(A) Localization of Pho4 around the ILV3, ASN1, CIS3, and YPS3 loci. Prospective Pho4 binding sites (X) in each region are shown in the schematic representation of the regions containing these genes at the top. Next two rows show Pho4 localization to these regions in the wild-type (WT) cells grown under high (H)- or low (L)-phosphate (Pi) conditions analyzed with high- and low-resolution (HR and LR) chips. The bottom two rows show Rpo21 localization to these regions analyzed with an HR chip.
(B) Northern analysis of genes showing Pi-independent but Pho4-dependent expression, as in the legend to Figure 1B. Pho4 dependency was either positive (ILV3 and ASN1) or negative (CIS3 and YPS3) for expression. ACT1 is the loading control.
(C) Enrichment of promoter regions in the ChIPed DNA fragments independent of Pi conditions analyzed by gene-specific PCR. Conditions for PCR are as described in the legend to Figure 1.
(D) A schematic representation of the ASN1 promoter showing Pho4 and Gcn4 binding sites (AACGTG and TGACTC, respectively) and activities of the wt and mutant (ASN1mut; lacking Pho4 binding site) promoters represented by β-galactosidase activity. The wt, Δpho85, Δpho4, or Δgcn4 cells harboring respective reporter plasmids were grown to the mid-log phase under high-Pi conditions before the preparation of cell extracts. The values are an average of three different assays, each of which contains measurements with three independent clones.

(A) Expression of the KCS1 sense and AS RNAs from the wt or mutant (Mut; lacking the three Pho4 binding sites) KCS1 gene in a YCp plasmid in Δkcs1 or Δkcs1 Δpho85 strain under high (H)- or low (L)-Pi conditions was analyzed by northern blotting with strand-specific RNA probes. Positions of the KCS1 sense (top panel) and AS RNA (second panel from the top) are designated by arrows and a vertical bar on the left-hand side of the panels. The positions of RNA size markers (nt) are on the right. ACT1 is the loading control. The Kcs1 protein tagged with the c-myc epitope at its C terminus was detected by western blotting using anti-myc antibody. The full-length and truncated Kcs1 proteins are designated by two arrows on the left-hand side of the second panel from the bottom. The positions of marker proteins are on the right. Actin is the loading control for western analysis. The faint band between 83 and 62 kDa markers observed in all lanes is nonspecific staining of yeast protein by the anti-myc antibody.
(B) Heterologous KCS1 AS RNA expression and production of the truncated Kcs1 protein. A Δkcs1 strain harboring KCS1mut-myc in a YCp vector and pGAL1-AS RNA in a YEp vector or vector alone was analyzed for KCS1 sense and AS RNA expression by northern blotting as designated. AS RNA expression was induced by incubating the transformants in galactose medium for 6 h (Gal) or repressed in glucose medium (Glc). Production of Kcs1-myc protein was detected as described above.
(C) An RNA hybrid formation of the KCS1 mRNA with the AS RNA detected by RNase protection assay and RT-PCR. A schematic representation of the KCS1 gene of interest is shown at the top. The arrows on the bar designate the major transcription start points of the mRNA (at −14) and of the AS RNA (+235). The boxes below the bar indicate the positions of the strand-specific primers used for the detection of dsRNA: the blank boxes are sense-strand-specific primers (sense, +243 to +223, and sense*, +415 to +395), and the black ones are primers for the AS strand (AS, −16 to +5, and AS*, −68 to −48). Total RNA was isolated from the wt, Δpho85, Δkcs1, Δkcs1 harboring KCS1mut, or Δkcs1 expressing the AS RNA under the TDH3 promoter, grown in high-Pi medium, except that Δkcs1 harboring KCS1mut was in low-Pi medium. About 10 μg of each RNA sample was incubated with (+) or without (–) RNase ONE. For negative control (–RNA), total RNA was replaced by yeast tRNA. The digestion products were then used as templates for first-strand cDNA synthesis with strand-specific primers as designated, and the cDNA products were subjected to PCR amplification with the appropriate reverse primer. The sense-strand-specific (sense) primer, from +243 to +223; antisense-strand-specific (AS), from −16 to +5; sense*, from +415 to +395; and AS*, from −68 to −48. For lanes 14, 16, 18, 20, 23, and 26, one-tenth of the amount of the cDNA template was used for PCR compared to those in the other lanes. The PCR products were analyzed by electrophoresis on a 5% polyacrylamide gel, and 10-fold dilutions of the PCR products were loaded in lanes 16, 20, 23, and 26. The positions of DNA size markers are shown on the right-hand side of each panel.
(D) Pho4-directed and Pi-dependent transcription within the KCS1 ORF. The N-terminally truncated wt or mutant (Mut) KCS1 ORF (+105 to +3150) placed in a YCp plasmid was introduced into Δkcs1 or Δkcs1 Δpho4 strain, and transcription of sense RNA under high (H)- or low (L)-Pi conditions was detected by northern blotting using a strand-specific probe (top panel). The positions of RNA size markers are shown on the right and ACT1 is the loading control.
(E) The intragenic RNA also can encode the truncated Kcs1-myc protein. The truncated wt or Mut KCS1 ORF (+105 to +3150) tagged with c-myc at its C terminus was introduced into the Δkcs1 strain, and the production of Kcs1-myc protein was analyzed by western blotting. WT* designates the full-length wt KCS1 gene tagged with c-myc, which produced the normal and truncated Kcs1-myc protein (lane 39) as in lane 5 of Figure 4A. Actin is the loading control for western analysis.

(A) Localization of Pho4 around the PHO8 and PST1 loci. Schematic representations of the regions of these genes (adopted from the results of pattern matching analysis provided by Saccharomyces Genome Database [SGD], http://www.yeastgenome.org/ATContents.shtml) showing prospective Pho4 binding sites (X) are at the top. The next two rows show Pho4 localization to these regions in the wild-type (WT) cells grown under high (H)- or low (L)-phosphate (Pi) conditions analyzed with high- and low-resolution (HR and LR) chips. Localization of Rpo21, the largest subunit of RNA polymerase II, to these regions in the WT or a Δpho4 mutant analyzed with HR chip is shown in the bottom two rows. Yeast cells containing Pho4-Flag × 3 or Rpo21-Flag × 3 were processed for chromatin immunoprecipitation (ChIP) as described in Methods, and enrichment in the immunoprecipitated fraction relative to whole genome DNA is shown in each panel. Blue bars above and below the x-axis represent genes on the Watson and Crick strands, respectively. The y-axis scales are log₂(signal ratio) and normalized ratio for HR and LR chips, respectively. In HR chip panels, brown bars represent signals that are judged as significant binding, whereas gray bars are not. The normalized ratio values of the peaks are shown in the LR chip panels.
(B) Northern analysis of genes showing Pho4- and Pi-dependent expression. Total RNA was isolated from the wt, Δpho4, or Δpho85 cells grown under high (H)- or low (L)-Pi conditions, subjected to northern blot analysis, and probed with a digoxigenin (DIG)-labeled probe for each gene as designated.
(C) Enrichment of promoter regions in the chromatin-immunoprecipitated (ChIPed) DNA fragments dependent on Pi conditions analyzed by gene-specific PCR. Total DNA in the whole cell extract (input) or ChIPed DNA fragments prepared from the wt cells grown under high- or low-Pi conditions were amplified by PCR for 25 or 30 cycles using primer pairs specific to the promoter regions of the genes designated on the left-hand side of the panel. The AIR2 ORF is the negative control for Pho4 binding.

(A) The positions of possible Pho4 binding sites within the KCS1 ORF (X) and Pi-dependent localization of Pho4 within the KCS1 ORF detected by HR- and LR-chip analyses are shown in the top two panels. In this case, the results using an HR chip bearing chromosome 3, 4, 5, and 6 sequences are shown for better resolution. Pi- and Pho4-dependent localization of Rpo21 to the KCS1 locus in the wt and Δpho4 strains under high- or low-Pi conditions using the HR chip is shown in the bottom two panels. The slight difference in the data processing programs between the chromosome 3–6 chip and the whole genome chip caused the different appearance of the figures as compared to Figures 1A and 2A.
(B) Demonstration of Pi-dependent enrichment of the KCS1 ORF fragment but not the 5′-upstream region by gene-specific PCR. Total DNA in the whole cell extract (input) or ChIPed DNA fragments prepared from the wt or Δpho85 cells grown under high- or low-Pi conditions were subjected to PCR using the primer set specific to the 5′-upstream region or ORF of KCS1 as designated. The conditions for PCR are the same as those described in the legend to Figure 1.
(C) Northern analysis of KCS1 sense and AS RNA expression in the wt and various pho mutant strains as designated under high (H)- or low (L)-Pi conditions using strand-specific DIG-RNA probes. For detection of KCS1 sense transcripts, an RNA probe specific to the 3′ region of KCS1 was employed (see Figure 3E). The arrows on the left-hand side of the panel indicate the positions of the KCS1 sense (the top panel) and AS transcripts (the middle panel), respectively. The vertical bar on the left-hand side of the top panel designates short KCS1 sense transcripts ranging from 2,300 to 1,800 nt. The positions of RNA size markers are shown on the right. ACT1 is the loading control.
(D) Effect of a Δrrp6 mutation on the levels of the KCS1 sense and AS RNA expression. Total RNA was isolated from the wt or Δrrp6 mutant cells grown under different Pi conditions as described above and subjected to northern analysis using strand-specific DIG-RNA probes as designated. The positions of RNA size markers are shown on the right. ACT1 is the loading control.
(E) A schematic representation of the structure of the KCS1 gene showing the positions of the ATG codon at +1 and +676, taking A of the initiating ATG as +1. The blank bar represents the promoter region, and the shaded one is the coding region. The white boxes in the coding region are the prospective Pho4 binding sites at +406, +1127, and +1393. The arrows on the bar designate the major transcription start points of the sense RNA in the 5′-upstream region (at −14) and in the ORF (at +537), and that below the bar is that of the AS RNA (+235), determined by the 5′-RACE method. The boxes below the bar indicate the positions of the strand-specific probes used for northern analysis: the blank box is a sense-strand-specific probe (+3150 to +1875), and the black one is the AS strand (−715 to +295).

The wt, various mutants, or those harboring plasmids expressing KCS1, KCS1mut, AS RNA, or a truncated Kcs1 protein (dKcs1) were incubated for 5 h in high (H)- or low (L)-Pi media before isolation of total RNA as described in Methods. ACT1 is the loading control.