Yichuan Mi

Ctenopharyngodon idella IRF2 plays an antagonistic role to IRF1 in transcriptional regulation of IFN and ISG genes

Interferon Regulatory Factors (IRFs) make up a family of transcription factors involved in transcriptional regulation of type I IFN and IFN-stimulated genes (ISG) in cells. In the present study, an IRF2 gene (termed CiIRF2, JX628585) was cloned and characterized from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiIRF2 is 1809 bp in length, with the largest open reading frame (ORF) of 981 bp encoding a putative protein of 326 amino acids. CiIRF2 contains a conserved DNA-binding domain (DBD) in N-terminal and a non-conserved C-terminal region. Protein sequence analysis revealed that CiIRF2 shares significant homology to the known IRF2 counterparts. Phylogenetic reconstruction confirmed its closer evolutionary relationship with other fish counterparts, especially with zebra fish IRF2. CiIRF2 was ubiquitously expressed at low level in all tested grass carp tissues and significantly up-regulated except in brain following poly I:C 6-12 h post stimulation. In order to understand fish innate immune and resistance to virus diseases, recombinant CiIRF2 with His-tag was over-expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. Promoter sequences of grass carp type I IFN gene (CiIFN) and two ISG genes (CiPKR and CiPKZ) were amplified and cloned. In vitro, gel mobility shift assays were employed to analyze the interaction of CiIRF2 protein with promoters of CiIFN, CiPKR and CiPKZ respectively. The results showed that CiIRF2 bound to these promoters with high affinity by means of its DBD. Afterwards, recombinant plasmids of pGL3-CiIFN, pGL3-CiPKR and pGL3-CiPKZ were constructed and transiently co-transfected with pcDNA3.1-CiIRF2 or pcDNA3.1-CiIRF1 respectively into C. idella kidney (CIK) cells. Dual-luciferase reporter assays demonstrated that CiIRF2 down-regulates the transcription activity of CiIFN, CiPKR and CiPKZ genes in CIK cells. To further understand the function of fish IRF2, expression plasmids (pcDNA3.1-IRF2 and pcDNA3.1-IRF1) were transiently co-transfected with pGL3-IFN or pGL3-CiPKZ into CIK cells, respectively. The results revealed that CiIRF2 plays an antagonistic role to CiIRF1 in transcriptional regulation of IFN and ISG genes.

The transcription regulation analysis of Ctenopharyngodon idellus PKR and PKZ genes.

Protein kinase R (PKR), the double-stranded RNA-activated protein kinase, exists in mammalian and fish. PKZ, a PKR-like protein kinase containing Z-DNA binding domains, just exists in fish. PKR and PKZ work synergistically in the antiviral defense by inhibiting intracellular protein translation. The transcriptional factor IRF3 (interferon regulatory factor 3) acts as a key regulator of type I IFN (Interferon) and ISG (interferon stimulated gene). On the basis of the cloned CiIRF3 previously, CiIRF3 with His-tag was over-expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. In this study, we have demonstrated that grass carp (Ctenopharyngodon idellus) PKR (CiPKR) and PKZ (CiPKZ) genes were inducible by Poly I:C in C. idella kidney (CIK) cells. So, they might be implicated in the intracellular antiviral activity. To understand the up regulatory mechanism of CiPKR and CiPKZ genes upon virus induction, we constructed wild type (pGL3-CiPKR-luc and pGL3-CiPKZ-luc) and the mutant (pGL3-CiPKR-nISRE-luc and pGL3-CiPKZ-nISRE-luc) reporter gene vectors according to the promoter sequences of CiPKR (KJ704845) and CiPKZ (KJ704844). In vitro, gel mobility shift assays demonstrated that CiIRF3 can combine CiPKR and CiPKZ promoters with high affinity. However, CiIRF3 bound to the mutants CiPKR-nISRE and CiPKZ-nISRE faintly. Whereafter, the recombinant plasmids of pGL3-CiPKR-luc, pGL3-CiPKZ-luc were transiently co-transfected with pcDNA3.1-CiIRF3, pcDNA3.1-CiIRF7 respectively into CIK cells. Cell transfection assays indicated that CiIRF3 and CiIRF7 up-regulated the transcriptional level of CiPKR and CiPKZ. The results also revealed that the consensus sequence of ISRE (interferon stimulated response element) is an important regulatory element for the transcriptional initiation of CiPKR and CiPKZ.

siRNA-mediated knockdown of CiGRP78 gene expression leads cell susceptibility to heavy metal cytotoxicity

Heavy metal ion is one of the critical environmental pollutants accumulated in living organisms and causes toxic or carcinogenic effects once passed threshold levels. As an important member of Hsp70 (heat shock protein 70) family, the 78-kDa glucose-regulated protein (GRP78) can enhance cell survival rates remarkably under thermal stress. Recent studies also demonstrated that the expression of GRP78 enhances the cell survival under heavy metal stress. In this study, three most representative heavy metal ions, Pb(2+), Hg(2+) and Cd(2+), were used to stimulate Ctenopharyngodon idella kidney (CIK) cells. The results showed that cell viability under Pb(2+), Hg(2+) and Cd(2+) stress decreased significantly. The longer and the greater the concentrations of stimulation from heavy metal ions, the higher the rate of cell death was observed. Among them, Hg(2+) is the most hazardous to cells. Under the same stress condition, Hg(2+) resulted in 50% of cell death, Cd(2+) (or Pb(2+)) led to 45% (or 35%) of cell death, respectively. Western immunoblotting indicated that C. idella GRP78 (CiGRP78) protein expression level was enhanced obviously in CIK cells under Pb(2+), Hg(2+) and Cd(2+) stress, meaning CiGRP78 is involved in heavy metal cytotoxicity. To further study the role of CiGRP78 in cytoprotection, we designed the siRNA against CiGRP78 (from nucleotides +788 to +806) and transfected it into CIK cells to silence endogenous CiGRP78. The viability rate of CIK cells transfected with or without siRNA incubated with HgCl2 for 12h showed a significant decrease from 50% to 21%. Our results showed that CiGRP78 protects cells against heavy metal stimuli to some extent.

Cloning, identification of the two cytokine receptor family B subunits CRFB1 and CRFB5 from grass carp (Ctenopharyngodon idella).

Similar to the mammalian counterparts, fish type I interferon (IFN) performs its potential biological activities via binding to the corresponding receptor on target cell membrane. Fish type I IFN receptor, a kind of enzyme-linked receptor, consists of two subunits and belongs to the class II cytokine receptor family B (CRFB). In the present study, we cloned and identified two putative grass carp (Ctenopharyngodon idella) type I interferon receptor subunits (termed CiCRFB1 and CiCRFB5) by homology cloning techniques. Phylogenetic tree analysis suggested that CiCRFB1 and CiCRFB5 shared highly homology to Danio rerio CRFB1 and CRFB5 respectively. CiCRFB1 and CiCRFB5 were up-regulated after the stimulation with Grass Carp Hemorrhagic Virus (GCHV) and Polyinosinic-polycytidylic acid (Poly I:C), indicating that they are related to the intracellular antiviral activity. In order to know more about the roles of CiCRFB1 and CiCRFB5 in the process, the extracellular domains of CiCRFB1 (CiCRFB1-EC) and CiCRFB5 (CiCRFB5-EC), as well as grass carp type I IFN (CiIFN) were expressed in Escherichia coli BL21, and purified by affinity chromatography with the Ni-NTA His-Bind resin. Cross-linking reactions were employed to analyze the affinity of the ligand (CiIFN) with the two putative receptor subunits (CiCRFB1-EC and CiCRFB5-EC). The result suggested the formation of (CiCRFB5)2 homodimer was more easily than that of (CiCRFB1)2 under the induction of CiIFN in vitro. However, CiIFN was inclined to bind to (CiCRFB1)2 homodimer. Interestingly, although CiIFN seemed unable to facilitate the formation of (CiCRFB1 + CiCRFB5) heterodimer in the absence of DSS cross linker, however it can bind to the heterodimer in the presence of DSS. This indicated that the homodimer and the heterodimer were the potential receptor for CiIFN.

ATF4 (activating transcription factor 4) from grass carp (Ctenopharyngodon idella) modulates the transcription initiation of GRP78 and GRP94 in CIK cells.

GRP78 and GRP94, belong to GRP (glucose-regulated protein) family of endoplasmatic reticulum (ER) chaperone superfamily, are essential for cell survival under ER stress. ATF4 is a protective protein which regulates the adaptation of cells to ER stress by modulating the transcription of UPR (Unfolded Protein Response) target genes, including GRP78 and GRP94. To understand the molecular mechanism of ATF4 modulates the transcription initiation of CiGRP78 and CiGRP94, we cloned ATF4 ORF cDNA sequences (CiATF4) by homologous cloning techniques. The expression trend of CiATF4 was similar to CiGRP78 and CiGRP94 did under 37 °C thermal stress, namely, the expression of CiATF4 was up-regulated twice at 2 h post-thermal stress and at 18 h post recovery from thermal stress. In this paper, CiATF4 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. On the basis of the cloned CiGRP78 and CiGRP94 cDNA in our laboratory previously, we cloned their promoter sequences by genomic walking approach. In vitro, gel mobility shift assays revealed that CiATF4 could bind to CiGRP78 and CiGRP94 promoter with high affinity. Subsequently, the recombinant plasmid of pGL3-CiGRPs and pcDNA3.1-CiATF4 were constructed and transiently co-transfected into Ctenopharyngodon idella kidney (CIK) cells. The impact of CiATF4 on CiGRP promoter sequences were measured by luciferase assays. These results demonstrated that CiATF4 could activate the transcription of CiGRP78 and CiGRP94. Whats more, for better understanding the molecular mechanism of CiATF4 modulate the transcription initiation of CiGRP, three mutant fragments of CiGRP78 promoter recombinant plasmids (called CARE-mut/LUC, CRE1-mut/LUC and CRE2-mut/LUC) were constructed and transiently co-transfected with CiATF4 into CIK cells. The results indicated that CRE or CARE elements were the regulatory element for transcription initiation of CiGRP78. Between them, CRE element would play more important role in it.

Promoter analysis and transcriptional regulation of a Gig2 gene in grass carp (Ctenopharyngodon idella).

Grass carp reovirus (GCRV)-induced gene 2 (Gig2) is recognized as a new antiviral factor involved in response to viral infection. However, little is known about the mechanisms behind the transcriptional regulation of Gig2 when infected by virus. In this study, the upstream promoter region of grass carp (Ctenopharyngodon idella) Gig2 gene (CiGig2) was identified by homology cloning strategy. CiGig2 promoter sequence was found to be 859 bp in length and contained three scattered IFN-stimulated response elements (ISRE). In addition, some grass carp IRFs (CiIRF1, CiIRF2 and CiIRF3) ORF sequences were subcloned into the expression plasmids pET-32a and expressed in Escherichia coli BL21, then the expressed proteins were purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assay was employed to screen the transcriptional regulatory factor for CiGig2. The results revealed that the recombinant polypeptides of CiIRF1, CiIRF2 and CiIRF3 bound to CiGig2 promoter with high affinity; indicating that IRF1, IRF2 and IRF3 could be the potential transcriptional regulatory factors for Gig2. Subsequently, CiGig2 promoter sequence was cloned into pGL3-Basic vector and the ORFs of CiIRF1, CiIRF2 and CiIRF3 were cloned into the expression plasmids pcDNA3.1 (+). Then, pGL3-CiGig2 promoter sequence and pcDNA3.1-CiIRFs were co-transfected into C. idella kidney (CIK) cells. The in vivo effects of CiIRFs on CiGig2 promoter were measured by dual-luciferase assays in the transfected CIK cells. Our results showed that the roles of CiIRFs were diversified in regulating CiGig2 transcription, e.g., CiIRF3 played a positive role in during this process; on the contrary CiIRF1 worked as a suppressor; however the effect of CiIRF2 on CiGig2 transcription was not obvious. For further study the roles of the three ISREs in CiGig2 transcription, we cloned three mutant CiGig2 promoters called ISRE1mut-luc (deleted ISRE1), ISRE2mut-luc (deleted ISRE2) and ISRE3mut-luc (deleted ISRE3), respectively. In vitro, gel mobility shift assays showed that all three mutant promoters also were combined with CiIRFs. CIK cells were co-transfected with CiGig2 promoter mutants (ISRE1mut-luc, ISRE2mut-luc or ISRE3mut-luc, respectively) and pcDNA3.1-IRFs. The results suggested that different ISRE played the diverse roles. ISRE2 is more important than ISRE1 and ISRE3 to the transcription of CiGig2 induced by CiIRF1. ISRE1 and ISRE3 are important to the transcription of CiGig2 induced by CiIRF2 and CiIRF3.

Identification of grass carp (Ctenopharyngodon idella) XBP1S as a primary member in ER stress

ABSTRACT X‐box binding protein 1 (XBP1), a vital basic leucine zipper transcription factor for the related gene transcription in endoplasmic reticulum (ER) stress, belongs to the CREB/ATF family. In mammals, XBP1S is the activated one of XBP1 isoform. In order to study the role of fish XBP1S, we cloned and identified the XBP1S (KU509247) from grass carp (Ctenopharyngodon idella) (named CiXBP1S) by homologous cloning and RACE technique. The full length of CiXBP1S is 1694 bp along with 124 bp of 5′ UTR, 418 bp of 3′ UTR and the longest open reading frame (1152 bp) encoding a polypeptide of 383 amino acids with a well conserved DNA binding domain (BRLZ domain). CiXBP1S shares significant homology to zebrafish XBP1S (˜90%) at amino acid level. RT‐PCR showed that the expression of CiXBP1S was ubiquitous in all tested grass carp tissues and was significantly up‐regulated under the stimulation with tunicamycin (Tm) in CIK (C. idellus kidney) cells. To study the molecular mechanism of transcriptional regulation for XBP1 signaling pathway in fish, we cloned grass carp XBP1 promoter sequence. Its promoter is 1036 bp in length and divided into two distinct regions in which an ER stress response element (ERSE) exists in the proximal region. Meanwhile, grass carp ATF6 (CiATF6N) and CiXBP1S were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni‐NTA His‐Bind resin. Gel mobility shift assay showed that CiATF6N and CiXBP1S had the high affinity with CiXBP1 promoter sequence in vitro. Co‐transfection of pcDNA3.1‐CiATF6 (or pcDNA3.1‐CiXBP1S respectively) with pGL3‐CiXBP1P2 (or pGL3‐CiXBP1P1 respectively) into epithelioma papulosum cyprini (EPC) cells showed that CiATF6 and CiXBP1S played a positive role in CiXBP1S transcription. CiXBP1S also had high affinity with CiGRP78 and CiGRP94 promoter sequences. In addition, recombinant plasmids of pGL3‐CiGRP78P and pGL3‐CiGRP94P were constructed and transiently co‐transfected with pcDNA3.1‐CiXBP1S (pcDN3.1‐CiXBP1S‐nBRLZ, respectively) into EPC cells. The result showed that CiXBP1S can activate CiGRP78 and CiGRP94 promoters. HIGHLIGHTSThe full‐length cDNA and promoter sequence of grass carp XBP1S was cloned and identified.CiXBP1S was ubiquitous expression in tested tissues and significantly up‐regulated in CIK cells after stimulation with Tm.CiATF6 and CiXBP1S can activate the transcription activity of CiXBP1 by binding to its promoter.CiXBP1S played a positive role in CiGRP78 and CiGRP94 transcription.