RESUMO
Transcriptional repressor DREAM, an EF-hand containing calcium-binding protein, blocks basal expression of target genes through specific interaction with DRE sites in the DNA. The sequence GTCA forms the central core of the DRE site, whereas flanking nucleotides contribute notably to the affinity for DREAM. Release of binding of DREAM from the DRE results in derepression, a process that is regulated by Ca(2+). Change of two amino acids within an EF-hand in DREAM blocks Ca(2+)-induced derepression and results in potent dominant negative mutants of endogenous DREAM.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA , Proteínas Repressoras/metabolismo , Fatores de Transcrição , Animais , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Motivos EF Hand , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Interatuantes com Canais de Kv , Mutação , Fatores de Transcrição NFI , Proteínas Nucleares , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transfecção , Células Tumorais Cultivadas , Proteína 1 de Ligação a Y-BoxRESUMO
Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, alphaCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of alphaCREM is not required for the interaction, but phospho-alphaCREM shows higher affinity for DREAM. The interaction with alphaCREM is independent of the Ca(2+)-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both alphaCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca(2+).
Assuntos
Motivos de Aminoácidos/genética , Proteínas de Ligação ao Cálcio , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Encefalinas/genética , Genes Reguladores/genética , Precursores de Proteínas/genética , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Linhagem Celular , Colforsina/farmacologia , Modulador de Elemento de Resposta do AMP Cíclico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Encefalinas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Humanos , Proteínas Interatuantes com Canais de Kv , Dados de Sequência Molecular , Mutação/genética , Neuroblastoma , Fosforilação , Precursores de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Alinhamento de Sequência , Transfecção , Células Tumorais CultivadasRESUMO
Fluxes in amounts of intracellular calcium ions are important determinants of gene expression. So far, Ca2+-regulated kinases and phosphatases have been implicated in changing the phosphorylation status of key transcription factors and thereby modulating their function. In addition, direct effectors of Ca2+-induced gene expression have been suggested to exist in the nucleus, although no such effectors have been identified yet. Expression of the human prodynorphin gene, which is involved in memory acquisition and pain, is regulated through its downstream regulatory element (DRE) sequence, which acts as a location-dependent gene silencer. Here we isolate a new transcriptional repressor, DRE-antagonist modulator (DREAM), which specifically binds to the DRE. DREAM contains four Ca2+-binding domains of the EF-hand type. Upon stimulation by Ca2+, DREAM's ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca2+. In addition to the prodynorphin promoter, DREAM represses transcription from the early response gene c-fos. Thus, DREAM represents the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator.