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Thiamine (vitamin B1) is essential for glucose catabolism. In the yeast species, Nakaseomyces glabratus (formerly Candida glabrata) and Saccharomyces cerevisiae , the transcription factor Pdc2 (with Thi3 and Thi2) upregulates pyruvate decarboxylase (PDC) genes and thiamine biosynthetic and acquisition (THI) genes during starvation. There have not been genome-wide analyses of Pdc2 binding. Previously, we identified small regions of Pdc2-regulated genes sufficient to confer thiamine regulation. Here, we performed deletion analyses on these regions. We observed that when the S. cerevisiae PDC5 promoter is introduced into N. glabratus , it is thiamine starvation inducible but does not require the Thi3 coregulator. The ScPDC5 promoter contains a 22-bp duplication with an AT-rich spacer between the 2 repeats, which are important for regulation. Loss of the first 22-bp element does not eliminate regulation, but the promoter becomes Thi3 dependent, suggesting cis architecture can generate a Thi3-independent, thiamine starvation inducible response. Whereas many THI promoters only have 1 copy of this element, addition of the first 22-bp element to a Thi3-dependent promoter confers Thi3 independence. Finally, we performed fluorescence anisotropy and chromatin immunoprecipitation sequencing. Pdc2 and Thi3 bind to regions that share similarity to the 22-bp element in the ScPDC5 promoter and previously identified cis elements in N. glabratus promoters. Also, while Pdc2 binds to THI and PDC promoters, neither Pdc2 nor Thi3 appears to bind the evolutionarily new NgPMU3 promoter that is regulated by Pdc2. Further study is warranted because PMU3 is required for cells to acquire thiamine from environments where thiamine is phosphorylated, such as in the human bloodstream. [ABSTRACT FROM AUTHOR]