Overexpression of transcription factor AP-2 stimulates the PA promoter of the human uracil-DNA glycosylase (UNG) gene through a mechanism involving derepression.
The PA promoter in the human uracil-DNA glycosylase gene (UNG) directs expression of the nuclear form (UNG2) of UNG proteins. Using a combination of promoter deletion and mutation analyses, and transient transfection of HeLa cells, we show that repressor and derepressor activities are contained within the region of DNA marked by PA. Footprinting analysis and electrophoretic mobility shift assays of PA and putative AP-2 binding regions with HeLa cell nuclear extract and recombinant AP-2alpha protein indicate that AP-2 transcription factors are central in the regulated expression of UNG2 mRNA. Chromatin immunoprecipitation with AP-2 antibody demonstrated that endogenous AP-2 binds to the PA promoter in vivo. Overexpression of AP-2alpha, -beta or -gamma all stimulated expression from a PA-luciferase reporter gene construct approximately 3- to 4-fold. Interestingly, an N-terminally truncated AP-2alpha, lacking the activation domain but retaining the DNA binding and dimerization domains, stimulated PA to a level approaching that of full-length AP-2, suggesting that AP-2 overexpression stimulates PA activity by a mechanism involving derepression rather than activation, possibly by neutralizing an inhibitory effect of endogenous AP-2 or AP-2-like factors.
Department of Cancer Research and Molecular Medicine, Children's and Women's Health, Faculty of Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway.
Per Arne
PA
Aas
Javier
J
Peña-Diaz
Nina Beate
NB
Liabakk
Hans E
HE
Krokan
Frank
F
Skorpen
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D050684:E2F Transcription Factors / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050656:Transcription Factor AP-2 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase / Q000235:genetics*
D023081:CCAAT-Binding Factor; D050684:E2F Transcription Factors; D011993:Recombinant Fusion Proteins; D050656:Transcription Factor AP-2; D014212:Tretinoin; D008156:Luciferases; D003850:Deoxyribonuclease I; D051981:Uracil-DNA Glycosidase