Overexpression of transcription factor AP-2 stimulates the PA promoter of the human uracil-DNA glycosylase (UNG) gene through a mechanism involving derepression

TY - JOUR

T1 - Overexpression of transcription factor AP-2 stimulates the PA promoter of the human uracil-DNA glycosylase (UNG) gene through a mechanism involving derepression

AU - Aas, Per Arne

AU - Pena Diaz, Javier

AU - Liabakk, Nina Beate

AU - Krokan, Hans E

AU - Skorpen, Frank

PY - 2009/7/4

Y1 - 2009/7/4

N2 - 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.

AB - 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.

KW - Base Sequence

KW - Binding Sites

KW - CCAAT-Binding Factor

KW - Cell Nucleus

KW - DNA Footprinting

KW - Deoxyribonuclease I

KW - E2F Transcription Factors

KW - Electrophoretic Mobility Shift Assay

KW - Gene Expression

KW - Gene Expression Regulation, Enzymologic

KW - HeLa Cells

KW - Humans

KW - Luciferases

KW - Molecular Sequence Data

KW - Mutation

KW - Promoter Regions, Genetic

KW - Protein Binding

KW - Recombinant Fusion Proteins

KW - Transcription Factor AP-2

KW - Transfection

KW - Tretinoin

KW - Uracil-DNA Glycosidase

U2 - 10.1016/j.dnarep.2009.03.008

DO - 10.1016/j.dnarep.2009.03.008

M3 - Journal article

C2 - 19411194

SN - 1568-7864

VL - 8

SP - 822

EP - 833

JO - DNA Repair

JF - DNA Repair

IS - 7

ER -