Two Adjacent Protein Binding Motifs in the cbh2(Cellobiohydrolase II-encoding) Promoter of the Fungus Hypocrea jecorina (Trichoderma reesei) Cooperate in the Induction by Cellulose

The cellulase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) consists of several cellobiohydrolases, endoglucanases, and beta-glucosidases, encoded by separate genes, which are coordinately expressed in the presence of cellulose or the disaccharide sophorose. Using cell-free extracts from sophorose-induced and noninduced mycelia and various fragments of the cbh2 promoter of H. jecorina in electrophoretic mobility shift assay (EMSA) analysis and performing in vitro and in vivo footprinting analysis, we detected the nucleotide sequence 5'-ATTGGGTAATA-3' (consequently named cbh2-activating element (CAE)) to bind a protein complex with different migration in EMSA of induced and noninduced cell-free extracts. EMSA analysis, employing oligonucleotide fragments containing specifically mutated versions of CAE, revealed that protein binding requires the presence of an intact copy of either one of two adjacent motifs: a CCAAT (=ATTGG) box on the template strand and a GTAATA box on the coding strand, whereas a simultaneous mutation in both completely abolished binding. H. jecorina transformants, containing correspondingly mutated versions of the cbh2 promoter fused to the Escherichia coli hph gene as a reporter, expressed hph in a manner paralleling the efficacy of CAE-protein complex formation in EMSA, suggesting that the presence of either of both motifs is required for induction of cbh2 gene transcription. Antibody supershift experiments with anti-HapC antiserum as well as EMSA competition experiments with CCAAT binding promoter fragments of the Aspergillus nidulans amdS promoter suggest that the H. jecorina CCAAT box binding complex contains a homologue of HapC. The nature of the adjacent, GTAATA-binding protein(s) and its cooperation with the HapC homologue in cbh2 gene induction is discussed.