Transcriptional regulation of the Drosophila catalase gene by the DRE/DREF system

Abstract

Reactive oxygen species (ROS) cause oxidative stress and aging. The catalase gene is a key component of the cellular antioxidant defense network. However, the molecular mechanisms that regulate catalase gene expression are poorly understood. In this study, we have identified a DNA replication-related element (DRE; 5'-TATCGATA) in the 5'-flanking region of the Drosophila catalase gene. Gel mobility shift assays revealed that a previously identified factor called DREF (DRE- binding factor) binds to the DRE sequence in the Drosophila catalase gene. We used site-directed mutagenesis and in vitro transient transfection assays to establish that expression of the catalase gene is regulated by DREF through the DRE site. To explore the role of DRE/DREF in vivo, we established transgenic flies carrying a catalase-lacZ fusion gene with or without mutation in the DRE. The beta-galactosidase expression patterns of these reporter transgenic lines demonstrated that the catalase gene is upregulated by DREF through the DRE sequence. In addition, we observed suppression of the ectopic DREF-induced rough eye phenotype by a catalase amorphic Cat(n1) allele, indicating that DREF activity is modulated by the intracellular redox state. These results indicate that the DRE/DREF system is a key regulator of catalase gene expression and provide evidence of cross-talk between the DRE/DREF system and the antioxidant defense system.

Figure 1

Complex formation between the DRE in the 5′-upstream region of the catalase gene and the Kc cell nuclear extract. (A) Structure of the 5′-upstream region of the catalase gene and base substitutions in the DRE. The transcription initiation site is indicated by the arrowhead and numbered +1. The closed box indicates the DRE sequence in the catalase gene promoter. The nucleotide sequences of the catalase-DRE site and its base- substituted mutants are shown in boxes below, with lower case letters for substituted nucleotides. (B) ³²P-Labeled catalase-DRE oligonucleotides (Probe) were incubated with Kc cell nuclear extracts in the absence of competitor (lane 2), in the presence of excess unlabeled catalase-DRE competitor (lane 3, wild-type; lane 4, mutant; see Materials and Methods) or in the presence of anti-DREF monoclonal antibody (MAb) (lane 5) (33). Nuclear extract was omitted from the assay shown in lane 1.

Figure 1

Complex formation between the DRE in the 5′-upstream region of the catalase gene and the Kc cell nuclear extract. (A) Structure of the 5′-upstream region of the catalase gene and base substitutions in the DRE. The transcription initiation site is indicated by the arrowhead and numbered +1. The closed box indicates the DRE sequence in the catalase gene promoter. The nucleotide sequences of the catalase-DRE site and its base- substituted mutants are shown in boxes below, with lower case letters for substituted nucleotides. (B) ³²P-Labeled catalase-DRE oligonucleotides (Probe) were incubated with Kc cell nuclear extracts in the absence of competitor (lane 2), in the presence of excess unlabeled catalase-DRE competitor (lane 3, wild-type; lane 4, mutant; see Materials and Methods) or in the presence of anti-DREF monoclonal antibody (MAb) (lane 5) (33). Nuclear extract was omitted from the assay shown in lane 1.

Figure 2

Effect of base substitution mutations in the DRE sequence on catalase gene promoter activity in Kc cells. Wild-type catalase-luc (300 ng) or selected point mutant catalaseDREmut-luc (300 ng) reporter plasmids were transiently transfected into Kc cells. After incubation for 48 h, cells were harvested for analyses of luciferase and β-galactosidase activity. All values were normalized for co-transfected β-galactosidase activity. The luciferase activity of the wild-type reporter gene alone was set at 1. Average values obtained from four independent experiments with SE values are given as luciferase activity relative to that of the wild-type reporter gene.

Figure 3

Effect of the DRE sequence on catalase gene promoter activity in vivo. (A) Quantitative β-galactosidase activities of the transgenic flies bearing two copies of a catalase–lacZ or catalaseDREmut–lacZ fusion gene. Crude extracts were prepared from third instar larvae and 3-day- old adult transgenic flies as described in Materials and Methods. The β-galactosidase activities are expressed as absorbance units at 574 nm/h/mg of protein. Averaged values obtained from four independent experiments with SE values are shown. (B) The histochemical staining of β-galactosidase activity in 3-day-old adult transgenic flies having one copy of a catalase–lacZ or catalaseDREmut–lacZ fusion gene. The adult tissues were dissected and stained with 0.2% X-gal solution in the dark. Foregut (a and b), hindgut (c and d), muscle (e and f), testis (g and h), ovaries (i and j).

Figure 3

Effect of the DRE sequence on catalase gene promoter activity in vivo. (A) Quantitative β-galactosidase activities of the transgenic flies bearing two copies of a catalase–lacZ or catalaseDREmut–lacZ fusion gene. Crude extracts were prepared from third instar larvae and 3-day- old adult transgenic flies as described in Materials and Methods. The β-galactosidase activities are expressed as absorbance units at 574 nm/h/mg of protein. Averaged values obtained from four independent experiments with SE values are shown. (B) The histochemical staining of β-galactosidase activity in 3-day-old adult transgenic flies having one copy of a catalase–lacZ or catalaseDREmut–lacZ fusion gene. The adult tissues were dissected and stained with 0.2% X-gal solution in the dark. Foregut (a and b), hindgut (c and d), muscle (e and f), testis (g and h), ovaries (i and j).

Figure 4

DREF over-expression can induce catalase gene expres sion in vivo. (A) β-Galactosidase activities of third instar larvae from +/+;catalase-lacZ/hs-GAL4 or UAS-DREF/+;catalase-lacZ/hs-GAL4 lines. Crude extracts were prepared after a 1 h heat shock at 37°C and incubation at 25°C for an additional 6 h. The β-galactosidase activity of the heat-shocked larvae +/+;catalase-lacZ/hs-GAL4 was set at 1. Relative levels of expression are shown as the mean ± SE of three independent experiments with three replicates per experiment. (B) Effect of DREF over-expression on catalase mRNA levels. Total RNA was prepared from the third instar larvae carrying UAS-DREF and hs-GAL4 following a 1 h heat shock at 37°C and incubation at 25°C for an additional 6 h, after which DREF and catalase mRNA levels were measured by RT–PCR.

Figure 4

DREF over-expression can induce catalase gene expres sion in vivo. (A) β-Galactosidase activities of third instar larvae from +/+;catalase-lacZ/hs-GAL4 or UAS-DREF/+;catalase-lacZ/hs-GAL4 lines. Crude extracts were prepared after a 1 h heat shock at 37°C and incubation at 25°C for an additional 6 h. The β-galactosidase activity of the heat-shocked larvae +/+;catalase-lacZ/hs-GAL4 was set at 1. Relative levels of expression are shown as the mean ± SE of three independent experiments with three replicates per experiment. (B) Effect of DREF over-expression on catalase mRNA levels. Total RNA was prepared from the third instar larvae carrying UAS-DREF and hs-GAL4 following a 1 h heat shock at 37°C and incubation at 25°C for an additional 6 h, after which DREF and catalase mRNA levels were measured by RT–PCR.

Figure 5

The Catⁿ¹ amorphic allele suppresses the rough eye phenotype caused by ectopic DREF expression Scanning electron microscope images of female eyes: (A) and (E) GMR-GAL4/+;+/+;+/+, (B) and (F) GMR-GAL4/+;UAS-DREF/+;+/+, (C) and (G) GMR-GAL4/+;UAS-DREF/+;Catⁿ¹/+, (D) and (H) GMR-GAL4/+;+/+;Catⁿ¹/+. These flies were developed at 28°C. Scale bars for 200 µm (A–D) and 50 µm (E–H) are indicated.