Identification Of Transcriptional Network For GAL Regulon Induction In Candida Albicans And Study On Its Regulatory Mechanism

Galactose is a monosaccharide that is abundant in nature and is found in many forms.The GAL pathway of the budding yeast Saccharomyces cerevisiae is a favorite textbook example of the regulation of gene expression in eukaryotes.This pathway is controlled through the Gal4 activator,as well as a sensor(Gal3)and a repressor(Gal80).While Gal4 mode has been well-characterized in Saccharomycetaceae clade,we know unexpectedly little about the regulation of the GAL pathway in other budding yeasts.Candida albicans is the most prevalent commensal-pathogenic fungus in humans.Although the GAL regulon structure and the overall logic of regulation have been preserved in this fungus,there are significant differences in the quantitative response of these genes to galactose between S.cerevisiae and C.albicans.For example,the GAL genes induction is faster and more sensitive to galactose concentration in C.albicans compared to that of S.cerevisiae.These findings imply that significant differences in regulatory mechanism for the induction of GAL genes exist between the two model yeasts.Since the GAL pathway in budding yeasts has often served as a model for studying gene regulation and evolution in eukaryotes,understanding how the GAL genes is regulated in C.albicans will add significantly to the literature of transcription regulatory network for galactose utilization in fungi.Considerable efforts have been made to identify numerous transcription factors involved in the galactose-inducible expression of three GAL genes in C.albicans.Rtg1,Rtg3 and Cph1 have been reported,none of which,however,are necessary for galactose utilization in this fungus.The molecular mechanism whereby C.albicans senses galactose and subsequent signaling that activates the expression of GAL genes are far less clear.Here,we find Rep1,a Ndt80-like family transcriptional factor,is required for galactose signaling in C.albicans.Rep1 is presented at the GAL gene promoters independent of the presence of galactose.Furthermore,it has a predicted “galectin-like”domain,and recognizes galactose via a direct physical interaction.Disrupting the interaction between Rep1 and galactose by mutating the binding central Y526 to alanine results in a severe growth defect on galactose,indicating that Rep1 binding to galactose is essential for galactose utilization in C.albicans.Given that Rep1 does not itself possess a transcriptional activity,we hypothesize that Rep1-mediated transcriptional activation upon galactose binding should be mediated by its interaction partner(s).We therefore identified proteins that interact with Rep1 specifically in the presence of galactose by Mass Spectrometry.Among these potential binding partners,Orf19.4959 is essential for galactose signaling in C.albicans.We therefore designated it as Cga1(Candida galactose gene activator).Through in vitro and in vivo immunoprecipitation assays,we find that Rep1 directly interacts with Cga1 in the presence of galactose.As such,we identify the Rep1-Cga1 mode for the GAL regulon induction in C.albicans.Upon bingding to galactose,Rep1 recruits the transcriptional activator Cga1 to the promoter of GAL genes,and then initiation of transcription can occur.Next,we investigated the conservation of Rep1-Cga1 regulation model in fungi.By protein sequence alignment,we found that Rep1 and Cga1 had clear homologous proteins only in CTG clade fungi,indicating that the regulation mode of Rep1-Cga1 is conserved only in fungi closely related to C.albicans,and the regulatoty mode of galactose metabolism in other fungal species needs to be further clarified.Although the transcriptional activation domain of Rep1 is not essential for galactose signaling pathway in C.albicans,it is required for the recruitment of the Nacetylglucosamine sensor Ngs1 to activate transcription of Glc NAc catabolic genes.In addition,we identify the target genes of Rep1 by Ch IP-Seq,and found that Rep1 had a slight inhibitory effect on other potential target genes except galactose-metabolized genes and N-acetylglucosamine metabolized genes.These results indicate that Rep1 acts as a scaffolding protein to recruit different transcriptional regulatory factors under different environmental or developmental signals for transcriptional regulation of downstream genes.In summary,we have identified Rep1-Cga1 mode for the GAL regulon induction in C.albicans,which represents a new example of network rewiring in fungi and provides insight into how C.albicans evolves transcriptional programs to colonize diverse host niches.