Interlocking Transcriptional Feedback Loops Control White-Opaque Switching in Candida albicans

Abstract

The human pathogen Candida albicans can assume either of two distinct cell types, designated “white” and “opaque.” Each cell type is maintained for many generations; switching between them is rare and stochastic, and occurs without any known changes in the nucleotide sequence of the genome. The two cell types differ dramatically in cell shape, colony appearance, mating competence, and virulence properties. In this work, we investigate the transcriptional circuitry that specifies the two cell types and controls the switching between them. First, we identify two new transcriptional regulators of white-opaque switching, Czf1 and white-opaque regulator 2 (Wor2). Analysis of a large set of double mutants and ectopic expression strains revealed genetic relationships between CZF1, WOR2, and two previously identified regulators of white-opaque switching, WOR1 and EFG1. Using chromatin immunoprecipitation, we show that Wor1 binds the intergenic regions upstream of the genes encoding three additional transcriptional regulators of white-opaque switching (CZF1, EFG1, and WOR2), and also occupies the promoters of numerous white- and opaque-enriched genes. Based on these interactions, we have placed these four genes in a circuit controlling white-opaque switching whose topology is a network of positive feedback loops, with the master regulator gene WOR1 occupying a central position. Our observations indicate that a key role of the interlocking feedback loop network is to stably maintain each epigenetic state through many cell divisions. The opportunistic fungal pathogen Candida albicans can switch between two heritable states—the “white” and “opaque” states. These two cell types differ in many characteristics, including cell structure, mating competence, and virulence. Recent studies of the molecular mechanism of regulating the white-opaque switch identified a master transcriptional regulator, Wor1. In this study, we identified two transcriptional regulators, Czf1 and Wor2, as new regulators of white-opaque switching. By constructing a series of single and double mutants and by examining where the master regulator Wor1 binds throughout the genome, we generated a molecular model of the bistable switch that regulates white-opaque switching. The regulatory model consists of interlocking positive feedback loops, which mutually reinforce one another and stabilize the opaque state. These results show how an organism can exist in two distinctive, heritable states without changes in the nucleotide sequence of its genome.