RESUMEN
Gene expression in higher eukaryotes appears to be regulated by specific combinations of transcription factors binding to regulatory sequences. The Ets factor PU.1 and the IRF protein Pip (IRF-4) represent a pair of interacting transcription factors implicated in regulating B cell-specific gene expression. Pip is recruited to its binding site on DNA by phosphorylated PU.1. PU.1-Pip interaction is shown to be template directed and involves two distinct protein-protein interaction surfaces: (i) the ets and IRF DNA-binding domains; and (ii) the phosphorylated PEST region of PU.1 and a lysine-requiring putative alpha-helix in Pip. Thus, a coordinated set of protein-protein and protein-DNA contacts are essential for PU.1-Pip ternary complex assembly. To analyze the function of these factors in vivo, we engineered chimeric repressors containing the ets and IRF DNA-binding domains connected by a flexible POU domain linker. When stably expressed, the wild-type fused dimer strongly repressed the expression of a rearranged immunoglobulin lambda gene, thereby establishing the functional importance of PU.1-Pip complexes in B cell gene expression. Comparative analysis of the wild-type dimer with a series of mutant dimers distinguished a gene regulated by PU.1 and Pip from one regulated by PU.1 alone. This strategy should prove generally useful in analyzing the function of interacting transcription factors in vivo, and for identifying novel genes regulated by such complexes.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Dimerización , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Cadenas lambda de Inmunoglobulina/genética , Factores Reguladores del Interferón , Ratones , Datos de Secuencia Molecular , Mutación/genética , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras/genética , Alineación de Secuencia , Activación Transcripcional/genética , Transfección/genética , Células Tumorales CultivadasRESUMEN
Pip is a lymphoid-restricted IRF transcription factor that is recruited to composite elements within immunoglobulin light-chain gene enhancers through a specific interaction with the Ets factor PU.1. We have examined the transcriptional regulatory properties of Pip as well as the requirements for its interaction with PU.1 and DNA to form a ternary complex. We demonstrate that Pip is a dichotomous regulator; it specifically stimulates transcription in conjunction with PU.1, but represses alpha/beta-interferon-inducible transcription in the absence of PU.1. Thus, during B-cell activation and differentiation, Pip may function both as an activator to promote B cell-specific gene expression and as a repressor to inhibit the antiproliferative effects of alpha/beta-interferons. Mutational analysis of Pip reveals a carboxy-terminal segment that is important for autoinhibition of DNA binding and ternary complex formation. A domain of Pip containing this segment confers autoinhibition and PU.1-dependent binding activity to the DNA-binding domain of the related IRF family member, p48. On the basis of these and other data we propose a model for PU.1/Pip ternary complex formation.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Factores Reguladores del Interferón , Factor 3 de Genes Estimulados por el Interferón , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón , Interferón-alfa/genética , Interferón-alfa/metabolismo , Interferón-alfa/farmacología , Interferón gamma/genética , Interferón gamma/metabolismo , Interferón gamma/farmacología , Ratones , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Androgens are required for the optimal growth and development of both the normal prostate and steroid-sensitive prostate cancer. PC3 prostate cancer cell lines stably expressing the human androgen receptor (AR) and possessing an androgen-sensitive phenotype (PC3-hAR) were used to examine the role of the epidermal growth factor receptor (EGFR) in androgen-stimulated prostate cancer cell growth. Epidermal growth factor (EGF) and dihydrotestosterone (DHT) independently induced the growth of PC3-hAR cells. Moreover, EGF and DHT in combination exerted a synergistic effect on PC3-hAR cell growth. DHT-exposed PC3-hAR cells expressed a greater than 2-fold increase in EGFR mRNA and 50% more EGFR protein than controls. Time course radioligand-binding assays confirmed these findings by showing an elevation in EGF binding in the DHT-exposed PC3-hAR cells. In addition, radioligand competition-binding studies revealed a 2-fold increase in EGFR-EGF binding affinity in the PC3-hAR cells after DHT treatment. However, no enhancement of transforming growth factor alpha or EGF expression was detected because DHT did not affect the levels of these cytokines in the PC3-hAR cell lysate or conditioned media. Our observations suggest that DHT increases both EGFR number and receptor-ligand affinity in androgen-sensitive prostate cancer cells and that these effects correlate with increased EGF binding and an enhanced mitogenic response to EGF.
