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ATF transcription factors are stress protein containing alkaline area leucine zipper and play an important role in the 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. To understand the molecular mechanism of ATF6 modulates the transcription initiation of CiGRP78 and CiGRP94, we cloned ATF6 ORF cDNA sequences (CiATF6) by homologous cloning techniques. CiATF6 (KT279356) is 4176 bp in length, comprising 159 nucleotides of 5′-untranslated sequence, a 1947nucleotide open reading frame and 2170 nucleotides of 3′untranslated sequence. The largest open reading frame of CiATF6 translate into 648 aa and contains a typical DNA binding domain—BRLZ domain. In vitro, the results showed that CiATF6 bound to the promoters of CiGRP78 and CiGRP94 by means of its BRLZ. The expression trend of CiATF6 was similar to CiGRP78 and CiGRP94 did under thermal stress. Afterwards, recombinant plasmids pGL-CiGPRP78P and pGL-CiGPRP94P were constructed and transiently co-transfected with pcDNA3.1-CiATF6 respectively into C. idella kidney (CIK) cells. Dual-luciferase reporter assays demonstrated that CiATF6 up-regulates the transcription activity of CiGRP78 and CiGRP94 genes in CIK cells. |
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