Title

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

Date of Award

2000

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Marietta Y. W. T. Lee, Committee Chair

Abstract

To understand roles of p53 in regulating DNA replication/repair, the DNA polymerase delta catalytic subunit gene (POLD1) was studied as a transcriptional target of p53 in response to DNA damage.Using a "tet-off" cell culture system, Northern blot results showed that POLD1 steady-state mRNA was repressed to about 23% of the control when ectopic wild-type p53 expression was induced to a physiologically relevant level. Cell culture transient transfection using a POLD1 promoter luciferase reporter (pGL-1758) demonstrated that POLD1 repression was regulated by transfected wild-type p53 plasmid at the level of POLD1 promoter in a dose-dependent manner. p53 inhibited the POLD1 promoter activity to a maximum of about 14% of the control. EMSA and footprinting assay results showed a specific physical interaction between p53 protein and the P4 p53-binding site in the POLD1 promoter sequence, using purified wild-type p53 overexpressed in insect cells. In EMSA, the interaction between p53 protein and the P4 p53-binding site was abrogated using a oligonucleotide (mP4) containing mutations in the P4 p53-binding site. A reporter construct (pGL-1758mP4) containing site-directed mutations in the P4 p53-binding site in POLD1 promoter was used. p53 repressed promoter activity of pG-L1758mP4 by about 35%. The combined data indicated that p53 mediated its primary transcriptional repression effects on the POLD1 promoter through a physical interaction between the p53 protein and the P4 p53-binding site. Finally, tumor-derived p53 mutations in the p53 DNA-binding domain (V143A, R175H, R248W, R273H and R282W) completely abolished the p53 transrepression activity as assayed using POLD1 promoter reporter. A double-mutation L22Q/W23S in the p53 transcription activation domain also eliminated the p53 transrepression activity. The DeltaPro p53 mutant with a deletion of the proline rich region partially inhibited the POLD1 promoter activity.An interaction between Sp1 protein and the P4 Sp1-binding site in POLD1 promoter sequence was detected by Southwestern assay and EMSA. In EMSA, the interaction was abolished using a oligonucleotide (mSp1) containing mutations in the P4 Sp1-binding site. Transfection assays demonstrated a 7-fold stimulation of POLD1 promoter activity by Sp1, which was reduced to 38% by the cotransfected wild-type p53 plasmid. Moreover, disruption of the P4 Sp1-binding site in the POLD1 promoter reporter construction resulted in a reduction of Sp1-stimulated promoter activity to 54%. These data indicated that the interaction between Sp1 protein and the P4 Sp1-binding site played a major role in the Sp1-stimulation of POLD1 promoter, and that the P4 Sp1-binding site was largely involved in the repression of the Sp1-stimulated promoter activity by p53.Finally, in EMSA and coimmunoprecipitation assays, p53 was demonstrated to compete with Sp1 protein for binding to the P4 sequence of the POLD1 promoter without the stable formation of a Sp1/p53/DNA complex or Sp1/p53 immuno-complex. A model is proposed wherein the p53 down-regulation of POLD1 promoter primarily involves a mechanism of molecular exclusion between p53 protein and Sp1 transcription factor at the overlapping P4 p53-binding site and P4 Sp1-binding site of the POLD1 promoter.

Keywords

Biology, Molecular

Link to Full Text

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