RESUMEN
Hepatocyte nuclear factor 1alpha (HNF1alpha) is a homeodomain transcription factor that is central to co-ordinated differentiation of a number of cell lineages, including hepatocytes in the liver and islet cells in the pancreas. HNF1alpha interacts directly with other transcription factors and co-factors and is involved in chromatin modification to alter gene expression. To further investigate the pivotal role of HNF1alpha in transcriptional control pathways we utilized RNA interference. An siRNA oligonucleotide specific for HNF1alpha reduced HNF1alpha protein levels by up to 70% in transient transfections of Caco2 cells. The same sequence incorporated into an shRNAi reduced protein levels by up to 90% in stable transfections. Microarray analysis of RNA from cell lines with stable RNAi-mediated down-regulation of HNF1alpha, identified genes known to be regulated by this transcription factor and also novel genes.
Asunto(s)
Regulación de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/antagonistas & inhibidores , Interferencia de ARN , Células CACO-2 , Factor Nuclear 1-alfa del Hepatocito/genética , Humanos , Oligonucleótidos/química , ARN Interferente Pequeño/químicaRESUMEN
Regulatory elements that lie outside the basal promoter of a gene may be revealed by local changes in chromatin structure and histone modifications. The promoter of the CFTR (cystic fibrosis transmembrane conductance regulator) gene is not responsible for its complex pattern of expression. To identify important regulatory elements for CFTR we have previously mapped DHS (DNase I-hypersensitive sites) across 400 kb spanning the locus. Of particular interest were two DHS that flank the CFTR gene, upstream at -20.9 kb with respect to the translational start site, and downstream at +15.6 kb. In the present study we show that these two DHS possess enhancer-blocking activity and bind proteins that are characteristic of known insulator elements. The DHS core at -20.9 kb binds CTCF (CCCTC-binding factor) both in vitro and in vivo; however, the +15.6 kb core appears to bind other factors. Histone-modification analysis across the CFTR locus highlights structural differences between the -20.9 kb and +15.6 kb DHS, further suggesting that these two insulator elements may operate by distinct mechanisms. We propose that these two DHS mark the boundaries of the CFTR gene functional unit and establish a chromatin domain within which the complex profile of CFTR expression is maintained.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas de Unión al ADN/fisiología , Elementos Aisladores/fisiología , Proteínas Represoras/fisiología , Factor de Unión a CCCTC , Células CACO-2 , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Regulación de la Expresión Génica/fisiología , Marcadores Genéticos , Humanos , Elementos Aisladores/genética , Unión Proteica/genética , Proteínas Represoras/genéticaRESUMEN
We have identified a novel motif which consists of the sequence (CCU)(n) as part of a polypyrimidine-rich tract and permits internal ribosome entry. A number of constructs containing variations of this motif were generated and these were found to function as artificial internal ribosome entry segments (AIRESs) in vivo and in vitro in the presence of polypyrimidine tract-binding protein (PTB). The data show that for these sequences to function as IRESs the RNA must be present as a double-stranded stem and, in agreement with this, rather surprisingly, we show that PTB binds strongly to double-stranded RNA. All the cellular 5' untranslated regions (UTRs) tested that harbor this sequence were shown to contain internal ribosome entry segments that are dependent upon PTB for function in vivo and in vitro. This therefore raises the possibility that PTB or its interacting protein partners could provide a bridge between the IRES-RNA and the ribosome. Given the number of putative cellular IRESs that could be dependent on PTB for function, these data strongly suggest that PTB-1 is a universal IRES-trans-acting factor.
Asunto(s)
Proteína de Unión al Tracto de Polipirimidina/metabolismo , Ribosomas , Regiones no Traducidas 5' , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteína de Unión al Tracto de Polipirimidina/química , Proteína de Unión al Tracto de Polipirimidina/genética , ARN Mensajero/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment and c-Myc translation can be initiated by cap-independent as well as cap-dependent mechanisms. In contrast to the process of cap-dependent initiation, the trans-acting factor requirements for cellular internal ribosome entry are poorly understood. Here, we show that members of the poly (rC) binding protein family, poly (rC) binding protein 1 (PCBP1), poly (rC) binding protein 2 (PCBP2) and hnRNPK were able to activate the IRES in vitro up to threefold when added in combination with upstream of N-ras and unr-interacting protein. The interactions of PCBP1, PCBP2 and hnRNPK with c-myc-IRES-RNA were shown to be specific by ultraviolet crosslinking analysis and electrophoretic mobility shift assays, while immunoprecipitation of the three proteins using specific antibodies followed by reverse transcriptase-polymerase chain reaction showed that they were able to bind c-myc mRNA. c-myc-IRES-mediated translation from the reporter vector was stimulated by cotransfection of plasmids encoding PCBP1, PCBP2 and hnRNPK. Interestingly, the mutated version of the c-myc IRES that is prevalent in patients with multiple myeloma bound hnRNPK more efficiently in vitro and was stimulated by hnRNPK to a greater extent in vivo.
Asunto(s)
Proteínas de Unión al ADN , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción , Sitios de Unión , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Biología Molecular/métodos , Familia de Multigenes , Mieloma Múltiple/genética , Mutación , Biosíntesis de Proteínas , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Unión al ARN , Ribosomas/genética , Ribosomas/metabolismo , Rayos UltravioletaRESUMEN
The 5'-untranslated region of Bag-1 mRNA contains an internal ribosome entry segment (IRES) and the translation of Bag-1 protein can be initiated by both cap-dependent and cap-independent mechanisms. In general, cellular IRESs require non-canonical trans-acting factors for their activity, however, very few of the proteins that act on cellular IRESs have been identified. Proteins that interact with viral IRESs have also been shown to stimulate the activity of cellular IRESs and therefore the ability of a range of known viral trans-acting factors to stimulate the Bag-1 IRES was tested. Two proteins, poly r(C) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB), were found to increase the activity of the Bag-1 IRES in vitro and in vivo. The regions of the Bag-1 IRES RNA to which they bind have been determined, and it was shown that PCBP1 binds to a short 66 nt section of RNA, whilst PTB interacts with a number of sites over a larger area. The minimum section of the RNA that still retained activity was determined and both PCBP1 and PTB interacted with this region suggesting that these proteins are essential for Bag-1 IRES function.
