RESUMO
Artificial transcription factors are powerful tools for regulating gene expression. Here we report results with engineered zinc-finger transcription factors (ZF-TFs) targeting four protein-coding genes, OCT4, SOX2, KLF4 and c-MYC, and one noncoding ribonucleic acid (RNA) gene, the microRNA (miRNA) miR302/367 cluster. We designed over 300 ZF-TFs whose targets lie within 1 kb of the transcriptional start sites (TSSs), screened them for increased messenger RNA or miRNA levels in transfected cells, and identified potent ZF-TF activators for each gene. Furthermore, we demonstrate that selected ZF-TFs function with alternative activation domains and in multiple cell lines. For OCT4, we expanded the target range to -2.5 kb and +500 bp relative to the TSS and identified additional active ZF-TFs, including three highly active ZF-TFs targeting distal enhancer, proximal enhancer and downstream from the proximal promoter. Chromatin immunoprecipitation (FLAG-ChIP) results indicate that several inactive ZF-TFs targeting within the same regulatory region bind as well as the most active ZF-TFs, suggesting that efficient binding within one of these regulatory regions may be necessary but not sufficient for activation. These results further our understanding of ZF-TF design principles and corroborate the use of ZF-TFs targeting enhancers and downstream from the TSS for transcriptional activation.
Assuntos
Transativadores/metabolismo , Ativação Transcricional , Dedos de Zinco , Linhagem Celular , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , MicroRNAs/biossíntese , MicroRNAs/genética , Fator 3 de Transcrição de Octâmero/biossíntese , Fator 3 de Transcrição de Octâmero/genética , Engenharia de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Transativadores/químicaRESUMO
BACKGROUND: The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. RESULTS: We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene. CONCLUSION: This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.
Assuntos
Escherichia coli/genética , Técnicas de Silenciamento de Genes/métodos , Poríferos/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , Actinas/análise , Actinas/genética , Actinas/metabolismo , Animais , Organismos Aquáticos/citologia , Organismos Aquáticos/efeitos dos fármacos , Organismos Aquáticos/genética , Escherichia coli/metabolismo , Comportamento Alimentar , Água Doce , Perfilação da Expressão Gênica , Histocitoquímica , Microscopia Confocal , Poríferos/citologia , Poríferos/efeitos dos fármacos , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/biossíntese , Água do MarRESUMO
Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that deaminate adenosines to create inosines in double-stranded RNA (dsRNA). Here we demonstrate that ADARs are not required for RNA interference (RNAi) and that they do not antagonize the pathway to a detectable level when RNAi is initiated by injecting dsRNA. We find, however, that transgenes expressed in the somatic tissues of wild-type animals are silenced in strains with deletions in the two genes encoding ADARs, adr-1 and adr-2. Transgene-induced gene silencing in adr-1;adr-2 mutants depends on genes required for RNAi, suggesting that a dsRNA intermediate is involved. In wild-type animals we detect edited dsRNA corresponding to transgenes, and we propose that editing of this dsRNA prevents somatic transgenes from initiating RNAi in wild-type animals.
