Supplemental Figures & Tables

Microarrays: A Global Network Model of the Arsenic Response

Transcript Profiling

Figure 1. Growth and Survival after Arsenite. [A - B]

Figure 1. Growth and Survival after Arsenite. [A - B]

S. cerevisiae strain BY4741 (MAT a, his3Δ, leu2Δ0, met15Δ0, uraΔ0) was used and grown in synthetic complete medium at 30°C. Treatment with 1mM, 2mM and 5mM AsIII resulted in a negligible effect on growth (after 18h) and survival (1h treatment followed by plating and colony formation counting), but still exhibited a pronounced transcriptional response (see Figure 2).

Download the PDF for Figure 1 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/supp-fig1.pdf)   (14KB)

Figure 2. Cluster analysis of Transcriptional Profiling Data.

Figure 2. Cluster analysis of Transcriptional Profiling Data.

A. Red = induced, green = repressed. Any genes found to be significant by MAPS analysis (see methods) were compiled across the four arrays, averaged, and subsequently clustered with Cluster/Treeview software (Eisen et al., 1998). The dendogram highlighted in pink depicts the zoomed in region shown to the right of the entire tree. Microarray analysis of biological replicates of the high dose treated cells (1mM AsIII) cluster well together, along with the lower dose (100uM AsIII) at 2h, or one cell division. The lower dose time-course (100uM AsIII) showed the beginning of gene expression changes at 30 min with the robust changes occurring at 2h. The 4h (100uM AsIII) treatment, or two cell divisions, was most different, reflecting either a cycling effect or the cell's return to homeostasis, which was further exemplified by a decrease in the transcriptional response.

B. This table shows the numbers of genes changing in each experiment at both the 95% and 99% confidence intervals (see Supplemental Materials and Methods).

Download the PDF for Figure 2 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/supp-fig2.pdf)   (640KB)

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Specific Deletion Profiling

Figure 3. Self-organized Clustering of Specific Deletion Strains.

Figure 3. Self-organized Clustering of Specific Deletion Strains.

Red = induced, green = repressed. Knockouts and the parent strain were subjected to 100uM AsIII for 2h. The experiments were repeated without arsenic to determine genes changing by the specific gene deletion alone. For tables of genes changing from the knockout alone, refer to KO_NoAsIII_Tables1-5 online. For a more detailed description of the specific deletion data under the effects of arsenic, go to Figure 2 in the paper.

FOFC_Tables 1-5

1. Intensity plots were generated from each specific deletion experiment after 100uM AsIII for 2h-- (yap1Δ vs yap1Δ 100uM AsIII, cad1Δ vs cad1Δ100uMAsIII, rpn4Δ vs rpn4Δ 100uM AsIII, arr1Δ vs arr1Δ 100uM AsIII, sod1Δ vs sod1Δ 100uM AsIII) as well as the parent vs parent 100uM AsIII in Resolver. A gene was considered a signature gene if the p value was less than 0.001 and if the fold change value was greater than or equal to 2 fold. At a p value of 0.001, one can expect around 10 false positives (10,000X0.001).

2. Lists were imported into GeneSpring. Each Deletion signature gene list was combined with the Parent signature gene list and saved as a new list.

3. The "Filter on Fold Change" function was used to compare the Parent control vs. Parent AsIII experiment with each deletion (AsIII) experiment. The gene list selected for each Filter on Fold Change analysis was a combination of the Parent signature gene list and the signature gene list of the AsIII Deletion being analyzed at the time. For example, if the comparison was being done between Parent AsIII and Yap1 AsIII, the list used in the analysis was the combination of the parent signature genes and the Yap1 signature genes. The Filter on Fold Change function reports genes were selected from the one condition (Parent) that had Normalized Data values that were greater or less than those in the other condition (Deletion under investigation) by a factor of 2 fold. Each resulting gene list was saved into the following tables.

   All genes combined:

   • FOFC_COMBINED_Table 1 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl1.xls)   (594KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl1.pdf)   (1.7MB)

   Knockouts:

   • FOFC_YAP1_Table 2 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl2.xls)   (80KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl2.pdf)   (190KB)
   • FOFC_CAD1_Table 3 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl3.xls)   (31KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl3.pdf)   (52KB)
   • FOFC_RPN4_Table 4 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl4.xls)   (167KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl4.pdf)   (375KB)
   • FOFC_ARR1_Table 5 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl5.xls)   (325KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl5.pdf)   (759KB)
   • FOFC_SOD1_Table 6 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl6.xls)   (493KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/fofc-tbl6.pdf)   (1.2MB)

MINUS_Tables 1-5
These tables are of genes failing to be induced or repressed (or showing such a decrease in expression that they no longer make significantly expressed gene lists), compared to the parent, in each knockout strain experiment after treatment with 100uM AsIII for 2h. See online Material and Methods for a detailed description on how these genes were identified.

• MINUS_YAP1_Table 1 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-yap1.xls)   (101KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-yap1.pdf)   (157KB)
• MINUS_CAD1_Table 2 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-cad1.xls)   (32KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-cad1.pdf)   (25KB)
• MINUS_RPN4_Table 3 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-rpn4.xls)   (155KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-rpn4.pdf)   (240KB)
• MINUS_ARR1_Table 4 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-arr1.xls)   (334KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-arr1.pdf)   (558KB)
• MINUS_SOD1_Table 5 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-sod1.xls)   (418KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/minus-sod1.pdf)   (765KB)

KO_NoAsIII_Tables1-5
Gene lists of differential expression in knockout strains compared to the parent without arsenic treatment. These were generated with a p value less than 0.001 and if the fold change value was equal to or greater than 2 fold.

• YAP1_Parent Table 1 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-yap1.xls)   (20KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-yap1.pdf)   (22KB)
• CAD1_Parent_Table 2 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-cad1.xls)   (19KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-cad1.pdf)   (25KB)
• RPN4_Parent_Table 3 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-rpn4.xls)   (81KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-rpn4.pdf)   (130KB)
• ARR1_Parent_Table 4 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-arr1.xls)   (54KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-arr1.pdf)   (83KB)
• SOD1_Parent_Table 5 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-sod1.xls)   (295KB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/parent-sod1.pdf)   (532KB)

Download the PDF for Figure 3 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/supp-fig3b.pdf)   (83KB)

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Figure 4. Yap1 likely regulates Arr2 and Arr3 after 2h 100uM AsIII. Yap1, however, does not regulate Rpn4 under arsenic-induced stress.

Figure 4. Yap1 likely regulates Arr2 and Arr3 after 2h 100uM AsIII. Yap1, however, does not regulate Rpn4 under arsenic-induced stress.

Induced = red, repressed = green.
Condition 1 = Parent wildtype;
Condition 2 = Yap1 deleted strain;
Condition 3 = Cad1 deleted strain;
Condition 4 = Rpn4 deleted strain;
Condition 5 = Arr1 (Yap8) deleted strain.
All conditions were treated with 100uM AsIII for 2 hours.

A. Self-organized heat map of 6172 genes selected from the various indicated conditions: AsIII-treated Parent wildtype strain with normalized data values that are greater or less than those in condition(s) — knocked out Yap1, Cad1 (Yap2) , Rpn4, Arr1 (Yap8) treated with AsIII -- by a factor of 2 fold.

B. The Yap1 knock-out strain fails completely to induce Arr2 (0.834 average fold-change) whereas the Arr1 (Yap8) knock-out induces Arr2 (2.90 average fold-change).

C. The Arr1 (Yap8) knock-out induction is more elevated compared to the Yap1 knock-out (1.8 and 1.1 average fold-change, respectively).

D. Yap1 is induced 2.7 fold in the Rpn4 knock-out. The wildtype parent strain shows an averaged induction of 4.7 fold. The presence of the Rpn4 binding motif in the Yap1 promoter may indicate some regulation by Rpn4.

E. Rpn4 is induced 3.7 fold in the Yap1 knock-out compared to 4.1 fold induction in the wildtype parent strain. Contrary to what has been reported in the literature, in the presence of arsenic, Yap1 does not appear to regulate Rpn4.

Download the PDF for Figure 4 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/supp-fig4.pdf)   (41KB)

Phenotypic Profiling Tables

Supplementary Table 1

The sensitivity data was ranked based on four experiments, 1mM (2X), 2mM, and 5mM AsIII, and assigned a new uniform distribution of p-values. Every gene in this table has a percentile rank. In the case that there was slow growth in the wild type, then a default value of 0.5 was assigned. The rankings on this table were used for the metabolic networking.

Download the Supplementary Table 1 (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/sup-tbl1.xls)   (1.7MB), pdf (http://www.niehs.nih.gov/research/atniehs/core/microarrays/model/docs/sup-tbl1.pdf)   (5.3MB)

To download the raw cel files used in this deletion pool study, please go to the http://www.brownlab.info/publications/Haugen2004/. (http://www.brownlab.info/publications/Haugen2004/)

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