RESUMEN
Interferon-regulatory factor 1 (IRF-1) is the first member of IRF family, which is involved in many biological processes such as immune response, antiviral defense, cell growth regulation, and apoptosis. In this study, an IRF-1 gene, EcIRF-1, was isolated and characterized from orange-spotted grouper (Epinephelus coioides). The full-length cDNA of EcIRF-1 is 1,730 bp, including an open reading frame of 906 bp, a 5'-terminal untranslated region (5'-UTR) of 153 bp, and a 3'-UTR of 671 bp. The EcIRF-1 gene consists of 10 exons and 9 introns, spanning over approximate 4.3 kb of genomic sequence. The 5'-UTR sequence contains an exon and an intron, and the 3'-UTR sequence is included in the last exon. Expression analysis by real-time PCR reveals that the EcIRF-1 gene is ubiquitously expressed in various healthy fish tissues, whereas its expression is upregulated in vivo in response to polyinosinic-polycytidylic acid or lipopolysaccharide stimulation. Subcellular localization analysis shows the EcIRF-1 is an intranuclearly localized and immobile protein in the cultured fish cells. Data presented in this paper provide an important base to further understand EcIRF-1 gene function and its regulation associated with interferon immune system in orange-spotted grouper.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Perciformes/genética , Filogenia , Animales , Secuencia de Bases , Análisis por Conglomerados , Cartilla de ADN/genética , ADN Complementario/genética , Componentes del Gen , Perfilación de la Expresión Génica , Lipopolisacáridos , Datos de Secuencia Molecular , Perciformes/inmunología , Poli I-C , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
JAK2(V617F) is the predominant mutation in myeloproliferative neoplasms (MPN). Modeling MPN in a human context might be helpful for the screening of molecules targeting JAK2 and its intracellular signaling. We describe here the derivation of induced pluripotent stem (iPS) cell lines from 2 polycythemia vera patients carrying a heterozygous and a homozygous mutated JAK2(V617F), respectively. In the patient with homozygous JAK2(V617F), additional ASXL1 mutation and chromosome 20 allowed partial delineation of the clonal architecture and assignation of the cellular origin of the derived iPS cell lines. The marked difference in the response to erythropoietin (EPO) between homozygous and heterozygous cell lines correlated with the constitutive activation level of signaling pathways. Strikingly, heterozygous iPS cells showed thrombopoietin (TPO)-independent formation of megakaryocytic colonies, but not EPO-independent erythroid colony formation. JAK2, PI3K and HSP90 inhibitors were able to block spontaneous and EPO-induced growth of erythroid colonies from GPA(+)CD41(+) cells derived from iPS cells. Altogether, this study brings the proof of concept that iPS can be used for studying MPN pathogenesis, clonal architecture, and drug efficacy.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Janus Quinasa 2/metabolismo , Trastornos Mieloproliferativos/metabolismo , Células Cultivadas , Eritropoyetina/farmacología , Humanos , Janus Quinasa 2/genética , Trastornos Mieloproliferativos/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Trombopoyetina/farmacologíaRESUMEN
Interferon regulatory factor 2 (IRF-2) is a multifunctional transcription factor which exhibits both transcriptional activating and repressing activities in the IRF family. In this study, we report an IRF-2 gene isolated from orange-spotted grouper (Epinephelus coioides). The 1854bp full-length cDNA sequence of EcIRF-2 has been cloned, encoding a putative peptide of 336 amino acids which is highly consistent with the feature of IRF family members. The genomic fragment of EcIRF-2 contains nine exons and eight introns, spanning over approximate 8.8kb. The expression of EcIRF-2 gene was detected in various tissues of healthy orange-spotted grouper and in four tissues after being challenged with poly I:C or LPS. EcIRF-2 gene is ubiquitously expressed in various healthy fish tissues and is up-regulated in vivo in response to poly I:C or LPS. Subcellular localization analysis of EcIRF-2 suggests it is an intranuclear protein in the fish cells. We believe this research is the first report of fish IRF-2 protein localization. The results in this research establish the base for further study of function mechanism of IRF family members in orange-spotted grouper.