THE GUT-ENRICHED KRUPPEL-LIKE FACTOR SUPPRESSES THE ACTIVITY OF THE CYP1A1 PROMOTER IN AN SP1-DEPENDENT FASHION

The gut-enriched Kruppel-like factor (GKLF) is a newly identified zinc finger-containing transcription factor. Recent studies indicate that GKLF binds to a core DNA sequence of 5'-(G/A) (G/A)GG(C/T)G(C/T)-3', w hich is found in an endogenous cis element, the basic transcription el ement (BTE) of the cytochrome P-450LA1 (CYP1A1) promoter. The present study characterizes the ability of GKLF to regulate CYP1A1 expression. By electrophoretic mobility gel shift assay (EMSA) and methylation in terference assay, GKLF was found to bind BTE in a manner similar to se veral other transcription factors known to interact with BTE including Spl and BTEB. Cotransfection studies in Chinese hamster ovary cells s howed that GKLF inhibited the CYP1A1 promoter in a dose and BTE depend ent manner. The same exper iments also revealed that BTE was responsib le for a significant portion of the CYP1A1 promoter activity. EMSA of nuclear extracts from Chinese hamster ovary cells showed that Spl and Sp3 were two major proteins that interacted with BTE. Additional cotra nsfection studies showed that GKLF inhibited Spl mediated activation o f the CYP1A1 promoter. In contrast, GKLF enhanced Sp3-dependent suppre ssion of the same promoter. Moreover, the ability of GKLF to inhibit S p1-dependent transactivation was in part due to physical interaction o f the two proteins. These findings indicate that GKLF is a negative re gulator of the CYP1A1 promoter in a BTE-dependent fashion and that thi s inhibitory effect is in part mediated by physical interaction with S p1.