The transcriptional repressor Ssn6 modulates phase separation to regulate fungal gene expression

The transcriptional regulation of cell fate plays a central role in eukaryotic cell differentiation. In the human fungal pathogen Candida albicans, the white-to-opaque cell fate switch is controlled by an interconnected network of eight transcription factors (TFs). These include Ssn6, a negative regulator of the opaque state that can function with its co-repressor Tup1; together, they form a global repressor complex that is conserved from yeast to mammals. Here, we evaluated the roles of four Ssn6 domains (N, TPR, M, and CTD) in white-opaque switching. Loss of the prion-like N or M domains had limited effects on Ssn6 phenotypes. In contrast, the tetratricopeptide repeat (TPR) domain was critical for function, consistent with this domain mediating interactions with Tup1 and DNA-binding TFs. The intrinsically disordered C-terminal domain (CTD) showed complex roles; deletion of this domain increased Ssn6 activity whereas substitution of acidic residues within this region abolished Ssn6 function. Notably, these phenotypes were linked to the phase separation capacity of Ssn6, as changes to the TPR or CTD altered the properties of Ssn6 condensates in human U2OS cells. Experiments using purified Ssn6 (± Tup1) demonstrated that this protein is recruited into condensates formed by other white-opaque-regulating TFs and alters condensate properties in a TPR- and CTD-dependent manner. Together, these experiments reveal how individual Ssn6 domains can modulate the phase separation properties of DNA-binding TFs and thereby regulate gene expression and cell fate.IMPORTANCEThe mechanisms by which transcription factors (TFs) and co-regulators control gene expression remain ill-defined. An interconnected network of TFs regulates an epigenetic switch between white and opaque states in Candida albicans and serves as a model to understand the transcriptional regulation of eukaryotic cell fate. Here, we examine the role of Ssn6, one of the eight core regulators of the white-opaque switch, and reveal key roles for both the structured TPR domain and the disordered C-terminal domain. We demonstrate that Ssn6 is readily incorporated into transcriptional condensates where it disrupts the liquid-like properties of these condensates, both in the presence and absence of the co-repressor Tup1. Together with studies in higher eukaryotes, these results suggest a conserved role for TPR-containing proteins in regulating gene expression via the modulation of the physical properties of transcriptional condensates.