A Pho7 UAS in the pho1+promoter is necessary expression. (A) Schematic of pho1+ promoter variants used for expression analysis. 2 kb pho1+pr-yfp+: full promoter, 280 bp pho1+pr-yfp+: promoter trimmed to only the Pho7-UAS, 180 bp pho1+pr-yfp+: promoter trimmed beyond the Pho7-UAS, UASΔ pho1+pr-yfp+: full promoter lacking only the 20 bp under the Pho7 ChIP-Seq peak. See text for vector construction. (B) FACS histogram profile for the 2 kb pho1+pr-yfp+ construct transformed into a pho7+ strain. Cells were grown in either high-Pi (blue) or no-Pi (pink) media for 4 hours prior to fixation and counting. Bar graph depicting mean YFP intensity for the 2 kb pho1+pr-yfp+ construct transformed into a pho7+, pho7 Δ, and csk1 Δ strain and grown in either high-Pi (blue) or no-Pi (pink) media are given as the average of three biological replicates ± SE. (C) The UASΔ pho1+pr-yfp+ construct was transformed into pho7+ or pho7 Δ strains and treated as in B. (D) The noted constructs were transformed into pho7+, pho7 Δ, or csk1 Δ backgrounds and treated as in B. (E) Model detailing the role of Csk1 in repressing Pho7 function at the pho1+ promoter. In high-Pi conditions Pho7 is not recruited to the UAS and Csk1 interacts near the UAS to prevent full Pho7 activity. When the external concentration of Pi drops, additional Pho7 is recruited to the promoter and Csk1 repression is relieved resulting in maximal induction. Proper regulation is controlled within a Pi sensing module located between bases -2000 and -280 in an unknown manner.