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.

Crystal structure of the WOPR-DNA complex and implications for Wor1 function in white-opaque switching of Candida albicans

“…The structural and biochemical data together indicate that the WOPR domain can recognize and bind a dsDNA containing the characteristic motif TAAACT with a strictly conserved T at position 1 and a highly conserved T at position 6 but some variations at the other positions with base preferences, which are largely in agreement with the biochemical results by Lohse et al [23]. This finding may explain why there are so many different CaWor1-binding sites (nearly 200 sites) in the C. albicans genome [15]. Based on these results, we predict the specific binding sites of the other four DNA fragments as TAAGGT for the MDR1 promoter, TAAAGT for the orf19.4394 promoter site 1, TAAAAA for the WOR1 promoter site 1, and TAGAGT for the WOR1 promoter site 3 (Supplementary information, Figure S2D).…”

“…Crystallization experiments of CaWor1 WOPR in complex with dsDNA of different lengths (13,15,17, and 20 bp) all yielded crystals with yet varied diffraction qualities. Finally, the crystal structure of CaWor1 WOPR in complex with a 17-bp dsDNA (5′-AAGT-TAAACTTTTTTGA-3′) (WOPR-17bp dsDNA) was determined to 3.0 Å resolution, and the crystal structure of CaWor1 WOPR in complex with a 13-bp dsDNA (5′-AAGTTAAACTTTT-3′) (WOPR-13bp dsDNA) to 2.1 Å resolution (Table 1).…”

“…The single-stranded oligonucleotides of different lengths (13,15,17, and 20 bases) corresponding to the template strands and their complementary strands were synthesized by Sangon Biotech (Shanghai). The dsDNAs were prepared by annealing of the template and complementary strands from 95 °C to 22 °C over a period of 6 h in the same buffer as for the protein.…”

“…White-opaque switching is controlled through expression of a master regulator, Wor1 (whiteopaque switching regulator 1), which is highly upregulated in opaque cells and is required for both the transition to and maintenance of the opaque cell type [10][11][12][13]. Wor1 can genetically interact with the other five key regulators, forming a network of positive and negative feedback loops to control white-opaque switching [14,15]. Recently, Wor1 is also found to regulate a phenotypic switch that promotes commensalism when passing through the mammalian gut in MTLa/α cell types [2].…”

“…As a transcriptional regulator, Wor1 can bind to more than 100 target promoters as well as its own upstream region (nearly 200 sites in the C. albicans genome) [15]. CaWor1 is a transcription regulator of 785 residues, consisting of a conserved N-terminal region (residues 1-325) termed WOPR box and a non-conserved and functionally and structurally undefined C-terminal region [23].…”