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

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. Keywords: ChIP-chip Chromatin-immunoprecipitation (ChIP) was performed using one of two different affinity purified antibodies (α-Wor1Nterm or α-Wor1Cterm ), raised against a peptides near the N- and C- termini of Wor1. Precipitated DNA was amplified, fluorescently labeled, and competitively hybridized against genomic DNA (input DNA) on custom DNA tiling microarrays containing 60-mer oligonucleotides tiled at 80bp intervals across the entire C. albicans genome (ChIP-chip). For each antibody, two microarrays were hybridized with DNA from two separate IPs of an opaque wild type a strain; a single ChIP was performed in a wor1Delete/wor1Delete (white) a strain as a control for the N-terminal antibody experiments and two ChIPs were performed in the deletion strain for the C-terminal antibody experiments.