HDA-2-Containing Complex Is Required for Activation of Catalase-3 Expression in Neurospora crassa

ABSTRACT

It is essential for aerobic organisms to maintain the homeostasis of intracellular reactive oxygen species (ROS) for survival and adaptation to the environment. In line with other eukaryotes, the catalase of Neurospora crassa is an important enzyme for clearing ROS, and its expression is tightly regulated by the growth phase and various oxidative stresses. Our study reveals that, in N. crassa, histone deacetylase 2 (HDA-2) and its catalytic activity positively regulate the expression of the catalase-3 (cat-3) gene. HDA-2, SIF-2, and SNT-1 may form a subcomplex with such a regulation role. As expected, deletion of HDA-2 or SIF-2 subunit increased acetylation levels of histone H4, indicating that loss of HDA-2 complex fails to deacetylate H4 at the cat-3 locus. Furthermore, loss of HDA-2 or its catalytic activity led to dramatic decreases of TFIIB and RNA polymerase II (RNAP II) recruitment at the cat-3 locus and also resulted in high deposition of H2A.Z at the promoter and transcription start site (TSS) regions of the cat-3 gene. Collectively, this study strongly demonstrates that the HDA-2-containing complex activates the transcription of the cat-3 gene by facilitating preinitiation complex (PIC) assembly and antagonizing the inhibition of H2A.Z at the cat-3 locus through H4 acetylation.

INTRODUCTION

Aerobic organisms produce reactive oxygen species (ROS) as by-products through the electron transport chain during intracellular and extracellular redox reactions. ROS mainly consist of singlet oxygen, superoxide radical, hydrogen peroxide, and hydroxyl radicals. Although ROS have pivotal roles in cell signal transduction and homeostasis (1–4), they also pose a serious threat to cells if the intracellular ROS levels deviate from the optimal amount (5–8). Excessive ROS can cause extensive oxidation damage to proteins, unsaturated fatty acids, and DNA, resulting in dysfunction or death of cells (5, 6, 8, 9). To counteract the damaging effects of ROS, eukaryotic cells have evolved various antioxidation strategies which convert harmful ROS into harmless constituents to cells. Superoxide dismutase (SOD), catalases (CAT), and peroxidases are three main types of enzymes involved in ROS clearance, among which SOD and CAT are the primary antioxidants in nearly all living cells exposed to oxygen (2, 3, 10, 11). These clearance mechanisms are highly conserved from bacteria to mammals (12).

The filamentous fungus Neurospora crassa possesses four catalase genes, cat-1, cat-2, cat-3, and cct-1/cat-4 (13–16). Among them, cat-1, cat-2, and cat-3 are inducible by oxidative stress and differentially expressed during the asexual life cycle (16, 17). Previous study showed that CAT-3 is the key catalase in growing hyphae whose function could not be replaced by other catalases (18). Furthermore, CAT-3 protein can be induced by various oxidative stresses (19). For example, H₂O₂ treatment can stimulate cat-3 expression by elevating histone acetylation levels of the cat-3 locus (20). Consistent with this, CPC1/GCN4 and the histone acetyltransferase GCN5 have been proved to positively regulate the expression of cat-3 (21). Furthermore, our previous research showed that histone variant H2A.Z is also a critical regulator for cat-3 transcription. SWR complex-mediated H2A.Z deposition and INO80 complex-mediated H2A.Z removal both significantly influence cat-3 transcription (22, 23). These results suggest that the chromatin structure and histone modifications play major roles in regulating the inducible expression of the cat-3 gene.

The dynamics of histone acetylation are controlled by the antagonistic roles of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In general, HDACs frequently serve as corepressors to inactivate transcription. Hos2 has been identified as a HDAC subunit in the Set3 complex, accompanied by Set3, Sif2, Snt1, YIL112w, Cpr1, and another histone deacetylase, Hst1 (24). Unlike the classical repressive role of most HDACs, Saccharomyces cerevisiae Hos2 can bind to and deacetylate histone in the coding region of active genes (25). Genetic evidence has shown that Set3 and the deacetylase activity of Hos2 promote the efficient activation of the GAL1 gene by deacetylating specific lysines in histones H3 and H4 (25, 26). Further study showed that the Set3 complex deacetylates histone at the 5′ ends of actively transcribed genes (27). In mammalian cells, the Set3 complex was proposed to be a functional homolog of the NCoR/SMRT complex, which forms a stable complex with histone deacetylase HDAC3 and TBL1. The deacetylases HDAC3 and TBL1 are mammalian homologs of Hos2 and Sif2, which are core subunits of the S. cerevisiae Set3 complex (28, 29). In addition, the Snt1 subunit of the Set3 complex shares the SANT domain with NCoR/SMRT (24, 30). Even though HDAC3 can act as a transcriptional corepressor, it is required for transcriptional activation in a class of retinoic acid response elements (31, 32). These results strongly suggest that the conserved enzymatic activity of the Set3 complex and NCoR/SMRT complex appear to be the Hos2 and HDAC3 subunits, which deacetylate histone H3 and H4 at the actively transcribed genes.

In budding yeast, Htz1/H2A.Z preferentially occupies the promoter regions of two transcriptionally inactive but inducible genes, GAL1 and PHO5 (33–37). Further study revealed that htz1Δ is synthetic sick with each core subunit of the Set3 complex, such as SET3, HOS2, SIF2, and SNT1, suggesting overlapping roles of them in some biological processes. Interestingly, the severe slow growth phenotype of htz1Δ set3Δ was partially suppressed by further deletion of the chromatin remodeler SWR1 gene (38). These results suggested that Set3/Hos2 histone deacetylase has previously unrecognized functions in the dynamic deposition or remodeling of nucleosomes containing H2A.Z. A recent study identified HDA-2 as a positive regulator for cat-3 transcription in Trichoderma atroviride (39). However, it is not clear whether HDA-2 is involved in regulation of cat-3 gene expression in N. crassa.

Here, we revealed that in N. crassa, histone deacetylase 2 (HDA-2) positively regulates cat-3 gene expression, and such a regulatory role depends on its catalytic activity. Immunoprecipitation data confirmed the interaction among N. crassa HDA-2, SIF-2, and SNT-1 proteins. Like HDA-2, deletion of the SIF-2 or SNT-1 subunit in HDA-2-containing complex downregulates cat-3 expression. The acetylation levels of histone H4 were increased in these mutants, indicating that loss of the HDA-2 complex fails to deacetylate H4 at the cat-3 locus. Chromatin immunoprecipitation (ChIP) assays revealed that loss of HDA-2 or its catalytic activity results in defective assembly of the preinitiation complex (PIC) and high deposition of H2A.Z at the cat-3 locus. Taken together, our results demonstrate that the HDA-2 complex is critical for cat-3 activation.

RESULTS

HDA-2, SIF-2, and SNT-1 may form a subcomplex for resisting H₂O₂-induced ROS stress.

To identify the factors that contribute to resisting H₂O₂-induced ROS stress, we performed H₂O₂ sensitivity assays to screen N. crassa knockout mutants. We found that a strain with deletion of the hda-2 gene (NCU02795) exhibited a severe H₂O₂ sensitivity phenotype compared to that of the wild-type (WT) strain (Fig. 1A and B). HDA-2 is a class I histone deacetylase and is the homolog of Saccharomyces cerevisiae Hos2 and Schizosaccharomyces pombe Phd1. In S. cerevisiae, Hos2p is the deacetylase subunit of the Set3 complex (24). Therefore, we searched N. crassa homologs of the other subunits in the S. cerevisiae Set3 complex through sequence alignment and identified SET-4 (SET3), NST-1 (HST1), NCU10346 (SNT1), NCU00388 (YIL112w), NCU06838 (SIF2), and NCU01200 (CPR1) (see Fig. S1A in the supplemental material).

FIG 1.

HDA-2, SIF-2, and SNT-1 may form a subcomplex for resisting H₂O₂-induced ROS stress. (A) Mycelial growth of the wild type (WT) and cat-3^KO, hda-2^KO, sif-2^KO, or snt-1^KO and HDA-2, SIF-2, or SNT-1 transformants in plates with 0, 5, or 10 mM H₂O₂ as indicated. Cultures were inoculated in plates at 25°C under constant light. (B) Quantitative results for relative growth rate of the WT, cat-3^KO, hda-2^KO, sif-2^KO, or snt-1^KO and HDA-2, SIF-2, or SNT-1 transformants. Error bars show standard deviations (SD; n = 3). Significance was assessed by using a two-tailed t test. ***, P < 0.001 versus WT. (C and D) Mapping of endogenous SIF-2 or HDA-2 responsible for the interaction with ectopically expressing wild-type Myc-HDA-2 in hda-2^KO strain or Myc-SIF-2 in sif-2^KO strain, respectively. In immunoprecipitation assays with anti-Myc antibody (α-Myc), the eluates were detected by Western blotting using anti-SIF-2 (α-SIF-2) and anti-Myc (α-Myc) antibodies (C) or anti-HDA-2 (α-HDA-2) and anti-Myc (α-Myc) antibodies (D), respectively. (E and F) Immunoprecipitation assays showing the interaction between Myc-SNT-1 and endogenous HDA-2 (E) or SIF-2 (F) protein, respectively.

FIG S1

Subunits of the Set3 complex in Saccharomyces cerevisiae and the interaction of their homologous proteins in Neurospora crassa. (A) Search for homologs of the S. cerevisiae Set3 complex in N. crassa by sequence alignment yielded information on seven proteins and their conserved domains. (B to I) Mapping of endogenous HDA-2 or SIF-2 responsible for the interaction with ectopically expressing Myc-SET-4, Myc-NST-1, Myc-YIL112w, or Myc-CPR-1 in the WT strain. For immunoprecipitation assays with anti-HDA-2 antibody (α-HDA-2) or anti-SIF-2 antibody (α-SIF-2), the eluates were detected by Western blotting using α-HDA-2 and α-Myc antibodies (B, D, F, and H) or α-SIF-2 and α-Myc antibodies (C, E, G, and I), respectively. Download FIG S1, TIF file, 1.1 MB ^{(1.1MB, tif)} .

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To systematically analyze the function of each subunit in H₂O₂-induced ROS stress, we tried to generate deletion mutants of these genes by gene replacement in the ku70^RIP strain. However, we could not obtain the homokaryotic deletion strains of the Yil112w (NCU00388) or Cpr1 (NCU01200) gene, suggesting that these two genes are essential to cell viability in N. crassa. As shown in Fig. 1A and B, deletion of sif-2 (NCU06838) or snt-1 (NCU10346) resulted in a similar H₂O₂ sensitivity phenotype with the hda-2^KO strain, indicating that these subunits probably cooperate to resist H₂O₂-induced ROS stress. To further confirm the H₂O₂ sensitivity phenotype of these knockout strains, we generated the complementary strains by transforming Myc-tagged constructs of subunit proteins into each corresponding knockout strain. As expected, ectopic expression of Myc-tagged proteins restored the H₂O₂ sensitive phenotypes of hda-2^KO, sif-2^KO, or snt-1^KO strains to those of WT strains (Fig. 1A and B), indicating that the H₂O₂ sensitivity phenotype of each mutant was due to the deletion of each subunit gene. However, the strains with deletion of nst-1 (NCU04737) or set-4 (NCU04389) exhibited similar H₂O₂ sensitivity as the WT strain (see Fig. S2A in the supplemental material), suggesting that NST-1 and SET-4 are not key regulators for resisting H₂O₂-induced ROS stress. Therefore, the HDA-2, SIF-2, and SNT-1 proteins play significant roles in responding to H₂O₂-induced ROS stress in N. crassa.

FIG S2

NST-1 and SET-4 are not key regulators for resisting H₂O₂-induced ROS stress. (A) Growth of the WT, nst-1^KO, and set-4^KO in plates under the treatment of 0 or 10 mM H₂O₂. (B) Western blot showing the levels of CAT-3 protein in the WT, nst-1^KO, and set-4^KO. The membrane stained by Coomassie blue represents the total protein in each sample and acted as loading control for Western blotting. (C) Catalase activity assay. Crude extracts from the WT, nst-1^KO, and set-4^KO were subjected to native-PAGE, and the catalase activities were determined with the in-gel assay. (D) RT-qPCR analysis showing the levels of cat-3 mRNA in the WT, nst-1^KO, and set-4^KO. Error bars show SD (n = 3). Download FIG S2, TIF file, 0.8 MB ^{(848.1KB, tif)} .

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To confirm the integrity of N. crassa HDA-2-containing complex, we generated HDA-2- and SIF-2-specific antibodies (see Fig. S3A and B in the supplemental material) and examined the interaction between HDA-2 and SIF-2 in hda-2^KO, Myc-HDA-2 and sif-2^KO, Myc-SIF-2 transformants. Immunoprecipitation assays revealed that the Myc-HDA-2 or Myc-SIF-2 protein strongly interacts with endogenous SIF-2 or HDA-2, respectively (Fig. 1C and D). The snt-1^KO strain exhibited similar H₂O₂ sensitivity as hda-2^KO and sif-2^KO strains, so we performed immunoprecipitation assays, which revealed that the Myc-SNT-1 protein strongly interacts with endogenous HDA-2 or SIF-2, respectively (Fig. 1E and F). To further confirm whether other homologous proteins of the S. cerevisiae Set3 complex also form complexes in N. crassa, we performed immunoprecipitation assays and found that Myc-SET-4, Myc-NST-1, Myc-YIL112w, and Myc-CPR-1 proteins interact with endogenous HDA-2 or SIF-2 (see Fig. S1B to I in the supplemental material). These results demonstrated that HDA-2, SIF-2, and SNT-1 may form a subcomplex to resist H₂O₂-induced oxidative stress in N. crassa.

FIG S3

Detection of HDA-2- and SIF-2-specific antibodies. (A and B) Immunodetection of HDA-2 or SIF-2 protein in WT, hda-2^KO (A), and sif-2^KO or sif-2^KO, SIF-2 (B) strains using the corresponding polyclonal antibody that specifically recognized the HDA-2 and SIF-2 or Myc-SIF-2 proteins in the WT or sif-2^KO, SIF-2 strain, respectively. Download FIG S3, TIF file, 0.4 MB ^{(389.7KB, tif)} .

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HDA-2-containing complex plays a key role in activation of cat-3 transcription.

Since CAT-3 is the major catalase in mycelia, the genetic analysis results above suggest that the HDA-2-containing complex may participate in the regulation of cat-3 expression in N. crassa. To test this possibility, we performed a Western blot assay to analyze the protein levels of CAT-3 in the WT and each mutant strain. As shown in Fig. 2A (see also Fig. S2B in the supplemental material), the protein levels of CAT-3 in hda-2^KO, sif-2^KO, and snt-1^KO strains were extremely lower than in the WT strain, whereas the protein levels of CAT-3 in the nst-1^KO and set-4^KO strains were similar to that in the WT strain. As expected, an in-gel assay showed that the stained bands corresponding to CAT-3 activity were weaker in hda-2^KO, sif-2^KO, and snt-1^KO mutants than in the WT, nst-1^KO, and set-4^KO strains (Fig. 2B; see also Fig. S2C). Consistent with the protein and catalase activity results, the levels of cat-3 mRNA in hda-2^KO, sif-2^KO, and snt-1^KO mutants were also much lower than those in the WT, nst-1^KO, and set-4^KO strains (Fig. 2C; see also Fig. S2D). These results suggest that the decreased expression of cat-3 in these mutants is responsible for their sensitivity to H₂O₂-induced ROS stress. In addition, ectopic expression of Myc-tagged HDA-2 or SIF-2 in each corresponding deletion strain restored the levels of CAT-3 activity (Fig. 2D and E) and cat-3 expression to WT levels (Fig. 2F to I). Taken together, these results demonstrated that HDA-2-containing complex is critical for transcriptional activation of the cat-3 gene.

FIG 2.

HDA-2-containing complex plays a key role for the activation of cat-3 transcription. (A) Western blot showing the levels of CAT-3 protein in the WT, hda-2^KO, sif-2^KO, snt-1^KO, and cat-3^KO strains. The membrane stained with Coomassie blue represents the total protein in each sample and acted as a loading control for Western blotting. (B) Catalase activity in-gel assay. Crude extracts from the WT, hda-2^KO, sif-2^KO, snt-1^KO, and cat-3^KO strains were subjected to native-PAGE, and the catalase activity was determined in the in-gel assay. (C) RT-qPCR analysis showing the levels of cat-3 mRNA in the WT, hda-2^KO, sif-2^KO, and snt-1^KO strains. Error bars show SD (n = 3). Significance was assessed by using a two-tailed t test. ***, P < 0.001 versus WT. (D and E) Catalase activity in-gel assays. Crude extracts from the WT, hda-2^KO, or sif-2^KO and HDA-2 or SIF-2 transformants were subjected to native-PAGE, and the catalase activity was determined with the in-gel assay. (F and G) Western blot showing the levels of CAT-3, HDA-2, or SIF-2 and Myc-HDA-2 or Myc-SIF-2 proteins in the WT, hda-2^KO, or sif-2^KO and HDA-2 or SIF-2 transformants. The membranes stained with Coomassie blue represent the total protein in each sample and acted as a loading control for Western blotting. (H and I) RT-qPCR analyses showing the levels of cat-3 mRNA in the WT, hda-2^KO, or sif-2^KO and HDA-2 or SIF-2 transformants. Error bars show SD (n = 3). Significance was assessed by using a two-tailed t test. ***, P < 0.001 versus WT.

The deacetylase activity of HDA-2 is required for activation of cat-3 transcription.

When the amino acid sequence of N. crassa HDA-2 was examined in a BLAST search against protein databases, its homologs were found to be highly conserved with Homo sapiens HDAC3, Saccharomyces cerevisiae Hos2, and Drosophila melanogaster HDAC3 (Fig. 3A). To determine whether the deacetylase activity of HDA-2 protein is required for activation of cat-3 transcription, we generated a series of HDA-2 mutants by introducing catalytic-dead point mutation (K89A, H202A/H203A, H240A/H241A, Y371F) or deletion of the zinc-binding sites (D238 V239 H240), respectively (40–42). A plate assay showed that ectopic expression of catalytic-dead Myc-HDA-2 failed to rescue the growth defect and H₂O₂ sensitive phenotypes of hda-2^KO strains (Fig. 3B and C), indicating that the deacetylase activity of HDA-2 plays an important role in regulation of H₂O₂ resistance. Consistent with the phenotypes of these transformants, expression of the catalytic-dead Myc-HDA-2 significantly decreased the CAT-3 activity, CAT-3 protein, and cat-3 mRNA levels compared to those of the WT and hda-2^KO, Myc-HDA-2 strains (Fig. 3D, E, and F). Taken together, these results demonstrated that the catalytic activity of HDA-2 protein is required for activation of cat-3 expression.

FIG 3.

The deacetylase activity of HDA-2 is required for activation of cat-3 transcription. (A) Amino acid sequence alignment of the conserved HDA-2 from Neurospora crassa, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus, and Homo sapiens. (B) Growth of the WT, hda-2^KO, and HDA-2 transformants in plates under the treatment of 0, 5, or 10 mM H₂O₂. The relative rate of growth was determined. (C) Quantitative results for relative growth rate of the WT, hda-2^KO, and HDA-2 transformants. Error bars show SD (n = 3). (D) Catalase activity assay. Crude extracts from the WT, hda-2^KO, and HDA-2 transformants were subjected to native-PAGE, and the catalase activities were determined by the in-gel assay. (E) Western blot analysis showing the levels of CAT-3, HDA-2, and Myc-HDA-2 proteins in the WT, hda-2^KO, and HDA-2 transformants. The membrane stained by Coomassie blue represents the total protein in each sample and acted as loading control for Western blotting. (F) RT-qPCR analysis showing the levels of cat-3 mRNA in the WT, hda-2^KO, and HDA-2 transformants. Error bars show SD (n = 3). Significance was assessed by using a two-tailed t test. ***, P < 0.001 versus WT.

HDA-2-containing complex can bind to the cat-3 locus and regulate H4 acetylation.

To test whether HDA-2-containing complex binds to the cat-3 locus, we carried out ChIP assays with HDA-2 or SIF-2 antibody. ChIP data revealed that HDA-2 and SIF-2 are highly enriched at the cat-3 locus in the WT strain compared to those in the hda-2^KO or sif-2^KO strains (Fig. 4A to C), confirming that HDA-2-containing complex could bind to the cat-3 locus. To test whether the binding of HDA-2 influences the histone acetylation state, we measured the levels of H3 and H4 acetylation at the cat-3 locus in the WT, hda-2^KO, and sif-2^KO strains. ChIP data showed that the H4 acetylation levels were increased at the cat-3 5′ end of the regulation region in these mutants compared to those of WT strain (Fig. 4D and E), while the H3 acetylation levels had no significant change (Fig. 4F and G). We also measured the levels of H2B and H3 at the cat-3 locus in the WT, hda-2^KO, and sif-2^KO strains. However, ChIP results showed that the occupancies of histone H2B and H3 at the cat-3 locus were identical in WT, hda-2^KO, and sif2^KO strains, indicating that the nucleosome density is not affected by the binding of HDA-2 and SIF-2 at the cat-3 locus (Fig. 4H and I). Taken together, these results suggest that HDA-2-containing complex is involved in regulation of H4 acetylation at the cat-3 locus but has no effect on the nucleosome density.

FIG 4.

HDA-2-containing complex can bind to the cat-3 locus and regulate H4 acetylation. (A) Schematic depiction of cat-3 (NCU00355) on the Neurospora genome. (primer pairs 5 to 8) under the schematic indicate the regions tested by ChIP-qPCR. TSS, transcription start site; ORF, open reading frame. (B and C) ChIP assays showing the binding levels of HDA-2 (B) and SIF-2 (C) at the cat-3 locus in the WT, hda-2^KO (B), and sif-2^KO (C) strains. (D and E) ChIP assays showing the acetylation of H4 at the cat-3 locus in the WT, hda-2^KO (D), and sif-2^KO (E) strains. (F and G) ChIP assays showing the acetylation of H3 at the cat-3 locus in the WT, hda-2^KO (F), and sif-2^KO (G) strains. (H and I) ChIP assays showing the occupancy levels of H2B (H) and H3 (I) at the cat-3 locus in the WT, hda-2^KO, and sif-2^KO strains. Error bars show SD (n = 3). Significance was assessed by using a two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

HDA-2-containing complex is required for PIC assembly at the cat-3 locus.

In S. cerevisiae, Rpd3 and Hos2 are required for RNA polymerase II (RNAP II) recruitment to the RNR3 gene for its transcription (43). To test whether the HDA-2-containing complex is required for the PIC assembly at the cat-3 promoter, we performed a ChIP assay using (TFIIB)-specific antibody in WT, hda-2^KO, and sif-2^KO strains. As shown in Fig. 5A, the enrichment of TFIIB at the cat-3 promoter, transcription start site (TSS), and 5′ end of the open reading frame (ORF) region was dramatically decreased in hda-2^KO and sif-2^KO strains compared to that in the WT strain, indicating that deletion of the hda-2 or sif-2 gene affects binding of TFIIB at the cat-3 locus. We further checked the enrichment of RNAP II at the cat-3 locus in WT, hda-2^KO, and sif-2^KO strains. ChIP assays using RPB-1-specific antibody revealed that RNAP II enrichment was also dramatically decreased at the cat-3 promoter and ORF regions in hda-2^KO and sif-2^KO strains compared to that in the WT strain (Fig. 5B), indicating that the HDA-2-containing complex facilitates the assembly of RNAP II at the cat-3 promoter/TSS region.

FIG 5.

HDA-2-containing complex is required for PIC assembly at the cat-3 locus. (A and B) ChIP assays showing the binding of TFIIB (A) and RPB-1 (B) at the cat-3 locus in the WT, hda-2^KO, and sif-2^KO strains. (C) ChIP assay showing the acetylation of H4 at the cat-3 locus in the WT, hda-2^KO, and HDA-2 transformants. (D and E) ChIP assays showing the binding of TFIIB (D) and RPB-1 (E) at the cat-3 locus in the WT, hda-2^KO, and HDA-2 transformants. Error bars show SD (n = 3). Significance was assessed by using a two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

A previous study showed that Rpd3 and Hos2 regulate the activation of RNR3 by deacetylating nucleosomes at the promoter in S. cerevisiae (43). To further confirm the catalytic activity of HDA-2 in regulation of PIC assembly at the cat-3 promoter and TSS regions, we examined the acetylation status of H4 at the cat-3 locus in WT, hda-2^KO, hda-2^KO, Myc-HDA-2 or hda-2^KO, Myc-HDA-2^ΔDVH strains. Consistent with the levels of cat-3 expression, ChIP assays revealed that ectopic expression of Myc-HDA-2 but not Myc-HDA-2^ΔDVH not only recovered the low levels of H4 acetylation at the cat-3 promoter seen in hda-2^KO strain (Fig. 5C), but also significantly rescued the TFIIB and RNAP II enrichment at the cat-3 gene region in the hda-2^KO strain (Fig. 5D and E). Taken together, these data suggested that the catalytic activity of the HDA-2 protein is required for its role in activation of cat-3 expression by regulating H4 acetylation and assembly of PIC at the cat-3 locus.

HDA-2-containing complex activates transcription of the cat-3 gene by antagonizing inhibition of H2A.Z at the cat-3 locus through H4 acetylation.

In S. cerevisiae, histone H3 and H4 tail acetylation is required for efficient recruitment of H2A.Z (36). We previously found that deposition of H2A.Z at the cat-3 gene promoter/TSS region negatively regulated transcription of cat-3 (22). To test whether the H4 hyperacetylation at the cat-3 promoter/TSS region promotes H2A.Z deposition, we examined the occupancies of H2A.Z at the cat-3 promoter/TSS region in WT, hda-2^KO, and sif-2^KO strains. ChIP assays using H2A.Z-specific antibody showed that H2A.Z occupancies were dramatically increased at the cat-3 promoter/TSS region in hda-2^KO and sif-2^KO strains compared to that of WT strain (Fig. 6A). Moreover, ectopic expression of WT Myc-HDA-2 but not catalytic-dead Myc-HDA-2^ΔDVH protein rescued the increased deposition of H2A.Z at the cat-3 promoter/TSS region in hda-2^KO strain (Fig. 6B). These results indicated that HDA-2-containing complex antagonizes H2A.Z excessive deposition to activate transcription of the cat-3 gene through H4 acetylation.

FIG 6.

HDA-2-containing complex activates transcription of the cat-3 gene by antagonizing the inhibition of H2A.Z at the cat-3 locus through H4 acetylation. (A) ChIP assay showing the occupancy levels of H2A.Z at the cat-3 locus in the WT, hda-2^KO, and sif-2^KO strains. The H2A.Z^KO strain was used as a negative control in the ChIP assay. (B) ChIP assay showing the occupancy levels of H2A.Z at the cat-3 locus in the WT, hda-2^KO, and HDA-2 transformants. The H2A.Z^KO strain was used as a negative control in the ChIP assay. (C and D) ChIP assays showing the binding of INO80 (C) and ARP8 (D) at the cat-3 locus in the WT, hda-2^KO, and sif-2^KO strains. The ino80^KO (C) and arp8^KO (D) strains were used as negative controls in the ChIP assays. (E) ChIP assay showing the binding of INO80 at the cat-3 locus in the WT, hda-2^KO, and HDA-2 transformants. The ino80^KO strain was used as a negative control in the ChIP assay. Error bars show SD (n = 3). Significance was assessed by using a two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Our previous study showed that the chromatin remodeling complex INO80C positively regulates cat-3 expression through removing H2A.Z around the cat-3 locus (23). To test whether the recruitment of INO80C to the cat-3 locus was affected by HDA-2-containing complex, we examined the enrichment of INO80 and ARP8, the catalytic and structural subunit of INO80C, at the cat-3 locus in WT, hda-2^KO, and sif-2^KO strains. ChIP data showed that the enrichment of INO80 and ARP8 at the cat-3 promoter/TSS region were dramatically decreased in hda-2^KO and sif-2^KO strains compared to those of WT strain (Fig. 6C and D). Ectopic expression of Myc-HDA-2 but not catalytic-dead Myc-HDA-2^ΔDVH protein significantly increased the INO80 enrichment around the cat-3 promoter region in the hda-2^KO strain (Fig. 6E). Taken together, these data demonstrated that the catalytic activity of HDA-2 protein is required for its role in activation of cat-3 expression by regulating the INO80C-mediated removal of H2A.Z around the cat-3 promoter/TSS region.

DISCUSSION

Our recent studies have shown that H₂O₂ treatment can induce cat-3 gene expression and increase H3 acetylation at the cat-3 locus (20). Biochemical analysis showed that the transcription factor CPC1/GCN4 and histone acetyltransferase GCN5 are required for maintenance of cat-3 expression and response to H₂O₂-induced oxidative stress in N. crassa (21). It is necessary to further identify the histone-modifying enzymes involved in regulation of cat-3 gene expression. Neurospora has 11 predicted proteins related to known or putative histone deacetylases (HDACs) (13). In this study, we generated most of the deletion mutants of these HDAC genes, but not the homokaryotic deletion strains of the nst-3 (NCU03059) or nst-6 (NCU05973) gene. According to the catalase activity in-gel assay and Western blot analysis, we found that only HDA-2 is involved in the positive regulation of cat-3 expression compared with the other eight histone deacetylases in N. crassa (see Fig. S4A and B in the supplemental material). In its catalytically dead mutants, the cat-3 expression level is significantly decreased, like that in hda-2^KO strains, indicating that expression of cat-3 is dependent on the catalytic activity of HDA-2. HDA-2 is a deacetylase of the class I type histone deacetylases and is the homolog of S. cerevisiae Hos2. Hos2 and Set3, which is the homologous protein of N. crassa SET-4, are central components of the Set3 complex (24). However, deletion of the hda-2, sif-2, or snt-1 subunit but not set-4 or nst-1 in the N. crassa genome leads to downregulation of cat-3 expression. Immunoprecipitation data confirmed the interaction among N. crassa HDA-2, SIF-2, and SNT-1 proteins. Taken together, our results suggest that HDA-2, SIF-2, and SNT-1 proteins may form a subcomplex for cat-3 activation.

FIG S4

Only HDA-2 is involved in positive regulation of cat-3 expression compared with the other eight histone deacetylases in N. crassa. (A) Catalase activity in-gel assay. Crude extracts from the WT, hda-1^KO, hda-2^KO, hda-3^KO, hda-4^KO, nst-1^KO, nst-2^KO, nst-4^KO, nst-5^KO, and nst-7^KO strains were subjected to native-PAGE, and the catalase activity was determined with the in-gel assay. (B) Western blot analysis showing the levels of CAT-3 protein in the WT, hda-1^KO, hda-2^KO, hda-3^KO, hda-4^KO, nst-1^KO, nst-2^KO, nst-4^KO, nst-5^KO, and nst-7^KO strains. The membrane stained with Coomassie blue represents the total protein in each sample and acted as a loading control for Western blotting. Download FIG S4, TIF file, 1.0 MB ^{(1,000.4KB, tif)} .

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The role of HDA-2-containing complexes in yeast and mammals in gene expression has been reported in previous studies. As a histone deacetylase, we found that HDA-2-containing complex positively regulates cat-3 transcription in N. crassa. In agreement with this result, HDA-2 was identified as a positive regulator for cat-3 transcription in Trichoderma atroviride (39). In yeast, Hos2 is important for activation of GAL1 and INO1 genes through binding to the coding regions of genes (25). In addition, Hos2 and an associated factor, Set3, are necessary for efficient gene transcription (25). In contrast to other class I histone deacetylases, which are frequently found as corepressors, Hos2 is directly required for gene activation. To identify the underlying molecular mechanism of HDA-2 for cat-3 transcription, we performed a series of experiments and found that histone H4 acetylation but not H3 acetylation at the cat-3 locus in hda-2^KO and sif-2^KO strains was increased. A previous study showed that H4 acetylation could affect the assembly of PIC (44). To be specific, a previous work reported that Cmr1, a largely uncharacterized nuclear protein in S. cerevisiae, is recruited to regulate RNAP II occupancy in transcribed coding regions, which is stimulated by the histone deacetylases Rpd3 and Hos2 (45). In this study, we found that deletion of HDA-2 or SIF-2 lead to a dramatic decrease of TFIIB and RPB-1 levels at the cat-3 promoter and TSS regions (Fig. 5A and B). These results strongly suggest that the HDA-2-mediated deacetylation of H4 is necessary for efficient recruitment of transcriptional machinery for activation of the cat-3 gene.

We previously showed that H2A.Z is immediately evicted from the chromatin at the cat-3 locus in response to oxidative stress with a corresponding accumulation of CPC1 at the cat-3 locus, and we suggested that H2A.Z antagonizes CPC1 binding to restrict cat-3 expression in a normal setting, whereas under oxidative stress H2A.Z is removed from chromatin, leading to a rapid and full activation of cat-3 transcription (22). In addition, our recent study found that the negative cofactor 2 (NC2) complex activates cat-3 expression by recruiting INO80 complex to remove H2A.Z from special H2A.Z-containing nucleosomes at the promoter and TSS of the cat-3 gene (23). Furthermore, NC2 is involved in removal of H2A.Z at hsp70/dnak and hsp90a genes (23). These results suggest that H2A.Z deposition may serve as a regulatory target for external stimuli and that the structure of H2A.Z-containing nucleosomes around these inducible genes is less stable than those of H2A-containing nucleosomes. In budding yeast, efficient deposition of H2A.Z is further promoted by histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the SWR1 remodeling complex that deposits H2A.Z (36). It has been found in previous studies that there is an interplay between the SWR1 complex and INO80 complex. For example, AtARP6, a specific subunit of SWR1-C that mediates the H2A.Z exchange in Arabidopsis, was found to have an inhibitory role in the local chromatin enrichment of AtINO80 (46). Moreover, both htz1Δ set3Δ and swr1Δ set3Δ exhibit a severe slow growth phenotype in S. cerevisiae, but the htz1Δ swr1Δ set3Δ triple mutant grows relatively well, indicating Set3/Hos2 histone deacetylase has previously unrecognized functions in dynamic deposition and remodeling of nucleosomes containing H2A.Z (38). Our ChIP assays showed that the abundance of H2A.Z at the cat-3 locus was dramatically increased in the absence of HDA-2 and SIF-2. In contrast, loss of HDA-2 and SIF-2 resulted in a decreased recruitment of INO80 and ARP8 subunits of the INO80 complex at the cat-3 locus compared to that in the WT strain. These results indicated that HDA-2-containing complex positively regulates transcription of the cat-3 gene by antagonizing inhibition of H2A.Z at the cat-3 locus through H4 acetylation.

According to our previous study, the H2A.Z-containing nucleosomes should be removed upon gene induction to provide access for the transcriptional machinery (22). In yeast, a genome-wide study showed that blocking PIC assembly resulted in promoter-specific H2A.Z accumulation, while H2A.Z eviction was unaffected upon depletion of INO80, indicating that the PIC is required to evict H2A.Z (47). In this study, we found that HDA-2-containing complex is required not only for PIC assembly but also for antagonizing the inhibition of H2A.Z at the cat-3 locus through H4 acetylation. Therefore, these results cannot completely exclude an interplay between PIC defective assembly and H2A.Z excessive deposition at the cat-3 locus in hda-2^KO and sif-2^KO strains.

MATERIALS AND METHODS

Strains and culture conditions.

The 87-3 (bd, a) strain was used as the wild-type strain in this study (48). The ku70 ^RIP (bd, a) strain, generated previously (49), was used as the host strain for creating the hda-2 (NCU02795), sif-2 (NCU06838), snt-1 (NCU10346), nst-1 (NCU04737), and set-4 (NCU4389) knockout mutants by deleting the entire ORF through homologous recombination using a protocol described previously (50). The plasmid containing the cfp promoter driven the HDA-2 ORF and its 3′-untranslated region (pcfp-5Myc-6×His-HDA-2) was used as the template for mutagenesis, and five mutations of HDA-2 (HDA-2^ΔDVH, HDA-2^K89A, HDA-2^H240A/H241A, HDA-2^H202A/H203A, and HDA-2^Y371F) were generated. Afterwards, plasmids pcfp-5Myc-6×His-HDA-2, pcfp-5Myc-6×His-HDA-2^ΔDVH, pcfp-5Myc-6×His-HDA-2^K89A, pcfp-5Myc-6×His-HDA-2^H240A/H241A, pcfp-5Myc-6×His-HDA-2^H202A/H203A, and pcfp-5Myc-6×His-HDA-2^Y371F were transformed into hda-2^KO (bd, his-3-deficient) strains to obtain the transformants hda-2^KO, Myc-HDA-2, hda-2^KO, Myc-HDA-2^ΔDVH, hda-2^KO, Myc-HDA-2^K89A, hda-2^KO, Myc-HDA-2^H240A/H241A, hda-2^KO, Myc-HDA-2^H202A/H203A, and hda-2^KO, Myc-HDA-2^Y371F. Applying the same method, a sif-2^KO, Myc-SIF-2 strain and snt-1^KO, Myc-SNT-1 strain were created. All strains used in this study possess the same bd background.

The medium for plate assays contained 1× Vogel’s salts, 3% sucrose, and 1.5% (wt/vol) agar with or without H₂O₂. Liquid cultures were grown at 25°C with shaking in minimal medium (1× Vogel’s and 2% glucose) for 18 h in constant light (LL).

Plate assay.

Age-appropriate conidia were inoculated in petri dishes with 50 mL liquid medium containing Vogel’s minimal medium (VM) and 2% glucose under static culture condition at 25°C in constant light (LL) until the exponential growth phase of mycelium. The disks of mycelium mat were cut with a cork borer for quantification. For each strain, an individual mycelium disk was transferred into the centers of VM plates containing 3% sucrose and 1.5% (wt/vol) agar and cultured at 25°C in constant light (LL). The response to oxidative stress was determined by analyzing disk diameters of strains on VM plates containing 3% sucrose and 1.5% (wt/vol) agar with or without H₂O₂ at the indicated concentrations. In order to exclude the effect of the growth rate of different strains on the H₂O₂ sensitivity, the calculation method used previously, which included the extent of relative growth rate to represent the extent of H₂O₂ sensitivity, was also used in this study (20–22). In addition, in order to visually analyze the growth phenotype, all plates were photographed until the disk diameters of the wild-type strain in medium without H₂O₂ exactly extended to the edge of the plate. Then, we directly analyzed the H₂O₂ sensitivity through visual observation of the disk diameters of strains in medium with oxidative stress.

In-gel assay for catalase.

Cell extracts of mycelium disks cultured for 18 h in liquid medium were used for the zymogram. Ground tissues were mixed with ice-cold extraction buffer containing 50 mM HEPES (pH 7.4), 137 mM NaCl, 10% glycerol, and protease inhibitors pepstatin A (1 μg/mL), leupeptin (1 μg/mL), and phenylmethylsulfonyl fluoride (PMSF; 1 mM), and centrifuged at 10,000 × g for 10 min at 4°C. The protein concentration was measured by Bio-Rad protein assay dye at 595 nm. For the in-gel assay, catalase activity was determined as described previously (51). Equal amounts of total protein (40 μg) were loaded into a 7.5% native polyacrylamide slab gel. After electrophoresis, the gel was immersed in 10 mM H₂O₂ with shaking for 10 min and then in a 1:1 mixture of freshly prepared 1% potassium hexacyanoferrate (III) and 1% iron (III) chloride hexahydrate. Catalase activity was visualized as a band where H₂O₂ was decomposed by catalase.

Protein analysis.

Protein extraction, quantification, and Western blot analysis were performed as described previously (52). Equal amounts of total protein (40 μg) were loaded into each lane. After electrophoresis, proteins were transferred onto a polyvinylidine difluoride (PVDF) membrane. Western blot analysis was performed using antibodies against the proteins of interest.

RNA analysis.

For quantitative real-time reverse transcriptase quantitative PCR (qPCR), total RNA was isolated with TRIzol reagent and treated with DNase I to remove genomic DNA, according to the manufacturer’s protocol. Each RNA sample (total RNA, 5 μg) was subjected to reverse transcription with Moloney murine leukemia virus reverse transcriptase (Promega) and then amplified by real-time PCR (ABI 7500). The primers used for qPCR are shown in Table S1 in the supplemental material. The relative values of gene expression were calculated using the threshold cycle (2^−ΔΔCT) method (53) by comparing the cycle number for each sample with that for the untreated control. The results were normalized to expression levels of the β-tubulin gene.

TABLE S1

Primers for RT-qPCR assays. Download Table S1, DOCX file, 0.01 MB ^{(14.9KB, docx)} .

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Generation of antiserum against HDA-2 and SIF-2.

Glutathione S-transferase (GST)–HDA-2 (amino acids T384 to R487) and GST-SIF-2 (amino acids E109 to N347) fusion proteins were expressed in BL21 cells, and soluble recombinant proteins were purified and used as the antigens to generate rabbit polyclonal antiserum, as described previously (54, 55).

ChIP analysis.

Chromatin immunoprecipitation (ChIP) assays were performed as described previously (56). Briefly, N. crassa tissues were fixed with 1% formaldehyde for 15 min at 25°C with shaking. Glycine was added at a final concentration of 125 mM, and samples were incubated for another 5 min. The cross-linked tissues were ground and resuspended at 0.5 g in 6 mL lysis buffer containing protease inhibitors (1 mM PMSF, 1 μg/mL leupeptin, and 1 μg/mL pepstatin A). Chromatin was sheared by sonication to approximately 500- to 1,000-bp fragments. A 1-mL aliquot of protein solution (2 mg/mL) was used for each immunoprecipitation reaction, and 10 μL was kept as the input DNA. The ChIP was carried out with 3 μL of anti-H4ac antibody (06-866; Millipore), 3 μL of anti-H3ac antibody (06-599; Millipore), 3 μL of anti-H3 antibody (2650; CST), 2 μL of anti-H2B antibody (1790; abcam), 10 μL of anti-HDA-2 antibody, 10 μL of anti-SIF-2 antibody, 10 μL of anti-TFIIB antibody, 10 μL of anti-RPB-1 antibody, 10 μL of anti-H2A.Z antibody, 10 μL of anti-INO80 antibody, 10 μL of anti-ARP8 antibody. Immunoprecipitated DNA was quantified by using real-time PCR with primer pairs. The primer pairs used are listed in Table S2 in the supplemental material. ChIP-quantitative PCR data were normalized by the input DNA and are presented as a percentage of input DNA. Each experiment was independently performed at least three times.

TABLE S2

Primers for ChIP-qPCR assays. Download Table S2, DOCX file, 0.01 MB ^{(13.2KB, docx)} .

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Coimmunoprecipitation.

Cell extracts from the adhered mycelium mat incubated for 18 h were used for performing coimmunoprecipitation (co-IP) analyses. Protein extraction, quantification, and coimmunoprecipitation assays were performed as described previously (55). Briefly, 4-mg/mL protein extracts in extraction buffer were incubated with 5 μL of monoclonal antibody to c-Myc (HT101-02; TransGen Biotech), 10 μL of antibody to HDA-2, and/or 10 μL of antibody to SIF-2 for 4 h at 4°C with rotation. Then, the 40 μL of precleaned protein G-Sepharose (17-0885-02; GE Healthcare) was added and incubated for 1 h at 4°C with rotation. The beads were washed three times with ice-cold extraction buffer, mixed with protein loading buffer, and boiled for 10 min, and the immunoprecipitated proteins were analyzed by Western blotting.