The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element

The 5 ' regulatory region of the cbh2 gene, encoding cellobiohydrolase II, of the filamentous fungus Hypocrea jecorina contains the cbh2 activating el ement (CAE) which is essential for cbh2 expression. The CAE consists of two separate, adjacent motifs, a CCAAT box on the template strand (ATTGG) and a GTAATA box on the coding strand, which co-operate in the induction of the gene by cellulose or sophorose. EMSA supershift experiments using an antib ody against Aspergillus nidulans HAPC suggested that the complex which bind s to the H. jecorina CCAAT box contains a HAPC homolog. To obtain direct ev idence for this, we have cloned the hap2, hap3 and hap5 genes from H. jecor ina. They encode proteins whose core regions display great similarity to As pergillus HAPB, HAPC and RAPE and to known HAP homologs from other organism s. All three genes are transcribed in a carbon source-independent manner. A . nidulans Delta hap strains were functionally complemented in vitro by the overexpressed H. jecorina HAP2, HAP3 and HAP5 proteins, and they thus repr esent subunits of the CCAAT-binding complex. Furthermore. all three protein s (HAP2, HAP3 and HAP5) were needed to bind to the CAE in the H. jecorina c bh2 gene promoter in vitro. We conclude that the CCAAT box on the template strand in CAE is bound by the H. jecorina equivalent of the HAP protein com plex.