RESUMEN
The promoter of the gene encoding the inducible nitric oxide synthase (iNOS) contains an octamer motif which is of importance for its activation by specific stimuli. We show that in contrast to the promoter of the neuronal nitric oxide synthase gene (nNOS) which is strongly activated by the Oct-2 octamer-binding POU family transcription factor, the iNOS gene is only weakly activated by Oct-2 via its octamer motif. Unlike the nNOS promoter, however, the iNOS promoter is strongly activated by the POU family transcription factors Brn-3a and Brn-3b. This activation is dependent upon the octamer motif in the iNOS promoter and requires the activation domain located within the POU domain of Brn-3a or Brn-3b but not the N-terminal activation domain of Brn-3a. Thus different but related POU proteins play important roles in the regulation of the genes encoding different forms of nitric oxide synthase.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Homeodominio/fisiología , Óxido Nítrico Sintasa/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/fisiología , Animales , Sitios de Unión/genética , Línea Celular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Vectores Genéticos/genética , Proteínas de Homeodominio/genética , Mutación , Óxido Nítrico Sintasa de Tipo II , Proteínas Recombinantes de Fusión/genética , Factor de Transcripción Brn-3 , Factor de Transcripción Brn-3B , Factores de Transcripción/genéticaRESUMEN
The Oct-2 POU family transcription factor contains three distinct regions whose deletion reduces its ability to inhibit transcription via its octamer binding site. Here we show that only one of these inhibitory domains is capable of also inhibiting the activity of activating molecules bound at adjacent sites upstream of a TATA box-containing promoter whereas the other two regions are inactive in this assay. None of the three regions is able to achieve this effect when located upstream of the same promoter containing an initiator motif. The mechanisms of action of these domains and their role in the functioning of the Oct-2 factor are discussed.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae , Animales , Sitios de Unión , Línea Celular , Cloranfenicol O-Acetiltransferasa/biosíntesis , Clonación Molecular , Cricetinae , Cartilla de ADN , Proteínas Fúngicas/biosíntesis , Neuronas , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , TATA Box , Factores de Transcripción/química , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
The aim of this investigation was to elucidate the roles of insulin-like growth factor-I (IGF-I) and transferrin in the survival and proliferation of Chinese hamster ovary (CHO) cells upon withdrawal of serum. For this purpose, we employed DNA analysis and flow cytometry to compare CHO cell lines expressing either IGF-I alone or IGF-I and transferrin. The ability of cells to cycle and the occurrence of apoptosis were monitored in these cells in serum-free medium. These results indicate that IGF-I alone is able to maintain the viability of CHO cells for an extended length of time in the absence of serum. Transferrin alone does not promote survival or proliferation. Only in the presence of both IGF-I and transferrin do cells survive and proliferate. Therefore, in attached CHO cultures, IGF-I alone does not stimulate cell proliferation but is a requirement for growth in serum-free medium in cooperation with transferrin. We report on the dual role of IGF-I as a survival factor in CHO cells and its interlocking role with transferrin to stimulate cell growth.