Trichoderma reesei ACE4, a Novel Transcriptional Activator Involved in the Regulation of Cellulase Genes during Growth on Cellulose

Abstract

The filamentous fungus Trichoderma reesei is a model strain for cellulase production. Cellulase gene expression in T. reesei is controlled by multiple transcription factors. Here, we identified by comparative genomic screening a novel transcriptional activator, ACE4 (activator of cellulase expression 4), that positively regulates cellulase gene expression on cellulose in T. reesei. Disruption of the ace4 gene significantly decreased expression of four main cellulase genes and the essential cellulase transcription factor-encoding gene ace3. Overexpression of ace4 increased cellulase production by approximately 22% compared to that in the parental strain. Further investigations using electrophoretic mobility shift assays, DNase I footprinting assays, and chromatin immunoprecipitation assays indicated that ACE4 directly binds to the promoter of cellulase genes by recognizing the two adjacent 5'-GGCC-3' sequences. Additionally, ACE4 directly binds to the promoter of ace3 and, in turn, regulates the expression of ACE3 to facilitate cellulase production. Collectively, these results demonstrate an important role for ACE4 in regulating cellulase gene expression, which will contribute to understanding the mechanism underlying cellulase expression in T. reesei. IMPORTANCET. reesei is commonly utilized in industry to produce cellulases, enzymes that degrade lignocellulosic biomass for the production of bioethanol and bio-based products. T. reesei is capable of rapidly initiating the biosynthesis of cellulases in the presence of cellulose, which has made it useful as a model fungus for studying gene expression in eukaryotes. Cellulase gene expression is controlled through multiple transcription factors at the transcriptional level. However, the molecular mechanisms by which transcription is controlled remain unclear. In the present study, we identified a novel transcription factor, ACE4, which regulates cellulase expression on cellulose by binding to the promoters of cellulase genes and the cellulase activator gene ace3. Our study not only expands the general functional understanding of the novel transcription factor ACE4 but also provides evidence for the regulatory mechanism mediating gene expression in T. reesei.

Keywords: ACE4; Trichoderma reesei; Zn(II)2Cys6 protein; cellulase gene; transcription factor.

FIG 1

ACE4 structure and phylogenetic analyses of ACE4. (A) The DNA-binding domain (red) of ACE4 is orthologous to that of Gal4 from Saccharomyces cerevisiae. The filamentous fungus-specific transcription factor domain (gray) regulates target gene expression. (B) Phylogenetic analyses of ACE4 protein and its orthologs. Representative ACE4 orthologs were first retrieved using BLASTP. Amino acid sequence alignment and phylogenetic analysis were then performed with ClustalW, and the maximum likelihood tree was generated using MEGA6.0. Numbers on the tree branches represent the bootstrap supports calculated per 1,000 bootstrap replicates.

FIG 2

Cellulase production of T. reesei strain QM6a and the Δace4, OEace4, and Race4 strains. (A to C) The pNPCase activity (A), CMCase activity (B), and total secreted protein (C) of T. reesei strains were separately measured using Avicel as the sole carbon source. (D) Dry biomass weights of T. reesei strains cultured with Avicel as the sole carbon source were measured. (E) SDS-PAGE of the secretomes in supernatants of the T. reesei QM6a, Δace4, and OEace4 strains. M, molecular mass marker. Values are means ± SD of the results from three independent experiments. Asterisks indicate significant differences compared with the parental strain (*, P < 0.05, Student's t test).

FIG 3

Transcription levels of major cellulase genes in T. reesei strains cultured on Avicel. The transcriptional levels of major cellulase genes cbh1 (A), cbh2 (B), egl1 (C), egl2 (D), xyr1 (E), and ace3 (F) were evaluated by real-time quantitative PCR (RT-qPCR). T. reesei strains were grown on Avicel for 72 or 96 h. The data are normalized to expression of QM6a at 72 h for each tested gene, with the sar1 gene used as an endogenous control in all samples. Values are means ± SD of the results from three independent experiments. Asterisks indicate significant differences compared with the parental strain (*, P < 0.05, Student's t test).

FIG 4

EMSAs of ACE4 binding to the cbh1 promoter regions. (A) The cbh1 promoter was divided into six parts (cbh1-P1 to -P6), and there was a 50- to 150-bp overlap between adjacent fragments, each of which was about 200 to 450 bp long. (B to G) The binding activity of GST-ACE4 to different cbh1 promoter fragments is indicated. Six biotin-labeled DNA fragments (∼10 ng) of the cbh1 promoter were incubated with the GST tag-fused Zn(II)₂Cys₆ domain of ACE4 expressed in E. coli and purified by GST affinity chromatography. For each EMSA, 10 nM biotin-labeled fragment and 0 to 1.0 μM (with the direction of increase pointing toward the right, as indicated by the triangle) recombinant GST-ACE4 were added. EMSAs with GST tag only (GST), a 200-fold excess of unlabeled specific fragments (S), or nonspecific (NS) competitor fragments (salmon sperm DNA) were performed as controls. ACE4-DNA complexes are indicated with an arrow. These results are representative of three experiments with similar results.

FIG 5

Identification of the sequence bound by ACE4 in the cbh1 promoter region. (A) Identification of the ACE4-protected cis elements in the FAM-labeled cbh1-D fragment in the DNase I footprinting assay. The underline represents the D9 fragment sequence. (B to E) The binding activity of GST-ACE4 to different cbh1 promoter fragments is indicated. Several biotin-labeled DNA fragments (Table S2 and Table S3) (∼10 nM) of the cbh1 promoter were incubated with the GST tag-fused Zn(II)₂Cys₆ domain of ACE4 expressed in E. coli and purified by GST affinity chromatography. For each EMSA, 10 nM biotin-labeled fragment and 0 to 0.4 μM (with the direction of increase pointing to the right, as indicated by the triangle) recombinant GST-ACE4 were added. ACE4-DNA complexes are indicated with an arrow. These results are representative of three experiments with similar results. (F) Alignment of fragments of the D9A-M sequence. The putative ACE4 binding sites are highlighted in yellow. The mutations contained in fragments are highlighted in red. The dashed lines indicate the splicing sites of the gels.

FIG 6

ACE4 binds to promoters in vivo. (A) Schematic representation of the localization of 5′-GGCC-3′ motifs (indicated with a diamond) within the 2.5-kb upstream regions of the cbh1, cbh2, ace3, and xyr1 genes. The motifs analyzed in the electrophoretic mobility shift assay (Fig. 4 and 7) are indicated with asterisks. The promoter regions for ChIP analysis are shown as CBH1-a/b/c, ACE3-a/b/c, CBH2-a, and XYR1-a (bottom lines). (B) Flag-tagged ACE4 is generated in the Flag-ACE4 strain. Expression was confirmed via Western blot analysis. Parental strain QM6a was used as a negative control. Total protein (30 μg) was loaded into each lane, and β-actin expression was used as a positive reference. (C) The ChIP assays were performed with Flag-ACE4 cells grown in Avicel. Immunoprecipitation was conducted using anti-Flag antibody. Relative IP levels were normalized to the IP/input ratio at the promoter regions of cellulase genes relative to the IP/input at the P_ACT1 region, which was used as a reference. Ratios higher than 1 were considered to be DNA enrichment due to ACE4 binding. The error bars represent the standard deviation among the three biological replicates. Asterisks indicate significant differences in IP/input ratio at the promoter regions of cellulase gene groups relative to the IP/input ratio at P_ACT1 groups in the corresponding samples (*, P < 0.05, Student's t test).

FIG 7

ACE4 activates the expression of ACE3. (A) EMSAs of ACE4 binding to ace3 promoter regions (P_ace3: −297 to −1). For each EMSA, 10 nM probe and 0 to 2.0 μM (with the direction of increase pointing toward the right, as indicated by the triangle) recombinant GST-ACE4 were added. ACE4-DNA complexes are indicated with an arrow. (B) ACE3 expression in the 6aFlag-ACE3 and OEace4-6aFlag-ACE3 mutants. Extracts prepared from each mutant were immunoblotted with anti-Flag and anti-β-actin antibodies. The intensities of Flag-ACE3 were measured and normalized to the actin level. Values are means ± SD of the results from three independent experiments. Asterisks indicate significant differences compared with the parental strain 6aFlag-ACE3 (*, P < 0.05, Student's t test).