, while the absence of Gcn5 permits a localized perturbation of chromatin structure immediately adjacent to the upstream transactivator binding site. However, this remodelling is not propagated to the proximal promoter, and no activation is observed under all conditions. Furthermore, Pho4 is bound to the PHO8 promoter in the absence of Snf2 or Gcn5, confirming a role for SWI-SNF and SAGA in chromatin remodelling independent of activator binding. These data provide new insights into the roles of the SWI-SNF and SAGA complexes in chromatin remodelling in vivo.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromatina/fisiologia , Proteínas Fúngicas/metabolismo , Regiões Promotoras Genéticas , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Acetiltransferases/metabolismo , Sítios de Ligação , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Histone acetyltransferase (HAT) activity has been demonstrated for several transcriptional activators, formally connecting chromatin modification with gene regulation. However, no effect on chromatin has been demonstrated. We have investigated the role of the HAT Gcn5 at the nucleosomally regulated PHO5 promoter. Under conditions of constitutive submaximal activation (i.e., in the absence of the negative regulator Pho80), deletion of Gcn5 determines a novel randomized nucleosomal organization across the promoter and leads to a dramatic reduction in activity. Furthermore, mutation of amino acids critical for Gcn5 HAT activity is sufficient to generate this structure. This intermediate state in chromatin opening gives way to the fully open structure upon maximal induction (phosphate starvation), even in the absence of Gcn5. Thus, Gcn5 is shown to affect directly the remodeling of chromatin in vivo.
