Gawky modulates MTF-1-mediated transcription activation and metal discrimination

Abstract

Metal-induced genes are usually transcribed at relatively low levels under normal conditions and are rapidly activated by heavy metal stress. Many of these genes respond preferentially to specific metal-stressed conditions. However, the mechanism by which the general transcription machinery discriminates metal stress from normal conditions and the regulation of MTF-1-meditated metal discrimination are poorly characterized. Using a focused RNAi screening in Drosophila Schneider 2 (S2) cells, we identified a novel activator, the Drosophila gawky, of metal-responsive genes. Depletion of gawky has almost no effect on the basal transcription of the metallothionein (MT) genes, but impairs the metal-induced transcription by inducing the dissociation of MTF-1 from the MT promoters and the deficient nuclear import of MTF-1 under metal-stressed conditions. This suggests that gawky serves as a 'checkpoint' for metal stress and metal-induced transcription. In fact, regular mRNAs are converted into gawky-controlled transcripts if expressed under the control of a metal-responsive promoter, suggesting that whether transcription undergoes gawky-mediated regulation is encrypted therein. Additionally, lack of gawky eliminates the DNA binding bias of MTF-1 and the transcription preference of metal-specific genes. This suggests a combinatorial control of metal discrimination by gawky, MTF-1, and MTF-1 binding sites.

Figure 1.

Initial assay conditions and RNAi screening. (A) The induction levels of endogenous MtnA under different copper-stressed conditions. S2 cells were treated with different concentrations of CuSO₄ (10 μM, 50 μM, 500 μM or 1 mM) for the indicated amounts of time (0, 2, 4, 8, 12, 18 or 24 h), and the expression range of MtnA mRNA was then measured by real-time RT-PCR following RNA isolation. Data were normalized to the unstressed sample and are shown as mean ± SEM. n = 3. (B) The effect of different concentrations of copper on cell growth. S2 cells were treated with different concentrations of CuSO₄ (10 μM, 50 μM, 500 μM or 1 mM) for the indicated amounts of time (0, 2, 4, 8, 12, 18 or 24 h), the number of copper treated cells was counted at different time points. The growth rate of unstressed cells was also measured as a control. Data are shown as mean ± SEM. n = 3. (C, D) RNAi screening results of 127 genes. After RNAi treatment, the basal (no treatment, NT) or copper-induced (Cu²⁺) expression of MtnA (C) and MtnD (D) was measured by real-time RT-PCR following RNA isolation. To induce copper stress, S2 cells were treated with 500 μM CuSO₄ for the final 12 h before collection. Loss of candidates such as MTF-1 (marked in green) resulted in a large reduction in MtnA and MtnD expression regardless of whether cells were stressed with copper or not. Instead, gawky (marked in red) was found to function solely under copper-stressed conditions (for details, see Supplementary Screening Data). Data were normalized to the β-gal dsRNA sample and are shown as Log2 fold change. n = 3. (E, F) The effect of gawky on MtnA and MtnD activation at different concentrations of copper_. After RNAi treatment, S2 cells were treated with increasing concentrations of CuSO₄ (1 μM, 10 μM, 50 μM, 500 μM, or 1 mM) for the final 12 h before collection. The copper-induced expression of MtnA (E) and MtnD (F) was measured by real-time RT-PCR following RNA isolation. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 3. ∗∗P < 0.01; ∗P < 0.05.

Figure 2.

Gawky is required for the metal-induced transcription but not the basal transcription of the MT genes. (A) Flow chart of the experimental setup for the data presented in (E, H and I). (B) Targeted positions of five independent dsRNAs that were used for RNAi depletion of gawky (for details, see Supplementary Table S1). (C, D) The knockdown efficiency of gawky dsRNAs. (C) S2 cells were treated with the indicated dsRNAs for 3 days, and western blotting was then applied to measure the expression of gawky protein with two different antibodies. Considering that the specificity of anti1 is better than that of anti2, anti1 was selected for the following experiment. (D) The level of gawky protein was quantified using ImageJ from three independent western blotting experiments. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 3. ∗∗P < 0.01; ∗P < 0.05. (E–H) S2 cells were treated with the indicated gawky dsRNAs for 3 days, and 500 μM copper was added for the final 12 h to induce metal stress. (E–G) MT expression was measured by real-time RT-PCR using RNA extracts from whole cells (E) or was visualized by FISH assays (F, G). Representative images are shown. Scale bars, 12 μm. (H) The transcription activities were also measured by real-time RT-PCR using nascent RNA extracts from nuclear run-on assays. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 3. ∗∗P < 0.01; ∗P < 0.05. (I) S2 cells were first treated with the gawky UTR dsRNAs (#4+5), and the rescue plasmid which is insensitive to dsRNA treatment was introduced to restore gawky expression. Real-time RT-PCR was then applied to quantify MT expression in unstressed or copper treated cells using RNA extracts from whole cells. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 3. ∗∗P < 0.01; ∗P < 0.05.

Figure 3.

Gawky moves into the nucleus and interacts with the MT promoters in response to metal stress. (A, B) S2 cells were treated with 500 μM copper or 50 μM cadmium for 12 h. (A) Proteins from nuclei (Nuc), cytoplasm (Cyto), or whole-cell extracts (Total) were subjected to western blotting analyses. HDAC1 served as a marker for the nuclear fraction. α-Tubulin served as a marker for the cytoplasmic fraction. Representative blots are shown. n = 3. (B) The level of gawky protein was quantified using ImageJ from three independent western blotting experiments. The Nuc/Cyto ratio (the relative nuclear gawky level divided by the relative cytoplasmic gawky level) of gawky was also calculated. Data were normalized to the unstressed sample and are shown as mean ± SEM. n = 3. (C–E) S2 cells were treated with 500 μM copper or 50 μM cadmium for 12 h and seeded on ConA-covered coverslips for the final 1 h. (C) Confocal microscopy analyses of gawky were performed with anti-gawky (anti1). Representative images are shown. Scale bars, 5 μm. n = 3. (D) Line profiles of fluorescence intensities in (C) showing changes in gawky subcellular localization. (E) Statistics of nuclear gawky signal in each condition. Data were normalized to the unstressed sample and are shown as mean ± SEM. n = 50 cells for each condition. ∗∗P < 0.01; ∗P < 0.05. (F) ChIP-real-time PCR analysis of the interaction between gawky and the MT promoters in unstressed cells or after metal treatment for 12 h. The ChIP data generated by a non-specific antibody were also included. Gawky binding sites were defined as regions enriched over the input DNA. The MT and Act5C loci with the locations of ChIP amplicons are shown below. Data are shown as mean ± SEM. n = 3.

Figure 4.

The regulatory effect of gawky on transcription depends on the promoter. (A) Schematic representation of FLAG-tagged firefly luciferase expression plasmids. The MT promoters or the Act5C promoter was inserted upstream of the transcriptional start site. Stable cell lines were generated using these plasmids. (B–D) S2 cell lines stably expressing FLAG-tagged firefly luciferase were treated with two independent gawky dsRNAs for 3 days followed by the addition of 500 μM copper for the final 12 h to induce metal stress. (B) The expression of firefly luciferase mRNA was subjected to real-time RT-PCR analyses following RNA isolation. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 3. ∗∗P < 0.01; ∗P < 0.05. (C) Firefly luciferase protein was subjected to western blotting analyses using equal amounts of protein extracts from whole cells. α-Tubulin served as a loading control. Representative blots are shown. n = 3. (D) The protein level of the copper-induced firefly luciferase in (C) was quantified using ImageJ from three independent western blotting experiments. To provide better representation of the levels of non-induced proteins, we increased the amount of protein used for western blotting analyses (see Supplementary Figure S6C and D). Data were normalized to the β-gal dsRNA sample and shown as mean ± SEM. ∗∗P < 0.01; ∗P < 0.05. n = 3. (E) Schematic representation of dati expression plasmids that can generate linear RNAs (exon 1, exon 2, and exon 3 are joined in a linear order) and circular RNAs (the end of exon 2 is joined to the beginning of exon 2). The MT promoters or the dati promoter was inserted upstream of the transcriptional start site. (F, G) After gawky RNAi, S2 cells were transfected with different dati expression plasmids for 2 days followed by the addition of 500 μM copper for the final 12 h to induce metal stress. The level of nascent linear (F) or circular (G) dati RNA was measured by real-time RT-PCR using RNA extracts from nuclear run-on experiments. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 3. ∗∗P < 0.01; ∗P < 0.05.

Figure 5.

Gawky is a partner of metal-activated MTF1 and is required for MTF1 recruitment to the MT promoters during metal stress. (A–D) Gawky binds to MTF-1 during metal stress. (A) The interaction between gawky and MTF-1 under normal or copper-stressed conditions was assayed by co-IP with an antibody against gawky (anti1), MTF-1, or a negative IgG using whole-cell extracts. (B) To examine whether the copper-induced interaction is in a DNA dependent manner, co-IP was performed using whole-cell extracts in the presence of DNase I. (C, D) To examine whether gawky interacts with MTF-1 in the nucleus or cytoplasm, co-IP was performed using nuclear or cytoplasmic extracts. All samples were subjected to western blotting analyses. α-Tubulin or Lam served as a negative control. Representative blots are shown. n = 3. (E) To examine the effect of gawky on MTF1 recruitment to the MT promoters, S2 cells were treated with two independent gawky dsRNAs for 3 days, and 500 μM copper was added for the final 12 h to induce metal stress. MTF1 recruitment to the MT genes was then measured using ChIP-real-time PCR. The ChIP data generated by a non-specific antibody were also included. MTF-1 binding sites were defined as regions enriched over the input DNA. The loci of the MT genes with the locations of ChIP amplicons are shown below. Data are shown as mean ± SEM. n = 3.

Figure 6.

Gawky controls nuclear import of metal-activated MTF-1. (A–D) S2 cells were treated with 500 μM copper or 50 μM cadmium for 12 h. (A, B) Proteins from nuclei (Nuc), cytoplasm (Cyto), or whole-cell extracts (Total) were subjected to western blotting analyses. HDAC1 served as a marker for the nuclear fraction. α-Tubulin served as a marker for the cytoplasmic fraction. Representative blots are shown. The level of MTF-1 protein was quantified using ImageJ from three independent western blotting experiments. The Nuc/Cyto ratio of MTF-1 was also calculated. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 3. (C, D) Confocal microscopy analyses of MTF-1 were performed with anti-MTF-1. Representative images are shown. Scale bars, 8 μm. n = 3. (D) Statistics of nuclear MTF-1 signal in each condition. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SEM. n = 40 cells for each condition. ∗∗P < 0.01; ∗P < 0.05.

Figure 7.

The regulatory effect of gawky on MTF-1-mediated metal discrimination. (A) Schematic representation of MRE plasmids. Four direct repeats of Cu or Cd MREs were inserted upstream of the dati promoter. (B–D) MRE plasmids were introduced into gawky dsRNA treated S2 cells for 2 days followed by the addition of 500 μM copper or 50 μM cadmium for the final 12 h to induce metal stress. (B) ChIP assay was performed with anti-MTF1 to examine its recruitment to the MRE containing promoter. (C, D) Real-time RT-PCR was performed to measure the levels of plasmid-derived dati mRNA (C) and circular dati RNA (D). The non-preferred condition is set to 1. Data are shown as mean ± SEM. n = 3. ∗∗P < 0.01; ∗P < 0.05. The same data were also processed in the way to separate the effect on the activation level from the metal specificity (see Supplementary Figure S13A–C). (E, F) S2 cells were treated with two independent gawky dsRNAs for 3 days followed by the addition of 500 μM copper or 50 μM cadmium for the final 12 h to induce metal stress. (E) ChIP assay was performed with anti-MTF1 to examine the MTF1 binding preference of six endogenous metal-specific genes. (F) The transcription preference was measured by real-time RT-PCR following RNA isolation. The non-preferred condition is set to 1. Data are shown as mean ± SEM. n = 3. ∗∗P < 0.01; ∗P < 0.05. The same data were also processed in the way to separate the effect on the activation level from the metal specificity (see Supplementary Figure S13D and E).

Figure 8.

A working model for the regulatory effects of gawky on metal-induced transcription. (A) In defense of heavy metals, gawky activates metal-responsive genes in concert with metal-activated MTF-1. (B) Loss of gawky eliminates the metal-specific transcription preference by impairing the DNA binding bias of MTF-1.