Transcriptional regulation of the human DNA polymerase delta catalytic subunit gene POLD1 by p53 tumor suppressor and Sp1.
The DNA polymerase delta catalytic subunit gene (POLD1) was studied as a transcriptional target of p53. Northern blotting showed that a significantly decreased steady-state level of POLD1 mRNA was associated with increased wild-type p53 expression in cells treated with methyl methanesulfonate. When ectopic wild-type p53 expression was induced to a physiologically relevant level in "tet-off" cultured cells in which p53 expression was tightly regulated by tetracycline, it was found that POLD1 steady-state mRNA was repressed by about 65%. Transient cotransfection experiments using a POLD1 promoter luciferase reporter construct showed that: (i) POLD1 promoter activity was inhibited by transfected wild-type p53 plasmid to a maximum of about 86%; (ii) p53 mediated a large part of the transcriptional repression through a sequence-specific interaction with a site identified as the P4 site of the POLD1 promoter; (iii) tumor-derived p53 mutations in the p53 DNA-binding domain completely abolished the p53 transrepression activity. Moreover, transfection assays demonstrated that p53 was able to repress Sp1-stimulated POLD1 promoter activity and that this repression was largely due to the loss of the sequence-specific interaction between Sp1 protein and the P4 Sp1-binding site, which overlaps the P4 p53-binding site. Finally, gel shift assays suggested that p53 competes with Sp1 protein for binding to the P4 sequence of the POLD1 promoter.
Li
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Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Lee
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MY
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D004249:DNA Damage; D004258:DNA Polymerase III / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D016158:Genes, p53; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D016159:Tumor Suppressor Protein p53; D008156:Luciferases; C434055:POLD1 protein, human; D004258:DNA Polymerase III; D013752:Tetracycline