Transcriptional regulation of the human DNA polymerase delta catalytic subunit gene POLD1 by p53 tumor suppressor and Sp1.

Abstract

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.

• Authors: B Li, M Y Lee
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 276 Issue 32 Pg. 29729-39 (Aug 10 2001) ISSN: 0021-9258 [Print] United States
• PMID: 11375983 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Protein Synthesis Inhibitors
• RNA, Messenger
• Sp1 Transcription Factor
• Tumor Suppressor Protein p53
• Luciferases
• POLD1 protein, human
• DNA Polymerase III
• Tetracycline

Topics

• Amino Acid Sequence
• Base Sequence
• Binding Sites
• Binding, Competitive
• Blotting, Northern
• Blotting, Southern
• Blotting, Western
• DNA Damage
• DNA Polymerase III (genetics, metabolism)
• Dose-Response Relationship, Drug
• Gene Expression Regulation, Enzymologic
• Genes, p53
• Humans
• Luciferases (metabolism)
• Models, Genetic
• Molecular Sequence Data
• Mutation
• Plasmids (metabolism)
• Promoter Regions, Genetic
• Protein Binding
• Protein Structure, Tertiary
• Protein Synthesis Inhibitors (pharmacology)
• RNA, Messenger (metabolism)
• Sequence Homology, Nucleic Acid
• Sp1 Transcription Factor (metabolism)
• Tetracycline (pharmacology)
• Time Factors
• Transcription, Genetic
• Transfection
• Tumor Cells, Cultured
• Tumor Suppressor Protein p53 (metabolism)