The yeast Cryptococcus neoformans uses 'mammalian' enhancer sites in the regulation of the virulence gene, CNLAC1

Transcriptional regulation in mammalian and plant cells is distinguished fr om fungi by the presence of blocks of multiple interacting DNA binding site s distributed over a relatively large upstream region of genes and the abil ity to use glutamine-rich enhancers such as Spl. We offer evidence that the haploid yeast Cryptococcus neoformans contains a virulence gene, CNLAC1, h aving regulatory properties more similar to mammalian systems than to that of yeast. We used a novel promoter plasmid, pVEW, and electromobility shift assay techniques adapted for the fungus for the first systematic structura l and functional study of a 5'-enhancer region of a basidiomycete fungus us ing the upstream region of CNLAC1. Two groups of interactive enhancer regio ns, located over a range of 1.5 kb from the mRNA start site are involved in CNLAC1 regulation (region 2: -1721 to -1615 and region 7) in addition to a TATA promoter at position -539. Region 2 contains a consensus Spl site and region 7 contains a consensus E2F site, each of which shows significant bi nding to nuclear proteins under derepressed conditions; cooperative binding was also suggested between DNA-binding protein of these sites and those bi nding nearby CCAAT sequences in each region. Two regions of repression were also evident under derepressed conditions (region 5: -1351 to -1207 and re gion 8: -991 to -971). Identification of functional Spl binding sites and t he presence of multiple interactive enhancer sites over a fairly large upst ream range suggests that cryptococcal transcriptional regulation contains f eatures often associated with higher eukaryotic regulation. C. neoformans t hus may provide a unique system for the study of certain aspects of higher eukaryotic transcription, using yeast genetic approaches. In addition, prop erties of basidiomycete yeast such as Cryptococcus exemplified in the prese nt study suggest an evolutionary progression in gene regulation within fung i toward properties exhibited in the kingdoms Animalia and Plantae. (C) 199 9 Elsevier Science B.V. All rights reserved.