ATF4(Activating Transcription Factor4) From Grass Carp(Ctenopharyngodon Idella) Modulates The Transcription Initiation Of GRP78and GRP94in CIK Cells

The endoplasmic reticulum (ER) is a factory in which secretory proteins aresynthesized and manufactured. ER dysfunction will disorder proteins folding whichcan induce ER stress. ER functions are sensitive to the stress. Mainly through the twopaths to response to ER stress timely in mammalian. First, the PERK-eIF2a pathwayprevents the accumulation of these aberrant unfolded and malfolded proteins byregulating the attenuation of protein translation and thus reducing the influx of newproteins into the ER. Second, activate the transcription of ER molecular chaperonesgenes, such as GRP78and GRP94. The phosphorylation of eIF2a inhibits total proteintranslation to response to different environmental stimuli. But it is an exception thatpreferred translation ATF4. ATF4is a main regulator which modulates thetranscription of key genes to adapt to the function of ER. ATF4is a member of theATF/CREB family of the basic leucine zipper (bZIP) transcription factor superfamily.ATF4is a protective protein which regulates the adaptation of cells to ER stress bymodulating the transcription of UPR (Unfolded Protein Response) target genes,including GRP78and GRP94. GRP78and GRP94, belong to GRP (glucose-regulatedprotein) family of endoplasmatic reticulum (ER) chaperone superfamily, are essentialfor cell survival under ER stress with spreading in the ER lumen. GRP is acharacterictic molecular chaperone in ER with its multiple functions in protein folding,assembly and trafficking.Our study found that CiATF4may participate in the similar tasks with GRP78and GRP94in response to cellular stress. The expression trend of CiATF4was similarto CiGRP78and CiGRP94did under37°C thermal stress, namely, the expression ofCiATF4was up-regulated twice at2hr post-thermal stress and at18hr post recoveryfrom thermal stress. They have same expression trends under the same stress whichimplies CiATF4was likely to strongly coordinate with CiGRP78and CiGRP94inresponse to cellular stress. ATF4has been reported as a key activating transcriptionfactor can regulate the transcription of some ER chaperone to adapt to ER stress inmammalian, which provides a theoretical basis for our speculation.To understand the molecular mechanism of ATF4modulates the transcriptioninitiation of CiGRP78and CiGRP94, we cloned ATF4ORF cDNA sequences(CiATF4) by homologous cloning techniques. CiATF4was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatographywith the Ni-NTA His-Bind Resin. At the same time, on the basis of the clonedCiGRP78and CiGRP94cDNA in our laboratory previously, we cloned their promotersequences by genomic walking approach. By analyzing, CiGRP78and CiGRP94promoters contain a multiple copies of the CCAAT (N9) CCACG element (ERSE)flanked by GC-rich sequences. ERSE is essential for the activation of unfoldedprotein response (UPR). In addition, CiGRP78promoter comprised a typical TATAbox(-26), three CCAAT box (-64,-97,-187), two Sp1(transcription factor specificityprotein1) binding site(-325,-656)，a TFIID (transcription factor IID) binding site(-462)，a YY1(Yin-Yang-1) binding site (-368)，a CARE binding site(-757) and twoCREB binding site(-30,-152). CRE and CARE are potential binding site for ATF4binding, but there has none of the two elements in CiGRP94promoter. In addition tothe ERSE, CiGRP94promoter comprised a TATA box (-53), two CCAAT box(212，-435)，a YY1binding site(-599) and two NF-Y (nuclear factor Y) binding site (-90,-205).In vitro, gel mobility shift assays revealed that CiATF4could bind to CiGRP78and CiGRP94promoter with high affnity. Compared to the control group, themigration rate of the experimental group (CiATF4and CiGRP78/CiGRP94promoterfragment recombinant expression in vitro) was clearly blocked. Even more, to acertain extent, this blocking effect enhanced with the increasing of ATF4concentration. This showed that CiATF4directly bind to CiGRP78and CiGRP94promoters and have the potential to regulate the transcription of CiGRP78andCiGRP94.To test and verify the function of CiATF4in vivo, the recombinant plasmid ofpGL3-CiGRPs and pcDNA3.1-CiATF4were constructed and transientlyco-transfected into Ctenopharyngodon idella kidney (CIK) cells. The impact ofCiATF4on CiGRP promoter sequences were measured by luciferase assays. Wefound that CiATF4could activate the activity of Luciferase Report Gene greatly. Itproved that CiATF4could activate the transcription of CiGRP78and CiGRP94.What’s more, to better understand the molecular mechanism of CiATF4modulate thetranscription initiation of CiGRP, three mutant fragments of CiGRP78promoterrecombinant plasmids (called CARE-mut/LUC, CRE1-mut/LUC and CRE2-mut/LUC)were constructed and transiently co-transfected with CiATF4into CIK cells. Theresults indicated that both CRE and CARE elements were the regulatory element fortranscription initiation of CiGRP78. CRE and CARE elements were important in the stimulation of CiATF4. Between them, CRE element would play more important rolein it.