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 switch is controlled by a network of eight transcription factors (TFs). These include Ssn6, a negative regulator of the opaque state that can function with its co-repressor Tup1, and together these 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 TPR (tetratricopeptide repeat) 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 while 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 observations reveal how individual Ssn6 domains can modulate the phase separation properties of DNA-binding TFs and thereby regulate gene expression and cell fate. Overall design: Exploration of global gene expression changes in Candida albicans where structured and unstructured domains of SSN6 have been deleted.