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.

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