Pi class glutathione S-transferase genes are regulated by Nrf 2 through an evolutionarily conserved regulatory element in zebrafish

Abstract

Pi class GSTs (glutathione S-transferases) are a member of the vertebrate GST family of proteins that catalyse the conjugation of GSH to electrophilic compounds. The expression of Pi class GST genes can be induced by exposure to electrophiles. We demonstrated previously that the transcription factor Nrf 2 (NF-E2 p45-related factor 2) mediates this induction, not only in mammals, but also in fish. In the present study, we have isolated the genomic region of zebrafish containing the genes gstp1 and gstp2. The regulatory regions of zebrafish gstp1 and gstp2 have been examined by GFP (green fluorescent protein)-reporter gene analyses using microinjection into zebrafish embryos. Deletion and point-mutation analyses of the gstp1 promoter showed that an ARE (antioxidant-responsive element)-like sequence is located 50 bp upstream of the transcription initiation site which is essential for Nrf 2 transactivation. Using EMSA (electrophoretic mobility-shift assay) analysis we showed that zebrafish Nrf 2-MafK heterodimer specifically bound to this sequence. All the vertebrate Pi class GST genes harbour a similar ARE-like sequence in their promoter regions. We propose that this sequence is a conserved target site for Nrf 2 in the Pi class GST genes.

Figure 1. Comparison of the vertebrate Pi class GST proteins

(A) Phylogenetic tree of Pi class GST proteins. The tree was constructed using the Clustal W program in the DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/search/clustalw-j.html). Scale bar, genetic distance. (B) Sequence alignment of various Pi class GST proteins. Conserved amino acids among Pi class GST proteins are highlighted in grey. Closed and open circles denote G- and H-sites respectively. Abbreviations: z, zebrafish; s, salmon; e, eel; x, Xenopus; b, bovine; h, human; r, rat; m, mouse.

Figure 2. Nrf 2-dependent induction of gstp1 and gstp2 in zebrafish larvae

(A) Confirmation of the primer specificity for RT-PCR. PCR was carried out using plasmid DNAs containing gstp1 (gstp1) or gstp2 (gstp2) cDNA as templates. Numbers indicate reaction cycles (Cyc). (B) Effect of nrf2-MO on the DEM-induced expression of gstp1 and gstp2. RT-PCR analysis of nrf2-MO (9 ng)- or mock-injected larvae, treated (+) or not treated (−) with 100 μM DEM for 6 h at day 5. The amounts of cDNA were standardized by the expression of ef1α. Each experiment was carried out at least three times and resulted in similar expression patterns. (C) Expression of the Pi class GST genes in the adult gills. RT-PCR analysis of the gills of adult male or female fish, treated (+) or not treated (−) with 100 μM DEM for 6 h.

Figure 3. Expression of gstp1 and gstp2 in Nrf 2-overexpressing embryos

RT-PCR analysis using total RNA isolated from embryos 8-h post-injection with or without 40 pg of Nrf 2 mRNA. Amounts of cDNA were standardized by the expression of ef1α.

Figure 4. Structure of the zebrafish gstp1 and gstp2 genes

Maps of contigurated sequences and λ phage clones containing gstp1 or gstp2 are displayed. E, X and S indicate enzyme sites for EcoRI, XhoI and SacI respectively. Black and white boxes denote the coding and non-coding regions of the Pi class GST cDNAs respectively. Nucleotide sequence data of 3.5-kbp regions of gstp1 and 0.61-kbp regions of gstp2 used for the GFP-reporter constructs have been deposited in the DDBJ, EMBL and GenBank® databases with accession numbers AB194129 and AB194130 respectively.

Figure 5. Induction of the GFP-reporter genes by Nrf 2 overexpression

(A) Scheme showing the analysing method. (B) GFP expression of the 3.5gstp1GFP and 0.61gstp2GFP constructs. 3.5gstp1GFP (20 pg) or 0.61gstp2GFP (100 pg) was injected with or without 40 pg of Nrf 2 mRNA into blastomere of one-cell stage embryos. GFP expression was analysed in 8-h embryos. Values indicate the percentages of embryos that showed more than ten GFP-positive cells. The number of embryos observed for each construct are indicated in parentheses.

Figure 6. Difference in GFP induction by Nrf 2 among various gstp1 constructs

(A) Transcriptional activity of deletion and mutation constructs. GFP expression was analysed in 8-h embryos that were injected with 40 pg of each construct and 40 pg of Nrf 2 mRNA. More than 30 embryos were examined for each construct. (B) Structure of the proximal ARE-like sequence in the gstp1 promoter and its mutation construct. Numbers indicate nucleotides from the transcriptional initiation site.

Figure 7. Binding of Nrf 2–MafK heterodimers to the ARE-like sequence

In vitro-translated Nrf 2 and MafK proteins were incubated with the ³²P-labelled oligonucleotide probe containing the ARE-like sequence in gstp1. Arrowheads indicate the positions of shifted bands containing the Nrf 2–MafK heterodimer, which were super-shifted (arrow) or reduced with the addition of anti-Nrf 2 (lane 5) or anti-MafK (lane 6) antibodies (Ab). Shifted complexes, including Nrf 2–MafK heterodimers, were specifically competed by the addition of a 25- or 100-fold molar excess of unlabelled oligonucleotide (lanes 8 and 9). Mutations (Mut) in the ARE-like sequence, as shown in Figure 6(B), eliminated the competition (lanes 10 and 11).

Figure 8. Alignment of various Pi class GST promoters

ARE-like sequences and the TATA box are highlighted in grey. Numbers indicate nucleotides from the transcriptional initiation site.