Purification and identification of positive regulators binding to a novel element in the c-Jun promoter.
A putative response element, GAGCCTC, was observed years ago in footprinting analysis of the c-jun promoter, and here we investigate its function in regulating c-jun expression and identify a protein complex that binds there. Electrophoretic mobility shift assays demonstrate a sequence-specific binding 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 show that the purified complex contains Ku80 and c-jun, which was further confirmed by antibody supershift, by immunoprecipitation with Southwestern blot or with UV cross-linking analysis in vitro as well as chromatin immunoprecipitation in vivo. c-Jun promoter activity and c-jun expression were decreased by Ku80 siRNA introduction. A mutant Ku80 plasmid 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 upregulate c-jun promoter activity. Thus, Ku80-c-jun activates c-jun expression by binding to this GAGCCTC element in the c-jun promoter and Ku70 may also serve a role.
Jiang
Daifeng
D
Department of Chemistry, University of Texas at San Antonio, San Antonio, Texas 78249, USA.
Zhou
YanWen
Y
Moxley
Robert A
RA
Jarrett
Harry W
HW
D034961:Antigens, Nuclear / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D014158:Transcription, Genetic; D014162:Transfection
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D016755:Proto-Oncogene Proteins c-jun; D034741:RNA, Small Interfering; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen