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-hydroxylase in mouse liver and shares a conserved regulatory element (-100TTCCGGGC-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 alpha and GABP beta 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 alpha-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.
Yokomori
N
N
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Kobayashi
R
R
Moore
R
R
Sueyoshi
T
T
Negishi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism; D005260:Female; D051268:GA-Binding Protein Transcription Factor; D006801:Humans; D008099:Liver / Q000737:chemistry; D008297:Male; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012733:Sex Differentiation / Q000235:genetics; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics*; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation
D001189:Aryl Hydrocarbon Hydroxylases; D003577:Cytochrome P-450 Enzyme System; D004247:DNA; D004268:DNA-Binding Proteins; D015226:Dinucleoside Phosphates; D051268:GA-Binding Protein Transcription Factor; D011993:Recombinant Fusion Proteins; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases; D014157:Transcription Factors; C495656:Gabpa protein, mouse; C495658:Gabpb1 protein, mouse; C015772:CpG dinucleotide