1. Volume 5, Issue 2, Pages 302-313 (October 2013)
Chromatin Modifications Sequentially Enhance ErbB2 Expression in ErbB2-Positive Breast Cancers 
Sathish Kumar Mungamuri, William Murk, Luca Grumolato, Emily Bernstein, Stuart A. Aaronson 
Cell Reports 
Volume 5, Issue 2, Pages (October 2013)
DOI: /j.celrep
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2. Cell Reports 2013 5, 302-313DOI: (10.1016/j.celrep.2013.09.009)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1
H3K4me3, Wdr5, RbBP5, and Ash2L Are Specifically Recruited onto the erbB2 Promoter in ErbB2-Overexpressing and ErbB2-Amplified Tumor Cells
(A) Western blot analysis showing the ErbB2 protein expression across the cell lines.
(B) Schematic representation of the erbB2 promoter, showing major and minor TSS (transcriptional start site) and the translational start site. The five regions used for chromatin immunoprecipitation (ChIP) are also indicated.
(C–F) ChIP analysis showing the H3K4me3 enrichment (C) and occupancy of Wdr5 (D), RbBP5 (E), and Ash2L (F) on the erbB2 promoter in B5/589, MCF-7, ZR-75-1, and SkBr3 cells. The relative H3K4me3 enrichment or occupancy each protein over the % input is shown in the form of a bar diagram. Region “C” of the promoter, which shows the most significant difference between different cell types, is highlighted. Gapdh TSS and MyoD TSS were used as positive and negative controls, respectively, in each ChIP experiment (Figure S2). The error bars represent SEM. Each ChIP experiment was done in triplicate and repeated at least three times, and representative experimental data are shown.
See also Figures S1 and S2.
Cell Reports 2013 5, DOI: ( /j.celrep )
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4. Figure 2
Silencing Wdr5 Inhibits ErbB2 Expression by Inhibiting AP-2 Recruitment, Both in ErbB2-Overexpressing and ErbB2-Amplified Cancer Cells
(A–D) Real–time quantitative PCR (A and C) and western blot analysis (B and D) of ZR-75-1 (A and B) and SkBr3 (C and D) cells stably transduced with inducible shRNA viruses and cultured in the presence of doxycycline for 48 hr.
(E) Schematic representation of the erbB2 promoter, showing major and minor TSS and the translational start site. The five regions used for chromatin immunoprecipitation (ChIP) are also indicated. The AP-2 binding site along with the recognition sequence is shown.
(F) ChIP analysis showing the AP-2 occupancy on the erbB2 promoter in B5/589, MCF-7, ZR-75-1, and SkBr3 cells. The target sequences (corresponding to region “C”) were detected by quantitative real-time PCR analysis of eluted DNA. The relative occupancy of AP-2 over the % input is shown in the form of a bar diagram.
(G and H) ChIP analysis showing AP-2 occupancy on erbB2 promoter in ZR-75-1 (G) or in SkBr3 (H) cells that were stably transduced with sh-Wdr5 (Seq 1) and cultured in the presence of doxycycline for 48 hr. The target sequences (corresponding to region “C”) were detected by quantitative real-time PCR analysis of eluted DNA. The relative AP-2 promoter occupancy over the % input is shown in the form of a bar diagram. The error bars represent SEM. Each experiment was repeated at least three times, and representative experimental data are shown.
Cell Reports 2013 5, DOI: ( /j.celrep )
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5. Figure 3
The erbB2 Promoter Is H3K9 Acetylated by Gcn5 in ErbB2-Amplified Cancers Only and Not in ErbB2-Overexpressing or ErbB2-Low Cells
(A) ChIP analysis showing the H3K9ac enrichment on the erbB2 promoter in B5/589, MCF-7, ZR-75-1 and SkBr3 cells. The relative H3K9ac enrichment over the % input is shown in the form of a bar diagram. Gapdh TSS, MyoD TSS and ALDOA TSS were used as positive and negative controls for H3K9ac ChIP assays. Note that ALDOA (aldolase A) TSS was positive for H3K4me3 and negative for H3K9ac histone mark (Figure S2).
(B and C) Quantitative real-time PCR (B) and western blot (C) analysis showing ErbB2 expression in the indicated cancer cell lines treated with either 0, 100, or 200 μM anacardic acid for 24 hr.
(D–G) Quantitative real-time PCR (D and F) and western blot (E and G) analysis of ZR-75-1 (D and E) and SkBr3 (F and G) cells stably transduced with inducible shRNA viruses and cultured in the presence of doxycycline for 48 hr. Doxycycline-inducible GFP shRNA was used as a negative control in all shRNA experiments to test for off-target effects of doxycycline and nonspecific shRNA effects. The error bars represent SEM. Each experiment was repeated at least three times, and representative experimental data areshown.
See also Figure S2.
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6. Figure 4
Wdr5 Silencing Cooperates with Chemotherapy- and Trastuzumab-Induced Growth Inhibition
(A) ChIP analysis showing H3K4me3 and H3K9ac enrichment on the erbB2 promoter in breast cancer patient samples, where P1 through P14 indicate different patients. The relative H3K4me3 and H3K9ac enrichment over the % input is shown in the form of a bar diagram. See also Figure S11.
(B) Cell growth of the indicated cell lines with or without doxycycline (to induce Wdr5-shRNA) in the culture medium.
(C and D) Cell viability of the indicated cells in the absence and presence of doxycycline and with increasing doses of either Cisplatin (C) or trastuzumab (D). The error bars represent SEM. Each experiment (except A: patient ChIPs; done once) was repeated at least three times, and representative experimental data are shown. The statistical significance was calculated by Student’s t test in MS Excel using two-tailed analysis. A p value <0.005 is indicated by one star, and < is indicated by two asterisks.
(E) Schematic model showing the regulation of chromatin conformation on the erbB2 promoter in different cancers. In ErbB2-nonamplified/overexpressing and ErbB2-amplified/overexpressing cancers, the promoter is H3K4 trimethylated, which opens chromatin structure and facilitates the recruitment of the AP-2 transcription factor, leading to ErbB2 transcription. In ErbB2-amplified/overexpressing cancers only, Gcn5 causes H3K9 acetylation, which further promotes this transcriptional enhancement. The proteins illustrated in green were specifically observed by ChIP in this study to occupy the erbB2 promoter.
Cell Reports 2013 5, DOI: ( /j.celrep )
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7. Figure S1
H3K4me3, Wdr5, RbBP5, and Ash2L Are Specifically Recruited onto the erbB2 Promoter in ErbB2-Overexpressing and ErbB2-Amplified Tumor Cells, Related to Figure 1
(A–D) ChIP analysis showing the enrichment of H3K4me3 (A), and occupancy of Wdr5 (B), RbBP5 (C), and Ash2L (D) on the erbB2 promoter in ErbB2-low (BT-20), ErbB2-overexpressing, nonamplified (MDA-MB-175 VII) and ErbB2-overexpressing, amplified (BT-474) tumor cell lines. The target sequences were detected by quantitative real–time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative H3K4me3 enrichment or occupancy of each protein over the % input is shown in the form of a bar diagram. Region “C” of the promoter, which showed the most significant difference between different cancer types, is highlighted. Gapdh TSS and MyoD TSS were used as positive and negative controls respectively in each ChIP experiment (Figure S2). The error bars represent the standard error of the mean. Each ChIP experiment was done in triplicate and repeated at least three times and a representative experimental data is shown.
Cell Reports 2013 5, DOI: ( /j.celrep )
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8. Figure S2
Positive and negative controls used in ChIP Experiments, Related to Figures 1C–1F and 3
ChIP analysis in different breast cancer cell lines showing the enrichment of H3K4me3 and H3K9ac as well as occupancy of Wdr5, RbBP5 and Ash2L on glyceraldehyde-3-phosphate dehydrogenase (Gapdh), myogenic differentiation 1 (MyoD) and aldolase A, fructose-bisphosphate (ALDOA) Transcription Start Sites (TSS). The target sequences were detected by real–time quantitative PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative histone mark enrichment or relative protein occupancy over the % input is shown in the form of a bar diagram. Gapdh TSS showed positive signal for all the ChIPs, while MyoD TSS worked as a negative control for all the ChIP experiments. Note while ALDOA TSS is positive for H3K4me3 and its readers, but negative for H3K9ac histone mark. The error bars represent the standard error of the mean.
Cell Reports 2013 5, DOI: ( /j.celrep )
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9. Figure S3
The erbB2 Promoter Has a High-CpG Island but Is Not Hypermethylated, Related to Results
(A) Schematic representation of erbB2 promoter showing the polypyrimidine / polypurine repeat. The actual sequence of nucleotides is also shown below. The CpG island prediction was done using Methprimer software using CpG island prediction tool ( and the CpG nucleotides are shown in red color.
(B) Western blot analysis of the given panel of breast cancer cell lines treated with 10 μM of 5-azacytidine for 48 hr.
Cell Reports 2013 5, DOI: ( /j.celrep )
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10. Figure S4
Wdr5 Silencing Reduces H3K4me3 Enrichment on the erbB2 Promoter Both in ErbB2-Overexpressing and ErbB2-Amplified Cells, Related to Results
(A) Western blot analysis of B5/589 cells stably transduced with inducible shRNA viruses and cultured in the presence of doxycycline for 48 hr.
(B and C) ChIP analysis showing H3K4me3 enrichment on erbB2 promoter in ZR-75-1 (B) and SkBr3 (C) cells that were stably transduced with sh-Wdr5 (Seq 1) and cultured in the presence of doxycycline for 48 hr. The target sequences were detected by quantitative real-time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative H3K4me3 enrichment over the % input is shown in the form of a bar diagram. The error bars represent the standard error of the mean. Each ChIP experiment was done in triplicate and repeated at least three times and a representative experimental data is shown.
Cell Reports 2013 5, DOI: ( /j.celrep )
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11. Figure S5 Wdr5 Is Required for ErbB2 Expression, Related to Results
(A and B) Real-time quantitative PCR (A) and western blot (B) analysis showing Wdr5 and ErbB2 mRNA and protein levels of SkBr3 cells that express inducible sh-Wdr5 (seq 2; targeting Wdr5 3′-UTR) and also either pCDNA3 or pCDNA3:Wdr5-ORF. Note that ErbB2 expression can be rescued in sh-Wdr5 expressing cells by Wdr5–ORF overexpression.
(C) Real–time quantitative PCR analysis of SkBr3 cells stably transduced with inducible sh–Wdr5 and cultured in the presence of doxycycline for the first 48 hr, followed by 24 hr or 48 hr without doxycycline, revealing that the reversal of Wdr5 expression is able to reverse ErbB2 mRNA expression. Note that there is no change in AP-2 expression in any of these conditions, even though Wdr5 affects AP-2 occupancy on the erbB2 promoter.
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12. Figure S6
Ash2L Is Required for ErbB2 Expression and H3K4me3 Enrichment on the erbB2 Promoter, Related to Results
(A and B) Real–time quantitative PCR (A) and Western blot (B) analysis showing Ash2L and ErbB2 mRNA and protein levels of SkBr3 cells that stably express inducible sh-Ash2L.
(C) ChIP analysis showing H3K4me3 enrichment on erbB2 promoter in SkBr3 cells that were stably transduced with sh-Ash2L (Seq 1) and cultured in the presence of doxycycline for 48 hr. The target sequences (corresponding to region “C”) were detected by quantitative real-time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative H3K4me3 enrichment over the % input is shown in the form of a bar diagram. The error bars represent the standard error of the mean. The experiment was done in triplicate and repeated three times and a representative experimental data is shown.
Cell Reports 2013 5, DOI: ( /j.celrep )
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13. Figure S7
Wdr5 Silencing Inhibits ErbB2 Transcription by Inhibiting AP-2 Promoter Occupancy, Related to Results
(A) ChIP analysis showing the AP-2 occupancy on the BCl-2 promoter in immortalized breast epithelial cells (B5/589), and ErbB2-low (MCF-7), ErbB2-overexpressing, nonamplified (ZR-75-1), and ErbB2-overexpressing, amplified (SkBr3) tumor cell lines. The target sequences were detected by quantitative real–time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative promoter occupancy of AP-2 over the % input is shown in the form of a bar diagram.
(B and C) ChIP analysis showing AP-2 occupancy (B) and H3K4me3 enrichment (C) on the BCl-2 promoter in SkBr3 cells that were stably transduced with sh-Wdr5 (Seq 1) and cultured in the presence of doxycycline for 48 hr. The target sequences (BCL–2 TSS for H3K4me3 ChIP and BCl–2 promoter for AP-2 ChIP) were detected by quantitative real–time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative AP-2 promoter occupancy (B) or H3K4me3 enrichment (C) over the % input is shown in the form of a bar diagram. The error bars represent the standard error of the mean. The experiment was done in triplicate and repeated three times and a representative experimental data is shown.
Cell Reports 2013 5, DOI: ( /j.celrep )
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14. Figure S8
The erbB2 Promoter Is H3K9 Acetylated by Gcn5 in ErbB2-Amplified Cancers Only, and Not in ErbB2-Overexpressing or ErbB2-Low Cells, Related to Results
(A) ChIP analysis showing the H3K9ac enrichment on the erbB2 promoter in ErbB2-low (BT-20), ErbB2-overexpressing, nonamplified (MDA-MB-175 VII) and ErbB2-overexpressing, amplified (BT-474) tumor cell lines. The target sequences were detected by quantitative real–time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative H3K9ac enrichment over the % input is shown in the form of a bar diagram.
(B and D) ChIP analysis showing enrichment of either H3K9ac (B) or H3K4me3 (D) on erbB2 promoter in SkBr3 cells that were stably transduced with sh–Gcn5 (Seq 1) and cultured in the presence of doxycycline for 48 hr. The target sequences were detected by quantitative real-time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative enrichment of H3K9ac (B) or H3K4me3 (C) over the % input is shown in the form of a bar diagram.
(C) ChIP analysis showing the Gcn5 recruitment on the erbB2 promoter in ErbB2-low (B5/589, MCF-7), ErbB2-overexpressing, nonamplified (ZR-75-1) and ErbB2-overexpressing, amplified (SkBr3) tumor cell lines. The target sequences were detected by quantitative real-time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative promoter occupancy over the % input is shown in the form of a bar diagram. e, ChIP analysis showing AP-2 occupancy on the erbB2 promoter in SkBr3 cells stably transduced with sh-Gcn5 (Seq 1) and cultured in the presence of doxycycline for 48 hr. The target sequences (corresponding to region “C”) were detected by quantitative real–time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative occupancy of AP-2 over the % input is shown in the form of a bar diagram. The error bars represent the standard error of the mean. The experiment was done in triplicate and repeated three times and a representative experimental data is shown.
Cell Reports 2013 5, DOI: ( /j.celrep )
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15. Figure S9
PCAF Silencing Does Not Block ErbB2 Expression, Related to Results
(A and B) Real-time quantitative PCR (A) and Western blot (B) analysis showing PCAF and ErbB2 mRNA and protein levels in SkBr3 cells that stably express inducible sh-PCAF.
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16. Figure S10
Wdr5 Silencing Affects H3K9ac Enrichment on erbB2 Promoter in ErbB2-Amplified Breast Cancer Cells, Related to Results
ChIP analysis showing enrichment of H3K9ac on the erbB2 promoter in SkBr3 cells that were stably transduced with sh-Wdr5 (Seq 1) and cultured in the presence of doxycycline for 48 hr. The target sequences were detected by quantitative real-time PCR analysis of eluted DNA. A known amount of chromatin from each sample was removed before immunoprecipitation and used for PCR amplification (Input). The relative H3K9ac enrichment over the % input is shown in the form of a bar diagram.
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17. Figure S11
Gene Copy Number Analysis in Primary Breast Cancer Patient Samples as well as Tumor Cell Lines, Related to Figure 4A
(A) Real–time quantitative PCR analysis of genomic DNA with primers spanning intron-exon junctions to identify erbB2 gene copy number. The samples were also analyzed for globin gene copy number, and the ratio of erbB2 versus globin copy number is plotted.
(B) Real-time quantitative PCR showing the ErbB2 expression from the RNA isolated from patient samples.
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18. Figure S12
ErbB2 and Grb7 Exhibit Different Mechanisms of Overexpression, Even Though They are Coamplified and Coexpressed, Related to Results
Western blot analysis of SkBr3 cells that were stably transduced with either sh–Wdr5 (Seq 1 and Seq 2) or sh–Gcn5 (Seq 1 and Seq 2) and cultured in the presence of doxycycline for 48 hr to induce shRNA expression.
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19. Figure S13
Wdr5 Silencing Cooperates with Trastuzumab in Inducing Growth Inhibition, Related to Results
(A and B) Soft agar assay in SkBr3 (A) and ZR-75-1 (B) cells stably expressing doxycycline inducible sh–Wdr5. The cells were plated along with 1 μg / ml of Herceptin. shRNA expression was induced by initially adding 1 μg ml-1 doxycycline to complete medium followed by overlay of 200 μl of fresh medium with doxycycline every alternative day for 3 weeks. Colonies were counted and microphotographed. The number of colonies observed was also represented on the right in the form of bar diagram.
Cell Reports 2013 5, DOI: ( /j.celrep )
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