P107
Chk1 kinase is a key signal transducer in the
checkpoint pathways responding to DNA damage. Chk1 has been shown to be transcriptionally down-regulated in response to cis-dichloro-diamino platinum treatment in a p53-dependent
manner. Now we isolated and characterised the 5'
flanking region of the Chk1 gene. We demonstrated that the isolated region has
promoter activity when cloned upstream to a reporter luciferase
gene and we could define the minimal promoter region. The modulation of the
transcriptional activity of the cloned Chk1 promoter
region by different transcription factors was investigated by co-transfection experiments and by using different isogenic systems. It was shown that p53 is indeed able to
down-regulate the promoter activity of the cloned region, providing a
mechanistic explanation to the observation that p53 decreases Chk1 protein
levels after DNA damage. Several E2F binding sites were detected in the genomic
sequence and E2F1 induced Chk1 promoter activity in co-transfection
experiments. This induction was abolished when a mutated form of E2F1, not able
to bind DNA, was used. Over-expression of E2F1 sustained an increased Chk1
promoter activity, whereas overexpression of Rb protein, which binds E2F factors, decreased Chk1
promoter activity. We discuss our results in the context of Chk1 involvment in DNA damage checkpoint pathways.