Glutathione S-transferase (GST) Ya subunit gene expression is induced in mammalian tissues by two types of chemical agents: (i) planar aromatic compounds (e.g., 3-methylcholanthrene, g3-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) and (i) electrophiles (e.g., trans-4-phenyl-3-buten-2-one and dimethyl fumarate) or compounds easily oxidized to electrophiles (e.g., tert-butylhydroquinone).To study the mechanism of this induction, we have introduced deletions in the 5' flanking region of a mouse GST Ya subunit gene, fused it to the coding sequence for chloramphenicol acetyltransferase (CAT) To elucidate the mechanisms of regulation of GST Ya subunit gene expression by the two types of inducers, we have previously isolated a mouse GST Ya gene (7, 8) and have shown the presence of xenobiotic responsive elements in the 5' flanking region (9). In the present study, we demonstrate that this region contains between nucleotides -754 and -713 an inducible element that activates Ya gene transcription in cis in response to a variety of inducers such as 3-methylcholanthrene, p-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), tert-butylhydroquinone, and trans4phenyl-3-buten-2-one. These results raise the question of the mechanisms proposed for the regulation of GST gene expression by planar aromatic and electrophilic inducers (4,5). Our results show that, in order to function as Ya gene inducers, the planar aromatics have to be metabolized by the cytochrome P1-450 system into electrophilic compounds. In this paper we bring evidence that the inducible expression of GST Ya subunit gene is controlled by a single electrophile-responsive element (EpRE), which is activated exclusively by an electrophilic signal. Protein (Weizmann Institute). The cell lines were propagated as described (9). Nuclear and cytosolic extracts were prepared from the cell lines as described by Dignam et al. (10), and DNase I protection patterns (footprint assays) have been described (9).Transfections. The plasmid constructions containing fragments of the 5' flanking region of the GST Ya subunit gene Abbreviations: GST, glutathione S-transferase; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; CAT, chloramphenicol acetyltransferase; EpRE, electrophile-responsive element. *Phase I xenobiotic metabolizing enzymes (e.g., cytochrome P1-450) introduce by oxidation or reduction functional groups into chemical compounds. 6258The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A mouse glutathione S-transferase gene encoding the Ya subunit was isolated and sequenced. The gene spans about 11 kb, contains seven exons, and encodes an mRNA of 841 nucleotides. Promoter elements, TATA and CAAT box sequences, were located 32 and 70 nucleotides upstream from the initiation of transcription site. The mRNA coding sequences of the mouse gene were highly homologous to a rat liver Ya mRNA species detected by cDNA cloning. The mouse Ya gene produces a 223-amino-acid polypeptide that differs from the 222-amino-acid rat Ya by 10 amino acid substitutions and a carboxyl terminus Phe-Lys-Ile-Gln instead of Phe-Lys-Phe. A genomic clone containing the last three exons of the rat Ya gene was also isolated, sequenced, and compared with the mouse Ya gene. An extensive sequence conservation (70-80%) in the 50 to 200 bases of introns at the exon-intron junctions as well as in the region beyond the cleavage-polyadenylation site of pre-mRNA was observed.

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