title: Grass carp (Ctenopharyngodon idella) ATF6 (activating transcription factor 6) modulates the transcriptional level of GRP78 and GRP94 in CIK cells.
abstract: ATF transcription factors are stress proteins containing alkaline area-leucine zipper and play an important role in endoplasmic reticulum stress. ATF6 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. In the present study, a grass carp (Ctenopharyngodon idella) ATF6 full-length cDNA (named CiATF6, KT279356) has been cloned and identified. CiATF6 is 4176 bp in length, comprising 159 nucleotides of 5'-untranslated sequence, a 1947 nucleotides open reading frame and 2170 nucleotides of 3'-untranslated sequences. The largest open reading frame of CiATF6 translates into 648 aa with a typical DNA binding domain (BRLZ domain) and shares significant homology to the known ATF6 counterparts. Phylogenetic reconstruction confirmed its closer evolutionary relationship with other fish counterparts, especially with Zebrafish ATF6. RT-PCR showed that CiATF6 was ubiquitously expressed and significantly up-regulated after stimulation with thermal stress in all tested grass carp tissues. In order to know more about the role of CiATF6 in ER stress, recombinant CiATF6N 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 shift assays were employed to analyze the interaction of CiATF6 protein with the promoters of grass carp GRP78 and GRP94, respectively. The result has shown that CiATF6 could bind to these promoters with high affinity by means of its BRLZ mainly. To further study the transcriptional regulatory mechanism of CiATF6, Dual-luciferase reporter assays were applied. Recombinant plasmids of pGL3-GRP78P and pGL3-CiGRP94P were constructed and transiently co-transfected with pcDNA3.1-CiATF6 (pcDN3.1-CiATF6-nBRLZ, respectively) into C. idella kidney (CIK) cells. The result has shown that CiATF6 could activate CiGRP78 and CiGRP94 promoters.
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang, 330031, China.
authors.forename: Xiangqin
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang, 330031, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang, 330031, China. Electronic address: huilinm6@163.com.
authors.forename: Huiling
authors.initials: H
authors.lastname: Mao
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang, 330031, China.
authors.forename: Yichuan
authors.initials: Y
authors.lastname: Mi
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang, 330031, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhong
authors.affiliation: Jiangxi Agricultural University, Nanchang, 330045, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Wei
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang, 330031, China.
authors.forename: Xiancheng
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Bioscience, College of Life Science, Nanchang University, Nanchang, 330031, China. Electronic address: hucy2008@163.com.
authors.forename: Chengyu
authors.initials: C
authors.lastname: Hu
mesh_terms: D051702:Activating Transcription Factor 6 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D015854:Up-Regulation
chemical_list: D051702:Activating Transcription Factor 6; D029941:Fish Proteins; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; C018551:glucose-regulated proteins
keywords: ATF6; Endoplasmic reticulum stress; GRP78 and GRP94; Transcriptional control; Unfolded protein respond