RESUMEN
High-level activity of the human PDGF-B promoter in choriocarcinoma cell lines depends upon an atypical, intronic enhancer-like element which does not function with heterologous promoters tested. An extensive series of mutant PDGF-B promoter-driven constructs identified a sequence flanking the TATA box which is required specifically for enhancer-mediated transcription in human choriocarcinoma cell lines. This element, which we here term an enhancer-dependent cis co-activator (EDC) contains an Inr (initiator) consensus sequence upstream of the TATA box which is required, but not sufficient for its function. Requirement for the EDC is cell type-specific, since it was dispensable for enhancer-mediated transcription in a human breast cancer cell line. Although it lies within the region defined, the TATA box itself is not required for EDC function, or for basal promoter function which may derive from a second Inr-like sequence situated at the transcriptional start site. These observations indicate that interactions between some promoter and enhancer elements may be more complex than that generally described for 'classical' enhancer systems and may suggest an additional function for the initiator motif.
Asunto(s)
Coriocarcinoma/genética , Elementos de Facilitación Genéticos , Factor de Crecimiento Derivado de Plaquetas/genética , Regiones Promotoras Genéticas , Proto-Oncogenes , Secuencias Reguladoras de Ácidos Nucleicos , TATA Box , Neoplasias Uterinas/genética , Secuencia de Bases , Neoplasias de la Mama/genética , Línea Celular , Secuencia de Consenso , Femenino , Humanos , Intrones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Embarazo , Proto-Oncogenes Mas , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The p21 GTPases, Rho and Cdc42, regulate numerous cellular functions by binding to members of a serine/threonine protein kinase subfamily. These functions include the remodeling of the cell cytoskeleton that is a feature of cell growth and differentiation. Two of these p21 GTPase-regulated kinases, the myotonic dystrophy protein kinase-related Cdc42-binding kinases (MRCKalpha and beta), have been recently characterized in rat. Both of these proteins phosphorylate nonmuscle myosin light chain, a prerequisite for the activation of actin-myosin contractility. Here we report the cDNA cloning of the human homologue of MRCKbeta, CDC42BPB, which was found by Northern blot analysis to be expressed in a wide range of tissues. The human CDC42BPB gene maps to cytogenetic band 14q32.3 by FISH analysis.
Asunto(s)
Cromosomas Humanos Par 14/genética , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Proteína Quinasa de Distrofia Miotónica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Distribución TisularRESUMEN
We have previously described a non-classical, promoter-specific enhancer for the human Platelet-Derived Growth Factor B (PDGF-B) gene. In JEG-3 choriocarcinoma cells the activity of the enhancer depends upon co-operation with a sequence (the Enhancer-Dependent cis Co-activator "EDC" element) within the promoter. The PDGF-B enhancer fails to activate heterologous promoters, indicating that promoter-specificity depends on an element within the enhancer that can recognise a target sequence within the promoter. Here we identify a sequence within the enhancer of the PDGF-B gene which directs activation of the PDGF-B promoter by distal cis-acting elements. This specifies the wild-type PDGF-B promoter as the target for the enhancer and has been designated the EDC specificity element (EDCse). The cell-type specific nature of this interaction is extended by the observation that the EDCse is also dispensable for enhancer activity in breast-cancer cells (ZR-75). Concomitant to this observation, JEG-3 and ZR-75 cells differ in the binding of nuclear factors to the EDCse. We discuss the relevance of the EDC/EDCse system in regulation of gene expression.