RESUMEN
B-lymphocyte-induced maturation protein (Blimp-1) is a transcriptional repressor that is considered to be a master regulator of terminal B-cell development because it is sufficient to trigger differentiation in the BCL(1)-cell model. Transcription of the c-myc gene is repressed by Blimp-1 during B-cell differentiation. In this study, we have explored the mechanism by which Blimp-1 represses transcription by using Gal4-fusion protein assays and assays in which Blimp-1 represses the natural c-myc promoter. The results show that Blimp-1 represses the c-myc promoter by an active mechanism that is independent of the adjacently bound activator YY1. Blimp-1 contains two regions that independently associate with histone deacetylase (HDAC) and endogenous Blimp-1 in nuclear extracts binds in vitro to the c-myc Blimp-1 site in a complex containing HDAC. The functional importance of recruiting HDAC for Blimp-1-dependent repression of c-myc transcription is supported by two experiments. First, the HDAC inhibitor tricostatin A inhibits Blimp-1-dependent repression in cotransfection assays. Second, a chromatin immunoprecipitation assay shows that expression of Blimp-1 causes deacetylation of histone H3 associated with the c-myc promoter, and this deacetylation depends on the Blimp-1 binding site in the c-myc promoter.
Asunto(s)
Histona Desacetilasas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Acetilación , Línea Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Factores de Unión al ADN Específico de las Células Eritroides , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Pruebas de Precipitina , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Transfección , Factor de Transcripción YY1RESUMEN
The c-Myc oncoprotein has previously been shown to associate with transcription regulator YY1 and to inhibit its activity. We show herein that endogenous c-Myc and YY1 associate in vivo and that changes in c-Myc levels, which accompany mitogenic stimulation or differentiation of cultured cells, affect the ratio of free to c-Myc-associated YY1. We have also investigated the mechanism by which association with c-Myc inhibits YY1's ability to regulate transcription. c-Myc does not block binding of YY1 to DNA. However, protein association studies suggest that c-Myc interferes with the ability of YY1 to contact basal transcription proteins TATA-binding protein and TFIIB.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Unión Competitiva , ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Regulación de la Expresión Génica , Ratones , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes , Proteína de Unión a TATA-Box , Factor de Transcripción TFIIB , Factor de Transcripción YY1 , Dedos de ZincAsunto(s)
Proteínas de Unión al ADN/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Unión al ADN Específico de las Células Eritroides , Humanos , Datos de Secuencia Molecular , Factor de Transcripción YY1RESUMEN
Yin-Yang-1 (YY1) regulates the transcription of many genes, including the oncogenes c-fos and c-myc. Depending on the context, YY1 acts as a transcriptional repressor, a transcriptional activator, or a transcriptional initiator. The yeast two-hybrid system was used to screen a human complementary DNA (cDNA) library for proteins that associate with YY1, and a c-myc cDNA was isolated. Affinity chromatography confirmed that YY1 associates with c-Myc but not with Max. In cotransfections, c-Myc inhibits both the repressor and the activator functions of YY1, which suggests that one way c-Myc acts is by modulating the activity of YY1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Células 3T3 , Proteínas E1A de Adenovirus/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/farmacología , Factores de Unión al ADN Específico de las Células Eritroides , Secuencias Hélice-Asa-Hélice , Humanos , Ratones , Proteínas Proto-Oncogénicas c-myc/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/farmacología , Transfección , Células Tumorales Cultivadas , Factores Estimuladores hacia 5' , Factor de Transcripción YY1RESUMEN
Previous studies on the murine c-myc promoter demonstrated that a ubiquitously present protein, common factor 1 (CF1), bound at two sites located -260 and -390 bp from the P1 transcription start site. CF1 has been purified to near homogeneity and shown to be identical to the zinc finger protein Yin-yang 1 (YY1) as judged by similarity of molecular weight and other biochemical properties, immunological cross-reactivity, and the ability of recombinant YY1 to bind to CF1 sites. In cotransfection experiments, YY1 is a strong activator of transcription from c-myc promoter-based reporters. Furthermore, in murine erythroleukemia cells, overexpressed YY1 causes increased levels of c-myc mRNA initiated from both major transcription initiation sites of the endogenous c-myc gene.