Regulatory networks often converge on very similar cis sequences to drive transcriptional programs due to constraints on what transcription factors are present. To determine the role of constraint loss on cis element evolution, we examined the recent appearance of a thiamine starvation regulated promoter in Candida glabrata. This species lacks the ancestral transcription factor Thi2, but still has the transcription factor Pdc2, which regulates thiamine starvation genes, allowing us to determine the effect of constraint change on a new promoter. We identified two different cis elements in C. glabrata - one present in the evolutionarily recent gene called CgPMU3, and the other element present in the other thiamine (THI) regulated genes. Reciprocal swaps of the cis elements and incorporation of the S. cerevisiaeThi2 transcription factor-binding site into these promoters demonstrate that the two elements are functionally different from one another. Thus, this loss of an imposed constraint on promoter function has generated a novel cis sequence, suggesting that loss of trans constraints can generate a non-convergent pathway with the same output.
Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially defective in C. glabrata, but the transcription factor CgPdc2 upregulates some thiamine biosynthetic and transport genes. One of these genes encodes a recently evolved thiamine pyrophosphatase (CgPMU3) that is critical for accessing external thiamine. Here, we demonstrate that CgPdc2 primarily regulates THI genes. In Saccharomyces cerevisiae, Pdc2 regulates both THI and pyruvate decarboxylase (PDC) genes, with PDC proteins being a major thiamine sink. Deletion of PDC2 is lethal in S. cerevisiae in standard growth conditions, but not in C. glabrata. We uncover cryptic cis elements in C. glabrata PDC promoters that still allow for regulation by ScPdc2, even when that regulation is not apparent in C. glabrata. C. glabrata lacks Thi2, and it is likely that inclusion of Thi2 into transcriptional regulation in S. cerevisiae allows for a more complex regulation pattern and regulation of THI and PDC genes. We present evidence that Pdc2 functions independent of Thi2 and Thi3 in both species. The C-terminal activation domain of Pdc2 is intrinsically disordered and critical for species differences. Truncation of the disordered domains leads to a gradual loss of activity. Through a series of cross species complementation assays of transcription, we suggest that there are multiple Pdc2-containing complexes, and C. glabrata appears to have the simplest requirement set for THI genes, except for CgPMU3. CgPMU3 has different cis requirements, but still requires Pdc2 and Thi3 to be upregulated by thiamine starvation. We identify the minimal region sufficient for thiamine regulation in CgTHI20, CgPMU3, and ScPDC5 promoters. Defining the cis and trans requirements for THI promoters should lead to an understanding of how to interrupt their upregulation and provide targets in metabolism for antifungals.
Pdc2 is critical for the transcriptional response to thiamine starvation in S. cerevisiae and C. glabrata. In this study, we determined how Pdc2 drives transcription of thiamine regulated (THI) promoters using genetic analyses, RNA‐sequencing, chromatin immunoprecipitation (ChIP)‐sequencing, and promoter‐YFP reporter constructs. We hypothesized that Pdc2 binds to THI promoters because the upregulation of THI genes requires Pdc2, but the Pdc2 binding site was unknown. Previously, we identified conserved sequences that are necessary for transcriptional upregulation in low thiamine conditions. Here, we determine that while PDC2 is essential in standard medium in S. cerevisiae, PDC2 in C. glabrata is not, but the essentiality of ScPDC2 is suppressible by overexpression of PDC1. We demonstrate that CgPDC2 is unable to suppress the Scpdc2Δ lethality, indicating that the two Pdc2 proteins have different specificities. We perform RNA‐seq on both species where is PDC2 is deleted, with either ScPDC2 or CgPDC2 supplied on a plasmid, and observe that Pdc2 regulates a relatively small subset of genes related to thiamine biosynthesis (THI genes), and both Proteins are partially functional in either species. Performing ChIP, we find that ScPdc2 binds weakly to DNA, whereas CgPdc2 strongly enriches at some THI promoters. Using ChIP‐seq, we demonstrate that CgPdc2 binds in the same location as a conserved sequence necessary for upregulation. With promoter‐YFP reporter constructs, we identify ~100 bp regions in the CgPMU3, CgTHI20, and ScPDC5promoters that are sufficient to confer regulation to a non‐thiamine regulated promoter in both species, and identify a DNA sequence that likely binds Pdc2. The sequence is repeated twice in the ScPDC5promoter and is a 20/22 bp duplicate containing a palindromic site. Finally, using an electrophoretic mobility shift assay (EMSA), we demonstrate that the DNA binding domain of Pdc2 from either species binds this sequence in the promoters of both species. This work defines the role of Pdc2 in transcription and opens up new questions. Specifically, it is unclear why the two species’ Pdc2 proteins have different affinities for DNA in vivo, and how CgPdc2 upregulates the CgPMU3 promoter. CgPMU3 is a recently evolved thiamine‐regulated gene which is PDC2 dependent, but ChIP‐seq data indicates that it is not closely bound by Pdc2. These results suggest that there are many ways for Pdc2 to regulate transcription.
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