(A) Histogram showing the number of Atf1/Pcr1-bound genes that exhibit various levels of average transcriptional response. Induced, repressed, and unresponsive genes are indicated by red, green, and grey, respectively. (B) Table showing the number of Atf1/Pcr1-bound H₂O₂-induced genes in various gene groups indicated. The p-value at 0.01 or 0.001 is indicated by 1 asterisk or 2 asterisks. Red and green indicate the enrichment and depletion, respectively. (C) Average expression profile of various gene groups: the Atf1/Pcr1 (major)-bound genes (in red), Atf1/Pcr1 (minor)-bound genes (in grey), and Atf1/Pcr1-unbound genes (in black). The purple dash line indicates the 4-fold increase in level of average transcription. Red and black arrows indicate the time when the average transcription level of the (major) Atf1/Pcr1-bound and unbound genes reaches 4-fold higher than the initial level at 0-min, respectively. (D) Scatter plot showing the level of transcript increase versus the deficient score (the level of requirement for growth fitness). Circle and cross indicate the Atf1/Pcr1-bound H₂O₂-induced genes and the unbound induced gene, respectively. (E) Box-plot indicating the distribution of the average level of Atf1 and Pcr1 enrichment at the H₂O₂-induced genes (red) and unresponsive genes (blue). (E) Scatter plot showing the level of Atf1/Pcr1 binding versus the deficient score. Circle and cross indicate the Atf1/Pcr1-bound H₂O₂-induced genes and Atf1/Pcr1-bound H₂O₂-unresponsive genes, respectively.

(A) Venn diagram showing the relationship between the Sty1 binding sites found in two independently repeated ChIP-chip experiments. (B) Scatter plot showing the relationship between the levels of Sty1 recruitment in two repeats. Black and grey dots indicate the overlapping and non-overlapping binding sites in two repeats. The diagonal and two-fold lines are indicated in black and purple, respectively. (C) Scatter plot showing the relationship between the levels of average Atf1/Pcr1 enrichment and average Sty1 enrichment at the 89 loci containing both major Atf1/Pcr1 and major Sty1 binding sites. The diagonal and two-fold lines are indicated in black and purple, respectively. (D) Box-plot showing the average levels of Sty1 enrichment at the loci that are overlapped with (OL w/) major Atf1/Pcr1 (in black) or not overlapped with (NOL w/) Atf1/Pcr1 (in grey) binding sites. (E) Box-plot showing the average levels of Atf1/Pcr1 enrichment at the loci that are overlapped with (OL w/) major Sty1 (in black) or not overlapped with (NOL w/) Sty1 (in grey) binding sites. (F) Scatter plot showing the ratio between Sty1 enrichment levels in atf1 or pcr1 and wild type cells. The solid line indicates the median and the dash line indicates the ratio at 50% reduction (−0.585 in log2 scale). (G) Histogram showing the number of Sty1-bound genes that exhibit various ratios between Sty1 enrichment levels in atf1/pcr1 and wild type cells. Sty1-bound genes that are also Atf1/Pcr1-bound (OL w/) or not bound (NOL w/) are indicated in black or grey. (H) Histogram showing the number of Sty1-bound Atf1/Pcr1-bound genes that exhibit various ratios between Sty1 enrichment levels in atf1/pcr1 and wild type cells. Genes that are induced or unresponsive upon H₂O₂ treatment are indicated by red or grey bars. The arrow indicates the cutoff for the Atf1/Pcr1-dependently Sty1-recrutied genes.

(A) Venn diagram showing the relationship between the Atf1 and/or Pcr1-dependent or independent H₂O₂-responsive genes. H₂O₂ responsive genes are defined as those whose transcript levels are changed for 2-fold or greater and FDR is less than 3% at least in two consecutive time points examined after H₂O₂ stress. The dependent genes are those whose transcript levels are changed for 2-fold or greater and FRD is less than 3% at least in two consecutive time points in mutant cells when compared to the wild type cells (see Materials and Methods). Number in the parentheses indicates the total number of genes. Numbers of induced and repressed genes are indicated in red and green, respectively. (B) Scatter plot showing the ratio between the levels of transcriptional response in atf1 or pcr1 cells and wild type cells. The scale is in log2. Each dot indicates a H₂O₂-induced gene. Genes whose transcriptional response is dependent on both Atf1 and Pcr1, one of Atf1 and Pcr1, or none are indicated by red dots, blue dots, or grey dots. Average transcriptional levels of three repeats at 30, 60, and 120min after H₂O₂ treatment are used. (C) Histogram showing the number of genes at various levels of average transcriptional induction upon H₂O₂ treatment.

(A) Venn diagram showing the relationship between Atf1 major binding sites and Pcr1 major binding sites. The intersection shows the Atf1/Pcr1 major∶major binding sites. Most of the Atf1 or Pcr1 Major binding sites in the non-intersection part are overlapped with the minor binding sites except for a few (see number in the parentheses) that are not overlapped with either major or minor binding sites indicated. (B) Scatter plot showing the relationship between enrichment levels of Atf1 and Pcr1 at the same loci. The display is identical to Figure 1D. (C) Profile of the Atf1 and Pcr1 occupancies. The heat-maps show the occupancy profile or the level of enrichment within a window of 1 Kb in length (∼33 probes) at each binding site. The profiles of binding sites are aligned at the apex. Average profiles of Atf1 and Pcr1 binding sites in cells prior to and after H₂O₂ treatment are shown at the bottom. (D) Position-specific scoring matrix (PSSM) showing the probability of nucleotide at each position. The most enriched motif matrix is shown: the column indicates the nucleotide position of the motif and the row indicates the nucleotide. (E) Motif logo based on the PSSM in (D). (F) ROC curve showing the PSSM scores of the Atf1/Pcr1 binding sequences (the true discovery) versus the randomly selected promoter sequences (the false positives). Black line indicates the randomly occurred motif and the red line indicates the enriched motif in the Atf1/Pcr1 binding sequences.

(A) A schematic view of the protein occupancy in the genome-wide ChIP-chip analysis. Level of binding affinity is estimated by the ratio between the ChIP-enriched DNA signal and non-enriched (WCE) DNA signal or level of enrichment. Significantly enriched regions (SER) define the protein occupancy. Apex of the occupancy indicates the binding site in the chromosome. (B) Relationship between level of enrichment and rank of percentiles. The array median (Median) is set to 1 or 0 in log scale. Array features' signal above 2.5 MAD (or Median Absolute Deviation) plus the array median is used as a cutoff for significantly enriched signals. (C) Venn diagram showing the relationship between Atf1 binding sites found in cells prior to and after H₂O₂ treatment. (D) Scatter plot showing the relationship between the Atf1 binding levels found in cells prior to and after H₂O₂ treatment. Each dot indicates a binding site. Black dot and grey dot indicate the binding site found in both conditions and in one of the two conditions, respectively. M indicates the median level of the overlapping (black) and non-overlapping (grey) binding sites. Correlation coefficient (R) is indicated. Black dash line indicates the diagonal. Purple lines indicate boundaries within 2-fold change. All overlapping binding sites (black dots) display level change for less than 2-fold except for a few. (E) Venn diagram showing the relationship between Pcr1 binding sites found in cells prior to and after H₂O₂ treatment. (F) Scatter plot showing the relationship between the Pcr1 binding levels found in cells prior to and after H₂O₂ treatment. All overlapping binding sites (black dots) display level change for less than 2-fold except for a few.

(A) Transcription units and Atf1/Pcr1 bindings at the chromosomal region containing pcr1. Chromosomal features are indicated at the top. Transcription units in the upper strand (up. str.) or bottom strand (bot. str.) and the Atf1/Pcr1 binding profiles after H₂O₂ stress are shown. TSS and BS indicate the transcription start site and binding sites, respectively. (B) Transcription units and Atf1/Pcr1 bindings at the chromosomal region containing atf1. Two 5′-end boundaries or TSS are shown. (C) Agarose gel showing the PCR fragments amplified on template cDNA, gDNA (genomic DNA), or the mixture of the two (mix). Molecular weight markers are shown on the left. (D) A magnified view from Figure 3B. atf1 transcript is shown on the top: the think line indicates the ORF and the grey line indicates the 5′-UTR. The position of primers A and B is indicated. cDNA signals and Atf1 binding profile are shown. At the bottom, 3 motifs at the binding site are indicated as Motif-1 (PSSM score = 18.9) and Motif-2 or 3 (PSSM score = 12.6). TSS predicted by Lantermann et al., (Lantermann et al., 2010) is indicated as TSS-1 and predicted in this study is indicated as TSS-2. The two predicted TSS are very close. The 5′-UTR intron and the start codon (+1) are also indicated. (E) Transcriptional profile of atf1 in response to H₂O₂ treatment. The atf1 transcript level determined by real-time quantitative PCR is shown at various time points in wild type or pcr1 cells. Error bar indicates the variation in three repeated measurements. (F) Transcriptional profile of pcr1 in response to H₂O₂ treatment.