RESUMO
Glucocorticoids influence numerous cell functions by regulating gene activity. The glucocorticoid receptor (GR) is a ligand-activated transcription factor and, like any other transcription factor, does not modulate gene activity just by binding to DNA. Interaction with other proteins is probably required to enhance the establishment of a functional transcription initiation complex. To identify such proteins, we analyzed the in vitro interaction of the glucocorticoid receptor bound to a double glucocorticoid response element with nuclear proteins and describe here three interacting proteins with different molecular weights. One of them, which we named GRIP 170 (GR-interacting protein), was purified and microsequenced, and it turned out to be an unknown protein. When tested in a cell-free transcription assay, the fraction highly enriched for GRIP 170 does not influence basal promoter activity but does enhance GR induction.
Assuntos
Proteínas de Transporte/metabolismo , Dexametasona/farmacologia , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Núcleo Celular/metabolismo , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Células HeLa , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/isolamento & purificação , Moldes Genéticos , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica/efeitos dos fármacosRESUMO
The influence of progesterone receptor (PR) and glucocorticoid receptor (GR) on transcription from the mouse mammary tumour virus (MMTV) promoter was analyzed using cell-free transcription of DNA templates with a G-free cassette. Preincubation of the templates with either PR or GR stimulates the rate of transcription initiation 10-50 fold, whereas the recombinant DNA binding domain of GR is inactive. Mutations that inactivate the nuclear factor I (NFI) binding site, or NFI depletion of the nuclear extract, decrease basal transcription without influencing receptor-dependent induction. Recombinant NFI, but not its DNA-binding domain, restores efficient basal transcription of the depleted extract. Recombinant OTF1 or OTF2, but not the POU domain of OTF1, enhance MMTV transcription independently of NF1. In agreement with this finding, NFI and OTF1 do not cooperate, but rather compete for binding to the wild type MMTV promoter, though they have the potential to bind simultaneously to properly oriented sites. Our results imply the existence of two independent pathways for MMTV transcription: one initiated by NFI and the other dependent on octamer transcription factors. Only the second pathway is stimulated by steroid hormone receptors in vitro.