RESUMEN
The far upstream element (FUSE) binding protein (FBP), a single-stranded nucleic acid binding protein, is recruited to the c-myc promoter after melting of FUSE by transcriptionally generated dynamic supercoils. Via interactions with TFIIH and FBP-interacting repressor (FIR), FBP modulates c-myc transcription. Here, we investigate the contributions of FBP's 4 K Homology (KH) domains to sequence selectivity. EMSA and missing contact point analysis revealed that FBP contacts 4 separate patches spanning a large segment of FUSE. A SELEX procedure using paired KH-domains defined the preferred subsequences for each KH domain. Unexpectedly, there was also a strong selection for the noncontacted residues between these subsequences, showing that the contact points must be optimally presented in a backbone that minimizes secondary structure. Strategic mutation of contact points defined in this study disabled FUSE activity in vivo. Because the biological specificity of FBP is tuned at several layers: (i) accessibility of the site; (ii) supercoil-driven melting; (iii) presentation of unhindered bases for recognition; and (iv) modular interaction of KH-domains with cognate bases, the FBP-FIR system and sequence-specific, single-strand DNA binding proteins in general are likely to prove versatile tools for adjusting gene expression.
Asunto(s)
ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Secuencia de Consenso , ADN Helicasas/genética , ADN de Cadena Simple/química , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Genes myc , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Mutación Puntual , Proteínas de Unión al ARN , Técnica SELEX de Producción de AptámerosRESUMEN
The upstream regulatory region of the Drosophila melanogaster hsp26 gene includes two DNase I-hypersensitive sites (DH sites) that encompass the critical heat shock elements. This chromatin structure is required for heat shock-inducible expression and depends on two (CT)n*(GA)n elements bound by GAGA factor. To determine whether GAGA factor alone is sufficient to drive formation of the DH sites, we have created flies with an hsp26/lacZ transgene wherein the entire DNA segment known to interact with the TFIID complex has been replaced by a random sequence. The replacement results in a loss of heat shock-inducible hsp26 expression and drastically diminishes nuclease accessibility in the chromatin of the regulatory region. Chromatin immunoprecipitation experiments show that the decrease in TFIID binding does not reduce GAGA factor binding. In contrast, the loss of GAGA factor binding resulting from (CT)n mutations decreases TFIID binding. These data suggest that both GAGA factor and TFIID are necessary for formation of the appropriate chromatin structure at the hsp26 promoter and predict a regulatory mechanism in which GAGA factor binding precedes and contributes to the recruitment of TFIID.
Asunto(s)
Cromatina/ultraestructura , Proteínas de Unión al ADN , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Proteínas de Choque Térmico/genética , Proteínas de Homeodominio/fisiología , TATA Box/genética , Factores de Transcripción TFII/fisiología , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Cromatina/genética , ADN/genética , ADN/metabolismo , ADN Recombinante/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Choque Térmico/biosíntesis , Calor , Larva , Sustancias Macromoleculares , Modelos Genéticos , Mutagénesis , Mutación Puntual , Unión Proteica , Factor de Transcripción TFIIDRESUMEN
NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF from a Drosophila nuclear extract reduced the level of polymerase that paused in the promoter proximal region of hsp70. Depletion of one NELF subunit in salivary glands using RNA interference also reduced the level of paused polymerase. In vivo protein-DNA cross-linking showed that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborated the cross-linking result and showed that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF were strongly recruited to chromosomal puffs harboring the hsp70 genes. We propose that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation.