AnCF, the CCAAT Binding Complex of Aspergillus nidulans, Contains Products of the hapB,hapC, and hapE Genes and Is Required for Activation by the Pathway-Specific Regulatory Gene amdR

Abstract

CCAAT binding factors (CBFs) positively regulating the expression of the amdS gene (encoding acetamidase) and two penicillin biosynthesis genes (ipnA and aatA) have been previously found in Aspergillus nidulans. The factors were called AnCF and PENR1, respectively. Deletion of the hapCgene, encoding a protein with significant similarity to Hap3p of Saccharomyces cerevisiae, eliminated both AnCF and PENR1 binding activities. We now report the isolation of the genes hapB and hapE, which encode proteins with central regions of high similarity to Hap2p and Hap5p of S. cerevisiae and to the CBF-B and CBF-C proteins of mammals. An additional fungus-specific domain present in HapE was revealed by comparisons with the homologs from S. cerevisiae,Neurospora crassa, and Schizosaccharomyces pombe. The HapB, HapC, and HapE proteins have been shown to be necessary and sufficient for the formation of a CCAAT binding complex in vitro. Strains with deletions of each of the hapB,hapC, and hapE genes have identical phenotypes of slow growth, poor conidiation, and reduced expression of amdS. Furthermore, induction of amdS by omega amino acids, which is mediated by the AmdR pathway-specific activator, is abolished in the hap deletion mutants, as is growth on γ-aminobutyric acid as a sole nitrogen or carbon source. AmdR and AnCF bind to overlapping sites in the promoters of the amdSand gatA genes. It is known that AnCF can bind independently of AmdR. We suggest that AnCF binding is required for AmdR binding in vivo.

ACKNOWLEDGMENTS

We gratefully acknowledge Norihiro Tsukagoshi and Masashi Kato for providing the expression plasmids pMalE-HapC and pMalE-HapE and Jack Kinsey for plasmid pCAAB1-1.5.

This work was supported by the Australian Research Council, by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 369), and by a DAAD Doktorandenstipendium im Rahmen des gemeinsamen Hochschulsonderprogramms III von Bund und Laendern granted to S.S.