Phosphine inhibits transcription of the catalase gene through the DRE/DREF system in Drosophila melanogaster

Abstract

Phosphine (PH₃) is a toxin commonly used for pest control. Its toxicity is attributed primarily to its ability to induce oxidative damage. Our previous work showed that phosphine could disrupt the cell antioxidant defence system by inhibiting expression of the catalase gene in Drosophila melanogaster (DmCAT). However, the exact mechanism of this inhibition remains unclear. Here, we implemented a luciferase reporter assay driven by the DmCAT promoter in D. melanogaster S2 cells and showed that this reporter could be inhibited by phosphine treatment. A minimal fragment of the promoter (-94 to 0 bp), which contained a DNA replication-related element (DRE) consensus motif (-78 to -85 bp), was sufficient for phosphine-mediated reporter inhibition, suggesting the involvement of the transcription factor DREF. Furthermore, phosphine treatment led to a reduction in DREF expression and consequent repression of DmCAT transcription. Our results provide new insights on the molecular mechanism of phosphine-mediated catalase inhibition. Phosphine treatment leads to reduced levels of the transcription factor DREF, a positive regulator of the DmCAT gene, thereby resulting in the repression of DmCAT at transcriptional level.

Figure 1

Establishment of phosphine (PH₃) treatment system in Drosophila melanogaster S2 cells. (A) Cell viability assays for S2 cells treated with different concentrations of PH₃ for various lengths of time. (B) Real-time PCR analysis of the DmCAT gene in S2 cells treated with 14 µg/L PH₃ for 0, 1, 2, or 4 h. CK: control. Lowercase letters indicate significant differences at p < 0.05. (C) Western blot against DmCAT in S2 cells treated with 14 µg/L PH₃ for 0, 1, 2, or 4 h (Full-length blots are presented in Supplementary Figs S1 and S2).

Figure 2

A minimal fragment containing a DNA replication-related element (DRE) motif is required for phosphine (PH₃)-mediated DmCAT repression. (A) Promoter activity of wild-type (−1,944 full length and −94) and mutant (−94 M) DRE DmCAT promoters treated with or without PH₃. (B) Luciferase assay comparing PH₃ inhibition efficiencies of full-length (−1,944) and various truncated fragments of DmCAT promoter. Relative promoter activity was defined as the ratio of luciferase activity in PH₃-treated cells to that in untreated cells. DRE-WT, wild-type DRE; DRE-M, mutant DRE. Lowercase letters indicate significant differences at p < 0.05.

Figure 3

Repression of DREF by phosphine (PH₃). (A) Western blot (Full-length blots are presented in Supplementary Figs S3 and S4) and (B) real-time PCR analysis of DREF in S2 cells treated with 14 µg/L PH₃ for 0, 1, 2, or 4 h. CK: control. (C) Chromatin immunoprecipitation (ChIP)-qPCR analysis of Pol II enrichment on the DREF promoter with or without PH₃ treatment. Lowercase letters indicate significant differences at p < 0.05. TSS, transcription start site.

Figure 4

Effect of DREF RNAi and overexpression on phosphine (PH₃)-mediated DmCAT repression. (A) Representative western blot validating the efficiency of DREF RNAi. CK: control (Full-length blots are presented in Supplementary Figs S4 and S5). (B) Luciferase assay comparing relative luciferase activities of control and DREF RNAi cells with or without PH₃ treatment. *p < 0.05 vs. untreated cells. (C) Representative western blot validating the overexpression of DREF (Full-length blots are presented in Supplementary Figs S4 and S6). (D) Luciferase assay comparing relative luciferase activities of control or DREF overexpressing cells with or without PH₃ treatment. *p < 0.05 vs. untreated cells.

Figure 5

Effect of phosphine (PH₃) on the expression of seven DREF target genes. *p < 0.05 vs. untreated cells.