The Upstream 1350~1250 Nucleotide Sequences of the Human ENDOU-1 Gene Contain Critical Cis-Elements Responsible for Upregulating Its Transcription during ER Stress

Abstract

ENDOU-1 encodes an endoribonuclease that overcomes the inhibitory upstream open reading frame (uORF)-trap at 5′-untranslated region (UTR) of the CHOP transcript, allowing the downstream coding sequence of CHOP be translated during endoplasmic reticulum (ER) stress. However, transcriptional control of ENDOU-1 remains enigmatic. To address this, we cloned an upstream 2.1 kb (−2055~+77 bp) of human ENDOU-1 (pE2.1p) fused with reporter luciferase (luc) cDNA. The promoter strength driven by pE2.1p was significantly upregulated in both pE2.1p-transfected cells and pE2.1p-injected zebrafish embryos treated with stress inducers. Comparing the luc activities driven by pE2.1p and −1125~+77 (pE1.2p) segments, we revealed that cis-elements located at the −2055~−1125 segment might play a critical role in ENDOU-1 upregulation during ER stress. Since bioinformatics analysis predicted many cis-elements clustered at the −1850~−1250, we further deconstructed this segment to generate pE2.1p-based derivatives lacking −1850~−1750, −1749~−1650, −1649~−1486, −1485~−1350 or −1350~−1250 segments. Quantification of promoter activities driven by these five internal deletion plasmids suggested a repressor binding element within the −1649~−1486 and an activator binding element within the −1350~−1250. Since luc activities driven by the −1649~−1486 were not significantly different between normal and stress conditions, we herein propose that the stress-inducible activator bound at the −1350~−1250 segment makes a major contribution to the increased expression of human ENDOU-1 upon ER stresses.