1. Volume 16, Issue 4, Pages 521-535 (November 2004)
Identification of Promoters Bound by c-Jun/ATF2 during Rapid Large-Scale Gene Activation following Genotoxic Stress 
Jun Hayakawa, Shalu Mittal, Yipeng Wang, Kemal S. Korkmaz, Eileen Adamson, Christopher English, Masahide Omichi, Michael McClelland, Dan Mercola 
Molecular Cell 
Volume 16, Issue 4, Pages (November 2004)
DOI: /j.molcel

2. Figure 1
Time Course of Phosphorylation and Transactivation following Genotoxic Stress
(A) Western analysis using anti-phospho-c-Jun or anti-phospho-ATF2 at various times following treatment of BT474 cells with 100 μM cisplatin or transplatin or mock treatment (control, “C”) (ATF2 data from Hayakawa et al. [2003]).
(B) Time course of transactivation of a reporter construct bearing five concatenated ATF2 binding sites from BT474 cells following addition to 100 μM cisplatin or transplatin or buffer (mock), left panel. The middle and right panels represent activation at the 3 hr time point following addition to 100 μM cisplatin. Dominant-negative c-Jun and ATF2 vectors were cotransfected with the reporter 24 hr prior to addition of cisplatin. SP600125, a JNK inhibitor, was added to 30 μM 30 min prior to cisplatin. Error bars were calculated based on three replicates of all experiments.
(C and D) Confirmation of specific precipitation of phosphorylated forms of c-Jun or ATF2 by Western analysis of ChIP products for BT474 cells treated with 100 μM cisplatin or transplatin or mock treated or untreated (control, “C”), or treated with 100 μM cisplatin in the presence of SP as described in (B).
(E) Confirmation of yield and size of DNA precipitated with anti-(apo)-c-cJun, anti-(apo)-ATF2, anti-phospho-c-Jun, and anti-phospho-ATF2 from BT474 cells following treatment with cisplatin (ethidium-Br-stained agarose gel [see Experimental Procedures for details]).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
siRNA JNK Blocks Synthesis, AP-1 Activation, Transactivation, and Anti-Phospho-Antibody-Mediated ChIP
(A) Inhibition of synthesis of JNK isoforms 1 and 2.
(B) Specific inhibition of phosphorylation of c-Jun and ATF2 following cisplatin stimulation.
(C) Inhibition of transactivation by siRNA following cisplatin stimulation.
(D) Inhibition of ChIP of c-Jun- or ATF2-containing DNA fragments by siRNA following cisplatin stimulation. All cell treatment conditions were as for Figure 1.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Analysis of Promoter Array Hybridization
(A) M-A plots of promoter array hybridization intensities of ChIP products from control BT474 cells (left) and cells treated with 100 μM cisplatin for 3 hr where M = log2 R − log2 G and A = (log2 R + log2 G)/2. where R is the intensity of the scanner signal for the experimental sample fluorophore (channel 2 [Ch2]) and G is the scanner signal for the reference sample fluorophore (channel 1 [Ch1]). Each DNA was competitively hybridized again to total genomic DNA from BT474 cells (i.e., left panel is self competition).
(B) Significance (“volcano”) plots (Jin et al., 2001; Wolfinger et al., 2001) of the hybridization intensity data for ChIP products of BT474 cells treated with 100 μM cisplatin or transplatin for 3 hr compared with control nontreatment ChIP sample. For the JNK inhibitor, 30 μM SP was added 30 min prior to cisplatin and compared with the control sample. The dashed line represents the significance threshold of B = 2.5. Genes of significant hybridization correspond to points with B > 2.5, M > log2 1.5, p < 0.05.
(C and D) Venn diagram and table of the distribution of 269 genes that meet the criteria of (B). Of 249 known or associated AP-1-regulated genes on the array, 86 exhibit significant binding by phospho-c-Jun and/or phospho-ATF2, whereas 183 are unrecognized new candidate AP-1-regulated genes.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Cluster Diagram Representing Competitive Hybridization to the Promoter Array
121 “common” genes of the 269 significant (as defined in Figure 3C) hybridizing genes are shown. Each column represents four independent preparations and hybridizations of the ChIP-captured DNA prepared from cells treated as indicated at the top of the column and analyzed by competitive hybridization with total genomic DNA. Red indicates high sample hybridization to the array at the named gene (right) compared to genomic DNA, whereas green indicates decreased hybridization compared to genomic DNA. Red font indicates known or potential AP-1-regulated genes; black indicates genes not previously associated with dominant AP-1 regulation. The cells were treated with cisplatin or transplatin for 3 hr and with SP and siRNA as described in the legends to Figures 1 and 2 and Experimental Procedures.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Validation of Hybridization Intensity
(A) Semiquantitative PCR analysis of phospho-ATF2 and phospho-c-Jun target regions. ChIP products for BT474 cells treated with cisplatin or untreated controls (“C”) were analyzed by PCR as described in the Experimental Procedures. The panels are images of agarose gels of the PCR products. The sequences on the right are the portions of promoter sequences amplified during the validation. Uppercase letters indicate putative AP-1 sites involved in binding of ATF2 and/or c-Jun.
(B) Quantitative PCR analysis of ChIP products. The results of quantitative PCR are compared to the promoter array hybridization results for six genes of the standard PCR reactions (A). ChIP products of phospho-c-Jun (asterisk) and phospho-ATF2 (cross) were analyzed by real-time PCR as described in the Experimental Procedures. The data are expressed as relative fold change of real-time PCR results to untreated control ChIP sample.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 Comparison of DNA Binding Profile with Expression Profile
Red cluster results indicate ChIP-captured DNA that exhibits increased binding to the corresponding gene upon cisplatin treatment compared to genomic DNA, whereas green indicates decreased DNA binding compared to genomic DNA.
121 of the 269 significantly hybridized genes (Figure 3C) are represented as a cluster diagram for cisplatin-treated and mock-treated cells as in Figure 4. Examples of genes significantly bound by only phospho-c-Jun or phospho-ATF2 are also shown. For comparison, the ratio of Affymetrix mRNA expression intensity for cisplatin-treated to mocked-treated cells is indicated. “+” indicates the 18 representative genes examined by quantitative PCR using total RNA made from the same cell preparations as used for the ChIP preparations. Red font indicates known or potential AP-1-regulated genes; black font indicates genes not associated with dominant AP-1 regulation.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7 mRNA Expression Correlates with Protein Expression
(Left) mRNA expression following genotoxic stress was examined by quantitative PCR for 15 representative genes at the indicated times after stimulation of cells. The mRNA level (ordinate) is expressed relative to quantitative PCR results for mock-treated cells. (Right) Corresponding protein expression at the indicated times was monitored by Western analysis using specific antibodies at the indicated times and for mock-treated, control (“C”), and transplatin-treated cells at 3 hr (see Experimental Procedures for details).
Molecular Cell  , DOI: ( /j.molcel )