Project Summary/Abstract Candida albicans is the number one cause of life-threatening fungal infections in hospitals. A key virulence attribute of C. albicans is the ability to switch between multiple distinct cellular forms that are adapted to unique environmental niches. The white-opaque phenotypic switch in C. albicans generates two distinct pathogenic cell types from a single genome, and each cell type is heritably maintained for hundreds of generations without any change in the underlying genomic sequence. Although heritable transcriptional programs underlie critical cell-fate decisions in organisms ranging from unicellular prokaryotes through multicellular higher eukaryotes, the underlying logic of these complex transcriptional circuits remains largely uncharacterized. The white-opaque switch has emerged as a powerful model system for understanding the transcriptional regulation of heritable cell-fate decisions in eukaryotes. Equivalent programs are not found in other model yeasts, like S. cerevisiae, making C. albicans an attractive ?simple? eukaryotic organism to study how cellular memory is regulated and inherited from one generation to the next. Many of the key transcriptional regulators have been identified, and the structures of the transcriptional networks that control switching have been elucidated. The high-dimensional interwoven structure of the opaque-specific regulatory circuit bears striking similarities to heritable transcriptional networks in higher eukaryotes, yet due to the complexity of these circuits, their logic remains elusive. This proposal seeks to uncover the logic of the transcriptional regulatory circuits that control heritable differentiation between the white and opaque cell types. Aim 1: Develop and validate a quantitative high-throughput assay for C. albicans white-opaque phenotypic switching. Using engineered fluorescent reporter strains and semi-automated flow cytometry, we will develop a high-throughput quantitative white-opaque switching assay. This assay will be verified for accuracy and robustness by analyzing a panel of previously-characterized switch regulatory mutants in a side- by-side comparison between our newly-developed assay and the traditional colony-plating assay. Aim 2: Determine the logic of the transcriptional circuits that control white and opaque cell-type heritability. Using CRISPR-mediated genome editing and high-throughput switch frequency analysis, we will systematically disrupt specific transcriptional regulatory interactions within the white and opaque transcriptional circuits and measure the resulting effects on switch dynamics. This data will be used to identify regulatory interactions that are critical for heritability and to model the logic of the white-opaque switch circuits. The completion of these aims will reveal the logic of the transcriptional circuits that control heritable cell-type differentiation in C. albicans. This work will also provide a paradigm for investigating heritable transcriptional circuits that control cell-fate decisions in higher eukaryotes.