Cell identity in eukaryotes is controlled by transcriptional regulatory networks that define cell-type-specific gene expression. In the opportunistic fungal pathogen Candida albicans, transcriptional regulatory networks regulate epigenetic switching between two alternative cell states, ‘white’ and ‘opaque’, that exhibit distinct host interactions. In the present study, we reveal that the transcription factors (TFs) regulating cell identity contain prion-like domains (PrLDs) that enable liquid–liquid demixing and the formation of phase-separated condensates. Multiple white–opaque TFs can co-assemble into complex condensates as observed on single DNA molecules. Moreover, heterotypic interactions between PrLDs support the assembly of multifactorial condensates at a synthetic locus within live eukaryotic cells. Mutation of the Wor1 TF revealed that substitution of acidic residues in the PrLD blocked its ability to phase separate and co-recruit other TFs in live cells, as well as its function in C. albicans cell fate determination. Together, these studies reveal that PrLDs support the assembly of TF complexes that control fungal cell identity and highlight parallels with the ‘super-enhancers’ that regulate mammalian cell fate.
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Multifactor transcriptional control of alternative oxidase induction integrates diverse environmental inputs to enable fungal virulence
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We thank R. Tjian for the gift of reporter cell lines, S. Sandler and J. Morschhauser for plasmids, L. Brossay for help with tissue culture, G. Williams for help with confocal microscopy, and members of the Bennett laboratory for helpful discussions. This work is supported by the National Institute of Allergy and Infectious Disease (grant nos. AI081704, AI135228 and AI141893 to R.J.B. and AI137975 to A.D.H.), the Burroughs Wellcome Fund (PATH award to R.J.B.), the National Heart, Lung and Blood Institute (grant no. T32HL134625 to M.I.S.), the National Institute of Dental and Craniofacial Research (grant no. F31DE02968001 to M.I.S.), the National Institute of Mental Health (grant no. T32MH020068 to V.H.R.), the National Institute of Neurological Disorders and Stroke (grant no. F31NS110301 to V.H.R.), a Howard Hughes Medical Institute International Student Fellowship (to Y.K.), the National Institute of General Medical Sciences (grant nos. GM120554 to I.J.F. and GM118530 to N.L.F.), the Welch Foundation (grant no. F-1808 to I.J.F.) and the National Science Foundation (grant nos. 1453358 to I.J.F. and 1845734 to N.L.F.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Top, ChIP-chip enrichment peaks shown for Wor1 (orange), Wor2 (pink), Wor3 (blue), Czf1 (green), Efg1 (purple) and Ahr1 (red). Solid lines indicate TF binding and dotted lines indicate controls. ORFs are represented by purple boxes and lighter purple boxes represent untranslated regions. Bottom, Positions of consensus DNA binding sites for each TF. The large circles represent motif hits with >75% of the maximum score, medium circles represent motif hits that have 50–75% of the maximum score, and small circles represent motif hits that have 25–50% of the maximum score. ChIP enrichment plot generated from data in refs. 27,30,36 and motif analysis performed using data from refs. 27,30.
a, Schematic of TF expression constructs, including 6x histidine tag, MBP, and TEV protease site. b, Purified proteins used in this study. SDS-PAGE gels of C. albicans Wor1, Efg1, Czf1 and Wor4 HIS6-MBP-TF fusion proteins, as well as proteins with different PrLD deletions and those where the DBD has been replaced with GFP. c, Image of a HIS6-MBP-Efg1 protein solution (30 μM) without (left) and with (right) the addition of TEV protease for 30 min at 22 °C. Cloudiness indicates formation of phase-separated condensates, as confirmed by microscopy. Protein droplets formed in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl at 22 °C. Scale bar; 5 μm. Representative data for an experiment repeated more than three times with similar results.
Extended Data Fig. 3 Hexanediol treatment selectively disrupts C. albicans TF condensates even during co-compartmentalization with other TFs.
a, Images of Efg1, Czf1, Wor1 (CaCmWor1), and Wor4 droplets at the indicated concentrations with or without 10% 1,6- or 2,5-hexanediol. For hexanediol treatment, proteins were incubated with TEV for 30 minutes in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, at 22 °C, and then mixed with 1,6- or 2,5-hexanediol in the same buffer, incubated for 10 minutes, and imaged. Wor1, Wor4, and Czf1 assays also included 5% PEG-8000. Where indicated for Wor4, hexanediol was added for 10 minutes and then TEV/PEG-8000 added and the sample incubated for an additional 30 minutes prior to imaging. Images represent a single experimental replicate with assays repeated at least twice with similar results. Scale bars; 10 μm. b, Representative images of fluorescently labeled Efg1, Wor1 (CaWor1), Wor4, and Czf1 proteins compartmentalized within Efg1 condensates, and treated with 10% 1,6- or 2,5-hexanediol. Unlabeled bulk protein (15 μM) was mixed with each of the fluorescently labeled proteins (37.5 nM) in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl. Proteins were then incubated at 22 °C with TEV for 30 minutes and treated with 1,6- or 2,5-hexanediol in the same buffer for 10 minutes prior to imaging. Dylight NHS-Ester labeling of the 4 proteins used fluors of 405, 488, 550 and 633 nm. Images represent a single experimental replicate with assays performed three times with similar results. Scale bar, 10 μm; images are maximum Z-stack projections. c, Representative images of fluorescently labeled Efg1, Wor1 (CaWor1), Wor4, and Czf1 proteins compartmentalized within Czf1, Wor1(CaCmWor1), or Wor4 condensates. Unlabeled bulk proteins (15 μM) were mixed with each of the fluorescently labeled proteins (37.5 nM) in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl. Proteins were then incubated at 22 °C with TEV for 30 min. Dylight NHS-Ester labeling of the 4 proteins used fluors of 488, 550, 405, and 633 nm. Images represent a single experimental replicate, with assays performed three times with similar results. Scale bars, 10 μm; images are maximum Z-stack projections.
Analysis of the ability of full-length or truncated TFs to co-partition within Efg1 condensates. a, Schematics of the GFP fusion proteins tested in phase separation assays. b, Efg1-GFP, Wor4-GFP, Czf1-GFP or Wor1-GFP variants were evaluated for their ability to co-partition with unlabeled Efg1 droplets. For each protein, the DBD was replaced with GFP. In all assays, proteins were incubated with TEV for 30 min at 22 °C in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl. Bulk (full-length) Efg1 was present at 30 μM with 3 μM TF-GFP fusion proteins included in each reaction. Box and whisker plots show all data points, maximum to minimum, and indicate enrichment ratios for each TF-GFP fusion protein with condensates formed by full-length Efg1. For each plot, data are median (line), mean (‘+’), 25–75th percentiles (box), and 5–95th percentiles (whiskers). Droplets were located in the DIC channel, and the intensity for the GFP signal inside the droplet compared to the signal intensity outside the droplet, following subtraction of fluorescence background. At least five images were used for quantification, with 25 total droplets measured for each construct. Statistical significance was performed using a two-tailed Mann-Whitney U-test; P-values: a, < 0.0001; ns, not significant. Scale bars; 5 μm.
Excel file of quantitative DNA curtain data.
Excel file of phenotypical switching in Candida data.
Excel file of quantitative analysis of microscopy data.
Excel file of quantitative analysis of microscopy data.
Excel file of quantitative analysis of microscopy data for Extended Data Fig. 4.
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Frazer, C., Staples, M.I., Kim, Y. et al. Epigenetic cell fate in Candida albicans is controlled by transcription factor condensates acting at super-enhancer-like elements. Nat Microbiol 5, 1374–1389 (2020). https://doi.org/10.1038/s41564-020-0760-7
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