A DNA METHYLATION SITE IN THE MALE-SPECIFIC P450 (CYP 2D-9) PROMOTER AND BINDING OF THE HETEROMERIC TRANSCRIPTION FACTOR GABP

The Cyp 2d-9 gene encodes the male-specific steroid 16 alpha-hydroxyla se in mouse liver and shares a conserved regulatory element ((-100)TTC CGGGC(-93)) with another male-specific Sip promoter, As shown with the Sip promoter (N. Yokomori, R. Moore, and M. Negishi, Proc. Natl. Acad . Sci. USA 92:1302-1306, 1995), the male-preferential demethylation al so occurs at CpG/-97 in the Cyp 2d-9 promoter, The transcription facto r which specifically binds to the demethylated element has been purifi ed, The peptide sequences reveal that the factor consists of GABP alph a and GABP beta 1 with Ets and Notch motifs, respectively, Both DNase I footprinting and gel shift assays indicate that the bacterially expr essed glutathione S-transferase-GABP fusion proteins hind to the regul atory element only when CpG/-97 is demethylated. Moreover, Cyp 2d-9 pr omoter is trans-activated hy coexpression of GABP proteins in HepG2 ce lls, Given the additional results that CpG/-50 of the female-specific steroid 15 alpha-hydroxylase (Cyp 2a-4) promoter is preferentially dem ethylated in the females, the sex-specific expressions of the P450 gen es correlate very well with DNA demethylation, We also conclude that G ABP is a methylation-sensitive transcription factor and is a potential transcription activator of the male-specific Cyp 2d-9 promoter.