The gut-enriched Krüppel-like factor (GKLF) suppresses the promoter activity of the cytochrome P450 1A1 (CYP1A1) gene in a SP1-dependent fashion*

Background: GKLF is a recently identified transcription factor that contains 3 C2H 2 zinc fingers with homology to the Drosophila Kriippel protein [Shields, Christy and Yang (1996) J. Biol. Chem. 271, 20009; Shields and Yang (1997) J. Biol. Chem. 277~2 18504]. Expression of GKLF is enriched in the gut epithelium and is associated with growth arrest. Recent studies have identified a consensus DNA sequence to which GKLF binds [Shields and Yang (1998) Nucl. Acids Res., in press]. This sequence is very similar to the basal transcription dement (BTE) this is essential for the promoter activity of many genes in the cytochrome P450 family, including CYP1A1. Aim: The aim of the present study is to investigate whether GKLF is involved in the regulation of the CYP1A1 promoter through the BTE. Methods: The binding of GKLF to BTE was analyzed by methylation interference and electrophoretic mobility shift assays (EMSA) using partially purified recombinant GKLF and a radioactively labeled double-stranded oligonucleotide containing the GKLF consensus binding sequence (named TDA) or BTE. The effect of GKLF on CYP1A1 promoter activity was examined by cotransfection experiments in Chinese hamster ovary (CHO) cells with an eukaryotic expression construct containing full-length or truncated GKLF and a chloramphenicol acetyl transferase (CAT) reporter gene driven by the CYP1A1 Y-flanking sequence including or not BTE. Results: EMSA of labeled TDA and BTE using recombinant GKLF demonstrated the formation of a single, identical DNA-protein complex. The minimal essential DNA sequences within TDA and BTE that bound GKLF were identical as defined by base-specific mutagenesis and methylation interference assays. In transiently transfected CHO cells, an expression construct containing full-length but not truncated GKLF was able to suppress, in a dose-dependent fashion, the activity of the CAT reporter driven by the wild-type CYP1A1 promoter but not by a mutated promoter in which the BTE was specifically deleted. As a deletion of the BTE from the CYP1A1 promoter also resulted in a significant decrease in the basal promoter activity, nuclear extracts from CHO cells were prepared and analyzed by EMSA. The results indicate that the primary protein in CHO cells binding to BTE was Spl as demonstrated by antibody super-shift experiments. Cotransfection of the CYP1A1 promoter-reporters with an expression construct containing Spl showed that Spl transactivated the CYP1A1 promoter in a BTE-dependent fashion. The further addition of GKLF to the above transfection condition resulted in a dose-dependent abrogation of the ability of Spl to activate CYP1A1 promoter. Conclusion: The results indicate that GKLF is an GASTROENTEROLOGY Vol. 114, No. 4