1. Volume 43, Issue 5, Pages 798-810 (September 2011)
Context-Specific Regulation of NF-κB Target Gene Expression by EZH2 in Breast Cancers 
Shuet Theng Lee, Zhimei Li, Zhenlong Wu, Meiyee Aau, Peiyong Guan, R.K. Murthy Karuturi, Yih Cherng Liou, Qiang Yu 
Molecular Cell 
Volume 43, Issue 5, Pages (September 2011)
DOI: /j.molcel
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2. Molecular Cell 2011 43, 798-810DOI: (10.1016/j.molcel.2011.08.011)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1
EZH2 Positively Regulates NF-κB-Dependent Gene Network in Aggressive Breast Cancer Cells
(A) Ingenuity Pathways Analysis (IPA) showing the top gene networks enriched in downregulated genes after EZH2 depletion in MDA-MB-231 cells.
(B) Gene network showing the connection to NF-κB (left) and downregulation of the genes following EZH2 depletion in the gene heatmap (right). Red molecules in the network represented the genes that are downregulated upon EZH2 depletion in MDA-MB-231 cells.
(C) Venn diagram showing the overlapping of genes downregulated upon depletion of EZH2, RelA, or RelB in MDA-MB-231 cells.
(D) NF-κB luciferase reporter activity in MDA-MB-231 and BT549 cells upon knockdown of EZH2, RelA, or RelB (top). Knockdown efficiency was shown in immunoblot analysis (bottom).
(E) qRT-PCR analysis of NF-κB target genes, IL8 and TNF, in MDA-MB-231 and BT549 cells upon EZH2 depletion.
(F) NF-κB luciferase reporter activity in MDA-MB-231 and BT549 cells upon EZH2 depletion and TNFα treatment for the indicated duration.
All the data in the graph bars represent + standard error of the mean (SEM), n = 3. See also Tables S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
EZH2 Positively Modulates NF-κB Target Gene Expression Independent of Histone Methyltransferase Activity
(A) NF-κB luciferase reporter activity in MDA-MB-231 and BT549 transfected with EZH2 5′-UTR siRNA followed by overexpression of EZH2 WT or SETΔ. ∗ represents p < 0.05; ∗∗ represents p <
(B) Western blot analysis of indicated proteins from cells treated in (A).
(C) NF-κB luciferase reporter activity in HMECs overexpressing EZH2 WT or SETΔ (left). Western blot analysis of indicated samples (right).
(D) qRT-PCR analysis of IL6, TNF, and IL8 in HMECs overexpressing EZH2 WT or SETΔ.
All the data in the graph bars represent + SEM, n = 3. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
EZH2 Physically Interacts with RelA/RelB Complex in Aggressive Breast Cancer Cells
(A) CoIP assay using nuclear extract of MDA-MB-231 cells stimulated with TNFα for 2 hr (left). The bands were quantified by densitometry (right).
(B) CoIP assay using nuclear extract of 293T cells overexpressed with RelA, RelB, and myc-tagged EZH2 constucts followed by TNFα stimulation for 2 hr.
(C) In vitro pull-down assay using recombinant MBP-RelA and MBP-RelB as well as GST and GST-EZH2 proteins.
(D) In vitro reimmunoprecipitation assay using recombinant RelA and RelB as well as GST and GST-EZH2 proteins.
(E) CoIP was performed in MDA-MB-231 depleted of RelB followed by TNFα treatment for 2 hr.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
EZH2, RelA, and RelB Coregulate a Subset of NF-κB Targets by Interdependent Promoter Recruitment
(A) Hierarchical clustering of NF-κB target genes that were coregulated with EZH2. Gene sets were obtained from microarray gene expression profiling of MDA-MB-231 cells depleted of EZH2, RelA, and RelB followed by TNFα treatment for 4 hr.
(B) qRT-PCR analysis of representative genes from each cluster of (A).
(C) qRT-PCR analysis of representative genes from MDA-MB-231 overexpressing RelA or RelB followed by depletion of EZH2 with or without TNFα treatment for 4 hr.
(D) ChIP assay of EZH2, RelA, RelB, and H3K27me3 on the promoters of TNF, IL6, IL8, and BIRC3 in MDA-MB-231 cells treated with TNFα for 2 hr. Primer pairs encompass a 2.5 kb surrounding the TSS (transcription start site) for TNF, IL8, and BIRC3 with approximately 500 bp interval. The percent input of H3K27me3 ChIP was plotted on the right y axis. Quantification of binding was determined as a percent of input DNAs.
(E) ChIP assay in MDA-MB-231 cells depleted with EZH2 or RelB. Quantification of binding was represented as fold enrichment over IgG control.
(F) Re-ChIP assay was performed to assess in vivo colocalization of EZH2, RelA, and RelB. First ChIP and second ChIP antibodies were indicated as the chart title and x axis labels, respectively. Quantification of binding was represented as fold change to control IgG second ChIP.
All the data in the graph bars represent + SEM, n = 3. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
NF-κB Target Gene Signature Coregulated by EZH2, RelA, and RelB Discriminates Basal versus Luminal Subtype of Breast Cancers and Is Associated with Poor Disease Outcome
(A) Nonsupervised clustering of 54 breast cancer cell lines based on 46 genes that were coregulated by EZH2, RelB, and RelA. A subset of 12 genes showing higher expression in basal- versus luminal-breast cancer cell lines in the heatmap. Expressions of ER gene signature (ESR1, FOXA1 and GATA3) as well as EZH2 and RELB were also shown.
(B) Box plots showing the average expression level of the 12 signature genes (left), EZH2, RelA and RelB (middle) and ER related genes (right) in basal and luminal breast cancer cell lines.
(C) Box plots showing the average expression level of the 12 signature genes (left), EZH2, RelA and RelB (middle) and ER related genes (right) in basal and luminal cancers of Farmer cohort (Farmer et al., 2005) (top) and ER-positive and ER-negative breast cancers in Netherlands cohort (Bos et al., 2009) (bottom).
(D) Kaplan-Meier analyses of disease-specific survival of breast cancer patients of Netherlands cohort. Patients whose tumors have overexpression of the 12 gene set were labeled in red, and those with underexpression of this gene set were labeled in green.
(E) Kaplan-Meier analyses of (D) by stratifying patients with quartiles partition.
See also Figures S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
EZH2 Negatively Regulates NF-κB Target Genes in ER-Positive Luminal-like Breast Cancer Cells
(A) Messenger RNA (mRNA) levels of NF-κB target genes (left) and ER-associated genes (right) in MDA-MB-231 and MCF7 gene expression data set.
(B) qRT-PCR analysis of IL6 and IL8 expression in MCF7 (left) and T47D (right) upon ER, RelA, or EZH2 knockdown followed by TNFα treatment for 4 hr. Relative mRNA expression level after EZH2 knockdown was plotted on the right y axis.
(C) CoIP of MCF7 cell lysates using the indicated antibodies followed by western blot analysis.
(D) ChIP assay of EZH2, H3K27me3, ER, or RelA on the promoters of IL6 and IL8 in MCF7 cells. Primer pairs spanning a 2.5 kb genomic region surrounding the TSS are indicated. P3 is located in the region within 500 bp upstream of TSS. Quantification of enrichment was determined as a percent of input DNAs.
(E) ChIP assay of EZH2, H3K27me3, ER, or RelA on the promoters of IL6 (P3) and IL8 (P3) in MCF7 cells treated with TNFα for indicated times. Quantification of binding was represented as fold enrichment over IgG.
(F) qPCR analysis of IL6 and IL8 mRNAs in MCF-7 cells overexpressing vector or RelB with or without EZH2 siRNA treatment.
All the data in the graph bars represent + SEM, n = 3. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Model for Roles of EZH2 in Modulating NF-κB Signaling in Breast Cancers
(A) In ER-negative basal-like breast cancer cells, EZH2 acts as a co-activator of RelA and RelB to promote the expression of NF-κB target genes such as TNF and IL6, which in turn activates NF-κB signaling through a positive feedback, leading to constitutive activation of NF-κB target gene expression.
(B) In ER-positive breast cancer cells, ER recruits PRC2 complex to the promoter of NF-κB target genes, leading to epigenetic silencing of NF-κB target genes.
Molecular Cell  , DOI: ( /j.molcel )
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