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. What's 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.
