AcFKH1, a novel member of the forkhead family, associates with the RFX transcription factor CPCR1 in the cephalosporin C-producing fungus Acremonium chrysogenum

Abstract

In the filamentous fungus Acremonium chrysogenum, a complex regulatory network of transcription factors controls the expression of at least seven cephalosporin C biosynthesis genes. The RFX transcription factor CPCR1 binds to regulatory sequences in the promoter region of cephalosporin C biosynthesis genes, and is involved in the transcriptional regulation of the pcbC gene which encodes isopenicillin N synthase. In this study, we used CPCR1 in a yeast two-hybrid screen to identify potential protein interaction partners. A cDNA was identified, encoding the C-terminal part (pos. 438-665) of the novel forkhead protein, AcFKH1. The full-length AcFKH1 amino acid sequence is 665 residues and shares between 31% and 60% identity with forkhead protein sequences in the genomes of Aspergillus nidulans, Fusarium graminearum, and Neurospora crassa. AcFKH1 is characterized by two conserved domains, the N-terminal forkhead-associated domain (FHA), which might be involved in phospho-protein interactions, and the C-terminal DNA-binding domain (FKH) of the winged helix/forkhead type. The two-hybrid system was also used to map the protein domains required for the interaction of transcription factors CPCR1 and AcFKH1. The observed interaction between CPCR1 and the C-terminus of AcFKH1 in the yeast system was verified in vitro in a GST pulldown assay. Using gel retardation analysis, the DNA-binding properties of the fungal forkhead protein AcFKH1 were investigated. AcFKH1 recognizes two forkhead consensus binding sites within the 1.2 kb promoter region of the divergently oriented cephalosporin biosynthesis gene pair pcbAB-pcbC from A. chrysogenum. Additionally, AcFKH1 is able to bind with high affinity to the SWI5-binding site of the yeast FKH2 protein.