A role for the mitotic proteins Bub3 and BuGZ in transcriptional regulation of catalase-3 expression

Abstract

The spindle assembly checkpoint factors Bub3 and BuGZ play critical roles in mitotic process, but little is known about their roles in other cellular processes in eukaryotes. In aerobic organisms, transcriptional regulation of catalase genes in response to developmental or environmental stimuli is necessary for redox homeostasis. Here, we demonstrate that Bub3 and BuGZ negatively regulate cat-3 transcription in the model filamentous fungus Neurospora crassa. The absence of Bub3 caused a significant decrease in BuGZ protein levels. Our data indicate that BuGZ and Bub3 interact directly via the GLEBS domain of BuGZ. Despite loss of the interaction, the amount of BuGZ mutant protein negatively correlated with the cat-3 expression level, indicating that BuGZ amount rather than Bub3-BuGZ interaction determines cat-3 transcription level. Further experiments demonstrated that BuGZ binds directly to the cat-3 gene and responses to cat-3 overexpression induced by oxidative stresses. However, the zinc finger domains of BuGZ have no effects on DNA binding, although mutations of these highly conserved domains lead to loss of cat-3 repression. The deposition of BuGZ along cat-3 chromatin hindered the recruitment of transcription activators GCN4/CPC1 and NC2 complex, thereby preventing the assembly of the transcriptional machinery. Taken together, our results establish a mechanism for how mitotic proteins Bub3 and BuGZ functions in transcriptional regulation in a eukaryotic organism.

Fig 1. Bub3 functions to suppress cat-3 gene expression.

(A) Mycelial growth of WT, bub3^KO, and bub3^KO,Bub3 (nbd) strains on plates with addition of 1x10^-3 M QA and 0, 10, or 20 mM H₂O₂. (B) Quantitation of growth relative to WT of bub3^KO and bub3^KO,Bub3 (nbd) strains under conditions described in panel A. (C) In-gel catalase activity assay of protein extracts from WT, bub3^KO, and bub3^KO,Bub3 strains. (D) The level of CAT-3 protein in WT, bub3^KO, and bub3^KO,Bub3 strains determined by western blot analyses. The membranes stained by Coomassie blue served as the loading control. (E) Levels of cat-3 mRNA in bub3^KO and bub3^KO,Bub3 strains relative to that in the WT strain as determined by RT-qPCR analyses.Cultures were inoculated at the center of petri dishes (9 cm in diameter) and grown at 25°C under constant light. Error bars indicate SD (n = 3). Significance was evaluated by two-tailed t-test. *P < 0.05, **P < 0.01, and ***P<0.001.

Fig 2. BuGZ and Bub3 function in the same pathway to repress cat-3 expression.

(A) Aerial hyphae and conidia of WT, bugz^KO, and bub3^KO strains on slants. Cultures were grown at 25°C under constant light after 1 day at 30°C. (B) Mycelial growth of WT and bugz^KO strains on plates with 0, 10, or 20 mM H₂O₂. (C) Quantitation of growth of bugz^KO strain relative to WT under conditions described in panel B. (D) In-gel catalase activity assays of protein extracts from WT and bugz^KO strains. (E) Western blot analysis of CAT-3 protein in WT and bugz^KO strains. The membranes stained by Coomassie blue served as the loading control. (F) Quantification of cat-3 mRNA in bugz^KO strain relative to WT. (G) In-gel catalase activity assays of protein extracts from WT, bugz^KO, bub3^KO, and bugz^KObub3^KO strains. (H) Western blot analysis of CAT-3 protein in WT, bugz^KO, bub3^KO, and bugz^KObub3^KO strains. The membranes stained by Coomassie blue served as the loading control. Error bars indicate SD (n = 3). Significance was evaluated by two-tailed t-test. *P < 0.05, **P < 0.01, and ***P<0.001.

Fig 3. BuGZ stability does not depend on the Bub3-BuGZ interaction.

(A) Western blot for BuGZ and Bub3 proteins in WT, bugz^KO, and bub3^KO strains. The membranes stained by Coomassie blue served as the loading control. (B) RT-qPCR quantification of bugz mRNA in bub3^KO strain relative to WT. (C) Amino acid sequence alignment of the conserved GLEBS domain of BuGZ from Neurospora crassa, Homo sapiens, Mus musculus, Xenopus laevis, and Danio rerio. The conserved EE residues are indicated by a black box, and G1~G5 segments are marked. (D) Co-immunoprecipitation assays analyzed by western blot to evaluate the interaction between Myc-BuGZ, Myc-BuGZ^ΔGLEBS, Myc-BuGZ^EE-AA, or Myc-BuGZ^ΔEE and endogenous Bub3. (E) Co-immunoprecipitation assays analyzed by western blot to evaluate the interaction between Myc-BuGZ, Myc-BuGZ^ΔGLEBS, Myc-BuGZ^ΔG1~ΔG5, or Myc-BuGZ^ΔEE and endogenous Bub3. (F) Western blot analyses of the levels of BuGZ protein in WT, bugz^KO, bugz^KO,BuGZ, bugz^KO,BuGZ^ΔGLEBS, bugz^KO,BuGZ^ΔG1~ΔG5, and bugz^KO,BuGZ^ΔEE strains. The membranes stained by Coomassie blue served as the loading control. A non-specific protein band is marked by an asterisk. Error bars indicate SD (n = 3). Significance was evaluated by two-tailed t-test. *P < 0.05, **P < 0.01, and ***P<0.001.

Fig 4. BuGZ protein level determines extent of cat-3 transcription inhibition.

(A) Mycelial growth of WT, bugz^KO, bugz^KO,BuGZ, bugz^KO,BuGZ^ΔGLEBS, bugz^KO,BuGZ^ΔG1~ΔG5, and bugz^KO,BuGZ^ΔEE strains on plates with 0, 10, or 20 mM H₂O₂. (B) Quantitation of growth of strains described in panel A relative to WT. (C) In-gel analyses of catalases activities in extracts from WT, bugz^KO, bugz^KO,BuGZ, bugz^KO,BuGZ^ΔGLEBS, bugz^KO,BuGZ^ΔG1~ΔG5, and bugz^KO,BuGZ^ΔEE strains. (D) Western blot analysis of CAT-3 protein in WT, bugz^KO, bugz^KO,BuGZ, bugz^KO,BuGZ^ΔGLEBS, bugz^KO,BuGZ^ΔG1~ΔG5, and bugz^KO,BuGZ^ΔEE strains. The membranes stained by Coomassie blue served as the loading control. (E) RT-qPCR analyses of cat-3 mRNA in bugz^KO, bugz^KO,BuGZ, bugz^KO,BuGZ^ΔGLEBS, bugz^KO,BuGZ^ΔG1~ΔG5, and bugz^KO,BuGZ^ΔEE strains relative to levels in the WT strain. (F) RT-qPCR analyses of cat-3 mRNA in bub3^KO, bub3^KO,Bub3, bub3^KO,BuGZ, and bub3^KO,BuGZ^ΔGLEBS strains relative to levels in the WT strain. Error bars indicate SD (n = 3). Significance was evaluated by two-tailed t-test. *P < 0.05, **P < 0.01, and ***P<0.001.

Fig 5. BuGZ binds to the cat-3 gene region specifically and its recruitment responses to cat-3 overexpression caused by oxidative stress.

(A) Schematic depicting the location of cat-3 (NCU00355) gene downstream of a 5-kb AT-rich heterochromatin region on linkage group III of Neurospora genome. Site of primer binding are indicated under the schematic. Primer H binds in a heterochromatin region; P in the promoter; T near the transcription start site; O₁, O₂, and O₃ in the open reading frame (ORF). (B) Quantification of recruitment of BuGZ to the cat-3 locus as well as control regions in WT and bub3^KO strains. The bugz^KO strain was used as a negative control. The prx1 gene promoter region (prx1 P) was used as a positive binding control for BuGZ, while the hyr1 gene promoter (hyr1 P) and a telomeric heterochromatin region on the right arm of chromosome VII (VIIR T) were used as negative control groups. (C, D) ChIP analysis of the binding of BuGZ after (C) H₂O₂ or (D) menadione (MD) treatment for 2 hours at cat-3 locus. Error bars indicate SD (n = 3). Significance was evaluated by two-tailed t-test. *P < 0.05, **P < 0.01, and ***P<0.001.

Fig 6. The intact zinc finger domains of BuGZ are essential for repressing cat-3 expression.

(A) Schematic of full-length BuGZ and mutants with deletions or alanine substitutions (indicated by gray diamonds) in zinc finger domains. The position of zinc finger 1 (red rectangle), zinc finger 2 (blue rectangle), GLEBS domain (yellow rectangle) and zinc ion binding sites (light purple diamonds) are indicated. (B) Mycelial growth of WT, bugz^KO, bugz^KO,BuGZ and transformants with various mutations in zinc finger domains on plates with 0, 10, or 20 mM H₂O₂. (C) Quantitation of growth rate of strains described in panel B relative to WT. (D) In-gel catalase activity assay of protein extracts from WT, bugz^KO, bugz^KO,BuGZ and transformants with various mutations in zinc finger domains. (E) Western blot for CAT-3 protein in WT, bugz^KO, bugz^KO,BuGZ and transformants with various mutations in zinc finger domains. The membranes stained by Coomassie blue served as the loading control. (F) RT-qPCR quantification of cat-3 mRNA in bugz^KO, bugz^KO,BuGZ and transformants with various mutations in zinc finger domains relative to WT. Error bars indicate SD (n = 3). Significance was evaluated by two-tailed t-test. *P < 0.05, **P < 0.01, and ***P<0.001.

Fig 7. BuGZ antagonizes the recruitment of transcription activators and RNAPII to the cat-3 gene.

ChIP analyses of binding of (A) CPC1, (B) NC2α, (C) NC2β, (D) TFIIB, (E) RPB-1, and (F) RNAPII phosphorylated at Ser2 at the cat-3 locus in WT and bugz^KO strains.