A DNA Methylation Site in the Male-Specific P450 ( Cyp 2d-9) Promoter and Binding of the Heteromeric Transcription Factor GABP

Abstract

The Cyp 2d-9 gene encodes the male-specific steroid 16␣-hydroxylase in mouse liver and shares a conserved regulatory element (؊100 TTCCGGGC ؊93) with another male-specific Slp promoter. As shown with the Slp promoter (N. Yokomori, R. Moore, and M. Negishi, Proc. Natl. Acad. Sci. USA 92:1302–1306, 1995), the male-preferential demethylation also occurs at CpG/؊97 in the Cyp 2d-9 promoter. The transcription factor which specifically binds to the demethylated element has been purified. The peptide sequences reveal that the factor consists of GABP␣ and GABP␤1 with Ets and Notch motifs, respectively. Both DNase I footprinting and gel shift assays indicate that the bacterially expressed glutathione S-transferase–GABP fusion proteins bind to the regulatory element only when CpG/؊97 is demethylated. Moreover, Cyp 2d-9 promoter is trans-activated by coexpression of GABP proteins in HepG2 cells. Given the additional results that CpG/؊50 of the female-specific steroid 15␣-hydroxylase (Cyp 2a-4) promoter is preferentially demethylated in the females, the sex-specific expressions of the P450 genes correlate very well with DNA demethylation. We also conclude that GABP is a methylation-sensitive transcription factor and is a potential transcription activator of the male-specific Cyp 2d-9 promoter.