Volume 43, Issue 5, Pages 811-822 (September 2011) Interplay between HDAC3 and WDR5 Is Essential for Hypoxia-Induced Epithelial- Mesenchymal Transition  Min-Zu Wu, Ya-Ping Tsai, Muh-Hwa Yang, Chi-Hung Huang, Shyue-Yih Chang, Cheng-Chi Chang, Shu-Chun Teng, Kou-Juey Wu  Molecular Cell  Volume 43, Issue 5, Pages 811-822 (September 2011) DOI: 10.1016/j.molcel.2011.07.012 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 43, 811-822DOI: (10.1016/j.molcel.2011.07.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Direct Activation of hdac3 Expression by HIF-1 (A) Upper view shows fold change of mRNA levels of hif-1α, vegf, hdac3, and hdac4 by real-time PCR analysis in FADU/MCF-7 cells under normoxia (N) or hypoxia (H); lower view illustrates western blot analysis of HDAC3 expression. The different bar graph symbols represent different species of molecules, which apply to (A)–(C). (B) Decreased expression of hif-1α/vegf/hdac3 mRNA levels and HDAC3 protein levels in H1299-HIF1α-si clones. Knockdown of unrelated protein topoisomerase 3α (H1299-top3α-si) and transfection with an empty vector (H1299-cont.) were used as controls. “1” and “2” indicate the samples from two different clones. (C) Knockdown of endogenous HIF-1α abolishes the induction of HDAC3 (mRNA and protein levels). Control experiments to knock down Top3α did not change the levels of HIF-1α or HDAC3 expression (Figure S1G). The asterisk (∗) indicates statistical significance (p < 0.05) between experimental and control clones. (D) Schematic representation of the hdac3 promoter region and the reporter constructs used in HIF-1α transfection experiments. The constructs contained wild-type (pXP2-HDAC3-HRE) or mutated (pXP2-HDAC3-mut) HRE located −37 to −33 bp upstream of the transcription start site of hdac3. (E) Luciferase activity of pXP2-HDAC3-HRE or pXP2-HDAC3-mut after cotransfection of different expression constructs under normoxia or hypoxia. The luciferase activity/β-galactosidase of 293T cells cotransfected with pXP2-HDAC3/pcDNA3 control vector under normoxia was applied as the baseline control. The asterisk (∗) indicates statistical significance (p < 0.05) between experimental and control transfections. The protein levels of different expression vectors are shown (Figure S1H). (F) ChIP analysis. Chromatin was incubated with IgG control or anti-HIF-1α antibody. The 253 bp fragment contains the HRE, whereas the 202 bp fragment does not contain any HRE in the hdac3 promoter (upper four lanes). The 262 bp fragment contains the HRE in the vegf promoter (lower two lanes). Input, 2% of total input lysate. Error bars indicate standard deviations (SDs) of duplicate mRNA levels by quantitative real-time PCR (A–C) or triplicate luciferase activity (E). Molecular Cell 2011 43, 811-822DOI: (10.1016/j.molcel.2011.07.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Knockdown of hdac3 in FADU-HIF1α(ΔODD) or H1299 Clones Reverts EMT and Metastasis (A) Western blot analysis of HIF-1α(ΔODD), HDAC3, epithelial, and mesenchymal markers in FADU-HIF1α(ΔODD)-HDAC3-si versus FADU-HIF1α(ΔODD) control clones. “1” and “2” indicate the samples from two different clones. (B) Fold change of migration and invasion activity of FADU-cDNA3, FADU-HIF1α(ΔODD), and FADU-HIF1α(ΔODD)-HDAC3-si clones. The FADU-HIF1α(ΔODD)-1 clone was selected as the control group. (C) In vivo metastatic ability of FADU-cDNA3, FADU-HIF1α(ΔODD), and FADU-HIF1α-HDAC3-si clones as assayed by tail vein injection (open bars) or orthotopic implantation (closed bars) methods. The FADU-HIF1α(ΔODD)-1 clone was selected as the control group. (D) EMT marker changes in H1299-control versus H1299-HDAC3-si clones. H1299-top3α-si clone was also used as a control. (E) Fold change of migration and invasion activity of H1299-control versus H1299-HDAC3-si clones. H1299-control clone was used as a control. (F) In vivo metastatic ability of H1299 control and H1299-HDAC3-si clones as assayed by tail vein injection methods. H1299-control clone was used as a control. The asterisk (∗) indicates statistical significance (p < 0.05) between experimental and control clones, which applies to (B), (C), (E), and (F). Error bars indicate standard deviations (SDs) of quadruple measurement of migration/invasion activity (B and E) or metastatic tumor nodules in mice experiments (n = 6) (C and F). Molecular Cell 2011 43, 811-822DOI: (10.1016/j.molcel.2011.07.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Inhibition of the EMT Phenotypes and Migration/Invasion Activity by hdac3 Knockdown in Two Cell Lines under Hypoxia (A and B) Western blot analysis showed the inhibition of EMT phenotypes in FADU-HDAC3-si or MCF7-HDAC3-si clones under hypoxia even in the presence of Snail and Twist1. “1” and “2” indicate the samples from two different clones. N, normoxia; H, hypoxia. (C and D) Inhibition of in vitro migration/invasion activity in FADU-HDAC3-si or MCF7-HDAC3-si clones versus the FADU or MCF-7 control clone (under normoxia [N] or hypoxia [H]). The asterisk (∗) indicates statistical significance (p < 0.05) between experimental and control clones (i.e., FADU or MCF-7 control clones in A and B). For (C) and (D), normoxic samples of each clone were used as controls versus their hypoxic counterparts. Error bars indicate standard deviations (SDs) of quadruple measurement of migration/invasion activity (C and D). Molecular Cell 2011 43, 811-822DOI: (10.1016/j.molcel.2011.07.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Specific Histone Marks Are Associated with Hypoxia-Induced EMT Using Western Blot Analysis and In Vitro Deacetylase Assay (A) Western blot analysis showed the decrease in H3K4Ac and increase in H3K4me2/me3 levels in hypoxic clones. Other histone marks are either not changed under hypoxia or not modulated by HDAC3. “1” and “2” indicate the samples from two different clones. Total histone 3, 4, or 2A levels were used as controls. N, normoxia; H, hypoxia. (B) Increased activity of HDAC3 to deacetylate the H3K4Ac peptide in hypoxic extracts using in vitro deacetylase assay. Anti-HDAC3 antibody was used to pull down the endogenous HDAC3 proteins and test its activity. WCE, whole-cell extracts. Molecular Cell 2011 43, 811-822DOI: (10.1016/j.molcel.2011.07.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Specific Histone Marks Are Associated with Hypoxia-Induced EMT Using qChIP Analysis of Promoters (A) qChIP results using different antibodies to measure the levels of different histone marks on the promoters of EMT marker genes. The covered regions for each gene are described in Table S9. The asterisk (∗) indicates statistical significance (p < 0.05) between experimental and control clones. (B and C) qChIP results showed the increased H3K9me2/me3 levels and EZH2 binding on the E-cadherin promoter. Error bars indicate standard deviations (SDs) of duplicate qChIP values for each sample (A–C). Molecular Cell 2011 43, 811-822DOI: (10.1016/j.molcel.2011.07.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Induction of WDR5 by Hypoxia, Interaction between WDR5 and HDAC3, and Increased HMT Activity in Hypoxic Cells (A) Western blot analysis showed the increased WDR5 levels in hypoxic FADU and MCF-7 cells. N, normoxia; H, hypoxia. (B) Coimmunoprecipitation assays showed that the anti-WDR5 antibody pulled down endogenous HDAC3 and other components of the HMT complex in FADU cells under hypoxia. IgG was used as a negative control. (C) Sequential qChIP assays showed the recruitment of WDR5 by HDAC3 on the N-cadherin/vimentin gene promoters. α-HDAC3 and α-WDR5, antibody against HDAC3 and WDR5, respectively. The asterisk (∗) indicates statistical significance (p < 0.05) between experimental and control clones. (D) Coimmunoprecipitation assay showed that the antibody against Twist1 pulled down HDAC3 and WDR5. IgG was used as a negative control. WCE, whole-cell extracts. (E) Coimmunoprecipitation assay showed that the antibody against WDR5 pulled down Twist1. IgG was used as a negative control antibody. WCE, whole-cell extracts. (F) Sequential qChIP assays showed that Twist1 recruited WDR5 on the N-cadherin promoter containing the E-box site in FADU or MCF-7 cells under hypoxia. IgG was used as a negative control. gapdh promoter was used as an input control. The asterisk (∗) indicates statistical significance (p < 0.05) between experimental (hypoxia) and control (normoxia) clones. (G) HMT assays showed the increase in H3K4-specific HMT activity in two cell lines under hypoxia. Peptides used in the assay are described in Supplemental Experimental Procedures. α-HDAC3, antibody against HDAC3. The assays were performed in the presence of DNase. Error bars indicate standard deviations (SDs) of duplicate qChIP values for each sample (C and F). Molecular Cell 2011 43, 811-822DOI: (10.1016/j.molcel.2011.07.012) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Regulation of WDR5 and Its Role in Hypoxia-Induced EMT and a Model to Depict the Role of Chromatin Modifiers in Hypoxia-Induced EMT (A) Western blot analysis showed that overexpression of HIF-1α(ΔODD) or HIF-2α induced WDR5 expression. (B) Transient transfection experiments identified a HRE (CGTG) located at the position from −643 to −640 bp upstream of the transcription start site of the wdr5 proximal promoter. Mutation of this site abolished activation by HIF-1α or HIF-2α. Open box represents the location of the HRE. The luciferase activity/β-galactosidase of 293T cells cotransfected with pXP2-WDR5-900/pcDNA3 control vector under normoxia was applied as the baseline control. The asterisk (∗) indicates statistical significance (p < 0.05) between experimental and control transfections. The protein levels of different expression vectors are shown (Figure S7C). (C) Knockdown of WDR5 in hypoxic clones abolished the activation of mesenchymal genes with the preservation of epithelial gene repression. (D) Knockdown of WDR5 in FADU clones decreased the in vitro migration/invasion activity induced by hypoxia. The asterisk (∗) indicates statistical significance (p < 0.05) between experimental and control clones. (E) A model of hypoxia-induced chromatin modifiers and the changes in associated histone marks that lead to hypoxia-induced EMT. Empty arrowheads indicate selective regulation of either epithelial or mesenchymal genes. Question mark indicates unknown mechanism. Upward or downward dark arrowheads indicate increased or decreased H3K27me3 levels. Error bars indicate standard deviations (SDs) of triplicate luciferase activity (B) or quadruple measurement of migration/invasion activity (D). Molecular Cell 2011 43, 811-822DOI: (10.1016/j.molcel.2011.07.012) Copyright © 2011 Elsevier Inc. Terms and Conditions