Purification and identification of positive regulators binding to a novel element in the c-Jun promoter

Biochemistry. 2008 Sep 2;47(35):9318-34. doi: 10.1021/bi800285q. Epub 2008 Aug 9.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antigens, Nuclear / chemistry
  • Antigens, Nuclear / isolation & purification
  • Antigens, Nuclear / metabolism*
  • Base Sequence
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Electrophoretic Mobility Shift Assay
  • Humans
  • Ku Autoantigen
  • Molecular Sequence Data
  • Promoter Regions, Genetic*
  • Proto-Oncogene Proteins c-jun / chemistry
  • Proto-Oncogene Proteins c-jun / genetics*
  • Proto-Oncogene Proteins c-jun / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Response Elements*
  • Transcription, Genetic
  • Transfection

Substances

  • Antigens, Nuclear
  • DNA-Binding Proteins
  • Proto-Oncogene Proteins c-jun
  • RNA, Small Interfering
  • Xrcc6 protein, human
  • Ku Autoantigen