PHO3, THI4, and THI20 were chosen as representatives because they have consensus Thi2p recognition sites in their upstream regions (Nosaka,

2006). We also tested the interaction PD98059 datasheet with PDC5, the expression of which is dependent on Pdc2p but not Thi2p (Nosaka et al., 2005). The Pdc2p with a V5-tag at the C-terminus was expressed from the GAL1 promoter in yeast cells grown in minimal medium containing 10 nM (low) thiamin, and tested for any association with upstream regions. Two or three different primer sets were initially designed for each gene, and, in the case of THI genes, one of these regions overlapped with the Thi2p-recognition site (Fig. 1a). The CYC1 promoter served as a negative control, as it was found not to

associate with Pdc2p and Thi2p in preliminary experiments. As shown in Fig. 1b, the V5-tagged Pdc2p associated with all the THI genes tested. Of note, the V5-tagged Pdc2p was concentrated in regions containing a Thi2p recognition site in PHO3 and TH20 whereas in THI4 it was concentrated a modest distance from the site. In addition, ChIP assays showed an association between Pdc2p and the PDC5 promoter with the strongest signal located about 400 bp upstream from the start codon. The primer sets that exerted the strongest signal for each gene were employed for further ChIP assays. We next investigated whether the associations between Pdc2p and the promoters of THI genes and PDC5 were influenced by the thiamin concentration in the medium and the absence of Thi2p or Thi3p. We found by Western Akt inhibitor analysis using an anti-V5 Cytoskeletal Signaling inhibitor antibody that Pdc2p was expressed to a similar degree under our experimental conditions (data not shown). As shown in Fig. 1c, when the yeast cells were grown in 1 µM (high) thiamin medium, the association with Pdc2p was decreased in PHO3 and THI20, and to a lesser extent in THI4. This result suggests that the interaction of Pdc2p with the THI gene promoters is sensitive to the intracellular TPP concentration. Furthermore, when the ChIP assay was carried out using thi2Δ and thi3Δ mutant

strains grown in low-thiamin medium, the coimmunoprecipitation of Pdc2p with THI genes was markedly decreased. Given that the association of Pdc2p with THI genes was not enhanced in the absence of Thi2p, it is unlikely that Pdc2p competes with Thi2p to bind to the target DNA. Conversely, Pdc2p’s association with the PDC5 promoter was unchanged by the thiamin concentration or absence of Thi2p and Thi3p. As Thi2p recognition sites exist in the promoters of THI genes and Thi3p interacts with both Pdc2p and Thi2p (Nosaka et al., 2005), it is probable that the recruitment of Pdc2p to these promoters is facilitated by Thi2p bound to its target DNA via interaction with Thi3p. Thus, not only the transactivation activity but also the recruitment of Pdc2p seems to be enhanced in response to thiamin starvation.