RESUMO
Increased histone acetylation has been associated with activated gene transcription and decreased acetylation with repression. However, there is a growing number of genes known, which are downregulated by histone deacetylase (HDAC) inhibitors through unknown mechanisms. This study examines the mechanism by which the mouse mammary tumor virus (MMTV) promoter is repressed by the HDAC inhibitor, trichostatin A (TSA). We find that this repression is transcriptional in nature and that it occurs in the presence and absence of glucocorticoids. TSA decreases MMTV transcription at a rapid rate, reaching maximum in 30-60 min. In contrast with previous reports, the repression does not correlate with an inhibition of glucocorticoid-induced nuclease hypersensitivity or NF1-binding at the MMTV promoter. Surprisingly, TSA does not induce sizable increases in histone acetylation at the MMTV promoter nor does it inhibit histone deacetylation, which accompanies deactivation of the glucocorticoid-activated MMTV promoter. Repression of MMTV transcription by TSA does not depend on the chromatin organization of the promoter because a transiently transfected MMTV promoter construct with a disorganized nucleoprotein structure was also repressed by TSA treatment. Mutational analysis of the MMTV promoter indicates that repression by TSA is mediated through the TATA box region. These results suggest a novel mechanism that involves acetylation of nonhistone proteins necessary for basal transcription.
Assuntos
Cromatina/ultraestrutura , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Vírus do Tumor Mamário do Camundongo/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Adenocarcinoma/patologia , Animais , Transformação Celular Viral , Cromatina/efeitos dos fármacos , Dexametasona/farmacologia , Feminino , Genes Reporter , Neoplasias Mamárias Experimentais/patologia , Camundongos , Nucleossomos/efeitos dos fármacos , Nucleossomos/ultraestrutura , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/biossíntese , Deleção de Sequência , TATA Box , Sequências Repetidas Terminais , TransfecçãoRESUMO
Repression and activation of the expression of homeotic genes are maintained by proteins encoded by the Polycomb group (PcG) and trithorax group (trxG) genes. Complexes formed by these proteins are targeted by PcG or trxG response elements (PREs/TREs), which share binding sites for several of the same factors. GAGA factor and Zeste bind specifically to PREs/TREs and have been shown to act as both activators and repressors. We have used purified proteins and complexes reconstituted from recombinant subunits to characterize the effects of GAGA and Zeste proteins on PcG function using a defined in vitro system. Zeste directly associates with the PRC1 core complex (PCC) and enhances the inhibitory activity of this complex on all templates, with a preference for templates with Zeste binding sites. GAGA does not stably associate with PCC, but nucleosomal templates bound by GAGA are more efficiently bound and more efficiently inhibited by PCC. Thus Zeste and GAGA factor use distinct means to increase repression mediated by PRC1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Proteínas de Homeodomínio/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Plasmídeos/genética , Complexo Repressor Polycomb 1 , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The nucleoprotein structure of the mouse mammary tumor virus (MMTV) promoter defines its response to cAMP signaling. A stably replicating MMTV template in highly organized chromatin is repressed in the presence of cAMP, whereas a transiently transfected template with a disorganized structure is activated. In this study, we investigate the nature of the cAMP-induced signal(s) by which these opposing responses occur to gain insight into their mechanism. We demonstrate that the transcriptional changes observed at both templates are mediated through cAMP-dependent protein kinase A (PKA). In addition, the MMTV promoter lacks a consensus cAMP response element (CRE) and neither template requires cAMP response element-binding protein (CREB) to elicit a response to cAMP signaling. However, the responses of the two templates differ mechanistically in that the CREB-binding protein p300 potentiates activation from the transient template in a manner dependent on its Cys/His-rich region 3, but does not appear to affect the repression of the replicating chromatin template. Chromatin immunoprecipitation assays show that cAMP treatment results in a decrease in acetylation of histone H4, and in multiple modifications of histone H3 at specific nucleosomes in the promoter region of the stable MMTV template. These findings suggest novel CREB-independent, chromatin-dependent pathways for transcriptional regulation by cAMP.