Characterization of the 5'flanking region of the human Chk1 gene: identification of E2F1 functional sites.

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 involvement in DNA damage checkpoint pathways.