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 tr anscriptional target of p53. Northern blotting showed that a significantly decreased steady-state level of POLD1 mRNA was associated with increased wi ld-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 regul ated by tetracycline, it was found that POLD1 steady-state mRNA was repress ed by about 65%. Transient cotransfection experiments using a POLD1 promote r luciferase reporter construct showed that: (i) POLD1 promoter activity wa s 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 d omain completely abolished the p53 transrepression activity. Moreover, tran sfection assays demonstrated that p53 was able to repress Sp1-stimulated PO LD1 promoter activity and that this repression was largely due to the loss of the sequence-specific interaction between Sp1 protein and the P4 Spl-bin ding site, which overlaps the P4 p53-binding site. Finally, gel shift assay s suggested that p53 competes with Sp1 protein for binding to the P4 sequen ce of the POLD1 promoter.