title: Interaction of IRF9 and STAT2 synergistically up-regulates IFN and PKR transcription in Ctenopharyngodon idella.
abstract: IRF9 is a key factor in the JAK-STAT pathway. Under the stimulation of type I IFN, IRF9 interacts with STAT1 and STAT2 to form the IFN-I-stimulated gene factor 3 (ISGF3) which activates the transcription of ISG. However, many studies also showed that the dimmer IRF9/STAT2 rather than the tripolymer IRF9/STAT1/STAT2 acts as the ISGF3 in cells in response to IFN signals. In the present study, the full-length cDNA sequence of IRF9 (termed CiIRF9, KT601055) and STAT2 (term CiSTAT2, KT781914) from grass carp were cloned and identified. A low level of constitutive expression of CiIRF9 was detected by RT-PCR in grass carp tissues, but it was significantly up-regulated by LPS and poly I:C stimulation. In vitro, a high-affinity interaction between CiIRF9 and the promoter of CiIFN or CiPKR was demonstrated by gel mobility shift assay. In vivo, the promoter activities of CiIFN and CiPKR were not only increased by transient transfection of CiIRF9, but also prominently increased by co-transfection of CiIRF9 and CiSTAT2. Moreover, the interaction of CiIRF9 and CiSTAT2 was further investigated by in vivo and in vitro protein interaction assays. Recombinant CiIRF9 and CiSTAT2, both tagged with FLAG (or HA), were expressed in HEK 293T cells by transient transfection experiment. Co-immunoprecipitation assays showed that CiIRF9 can interact with CiSTAT2 in vivo. Soluble GST-ST2-936 (containing the N-terminal and coiled-coil domain of CiSTAT2) was expressed and purified from E. coli. A GST pull-down assay suggested that GST-tagged ST2-936 efficiently bound to FLAG-tagged IRF9. The data indicated that interaction of IRF9 and STAT2 synergistically up-regulated the transcriptional level of IFN and ISG genes.
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wu
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Liqiang
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Xu
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Lin
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Huiling
authors.initials: H
authors.lastname: Mao
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Ran
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Keyi
authors.initials: K
authors.lastname: Huang
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Haizhou
authors.initials: H
authors.lastname: Wang
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Qingli
authors.initials: Q
authors.lastname: Huang
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Xu
authors.affiliation: College of Life Science, Key Lab of Aquatic Resources and Utilization of Jiangxi Province, Nanchang University, Nanchang 330031, China. Electronic address: hucy2008@163.com.
authors.forename: Chengyu
authors.initials: C
authors.lastname: Hu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics* / Q000378:metabolism; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism*; D015151:Immunoblotting; D047468:Immunoprecipitation; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit / Q000235:genetics / Q000378:metabolism; D007372:Interferons / Q000096:biosynthesis; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D050795:STAT2 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology*; D059467:Transcriptome; D015854:Up-Regulation; D019892:eIF-2 Kinase / Q000096:biosynthesis
chemical_list: D029941:Fish Proteins; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D050795:STAT2 Transcription Factor; D007372:Interferons; D019892:eIF-2 Kinase
keywords: Fish; IRF9; Interaction; STAT2; Transcriptional control