DeltaN133p53 expression levels in relation to haplotypes of the TP53 internal promoter region.
The transcription of the DeltaN133p53 isoform of the TP53 gene is controlled by an internal promoter region (IPR) containing eight polymorphisms in 11 common haplotypes, following a resequencing of 47 Caucasians. We assayed the functional effects of the commonest six haplotypes on the promoter activity with a luciferase reporter system, in HeLa and 293T cells. These studies showed that different IPR haplotypes are associated with differences in the promoter activity resulting in marked variation in the baseline expression of DeltaN133p53. In vivo quantitative-polymerase chain reaction (PCR) on human tissues confirmed that the baseline levels of DeltaN133p53 showed haplotype specific differences that paralleled those seen in vitro. When cell lines were treated with camptothecin, the fold-increase in DeltaN133p53 levels was dose-dependent but haplotype-independent (i.e., similar for all the haplotypes). Finally, we used an electrophoretic mobility shift assay to analyze the rs1794287 polymorphism and found changes in the pattern of protein binding. This partially confirmed our in silico analysis showing that the polymorphism rs1794287 can affect the function of the internal promoter by changing its affinity for several transcription factors. Thus, we showed that the expression of DeltaN133p53 is under genetic control, and suggested the presence of interindividual differences underlying this mechanism.
Bellini
Ilaria
I
Department of Biology, University of Pisa, Via Derna 1, Pisa, Italy.
Pitto
Letizia
L
Marini
Maria G
MG
Porcu
Loredana
L
Moi
Paolo
P
Garritano
Sonia
S
Boldrini
Laura
L
Rainaldi
Giuseppe
G
Fontanini
Gabriella
G
Chiarugi
Massimo
M
Barale
Roberto
R
Gemignani
Federica
F
Landi
Stefano
S
D019295:Computational Biology; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006239:Haplotypes / Q000235:genetics*; D006367:HeLa Cells; D006720:Homozygote; D006801:Humans; D007438:Introns / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D050505:Mutant Proteins; D020033:Protein Isoforms; D016159:Tumor Suppressor Protein p53; D004247:DNA; D008156:Luciferases