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

J Biol Chem. 2001 Aug 10;276(32):29729-39. doi: 10.1074/jbc.M101167200. Epub 2001 May 25.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Binding, Competitive
  • Blotting, Northern
  • Blotting, Southern
  • Blotting, Western
  • DNA Damage
  • DNA Polymerase III / genetics*
  • DNA Polymerase III / 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*

Substances

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