Recognition of galactose by a scaffold protein recruits a transcriptional activator for the GAL regulon induction in Candida albicans
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This important manuscript investigates the circuitry connecting the galactose utilization regulon of the human pathogen and model organism Candida albicans to the sensing of galactose. In the non-pathogenic model yeast Saccharomyces cerevisiae this circuit represents a textbook model that rivals the lac operon as a teaching tool. Using a broad array of mainly classical approaches, this study convincingly demonstrates the transcriptional activators that are required for galactose (and GlcNAc) responsive galactose metabolic genes in C. albicans. The recognition of just how different the regulation of the galactose pathway across fungal species represents an important advance in our understanding of the evolution of the regulatory control of these circuits, and would make a nice addition to the textbook version of eukaryotic gene regulation.
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Abstract
The GAL pathway of yeasts has long served as a model system for understanding of how regulatory mode of eukaryotic metabolic pathways evolves. While Gal4 mode has been well-characterized in Saccharomycetaceae clade, little is known about the regulation of the GAL pathway in other yeasts. Here, we find that Rep1, a Ndt80-like family transcription factor, serves as a galactose sensor in the commensal-pathogenic fungus Candida albicans . It is presented at the GAL gene promoters independent of the presence of galactose. Rep1 recognizes galactose via a direct physical interaction. The net result of this interaction is the recruitment of a transcriptional activator Cga1 (Candida galactose gene activator, orf19.4959) and transcription of the GAL genes proceeds. Rep1 and Cga1 are conserved across the CTG species. Rep1 itself does not possess transcriptional activity. Instead, it provides a scaffold to recruit different factors for transcriptional regulation. Rep1-Cga1 mode of regulation represents a new example of network rewiring in fungi, which provides insight into how C. albicans evolves transcriptional programs to colonize diverse host niches.
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eLife assessment
This important manuscript investigates the circuitry connecting the galactose utilization regulon of the human pathogen and model organism Candida albicans to the sensing of galactose. In the non-pathogenic model yeast Saccharomyces cerevisiae this circuit represents a textbook model that rivals the lac operon as a teaching tool. Using a broad array of mainly classical approaches, this study convincingly demonstrates the transcriptional activators that are required for galactose (and GlcNAc) responsive galactose metabolic genes in C. albicans. The recognition of just how different the regulation of the galactose pathway across fungal species represents an important advance in our understanding of the evolution of the regulatory control of these circuits, and would make a nice addition to the textbook version of …
eLife assessment
This important manuscript investigates the circuitry connecting the galactose utilization regulon of the human pathogen and model organism Candida albicans to the sensing of galactose. In the non-pathogenic model yeast Saccharomyces cerevisiae this circuit represents a textbook model that rivals the lac operon as a teaching tool. Using a broad array of mainly classical approaches, this study convincingly demonstrates the transcriptional activators that are required for galactose (and GlcNAc) responsive galactose metabolic genes in C. albicans. The recognition of just how different the regulation of the galactose pathway across fungal species represents an important advance in our understanding of the evolution of the regulatory control of these circuits, and would make a nice addition to the textbook version of eukaryotic gene regulation.
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Reviewer #1 (Public Review):
In 2007 it was observed that, although the central elements of galactose utilization are similar in both S. cerevisiae and C. albicans (clustered metabolic genes, transcriptional induction in the presence of galactose) the induction mechanisms were different. Until now, however, although the way the presence of galactose was sensed and this information transmitted to the induction of gene expression was well understood in S. cerevisiae, it was quite mysterious in C. albicans. This work proposes that in C. albicans, the general transcription regulator Rep1 serves as a direct galactose binding protein and that the binding of galactose to Rep1 allows it to serve as a scaffold to collect the transcriptional machinery necessary to induce the elements of the Gal regulon.
The first line of evidence for the Rep1 …
Reviewer #1 (Public Review):
In 2007 it was observed that, although the central elements of galactose utilization are similar in both S. cerevisiae and C. albicans (clustered metabolic genes, transcriptional induction in the presence of galactose) the induction mechanisms were different. Until now, however, although the way the presence of galactose was sensed and this information transmitted to the induction of gene expression was well understood in S. cerevisiae, it was quite mysterious in C. albicans. This work proposes that in C. albicans, the general transcription regulator Rep1 serves as a direct galactose binding protein and that the binding of galactose to Rep1 allows it to serve as a scaffold to collect the transcriptional machinery necessary to induce the elements of the Gal regulon.
The first line of evidence for the Rep1 scaffold model is the observation that Rep1 is needed for C. albicans to both grow on galactose and to induce the genes encoding the galactose processing proteins Gal1 and Gal10. Previous candidate regulators Rtg1 and Rtg3 only blocked growth on galactose in the presence of Antimycin A, so Rep1 represents a first element specifically required for galactose growth. Further analysis of Rep1 function involved the observation that Rep1 was a member of the family of transcription factors including Ntd80, a TF that has been implicated in a variety of cellular controls. The authors investigated a specific unique domain of Rep1 by moving it to Ndt80 - the fusion protein did not allow complementation of the galactose growth defect, suggesting this domain was not critical to the Rep1 involvement in galactose growth. Further analysis of Rep1 domains by deletions showed that removal of the putative transcriptional activation domain of the protein also did not block either growth on galactose medium or galactose-mediated induction of GAL1 and GAL10 expression. The Rep1 protein was found to be constitutively bound to the promoters of GAL1 and GAL10, and not really influenced in this binding by carbon source.
To attempt to determine the connection between the apparent constitutive binding and the galactose-mediated induction of gene expression the authors investigated the relationship between sugars and the Rep1 protein. Modelling suggested a possible galactose binding pocket, binding was shown biochemically, and mutations within the presumed binding site disrupted galactose binding and protein function.
The authors next assess how the binding of galactose to Rep1 leads to gene induction because the binding to the regulated promoters seems constitutive, and the activation domain seems unimportant for protein function, and in fact, doesn't act as an activation domain in a 1 hybrid assay. They speculate protein binding and search for interacting proteins by mass spec after IP with a tagged Rep1 protein in the presence of galactose. Orf19.4959 is identified and tested. The binding data is presented as a supplementary table and includes many hits that do not appear promising candidates. Inactivation of the TF Orf19.4959 blocks growth on galactose and induction of the GAL1 and GAL10 genes, and the protein, called Cga1, does have transactivating ability in a 1 hybrid assay. The authors thus propose that galactose binding to Rep1 facilitates the binding of Cga1 and leads to the activation of gene expression for galactose metabolism.
This model is tested by immunoprecipitation assays that showed Cga1-Rep1 interaction only in the presence of galactose, and that DNA association of Cga1 to GAL promoters was galactose and Rep1 dependent. Further experiments provide a framework for Rep1 function in other pathways and suggest a candidate polyA binding motif for the Rep1 protein. The generalization of the model is proposed by noting a pattern of Rep1/Cga1 presence in other fungal species.
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Reviewer #2 (Public Review):
Despite previous studies, regulation of the genes required for galactose metabolism in Candida albicans has remained murky. For example, previous work had highlighted Rtg1 and Rtg3 as the key regulator components, an interesting finding given that these factors are important for glucose not galactose regulation in S. cerevisiae. As galactose metabolism is one of the best-understood regulatory systems, the evolutionary difference in the regulation of the galactose response has the potential to teach us about regulatory evolution in general.
The authors initially sought to understand how GlcNAc signaling cross-reacted with galactose gene induction, but they quickly discovered that Reg1, the mediator of GlcNAc signaling was essential for galactose metabolism while Rtg1 and Rtg3 were not. Overturning previous …
Reviewer #2 (Public Review):
Despite previous studies, regulation of the genes required for galactose metabolism in Candida albicans has remained murky. For example, previous work had highlighted Rtg1 and Rtg3 as the key regulator components, an interesting finding given that these factors are important for glucose not galactose regulation in S. cerevisiae. As galactose metabolism is one of the best-understood regulatory systems, the evolutionary difference in the regulation of the galactose response has the potential to teach us about regulatory evolution in general.
The authors initially sought to understand how GlcNAc signaling cross-reacted with galactose gene induction, but they quickly discovered that Reg1, the mediator of GlcNAc signaling was essential for galactose metabolism while Rtg1 and Rtg3 were not. Overturning previous work requires strong evidence, and the authors deliver with a series of growth assays, qPCR, and chromatin IP in wt and mutant backgrounds.
The authors go further to demonstrate that the factor itself interacts with galactose based on isothermal calorimetry (although it would be nice to have seen if this was specific to galactose over glucose or GlcNAc). They show glucose regulation occurs by Reg1 recruiting Cga1 in a manner independent of the activation domain of Reg1 (immunoprecipitation, reporter assays, and chromatin IP). In contrast, Reg1 activation mediated by GlcNAc requires Reg1's activation domain and employs Rgs1. Once again, the experimental evidence for these two regulatory mechanisms is strong.
Evolutionary analysis shows that this specific instantiation of this mechanism, the combination of Reg1 and Cga1, is probably restricted to the CTG clade. While the paper does not explain how these regulatory changes happen, it sets the foundation for future work to tease this apart; work that before this paper would have been unlikely to have been successful.
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