The Aspergillus nidulans multimeric CCAAT binding complex AnCF is negatively autoregulated via its hapB subunit gene

Cis-acting CCAAT elements are frequently found in eukaryotic promoter regio ns. Many of them are bound by conserved multimeric complexes. In the fungus Aspergillus nidulans the respective complex was designated AnCF (A, nidula ns CCAAT binding factor). AnCF is composed of at least three subunits desig nated HapB, HapC and HapE. Here, we show that the promoter regions of the h apB genes in both A. nidulans and Aspergillus oryzae contain two inversely oriented, conserved CCAAT boxes (box a and box beta). Electrophoretic mobil ity shift assays (EMSAs) using both nuclear extracts and the purified, reco nstituted AnCF complex indicated that AnCF binding in vitro to these boxes occurs in a non-mutually exclusive manner. Western and Northern blot analys es showed that steady-state levels of HapB protein as well as hapB mRNA wer e elevated in hapC and hapE deletion mutants, suggesting a repressing effec t of AnCF on hapB expression. Consistently, in a hapB deletion background t he hapB-lacZ expression level was elevated compared with the expression in the wild-type. This was further supported by overexpression of hapB using a n inducible alcA-hapB construct. Induction of alcA-hapB expression strongly repressed the expression of a hapB-lacZ gene fusion. However, mutagenesis of box beta led to a fivefold reduced expression of a hapB-lacZ gene fusion compared with the expression derived from a wild-type hapB-lacZ fusion. Th ese results indicate that (i) box beta is an important positive cis-acting element in hapB regulation, (ii) AnCF does not represent the corresponding positive trans-acting factor and (iii) that AnCF is involved in repression of hapB. (C) 2001 Academic Press.