RESUMO
Regulated expression of miRNAs influences development in a wide variety of contexts. We report here that miR290-5p (100049710) and miR292-5p (100049711) are induced at the pre-B stage of murine B cell development and that they influence assembly of the Igκ light chain gene (243469) by contributing to the activation of germline Igκ transcription (κGT). We found that upon forced over-expression of miR290-5p/292-5p in Abelson Murine Leukemia Virus (AMuLV) transformed pro-B cells, two known activators of κGT, E2A (21423) and NF-κB (19697), show increased chromosomal binding to the kappa intronic enhancer. Conversely, knockdown of miR290-5p/292-5p in AMuLV pro-B cells blunts drug-induced activation of κGT. Furthermore, miR290-5p/292-5p knockdown also diminishes κGT activation, but not Rag1/2 (19373, 19374) expression, in an IL-7 dependent primary pro-B cell culture system. In addition, we identified a deficiency in κGT induction in miR290 cluster knockout mice. We hypothesize that increased expression of miR290-5p and miR292-5p contributes to the induction of κGT at the pre-B stage of B cell development through increased binding of NF-κB and E2A to kappa locus regulatory sequences.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Loci Gênicos/genética , Cadeias kappa de Imunoglobulina/genética , MicroRNAs/metabolismo , Vírus da Leucemia Murina de Abelson/fisiologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/virologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzamidas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Transformação Celular Viral/efeitos dos fármacos , Transformação Celular Viral/genética , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Mesilato de Imatinib , Íntrons/genética , Camundongos , MicroRNAs/genética , NF-kappa B/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Escherichia coli, a facultative aerobe, expresses two distinct respiratory nitrate reductases. The periplasmic NapABC enzyme likely functions during growth in nitrate-limited environments, whereas the membrane-bound NarGHI enzyme functions during growth in nitrate-rich environments. Maximal expression of the napFDAGHBC operon encoding periplasmic nitrate reductase results from synergistic transcription activation by the Fnr and phospho-NarP proteins, acting in response to anaerobiosis and nitrate or nitrite, respectively. Here, we report that, during anaerobic growth with no added nitrate, less-preferred carbon sources stimulated napF operon expression by as much as fourfold relative to glucose. Deletion analysis identified a cyclic AMP receptor protein (Crp) binding site upstream of the NarP and Fnr sites as being required for this stimulation. The napD and nrfA operon control regions from Shewanella spp. also have apparent Crp and Fnr sites, and expression from the Shewanella oneidensis nrfA control region cloned in E. coli was subject to catabolite repression. In contrast, the carbon source had relatively little effect on expression of the narGHJI operon encoding membrane-bound nitrate reductase under any growth condition tested. Carbon source oxidation state had no influence on synthesis of either nitrate reductase. The results suggest that the Fnr and Crp proteins may act synergistically to enhance NapABC synthesis during growth with poor carbon sources to help obtain energy from low levels of nitrate.