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. 2015 Jul 3;290(27):16906-17.
doi: 10.1074/jbc.M115.642363. Epub 2015 May 20.

Hippo Component TAZ Functions as a Co-repressor and Negatively Regulates ΔNp63 Transcription through TEA Domain (TEAD) Transcription Factor

Ivette Valencia-Sama  1 , Yulei Zhao  1 , Dulcie Lai  1 , Helena J Janse van Rensburg  1 , Yawei Hao  1 , Xiaolong Yang  2
Affiliations

Hippo Component TAZ Functions as a Co-repressor and Negatively Regulates ΔNp63 Transcription through TEA Domain (TEAD) Transcription Factor

Ivette Valencia-Sama et al. J Biol Chem. .

Abstract

Transcriptional co-activator with a PDZ binding domain (TAZ) is a WW domain-containing transcriptional co-activator and a core component of an emerging Hippo signaling pathway that regulates organ size, tumorigenesis, metastasis, and drug resistance. TAZ regulates these biological functions by up-regulating downstream cellular genes through transactivation of transcription factors such as TEAD and TTF1. To understand the molecular mechanisms underlying TAZ-induced tumorigenesis, we have recently performed a gene expression profile analysis by overexpressing TAZ in mammary cells. In addition to the TAZ-up-regulated genes that were confirmed in our previous studies, we identified a large number of cellular genes that were down-regulated by TAZ. In this study, we have confirmed these down-regulated genes (including cytokines, chemokines, and p53 gene family members) as bona fide downstream transcriptional targets of TAZ. By using human breast and lung epithelial cells, we have further characterized ΔNp63, a p53 gene family member, and shown that TAZ suppresses ΔNp63 mRNA, protein expression, and promoter activity through interaction with the transcription factor TEAD. We also show that TEAD can inhibit ΔNp63 promoter activity and that TAZ can directly interact with ΔNp63 promoter-containing TEAD binding sites. Finally, we provide functional evidence that down-regulation of ΔNp63 by TAZ may play a role in regulating cell migration. Altogether, this study provides novel evidence that the Hippo component TAZ can function as a co-repressor and regulate biological functions by negatively regulating downstream cellular genes.

Keywords: Hippo pathway; cell migration; oncogene; transcription coactivator; transcription corepressor.

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Figures

FIGURE 1.
FIGURE 1.
Validation of target genes negatively regulated by TAZ using real time qRT-PCR. A, Western blot analysis of TAZ expression in MCF10A cells. MCF10A cells were stably infected with lentivirus expressing WPI vector control or TAZ-HA. Western blot analysis was performed by using anti-TAZ antibody. β-Actin was used as an internal loading control. B and D, qRT-PCR analysis of TAZ (B) and its down-regulated cellular gene mRNA expression (D). Total RNAs were extracted from MCF10A-WPI and MCF10A-TAZ cells. mRNA levels were measured by real time qRT-PCR using gene-specific primers (Table 1). Relative expression levels of mRNA in MCF10A-TAZ (black bars) were compared with MCF10A-WPI control cells (white bars). Data are represented as relative -fold decrease. The experiment was performed in duplicate, and error bars represent S.D. from each set of duplicates. Statistical differences in mRNA levels between MCF10A-WPI and MCF10A-TAZ cells were analyzed by Student's t test. *, statically significant difference (p < 0.05). C, heat map for genes down-regulated by TAZ.
FIGURE 2.
FIGURE 2.
Validation of ΔNp63 as a downstream transcriptional target of TAZ. A, p63 protein expression levels were assessed in MCF10A cells overexpressing WPI control or TAZ using anti-p63 antibody. MCF-7 protein cell lysate was used as a negative control for p63 staining. COS7 cells transfected with TAp63-FLAG or ΔNp63-FLAG were used as positive controls. β-Actin was used as an internal loading control. B, ΔNp63 and TAZ protein levels were assessed in MCF10A cells in the presence (+) or absence (−) of Dox-mediated inducible expression of wild-type (TAZ-WT) or constitutively active TAZ (TAZ-S89A). C, expression of ΔNp63 in MCF10A mammary and HBE135 lung epithelial cells. TAZ expression was induced with Dox (+) in MCF10A-TAZ or HBE135-TAZ-S89A cells. D, knockdown of TAZ in breast and lung cancer cells caused enhanced protein expression of ΔNp63. HCC38 breast and A549 lung cancer cells were transiently transfected with control siRNA (siCtrl) or siRNA against TAZ (siTAZ). Three days after transfection, cells were subjected to protein extraction and Western blot analysis using anti-TAZ or anti-p63 antibody. E, TAZ suppresses ΔNp63 promoter activity. SK-BR3 cells grown in a 12-well plate were transfected with ΔNp63-luc alone (0.1 μg) or in combination with increasing amounts (0, 0.1, and 0.4 μg) of TAZ followed by the Dual-Luciferase assay. -Fold changes were calculated by normalizing SK-BR3 cells transfected with ΔNp63-luc alone to those transfected with TAZ. The experiment was performed in triplicate. The experiment was performed in triplicate, and error bars represent S.D. from each set of triplicates. *, statistically significant difference (p < 0.05).
FIGURE 3.
FIGURE 3.
TEAD binding domain is essential for TAZ-induced transcriptional repression of ΔNp63. A, TEAD binding domain is critical for ΔNp63 repression. Constitutively active TAZ-S89A with mutations in TEAD binding (S89A-F52A/F53A) or WW (S89A-WWm) domains were induced with Dox in MCF10A cells. ΔNp63 and TAZ protein expression was assessed and compared with MCF10A cells with inducible expression of TAZ-S89A (S89A). β-Actin was used as an internal loading control. B, qRT-PCR analysis of ΔNp63 mRNA in MCF10A cells expressing WPI, TAZ, or TAZ-F52A/F53A. C, TEAD binding mutant of TAZ abolishes TAZ-mediated suppression of ΔNp63 promoter. SK-BR3 cells were transfected with ΔNp63-luc alone or in combination with wild-type TAZ (TAZ) or its TEAD binding mutant (TAZ-F52A/F53A). Promoter activity was measured as described in Fig. 2E. The experiment was performed in triplicate. D, qRT-PCR analysis of TAZ-down-regulated genes in MCF10A-WPI, TAZ, or TAZ-F52A/F53A cells. Procedures and data analyses were performed as described in Fig. 1B. The experiment was performed in triplicate, and error bars represent S.D. from each set of triplicates. *, statistically significant difference (p < 0.05).
FIGURE 4.
FIGURE 4.
TEAD-dependent suppression of ΔNp63 by TAZ. A, TEAD knockdown diminishes TAZ-induced repression of ΔNp63 protein. Transient siRNA knockdown of TEAD1/3/4 (siTEAD) was performed in MCF10A cells with inducible expression of TAZ-S89A. An siRNA targeting a nonspecific sequence was used as a negative control (siCtrl). Twenty-four hours post-transfection, cells were induced (+) or not (−) with Dox. Protein was extracted 48 h postinduction, and ΔNp63 expression was assessed in cells with and without TAZ-S89A expression. β-Actin was used as an internal loading control. B, knockdown of TEAD abolishes TAZ-induced suppression of ΔNp63 mRNA. qRT-PCR analysis of ΔNp63 mRNA was performed. Cell lines and treatment conditions were as described in A. C, knockdown of TEAD by siRNA partially blocks TAZ-induced suppression of ΔNp63 promoter activity. D, expression of TEAD in MCF10A-WPI, MCF10A-TAZ, SK-Luci6, and SK-BR3 cells. E, TAZ fails to inhibit TAZ promoter in TEAD-negative SK-Luci6 cells. Luciferase analysis was performed as described in Fig. 2E. The experiment was performed in triplicate, and error bars represent S.D. from each set of triplicates. *, statistically significant difference (p < 0.05).
FIGURE 5.
FIGURE 5.
TAZ and TEAD are recruited on ΔNp63 promoter through TREs to directly suppress ΔNp63 transcription. A, TEADs repress ΔNp63 promoter activity. SK-BR3 cells were transfected with ΔNp63-luc alone or in combination with TEAD1, TEAD2, TEAD3, or TEAD4, and luciferase assay was performed as described in Fig. 2E. B, ChIP analysis of TAZ interaction with the ΔNp63 promoter. DNA and protein were cross-linked after treating MCF10A-WPI and MCF10A-TAZ-S89A-HA cells with 1% formaldehyde. Chromatin and DNA-binding protein were subjected to immunoprecipitation using mouse monoclonal α-HA (F7) antibody followed by PCR and electrophoresis on a 3% agarose gel. 0.2% input chromatin extracted from MCF10A-WPI or MCF10A-TAZ-S89A-HA cells was used as a positive PCR control. C, mapping the TRE in the ΔNp63 promoter. Three potential TREs (TRE1 (GGAAT), TRE2 (CATGCC), and TRE3 (GGTAT)) in the ΔNp63 promoter were mutated (TRE1M (AAAAA), TRE2M (AAAAAA), and TRE3M (AAAAA)) alone or in combination (TRE1/2M). ΔNp63-luc containing WT, TRE1M, TRE2M, TRE3M, or TRE1/2M was transfected alone (−TAZ; control) or together with TAZ (+TAZ) into SK-BR3 cells followed by the Dual-Luciferase assay. Promoter activity is shown relative to control and was calculated as the ratio of relative luciferase units of +TAZ to −TAZ. The mean and S.D. of three experiments are shown. The mean and S.D. (error bars) of three experiments are shown. *, statistically significant difference (p < 0.05) between relative luciferase units of +TAZ and −TAZ.
FIGURE 6.
FIGURE 6.
Suppression of ΔNp63 transcription by TAZ through modulation of chromatin acetylation rather than VGLL4. A, knockdown of VGLL4 by siRNA. MCF10A-TAZ-S89A cells were transfected with control siRNA (siCtrl) or siRNA against VGLL4 (siVGLL4) followed by incubation in the absence (−) or presence (+) of Dox for 1 day. Cells were subjected to protein extraction and Western blot analysis using anti-p63 and anti-VGLL4 antibodies. B, qRT-PCR analysis of ΔNp63 mRNA. Experimental procedures were as described in A. C, levels of ΔNp63 mRNA after treatment of cells with histone deacetylase (HDAC) and histone methyltransferase inhibitors. MCF10A-TAZ-S89A cells were untreated or treated with trichostatin A (TSA) (300 nm) or UNC0631 (20 μm) in the absence (−) or presence (+) of Dox for 1 day followed by qRT-PCR analysis. Data analysis was described in Fig. 1B. D, interaction of TAZ with histone deacetylase complex. Co-immunoprecipitation analysis was performed by immunoprecipitation (IP) of TAZ-HA in 250 μg of protein lysates extracted from MCF10A-WPI and MCF10A-TAZ-S89A-HA cells using anti-HA antibody followed by Western blotting using each specific antibody against each protein of the histone deacetylase complex. The membrane was stripped and reprobed with anti-HA antibody to see whether TAZ-S89A-HA was pulled down from MCF10A-TAZ-S89A-HA rather than WPI cells. About 1100 of input protein lysate (2.5 μg) was also subjected to Western blotting. The experiment was performed in triplicate, and error bars represent S.D. from each set of triplicates. *, statistically significant difference (p < 0.05).
FIGURE 7.
FIGURE 7.
Reintroduction of ΔNp63 partially recues TAZ-mediated cell migration. A, Western blot analysis of ΔNp63 expression. MCF10A-TAZ cells were infected with lentivirus expressing ΔNp63 (MCF10A-TAZ-ΔNp63). Protein was extracted from these cells, and ΔNp63 expression was compared with MCF10A-WPI and MCF10A-TAZ cells. β-Actin was used as an internal loading control. B, ΔNp63 reintroduction in TAZ-overexpressing cells partially rescues TAZ-induced increased cell migration. MCF10A-WPI, MCF10A-TAZ, and MCF10A-TAZ-ΔNp63 cells were plated to confluence and starved in 2% horse serum overnight. A wound healing assay was performed, and cell migration was analyzed between cells at different time points (0, 20, and 40 h). C, quantification of cell migration. Cell migration distance (pixels) was quantified in all cells as described in B. The experiment was performed in triplicate, and error bars represent S.D. from each set of triplicates. *, statistically significant difference (p < 0.05) between MCF10A-TAZ and MCF10A-WPI or MCF10A-TAZ-ΔNp63. D, TEAD-dependent increased cell migration by TAZ. Cell migration analyses were performed using the established cell lines and conditions described in Fig. 3A. E, overexpression of TAZ-S89A causes increased cell migration in HBE135 cells. Wound healing analyses were performed in cell lines described in Fig. 2C. F, knockdown of TAZ in HCC38 and A549 cells decreases cell migration. G and H, overexpression of TAZ-S89A induces EMT in both MCF10A (G) and HBE135 (H) cells.
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