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 ScPDC5 promoters 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 ScPDC5 promoter 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.
Support or Funding Information
This work was supported by the National Science Foundation grant (MCB 1921632), the Dennis M. Cook Endowed Gregor Mendel Chair in Genetics Endowment, the Villanova College of Liberal Arts and Sciences, and the Villanova Department of Biology.
This work was supported by the National Science Foundation grant (MCB 1921632), the Dennis M. Cook Endowed Gregor Mendel Chair in Genetics Endowment, the Villanova College of Liberal Arts and Sciences, and the Villanova Department of Biology.amp;nbsp;
