NC2 complex is a key factor for the activation of catalase-3 transcription by regulating H2A.Z deposition

Abstract

Negative cofactor 2 (NC2), including two subunits NC2α and NC2β, is a conserved positive/negative regulator of class II gene transcription in eukaryotes. It is known that NC2 functions by regulating the assembly of the transcription preinitiation complex. However, the exact role of NC2 in transcriptional regulation is still unclear. Here, we reveal that, in Neurospora crassa, NC2 activates catalase-3 (cat-3) gene transcription in the form of heterodimer mediated by histone fold (HF) domains of two subunits. Deletion of HF domain in either of two subunits disrupts the NC2α-NC2β interaction and the binding of intact NC2 heterodimer to cat-3 locus. Loss of NC2 dramatically increases histone variant H2A.Z deposition at cat-3 locus. Further studies show that NC2 recruits chromatin remodeling complex INO80C to remove H2A.Z from the nucleosomes around cat-3 locus, resulting in transcriptional activation of cat-3. Besides HF domains of two subunits, interestingly, C-terminal repression domain of NC2β is required not only for NC2 binding to cat-3 locus, but also for the recruitment of INO80C to cat-3 locus and removal of H2A.Z from the nucleosomes. Collectively, our findings reveal a novel mechanism of NC2 in transcription activation through recruiting INO80C to remove H2A.Z from special H2A.Z-containing nucleosomes.

Figure 1.

Nc2 mutants are extremely sensitive to H₂O₂–induced ROS stress. (A, B) Plate assay analyzing mycelial growth on plates with 0, 8, or 10 mM H₂O₂ as indicated. Cultures were inoculated in plates at 25°C under constant light. (C, D) Amino acid sequence alignment of the conserved histone fold domain of NC2α (C) and NC2β (D) from Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Mus musculus, Homo sapiens, and Drosophila melanogaster. (E) Plate assay analysis showing mycelial growth of wild type (WT), Nc2α^KO, Nc2α^KO, pqa-Myc-NC2α and cat-3^KO strains on plates with or without 10 mM H₂O₂ and in the absence or presence of 10⁻³ M QA. Cultures were inoculated in plates at 25°C under constant light. (F) The analysis of mycelial growth using plate assay in WT, Nc2β^KO, Nc2β^KO, pqa-Myc-NC2β and cat-3^KO strains. The method tested in plates is same with (E).

Figure 2.

NC2 complex is a key factor for the activation of cat-3 gene transcription. (A, B) In-gel assay analysis of the CAT-3 activity levels in WT, Nc2α^KO, Nc2β^KO, Nc2α^KO, pqa-Myc-NC2α and Nc2β^KO, pqa-Myc-NC2β strains. (C, D) Western blot analyses showing the levels of CAT-3 protein in the WT, Nc2α^KO, Nc2β^KO, Nc2α^KO, pqa-Myc-NC2α and Nc2β^KO, pqa-Myc-NC2β strains. The membranes stained by coomassie blue represent the total protein in each sample and act as loading control for western blot. (E, F) RT-qPCR assays analyzing the levels of cat-3 mRNA in the WT, Nc2α^KO and Nc2β^KO strains. Error bars show s.d. (n = 3). Significance was evaluated by using a two-tailed t-test. ***P < 0.001 versus WT.

Figure 3.

Conserved regions of NC2 subunits are required for transcriptional activation of cat-3. (A, B) Schematic drawing of N. crassa NC2α (A) and NC2β (B) subunits and their various deletion mutants. The position of the histone fold (red rectangle), acidic (green rectangle), TBP-binding (blue rectangle) and repression (gray rectangle) regions are indicated. (C) Plate assays analyzing mycelial growth of different deletion strains across NC2α coding region at endogenous locus on plates with 0 or 10 mM H₂O₂. Cultures were inoculated in plates at 25°C under constant light. (D) In-gel assay analysis of the CAT-3 activity levels in different deletion strains across NC2α coding region at endogenous locus. (E) Western blot analyses showing the levels of CAT-3 protein in different deletion strains across NC2α coding region at endogenous locus. The membrane stained by coomassie blue represents the total protein in each sample and acts as loading control for western blot. (F) RT-qPCR assays analyzing the levels of cat-3 mRNA in different deletion strains across NC2α coding region at endogenous locus. Error bars show s.d. (n = 3). Significance was evaluated by using a two-tailed t-test. **P < 0.01 and ***P < 0.001 versus WT. (G) Plate assays analyzing mycelial growth of different deletion strains across NC2β coding region at endogenous locus on plates with 0 or 10 mM H₂O₂. Cultures were inoculated in plates at 25°C under constant light. (H) In-gel assay analysis of the CAT-3 activity levels in different deletion strains across NC2β coding region at endogenous locus. (I) Western blot analyses showing the levels of CAT-3 protein in different deletion strains across NC2β coding region at endogenous locus. The membrane stained by coomassie blue represents the total protein in each sample and acts as loading control for western blot. (J) RT-qPCR assays analyzing the levels of cat-3 mRNA in different deletion strains across NC2β coding region at endogenous locus. Error bars show s.d. (n = 3). Significance was evaluated by using a two-tailed t-test. **P < 0.01 and ***P < 0.001 versus WT.

Figure 4.

The integrity of NC2 heterodimer is a prerequisite for NC2 to bind to cat-3 locus and to activate its expression. (A, B) ChIP assays showing the binding levels of NC2α (A) and NC2β (B) at cat-3 locus in Nc2α^KO, Nc2β^KO and wild-type strains. Short black lines (primer pairs 3–7) under the schematic diagram of cat-3 gene represent the regions detected by ChIP-qPCR. TSS, transcription start site; ORF, open reading frame. Error bars show s.d. (n = 3). Significance was assessed by using a two-tailed t-test. **P < 0.01 and ***P < 0.001 versus WT. (C, D) Immunoprecipitation assays showing the interaction between Myc-NC2α or Myc-NC2β and endogenous NC2β or NC2α protein, respectively.

Figure 5.

HF domains in NC2 subunits are essential for its integrity and ability to bind at cat-3 locus. (A, B) Mapping of the NC2α region responsible for the interaction with NC2β in Nc2α^KO strain with ectopically expressing wild-type Myc-NC2α or its various deletion mutants. Immunoprecipitation assays with anti-Myc antibody (A) or anti-NC2β antibody (B), the eluates were detected by western blot analysis using anti-Myc (α-Myc) and anti-NC2β (α-NC2β) antibodies. The red arrows denote specific bands, and the black arrow shows heavy chain. (C, D) Mapping of the NC2β region responsible for the interaction with NC2α in Nc2β^KO strain with ectopically expressing Myc-NC2β or its various deletion mutants. Immunoprecipitation assays with anti-Myc antibody (C) or anti-NC2α antibody (D), the eluates were detected by western blot analysis using anti-Myc (α-Myc) and anti-NC2α (α-NC2α) antibodies. The blue arrow denotes heavy chain of IgG, the black arrow shows light chain. (E, F) ChIP assays showing the binding levels of NC2α (E) and NC2β (F) at cat-3 locus in different deletion strains across NC2α coding region at endogenous locus. (G, H) ChIP assays showing the binding levels of NC2α (G) and NC2β (H) at cat-3 locus in different deletion strains across NC2β coding region at endogenous locus. Error bars show s.d. (n = 3). Significance was assessed by using a two-tailed t-test. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. WT.

Figure 6.

NC2 complex activates the transcription of cat-3 gene by antagonizing the inhibition of H2A.Z at cat-3 locus. (A, B) ChIP assays showing the occupancy levels of H2B (A) and H3 (B) at cat-3 locus in Nc2α^KO and Nc2β^KO as well as wild type strains. (C, D) ChIP assays showing the relative occupancy levels of H2A.Z at cat-3 locus in WT, Nc2α^KO (C) and Nc2β^KO (D) strains. The relative occupancy levels of H2A.Z represent the ratio of H2A.Z to H2B. (E) ChIP assays analyzing the effects of deletion of HF domain of NC2α on the binding of H2A.Z at cat-3 locus. The H2A.Z^KO strain was used as a negative control. (F) ChIP assays analyzing the effects of deletion of C-terminal repression or HF domain of NC2β on the binding of H2A.Z at cat-3 locus. The H2A.Z^KO strain was used as a negative control. Error bars show s.d. (n = 3). Significance was assessed by using a two-tailed t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 7.

INO80C is involved in activating cat-3 transcription by mediating the removal of H2A.Z from nucleosomes around cat-3 locus. (A) In-gel assay analysis of the CAT-3 activity levels in WT, ino80^KO and arp8^KO strains. (B) Western blot analyses showing the levels of CAT-3 protein in the WT, ino80^KO and arp8^KO strains. The membranes stained by coomassie blue represent the total protein in each sample and act as loading control for western blot. (C) RT-qPCR assays analyzing the levels of cat-3 mRNA in the WT, ino80^KO and arp8^KO strains. Error bars show s.d. (n = 3). Significance was evaluated by using a two-tailed t-test. ***P < 0.001 versus WT. (D, E) ChIP assays revealing the binding of INO80 (D) and ARP8 (E) at cat-3 locus. (F, G) ChIP assays analyzing the effects of deletion of INO80 (F) and ARP8 (G) on the binding of H2A.Z at cat-3 locus. The H2A.Z^KO strain was used as a negative control. Error bars show s.d. (n = 3). Significance was evaluated by using a two-tailed t-test. **P < 0.01 and ***P < 0.001.

Figure 8.

NC2 complex mediates the recruitment of INO80C to cat-3 locus to activate its expression. (A, B) ChIP assays analyzing the effects of deletion of full-length NC2α or HF domain of NC2α on the binding of INO80 (A) and ARP8 (B) at cat-3 locus. (C, D) ChIP assays analyzing the effects of deletion of full length, C-terminal repression or HF domain of NC2β on the binding of INO80 (C) and ARP8 (D) at cat-3 locus. Error bars show s.d. (n = 3). Significance was assessed by using a two-tailed t-test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus WT. (E, F) Immunoprecipitation assays showing the interaction between Myc-INO80 and endogenous NC2β. (G, H) Immunoprecipitation assays showing the interaction between Myc-NC2β and endogenous INO80 (G) or ARP8 (H). (I) GST pull-down assay showing the interaction between NC2β and ARP8 in vitro. Purified GST-NC2β or GST-GFP was incubated with His-ARP8. After being immunoprecipitated with glutathione-sepharose beads, the proteins were detected by western blot analysis with anti-His and anti-GST antibodies. (J) A model depicting the mechanism of NC2 regulating cat-3 expression. On the one hand, NC2 recruits INO80C to remove H2A.Z at cat-3 promoter and TSS, on the other hand, NC2 promotes the PIC assembly at cat-3 TSS region, ultimately resulting in the transcription activation of cat-3.