Analysis Of Transcriptional Regulation Of GRP78 And GRP94 Mediated By ATF6 And XBP1 In Grass Carp (Ctenopharyngodon Idella)

ATF6(activating transcription factor 6)and XBP1S(X-box binding protein 1)are vital basic leucine zipper transcription factor for related gene transcription in endoplasmic reticulum(ER)stress,and belong to the CREB/ATF family.ATF6 and XBP1 S are protective protein which regulate the adaptation of cells to ER stress by modulating the transcription of UPR(Unfolded Protein Response)target genes.Under the ER stress,the cells will produce a core activity form of ATF6(ATF6N)in the nucleus,ATF6 N activates the expression of guucose regulated protein(GRP)(mainly GRP78 and GRP94)and activates unspliced XBP1(XBP1U)mRNA.At the same time IRE1α dimerization and phosphorylation,and subsequently cleaves the 26 nucleotides in specific intron-exon area of XBP1 U mRNA to generate the open reading frame coding longer XBP1(spliced XBP1,XBP1S).XBP1 S transits into the incleus and and alleviates the ER stress through activating the related proteins and ERAD in the ER stress response,finally promoting cells maintain ER homeostesis.In order to understand the molecular mechanism of fish ATF6,as a transcriptional regulation factor,regulating the transcription of downstream target genes.Grass carp(Ctenopharyngodon idella)ATF6 full-length cDNA(named CiATF6,KT279356)has been cloned and identified,we analyzed its protein structure and found that the N-termianl containing a very conservative BRLZ domain——DNA structure domain.And,the promoter of CiGRP78 and CiGRP94 were cloned.We analyzed the the expression of CiATF6 in different tissues and CIK cells under tunicamycin stimulation by RT-PCR.The results showed that CiATF6 was ubiquitously expressed in all tested grass carp tissues and the highest expression was in liver tissue.And TM upregulated the expression of CiATF6.Recombinant CiATF6 N with His-tag was over-expressed in Rosetta Escherichia coli,and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin.In vitro,gel mobility shifts assays showed that CiATF6 N have obvious blocking effect with the promoter of CiGRP78 and CiGRP94,those illustrated that CiATF6 is potential transcriptional regulator factor for CiGRP78 and CiGRP94.Then the recombinant plasmid of pcDNA3.1-CiATF6 and pcDNA3.1-CiATF6 were constructed and transiently co-transfected into CIK cells with pGL-CiGRP78 P or pGL-CiGRP94 P,respectively.The results of luciferase assays showed that CiATF6 significantly up-regulated the activity of luciferase,demonstrating that CiATF6 could activate the transcription of CiGRP78 and CiGRP94.To further explore of grass crap ATF6 regulating the transcription of XBP1,the promoter of grass carp XBP1(CiXBP1)and the grass carp XBP1S(CiXBP1S)(KU509247)full-length cDNA were cloned and identified.We analyzed the expression of CiXBP1 S in different tissues and CIK cells under TM stimulation by RT-PCR.The results showed that CiXBP1 S was ubiquitously expressed in all tested grass carp tissues and the highest expression was in liver tissue.And TM upregulated the expression of CiXBP1 S.The promoter of CiXBP1 contains an ER stress element(ERSE)by using online software,and we constructed the luciferase reporter gene recombinant plasmid of pGL-CiXBP1 P and its mutant pGL-CiXBP1P-P1 and pGL-CiXBP1P-P2.The dual luciferase assays found that the pGL-Ci XBP1 P and pGL-CiXBP1P-P2 had high promoter activity,while pGL-CiXBP1P-P1 almost has no activity.In vitro,gel mobility shifts assays showed that CiATF6 N have obvious blocking effect with the CiXBP1P-P2 rather than CiXBP1P-P1.And the same time,we found that CiXBP1 S also have the same phenomenon.The dual luciferase assays further confirmed that Ci ATF6 can activate the transcription of CiXBP1,more interesting is that CiXBP1 S can increase the luciferase activity value of pGL-CiXBP1P-P2,while the luciferase activity value of pGL-CiXBP1P-P1 has no changed.The results show that CiXBP1 S has self-regulation function.To study the molecular mechanism of CiXBP1 S regulating the transcription of CiGRP78 and CiGRP94.Gel mobility shift assays were employed to analyze the interaction of CiXBP1 S protein with the promoters of grass carp GRP78 and GRP94,respectively.The results showed that CiXBP1 S have obviously blocking for the promoter of CiGRP78 and CiGRP94,but Ci XBP1S-nBRLZ has no blocking phenomenon.Those suggested that the BRLZ domain of CiXBP1 S is very important for regulating the downstream target genes.The dual luciferase assays further confirmed that CiXBP1 S can significantly increase the promoter activity of CiGRP78 and CiGRP94,but CiXBP1S-nBRLZ has no effect.