Distinct class of DNA-binding domains is exemplified by a master regulator of phenotypic switching in Candida albicans.

Among the most important classes of regulatory proteins are the sequence-specific DNA-binding proteins that control transcription through the occupancy of discrete DNA sequences within genomes. Currently, this class of proteins encompasses at least 37 distinct structural superfamilies and more than 100 distinct structural motifs. In this paper, we examine the transcriptional regulator Wor1, a master regulator of white-opaque switching in the human fungal pathogen Candida albicans. As assessed by a variety of algorithms, this protein has no sequence or structural similarity to any known DNA-binding protein. It is, however, conserved across the vast fungal lineage, with a 300aa region of sequence conservation. Here, we show that this 300aa region of Wor1 exhibits sequence-specific DNA binding and therefore represents a new superfamily of DNA-binding proteins. We identify the 14-nucleotide-pair DNA sequence recognized by Wor1, characterize the site through mutational analysis, and demonstrate that this sequence is sufficient for the Wor1-dependent activation of transcription in vivo. Within the 300aa DNA-binding conserved region, which we have termed the WOPR box, are two domains (WOPRa and WOPRb), dissimilar to each other but especially well-conserved across the fungal lineage. We show that the WOPR box binds DNA as a monomer and that neither domain, when expressed and purified separately, exhibits sequence-specific binding. DNA binding is restored, however, when the two isolated domains are added together. These results indicate that the WOPR family of DNA-binding proteins involves an unusual coupling between two dissimilar, covalently linked domains.

A conserved transcriptional regulator governs fungal morphology in widely diverged species.

Fungi exhibit a large variety of morphological forms. Here, we examine the functions of a deeply conserved regulator of morphology in three fungal species: Saccharomyces cerevisiae, Candida albicans, and Histoplasma capsulatum. We show that, despite an estimated 600 million years since those species diverged from a common ancestor, Wor1 in C. albicans, Ryp1 in H. capsulatum, and Mit1 in S. cerevisiae are transcriptional regulators that recognize the same DNA sequence. Previous work established that Wor1 regulates white-opaque switching in C. albicans and that its ortholog Ryp1 regulates the yeast to mycelial transition in H. capsulatum. Here we show that the ortholog Mit1 in S. cerevisiae is also a master regulator of a morphological transition, in this case pseudohyphal growth. Full-genome chromatin immunoprecipitation experiments show that Mit1 binds to the control regions of the previously known regulators of pseudohyphal growth as well as those of many additional genes. Through a comparison of binding sites for Mit1 in S. cerevisiae, Wor1 in C. albicans, and Wor1 ectopically expressed in S. cerevisiae, we conclude that the genes controlled by the orthologous regulators overlap only slightly between these two species despite the fact that the DNA binding specificity of the regulators has remained largely unchanged. We suggest that the ancestral Wor1/Mit1/Ryp1 protein controlled aspects of cell morphology and that movement of genes in and out of the Wor1/Mit1/Ryp1 regulon is responsible, in part, for the differences of morphological forms among these species.