Purification and identification of positive regulators binding to a Novel element in the c‐jun promoter

Abstract

A putative response element, GAGCCTC, was investigated to regulate c‐jun expression in this study. Electrophoretic mobility shift assays demonstrate that sequence‐specific binding proteins form a specific complex with this element in HEK293 cells. Additionally, unlabeled consensus AP‐1 element DNA, but not a similar NF‐jun element DNA, competes with complex formation. Mutations of this element decrease c‐jun promoter reporter activity by nearly 5‐fold in HEK293 cells. A new, two‐step oligonucleotide trapping technique was developed to purify the element binding proteins. LC‐Nanospray‐ESI‐MS/MS identification and western blotting analysis show that the purified complex contains Ku80, Ku70 and c‐jun, which was further confirmed by antibody supershift and immunoprecipitation analysis, and by southwestern blot and UV crosslinking analysis in vitro as well as chromatin immunoprecipitation in vivo. c‐Jun promoter activity decreases after Ku80 was significantly decreased by its siRNA, and c‐jun expression decreased at both the mRNA and protein levels. A mutant Ku80 with normal amino acid sequence but immune to the siRNA recovers c‐jun promoter activity from siRNA inhibition. Similarly, Ku70 wild type transfection can also up‐regulate c‐jun promoter activity. Thus, we speculate that Ku80‐Ku70‐c‐jun trimer activates c‐jun expression by binding to this GAGCCTC element in the c‐jun promoter.