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

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 27, pp. 16906 –16917, July 3, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Hippo Component TAZ Functions as a Co-repressor and Negatively Regulates ⌬Np63 Transcription through TEA Domain (TEAD) Transcription Factor*□ S

Received for publication, January 30, 2015, and in revised form, May 6, 2015 Published, JBC Papers in Press, May 20, 2015, DOI 10.1074/jbc.M115.642363

Ivette Valencia-Sama1,2, Yulei Zhao1,3, Dulcie Lai, Helena J. Janse van Rensburg, Yawei Hao, and Xiaolong Yang4 From the Department of Pathology and Molecular Medicine, Queen’s University, Kingston, Ontario K7L 3N6, Canada Background: TAZ functions as a co-activator by up-regulating downstream transcriptional targets. Results: TAZ can negatively regulate transcription of many genes such as ⌬Np63 through TEAD transcription factor. Conclusion: TAZ can also function as a transcriptional co-repressor. Significance: Our findings open a new avenue for TAZ function in cancer.

* This work was supported in part by Canadian Institute of Health Research (CIHR) Grant 119325 and Canadian Breast Cancer Foundation, Canada (to X. Y.). □ S This article contains supplemental Table S1. 1 Both authors contributed equally to this work. 2 Supported by the CIHR/Terry Fox Foundation Training Program in Transdisciplinary Cancer Research and a Queen’s graduate studentship. 3 Supported by an Ontario Trillium graduate scholarship. 4 To whom correspondence should be addressed: Dept. of Pathology and Molecular Medicine, Queen’s University, Richardson Lab 201, 88 Stuart St., Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-6000 (ext. 75998), Fax: 613-533-2970; E-mail: [email protected].

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The Hippo pathway was originally discovered in Drosophila as an evolutionarily conserved tumor suppressor pathway that acts as a key regulator of organ size control (1, 2). This signaling pathway has been shown to control many biological functions such as cell proliferation, apoptosis, cell-cell contact inhibition, stem cell self-renewal, and tissue regeneration (2–10). In mammals, cell-cell contact or increased actin polymerization can activate mammalian sterile-20-like kinase 1/2, which subsequently activates adaptor proteins Mob1A/1B and scaffold protein salvador (Sav1) to promote the phosphorylation and activation of large tumor suppressor 1/2 kinases. In turn, large tumor suppressor 1/2 phosphorylate downstream transcriptional co-activators transcriptional co-activator with a PDZ binding domain (TAZ)5 and its paralog yes-associated protein (YAP) to promote their cytoplasmic retention and subsequent degradation (11–14). Conversely, dephosphorylated YAP and TAZ are able to enter the nucleus where they interact with multiple transcription factors and exert high transactivation activity. TAZ is a widely characterized oncogene that is overexpressed or dysregulated in several cancer types including breast (15, 16), lung (17, 18), colorectal (19), and thyroid (20). It is proposed as a major regulator of cell proliferation, cell migration and invasion, epithelial-mesenchymal transition (EMT), human embryonic stem cell renewal, and drug resistance (21– 27). Within the N terminus of TAZ lies a TEAD binding domain (TBD) responsible for the interaction with the TEAD family of transcription factors. Mounting evidence over the years has supported TEAD family members as one of the most common binding partners of TAZ; they play crucial roles in mediating many TAZ functions including cellular growth, proliferation, and oncogenic transformation (28 –31). The mechanisms underlying TAZ-mediated transcriptional activation of downstream genes through its interaction with transcription factors have been often studied and observed by many research groups. However, there has been little interest in elucidating novel tar5

The abbreviations used are: TAZ, transcriptional co-activator with a PDZ binding domain; YAP, yes-associated protein; EMT, epithelial-mesenchymal transition; TBD, TEAD binding domain; Dox, doxycycline; VGLL4, vestigial-like protein 4; qRT-PCR, quantitative reverse transcription-PCR; TRE, TEAD response element.

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Downloaded from http://www.jbc.org/ at Duke University on November 14, 2015

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 ⌬Np63, a p53 gene family member, and shown that TAZ suppresses ⌬Np63 mRNA, protein expression, and promoter activity through interaction with the transcription factor TEAD. We also show that TEAD can inhibit ⌬Np63 promoter activity and that TAZ can directly interact with ⌬Np63 promoter-containing TEAD binding sites. Finally, we provide functional evidence that down-regulation of ⌬Np63 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.

TAZ Suppresses ⌬Np63 Transcription as a Co-repressor gets negatively regulated by TAZ and addressing their molecular mechanisms and functional implications in tumorigenesis. In this study, we have identified ⌬Np63, a member of the p53 tumor suppressor family, as a significantly down-regulated target in TAZ-overexpressing breast and lung epithelial cells. Moreover, we show that TAZ-induced repression of ⌬Np63 transcription is mediated by the TEAD family of transcription factors and that reintroduction of ⌬Np63 into TAZ-overexpressing cells partially rescues TAZ-induced cell migration. Together, our findings provide the first evidence that TAZ can directly negatively regulate cellular gene transcription by interacting with TEAD transcription factor.

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Experimental Procedures Plasmid Construction and Site-directed Mutagenesis—The promoter region of ⌬Np63 (nucleotide positions ⫺1500 to ⫹40) was amplified by PCR from genomic DNA extracted from MCF10A human immortalized mammary cells using the following primers: ⌬Np63-pr sense primer (5⬘-ATGGTACCTATGTGTGAAGAAATGAATGTTTTGTCTG-3⬘; KpnI site is underlined) and ⌬Np63-pr antisense primer (5⬘-AATCTCGAGAAGATAACAGAACTCAAGTCCCTCTCTCTC-3⬘; XhoI site is underlined). The PCR products were digested with KpnI/XhoI and subsequently cloned into the KpnI/XhoI sites of the pGL3-Basic luciferase reporter vector (Promega). Human ⌬Np63 cDNA (Addgene) was cloned into the XhoI/MluI sites of the doxycycline (Dox)-inducible pTRIPZ lentiviral vector (Open Biosystems). Mutation of TAZ-F52A/F53A (F, phenylalanine; A, alanine) was performed by overlapping PCR using TAZ-mutagenic primers. TAZ-S89A-F52A/F53A mutant was created using overlapping PCR and subsequently cloned into the XhoI/MluI sites of the pTRIPZ lentiviral vector. Cell Culture—MCF10A (human immortalized epithelial breast) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture F-12 Ham (Sigma-Aldrich) supplemented with 5% horse serum, 1% penicillin-streptomycin, 2.5 mM L-glutamine, 10 ␮g/ml insulin, 0.5 ␮g/ml hydrocortisone, 100 ng/ml cholera toxin, and 20 ng/ml human EGF. SK-BR3 (human breast cancer) cells were cultured in McCoy’s 5A modified medium (Sigma-Aldrich) supplemented with 2.2 g/liter sodium bicarbonate, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin. SK-Luci6 (human anaplastic lung cancer), HEK293T (human embryonic kidney), COS7 (monkey fibroblast-like kidney), A549 (lung adenocarcinoma), and HCC38 (human ductal breast carcinoma) cells were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37 °C with 5% CO2. Lentiviral Production, Infection, and Establishment of Cell Lines with Stable Overexpression of Cellular Genes—Lentiviral production, purification, titration, and infection of overexpressing constructs were performed as described (11). Generation of ⌬Np63-overexpressing stable cell lines was performed by infecting TAZ-overexpressing MCF10A cells with lentivirus expressing Dox-inducible ⌬Np63 (pTRIPZ vector) at a multiplicity of infection of 2. Generation of stable cell lines with overexpression of TEAD binding mutants of TAZ was performed by infecting TAZ-low MCF10A cells with lentivirus expressing

TAZ-F52A/F53A-HA (WPI vector) or Dox-inducible TAZS89A-F52A/F53A-HA at a multiplicity of infection of 2. Cells were selected 48 h postinfection using 1 ␮g/ml puromycin. Microarray and Data Analysis—Gene expression profile analysis by microarray and data analysis were as described (32). Transient Knockdown of Gene Expression by Small Interfering RNA (siRNA)—To knock down TEAD1/3/4, 5 ⫻ 104 SK-BR3 cells were transfected with 50 nM TEAD1/3/4 siRNA (5⬘-CACAAGACGUCAAGCCUUU-3⬘ (sense)/5⬘-UUGUGGAUGAAGUUGAUCAUU-3⬘ (antisense)) (GE Healthcare) using Lipofectamine RNAiMAX transfection reagent (Life Technologies) according to the manufacturer’s protocol. Efficiency of knockdown was assessed by Western blotting 48 h post-transfection. Reagents, Antibodies, Western Blotting, and Co-immunoprecipitation—Trichostatin A and UNC0631 were purchased from Sigma. The mouse monoclonal antibodies used in this study were obtained from the following companies: anti-TAZ from BD Pharmingen; anti-p63 (4A4), anti-vestigial-like protein 4 (VGLL4), and anti-FLAG (M2) from Sigma-Aldrich; antiTEAD (TEF-1) from Abcam; and anti-HA (F7) from Santa Cruz Biotechnology. Anti-histone acetylation component antibodies were obtained from Cell Signaling Technology. Protein extraction, Western blot analyses, and co-immunoprecipitation were performed as described (11). RNA Isolation and Quantitative Reverse Transcription-PCR (qRT-PCR)—Cells were grown to about 70 –90% confluence. RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. RNA quantitation and quality were assessed by spectrophotometry and RNase-free gel electrophoresis. qRT-PCR analyses were performed in duplicates of 200 ng/␮l RNA/sample/reaction with 200 nM genespecific forward and reverse primers (Table 1) using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen) and the Applied Biosystems ViiA 7 Real-Time PCR System. 18S ribosomal RNA (rRNA) expression was used as an internal control. mRNA expression levels were calculated as described (11) and are shown as -fold change. Dual-Luciferase Assay—Triplicates of 5 ⫻ 104 cells/well SK-BR3 or SK-LuCi6 cells were seeded in 12-well plates and transfected with ⌬Np63-luc or its mutants (0.1 ␮g) alone or in combination with TAZ (0.2 ␮g), TAZ (0.2 ␮g) plus TEAD (0.1 ␮g), or their respective mutants using PolyJet reagent (SignaGen). 10 ng/well Renilla luciferase vector (pRL-TK) was used as an internal transfection control. Luciferase activity was assessed 48 h post-transfection using a Turner Biosystem 20/20 luminometer and the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Chromatin Immunoprecipitation (ChIP) Assay—A ChIP-IT Express enzymatic kit (Active Motif) was used for ChIP analysis of TAZ-S89A and ⌬Np63 promoter interaction. MCF10A cells expressing WPI or TAZ-S89A were grown to 70 – 80% confluence on 150-mm dishes. Cells were treated with 1% formaldehyde, lysed, harvested, and homogenized using a Dounce homogenizer according to the manufacturer’s protocol. DNA was enzymatically sheared, and the fragmented chromatin was incubated with 2 ␮g of mouse anti-HA (F7) monoclonal anti-

TAZ Suppresses ⌬Np63 Transcription as a Co-repressor TABLE 1 Primers used for qRT-PCR F, forward; R, reverse. F R

GGACTGTATCCGCATGCA GACCTGGGCTGTGCGTAG GAGTTCTGTTATCTTCTTAAG TGTTCTGCGCGTGGTCTG

BMP2 F R

TGCGCATGCTTCCACCATGAAG TCTGCTGSGGTGATAAACTCC

CXCL1 F R

AGTCATAGCCACACTCAAGAATGG GATGCAGGATTGAGGCAAGC

CXCL2 F R

CGCCCAAACCGAAGTCATAG AGACAAGCTTTCTGCCCATTCT

CXCL3 F R

TCCCCCATGGTTCAGAAAATC GGTGCTCCCCTTGTTCAGTAT

IL1␣ F R

TGTGACTGCCCAAGATGAAG CTTAGCGCCGTGAGTTTCCC

IL1␤ F R

GAAGCTGATGGCCCTAAACAG GAAGCCCTTTGCTGTAGTGGT

IL6 F R

TCCTCGACGGGCATCTCAGCC ATCTTTGGAAGGTTCAGGTTG

IL8 F R

CGGAAGGAACCATCTCACTG AGCACTCCTTGGCAAAACTG

GJA1 F R

ACACCTTCCCTCCAGCAGTT GGAGTTCAATCACTTGGCGT

body. Chromatin was eluted, reverse cross-linked, and treated with Proteinase K. Amplification of the ⌬Np63 promoter was performed by PCR using the following primers: (5⬘-ATGGTACCGTCTGTCTCCTGGGTTTG-3⬘ (sense) and 5⬘-GTGCACTTTCTTATGAAAGAGAC-3⬘ (antisense)). The PCR products were run on a 3% ethidium bromide-agarose gel and visualized under UV light using the Gel Doc system. Wound Healing Cell Migration Assay—MCF10A-WPI, MCF10A-TAZ, and MCF10A-TAZ-⌬Np63 cells were grown to 80% confluence, serum-starved overnight in 2% horse serum, and scratch-wounded 24 h later using a P20 pipette tip. Cell migration was monitored, and pictures were taken at 0, 20, or 40 h under white light at 10⫻ magnification using a Nikon Eclipse TE-2000U inverted microscope and a Nikon Coolpix 990 camera. Distance migrated (pixels) was measured with Adobe Photoshop software. MCF10A-TAZ-S89A, MCF10ATAZ-S89A-F52A/F53A, MCF10A-TAZ-S89A-WWm, and HBE135-TAZ-S89A cells were untreated (⫺) or treated (⫹) with Dox (1 ␮g/ml); A549 and HCC38 cells were infected with siRNA against TAZ; and cells were subsequently plated and scratched-wounded as described above. Pictures were taken at 24 or 48 h. Statistical Analysis—Significant differences were analyzed by Student’s t test, and differences in mRNA levels between MCF10A-TAZ and its mutants and in promoter activities were calculated using analysis of variance tests. A p value ⬍0.05 was regarded as statistically significant.


6

X. Yang, unpublished data.



⌬Np63 F R

Results Identification and Validation of Target Genes Negatively Regulated by TAZ—To identify downstream genes mediating TAZ function, we performed a 44,000 whole human genome microarray profiling (32) using RNAs from MCF10A stably expressing WPI empty vector control (MCF10A-WPI) or wildtype TAZ (MCF10A-TAZ). Enhanced TAZ mRNA and protein expression levels were confirmed by qRT-PCR and Western blotting (Fig. 1, A and B). Although 390 genes were up-regulated by TAZ (32), surprisingly, about 328 cellular genes were also found to be down-regulated by TAZ (Fig. 1C and supplemental Table S1). Based on their functional relevance in tumorigenesis, we confirmed several target genes from our DNA microarray results using real time qRT-PCR. Interestingly, we identified several proinflammatory cytokines and chemokines including IL1␣/␤, IL6, IL8, CXCL1/2/3, and BMP2 as well as p63 isoforms ⌬Np63 and TAp63 as significantly down-regulated (1.5–10 ⫾ 0 – 0.05-fold) targets in MCF10A-TAZ cells (Fig. 1D). Among these, proinflammatory cytokines IL1␣ and IL1␤ showed the most significant decrease in their relative mRNA expression, suggesting a possible involvement of TAZ in immune and inflammatory responses. Surprisingly as well, the p53 family members TAp63 and its N-terminal truncated form ⌬Np63 showed an important TAZ-induced suppression in mRNA expression levels. Because p63 has been previously suggested as a marker of epithelial breast carcinoma and plays important roles in tumorigenesis and metastasis (33, 34), we sought to elucidate the molecular mechanisms and functional implications of TAZ-mediated repression of p63. ⌬Np63 Is a Novel Down-regulated Target of TAZ—To further confirm TAZ-mediated suppression of p63 in MCF10A cells, we performed protein analysis using Western blotting. Protein lysates from MCF10A-WPI and MCF10-TAZ cells were analyzed together with MCF-7 p63 negative control and COS7 cells transfected with TAp63-FLAG and ⌬Np63-FLAG plasmids. We identified ⌬Np63 rather than TAp63 as the p63 protein isoform predominantly suppressed by TAZ in MCF10A-TAZ cells (Fig. 2A). Similarly, ⌬Np63 protein expression was highly decreased in MCF10A cells after transient induction of wildtype TAZ (TAZ-WT) or constitutively active TAZ (TAZ-S89A) by Dox (Fig. 2B), suggesting that suppression of ⌬Np63 by TAZ is not caused by viral infection or puromycin selection during establishment of stable lines. As expected, inducible expression of TAZ-S89A displayed a stronger effect on ⌬Np63 repression compared with TAZ-WT. To characterize ⌬Np63 as a bona fide down-regulated target gene of TAZ in other cell type, we analyzed ⌬Np63 protein expression levels in HBE135 human bronchial epithelial lung cells containing inducible expression of TAZ-S89A (Fig. 2C). Interestingly, ⌬Np63 expression was also repressed in HBE135 cells after TAZ induction, similar to the effect observed in MCF10A cells. Furthermore, we performed transient TAZ knockdown using siRNAs (siTAZ) in HCC38 human epithelial breast cancer cells and A549 human lung adenocarcinoma cells that expressed high levels of TAZ (35).6

TAZ Suppresses ⌬Np63 Transcription as a Co-repressor

FIGURE 2. Validation of ⌬Np63 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 ⌬Np63-FLAG were used as positive controls. ␤-Actin was used as an internal loading control. B, ⌬Np63 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 ⌬Np63 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 ⌬Np63. 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 ⌬Np63 promoter activity. SK-BR3 cells grown in a 12-well plate were transfected with ⌬Np63-luc alone (0.1 ␮g) or in combination with increasing amounts (0, 0.1, and 0.4 ␮g) of TAZ followed by the Dual-Luciferase assay. -Fold changes were calculated by normalizing SK-BR3 cells transfected with ⌬Np63-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).



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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. ␤-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.


⌬Np63 protein levels were significantly increased after TAZ was knocked down in both cell lines (Fig. 2D). Finally, we sought to elucidate the effects of TAZ overexpression on ⌬Np63 promoter activity using the Dual-Luciferase assay. The promoter region of ⌬Np63 was cloned from MCF10A genomic DNA into a luciferase reporter vector (⌬Np63-luc), which was transiently transfected into TAZ-low SK-BR3 breast cancer cells alone or in combination with increasing dosages of TAZ (Fig. 2E). Concordant with our previous results, TAZ showed a dosage-dependent suppression of ⌬Np63-luc activity, suggesting that TAZ causes reduced ⌬Np63 expression by suppressing its promoter activity. Together, these results strongly suggest ⌬Np63 as a bona fide downstream target negatively regulated by TAZ in several breast and lung cell lines. TBD Is Necessary for TAZ-induced Negative Regulation of ⌬Np63—TAZ was originally identified as a transcriptional coactivator that lacks a DNA binding domain. Thus, to modulate transcription of downstream cellular genes, TAZ requires interactions with transcription factors through its TBD or WW (W, tryptophan) domain. Previous studies have shown that TAZ interacts with members of the TEAD family of transcription factors (TEAD1– 4) through seven conserved residues in the TBD of TAZ (23, 28). Among these, two phenylalanine residues located at positions 52 and 53 (Phe-52/53) were shown to be critical for TAZ-TEAD binding. Therefore, we examined whether the effect of TAZ on ⌬Np63 repression could be abolished in TBD mutants of TAZ. We generated a missense mutation in TAZ Phe-52 and Phe-53 residues (TAZ-F52A/F53A) to


abolish TEAD interaction with TAZ. We then established MCF10A cells stably expressing TAZ-F52A/F53A and examined the effect of this TAZ mutant on mRNA and protein expression of ⌬Np63. qRT-PCR and Western blot analysis showed that loss of interaction with TEAD in TAZ-F52A/F53A mutant completely abolished TAZ-induced suppression of both protein and mRNA of ⌬Np63 (Fig. 3, A and B). The TAZF52A/F53A mutant also showed inability to suppress ⌬Np63 promoter activity (Fig. 3C). Interestingly, loss of interaction with TEAD in TAZ-F52A/F53A had a dominant-negative effect and activated transcription (1–28 ⫾ 0 –1-fold) of many down-regulated genes including ⌬Np63, BMP2, CXCL2, CXCL3, IL1␣, and IL1␤ (Fig. 3, B and D). Conversely, overexpression of TAZ-F52A/F53A can still suppress TAp63 mRNA, suggesting that TAZ regulates TAp63 and ⌬Np63 differently and that TAZ may down-regulate TAp63 independently of TEAD. Next, we sought to explore whether the TBD of TAZ was the only domain responsible for p63 suppression. Because the TAZ WW domain has been shown to be critical for gene transcription regulation through interaction with (L/P)PXY (L, lysine; P, proline; X, any amino acid; Y, tyrosine) motif-containing transcription factors such as TTF1 and Pax8 (36 –38), we tested whether a WW domain TAZ mutant (TAZ-WWm) containing two residue mutations, W152A and P155A, had any effect on TAZ-induced suppression of ⌬Np63. MCF10A cells stably expressing TAZ-S89A-WWm were established by infecting MCF10A cells with lentivirus expressing inducible TAZ-S89A-WWm (MCF10A-TAZ-S89A-WWm). Similarly to VOLUME 290 • NUMBER 27 • JULY 3, 2015


FIGURE 3. TEAD binding domain is essential for TAZ-induced transcriptional repression of ⌬Np63. A, TEAD binding domain is critical for ⌬Np63 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. ⌬Np63 and TAZ protein expression was assessed and compared with MCF10A cells with inducible expression of TAZ-S89A (S89A). ␤-Actin was used as an internal loading control. B, qRT-PCR analysis of ⌬Np63 mRNA in MCF10A cells expressing WPI, TAZ, or TAZ-F52A/F53A. C, TEAD binding mutant of TAZ abolishes TAZ-mediated suppression of ⌬Np63 promoter. SK-BR3 cells were transfected with ⌬Np63-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).


the effect observed in MCF10A-TAZ-S89A, in the presence of Dox, the WW domain mutant TAZ-WWm effectively suppressed ⌬Np63 expression (Fig. 3A). Together, these findings suggest that the TBD, but not the WW domain, of TAZ is essential for TAZ-induced repression of ⌬Np63. TEAD Is Essential for TAZ-induced Suppression of ⌬Np63— To directly confirm whether TAZ suppresses ⌬Np63 through TEAD, we performed transient knockdown of TEAD using a previously used siRNA simultaneously targeting TEAD1, TEAD3, and TEAD4 (siTEAD) in MCF10A cells with inducible expression of constitutively active TAZ-S89A. Although ⌬Np63 protein and mRNA were significantly repressed by TAZ-S89A in the presence of Dox in cells expressing a siRNA negative control (siCtrl), TAZ-S89A-mediated repression of ⌬Np63 seemed to be abolished in MCF10A cells with TEAD knockdown (Fig. 4, A and B). In addition, TEAD knockdown also diminished TAZ-induced suppression of ⌬Np63 promoter (Fig. 4C). Moreover, although TAZ can suppress ⌬Np63-luc activity in TEAD-positive SK-BR3 cells, its suppression on ⌬Np63 promoter was abolished in TEAD-negative SK-Luci6 lung cancer cells (Fig. 4, D and E). Together, these studies strongly suggest that TEAD is essential for TAZ-induced suppression of ⌬Np63. TAZ Suppresses ⌬Np63 by Directly Binding to the ⌬Np63 Promoter through TEAD—Next, we tested whether TAZ suppresses ⌬Np63 transcription by directly interacting with ⌬Np63 promoter through TEAD. Because TEAD is required JULY 3, 2015 • VOLUME 290 • NUMBER 27

for TAZ-induced suppression of ⌬Np63 transcription, we first tested whether TEAD can directly suppress ⌬Np63 promoter activity by transfecting ⌬Np63-luc reporter alone or together with TEAD1, TEAD2, TEAD3, or TEAD4 into SK-BR3 cells. Significantly, all TEADs showed ⌬Np63 promoter repression (Fig. 5A), although TEAD1/3/4 exerted a more dramatic effect than TEAD2 in ⌬Np63 repression with over 3.5 ⫾ 1.2-fold decrease in the promoter activity, suggesting that TEADs are involved in ⌬Np63 repression. To confirm that TAZ-TEAD indeed directly binds to ⌬Np63 promoter, we performed a ChIP assay in MCF10A cells expressing WPI vector or TAZS89A-HA using anti-HA antibody and primers flanking a TEAD response element (TRE) (Fig. 5C, TRE1). Interestingly, our ChIP assay showed that TAZ-S89A could indeed be coimmunoprecipitated with the ⌬Np63 promoter DNA in vivo (Fig. 5B). After further examination of ⌬Np63 promoter sequences, we identified three potential TREs (TRE1, TRE2, and TRE3) in the ⌬Np63 promoter and mutated them individually (TRE1M, TRE2M, or TRE3M) or in combination (TRE1/2M; Fig. 5C). Although mutation of TRE1 or TRE2 rather than TRE3 partially blocked TAZ-induced suppression of ⌬Np63 promoter, combined mutations of both TRE1 and TRE2 (TRE1/2M) completely abolished TAZ-induced suppression of ⌬Np63 promoter (Fig. 5C), thus suggesting that TAZ-TEAD complex binds to TRE1 and TRE2 to suppress ⌬Np63 transcription. JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 4. TEAD-dependent suppression of ⌬Np63 by TAZ. A, TEAD knockdown diminishes TAZ-induced repression of ⌬Np63 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 ⌬Np63 expression was assessed in cells with and without TAZ-S89A expression. ␤-Actin was used as an internal loading control. B, knockdown of TEAD abolishes TAZ-induced suppression of ⌬Np63 mRNA. qRT-PCR analysis of ⌬Np63 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 ⌬Np63 promoter activity. D, expression of TEAD in MCF10A-WPI, MCF10ATAZ, 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).


Modulation of Deacetylation Is Critical for TAZ-TEAD-induced Suppression of ⌬Np63—Two recent studies suggest that TEAD or its Drosophila homolog Scallop can suppress cellular gene transcription through interaction with transcription cofactor VGLL4 or Tgi, respectively (39, 40). However, VGLL4 knockdown in MCF10A cells could not block TAZ-induced suppression of ⌬Np63 (Fig. 6, A and B), suggesting that VGLL4 is not involved in TAZ-TEAD-induced transcriptional repression of ⌬Np63. Previous studies also suggest that transcriptional suppression of some genes may depend on DNA methylation or histone deacetylation of chromatin of their promoter regions (41– 43). To explore whether TAZ-TEAD-induced ⌬Np63 transcriptional repression is due to chromosome methylation/ acetylation, we treated breast cancer cells with inhibitors of histone modification. Significantly, treatment of cells with histone deacetylase inhibitor trichostatin A rather than histone methyltransferase inhibitor UNC0631 partially rescued TAZinduced suppression of ⌬Np63 transcription (Fig. 6C). Moreover, we have further shown that TAZ directly interacts in vivo with some components (CHD4, MTA1, and RBP46) of the histone deacetylase complex (Fig. 6D).


Functional Implications of TAZ-TEAD Interactions and ⌬Np63 Suppression—Our results have strongly elucidated the role of TEAD and its co-activator TAZ in p63 transcriptional repression, particularly ⌬Np63, in breast and lung epithelial cells. Because TAZ overexpression has been previously correlated with enhanced cell migration and invasion (14, 15, 44) and ⌬Np63 knockdown causes EMT and increased cell migration and metastasis in MCF10A cells or breast cancer (45– 47), we sought to elucidate the functional consequences of TAZTEAD-mediated repression of ⌬Np63 in breast cell migration. First, we reintroduced ⌬Np63 expression in MCF10ATAZ cells by using lentiviral infection. Assessment of similar TAZ and ⌬Np63 protein expression levels was performed by Western blotting (Fig. 7A). Second, we sought to examine the functional implications of ⌬Np63 suppression on cell migration by performing a wound healing assay in MCF10A cells expressing WPI vector control, TAZ, or TAZ plus ⌬Np63. Cell migration was compared at different time points after wound induction. Compared with the WPI control (MCF10A-WPI), TAZ-overexpressing cells (MCF10ATAZ) increased cell migration 9.0 ⫾ 1.5- and 8.8 ⫾ 3.5-fold at 20 and 40 h, respectively. However, this effect was partially VOLUME 290 • NUMBER 27 • JULY 3, 2015


FIGURE 5. TAZ and TEAD are recruited on ⌬Np63 promoter through TREs to directly suppress ⌬Np63 transcription. A, TEADs repress ⌬Np63 promoter activity. SK-BR3 cells were transfected with ⌬Np63-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 ⌬Np63 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 ␣-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 ⌬Np63 promoter. Three potential TREs (TRE1 (GGAAT), TRE2 (CATGCC), and TRE3 (GGTAT)) in the ⌬Np63 promoter were mutated (TRE1M (AAAAA), TRE2M (AAAAAA), and TRE3M (AAAAA)) alone or in combination (TRE1/2M). ⌬Np63-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.


abolished in TAZ-⌬Np63-expressing cells (MCF10A-TAZ⌬Np63) (Fig. 7, B and C). Interestingly, further examination of TAZ and its two domain mutants showed that although Dox induction of TAZS89A (⫹Dox) caused increased cell migration TBD mutant TAZ-F52A/F53A completely abolished TAZ-induced increased cell migration in MCF10A cells (Figs. 3A and 7D). Conversely, WW domain mutant TAZ-WWm can still cause increased cell migration (Fig. 7D), suggesting that TEAD binding domain, rather than WW domain, is essential for TAZinduced increased cell migration. Furthermore, enhanced cell migration is also observed when TAZ-S89A is overexpressed in the presence of Dox (⫹Dox) in HBE135 cells (Figs. 2C and 7E). Moreover, down-regulation of TAZ in A549 and HCC38 cells inhibits cell migration (Fig. 7F). Finally, we have further shown that overexpression of TAZ-S89A in both breast MCF10A and lung HBE135 cells causes EMT, thus suggesting that this TAZinduced increased cell migration is due to loss of cell-cell adhesion (Fig. 7, G and H). In summary, our results suggest that TAZ interacts with TEAD to promote ⌬Np63 suppression and reduced cell-cell adhesion, thus increasing the migratory capacity of cells, which can further lead to metastatic progression in breast and lung cancer cells. JULY 3, 2015 • VOLUME 290 • NUMBER 27

Discussion TAZ Is a Dual Regulator of Gene Transcription—Studies have widely characterized TAZ as a transcriptional co-activator of gene expression. Its ability to interact with a wide range of transcription factors accounts for its multifunctional effects in tumor development and progression. Moreover, TAZ-induced activation of protumorigenic genes such as Cyr61, CTGF, BMP4 (32, 44) and many others has been often reported in the literature. However, our DNA microarray data have uncovered a whole new perspective on TAZ transcriptional regulation. Besides its well studied role as a transcriptional co-activator, our results have suggested a novel function of TAZ in transcriptional repression. This transcriptional duality of TAZ has been previously questioned after observing that TAZ-induced activation of RUNX2 and repression of the transcriptional activity of peroxisome proliferator-activated receptor-␥ were critical for mesenchymal stem cell differentiation (48). Recently, phosphorylation of Tyr-316 of TAZ has been shown to promote TAZ interaction and repression of the transcriptional activity of NFAT5 in response to hyperosmotic stress (49). Although these studies have shed light on the transcriptional repressing potential of TAZ, they have failed to elucidate the molecular mechanisms and oncogenic functions underlying TAZ-inJOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 6. Suppression of ⌬Np63 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 ⌬Np63 mRNA. Experimental procedures were as described in A. C, levels of ⌬Np63 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 ␮M) 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 ␮g 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-TAZS89A-HA rather than WPI cells. About 1⁄100 of input protein lysate (2.5 ␮g) 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).


duced suppression. The fact that our microarray results have shown that transcription of over 320 cellular genes can be suppressed by TAZ has uncovered a new layer of the signaling complexity of TAZ and the Hippo pathway. Furthermore, validation analysis of several target genes including p63 and proinflammatory cytokines and chemokines has indeed confirmed that TAZ expression is also critical for transcriptional inhibition. Moreover, the strong repression of IL1␣/␤ exerted by TAZ has suggested a novel role in modulating the inflammatory response and tumor microenvironment. In fact, TAZ was found to indeed repress IL1␤ promoter in the same manner as shown for ⌬Np63 (data not shown). Collectively, our results have elucidated an underrated role of TAZ as a negative regulator of transcription in breast and lung epithelial cells. Moreover, we have uncovered the duality of TAZ in gene transcription regulation, a trait that has been reported previously for other transcriptional cofactors such as CCAAT-enhancerbinding protein or cAMP-response element-binding protein (CREB)-binding protein and its paralog p300 (50, 51). ⌬Np63 Is a Novel Downstream Target Negatively Regulated by TAZ—Despite the progress made toward elucidating the molecular mechanisms involved in breast cancer development and progression, metastatic cancer cells remain a major obstacle for successful breast cancer treatments. In this context, TAZ has been highly associated with cell acquisition of EMT phenotypes and subsequent metastatic dissemination of breast cells


(14, 15, 32, 52). By regulating gene expression, TAZ has been shown to modulate oncogenic traits in cells. However, the transcriptional downstream targets mediating these TAZ-induced phenotypes remain mostly unexplored. By using a DNA microarray and real time qRT-PCR, we identified p63 isoforms TAp63 and ⌬Np63 as transcriptional targets negatively regulated by TAZ in mammary tumorigenesis. Of these, ⌬Np63 showed the most significant repression and was shown to be the predominant isoform expressed in MCF10A cells. Therefore, we characterized ⌬Np63 as a bona fide negative transcriptional target of TAZ involved in cell migration. First, we have shown that TAZ overexpression in MCF10A non-tumorigenic breast cells causes a significant decrease in ⌬Np63 mRNA expression levels (Fig. 1D). Second, we have shown that overexpression of both wild-type TAZ and its constitutively active mutant TAZS89A suppresses ⌬Np63 protein expression in MCF10A cells, whereas TAZ knockdown caused increased ⌬Np63 in breast and lung cancer cells (Fig. 2, A, B, and D). Finally, we have confirmed that TAZ physically interacts with ⌬Np63 promoter and causes ⌬Np63 transcriptional repression through interaction with TEAD and TRE (Fig. 2E). After confirming that ⌬Np63 is indeed a real downstream target negatively regulated by TAZ, we examined the functional implications of ⌬Np63 down-regulation in mammary epithelial cells. After reintroducing ⌬Np63 protein expression into TAZoverexpressing MCF10A cells, we found that ⌬Np63 could parVOLUME 290 • NUMBER 27 • JULY 3, 2015


FIGURE 7. Reintroduction of ⌬Np63 partially recues TAZ-mediated cell migration. A, Western blot analysis of ⌬Np63 expression. MCF10A-TAZ cells were infected with lentivirus expressing ⌬Np63 (MCF10A-TAZ-⌬Np63). Protein was extracted from these cells, and ⌬Np63 expression was compared with MCF10AWPI and MCF10A-TAZ cells. ␤-Actin was used as an internal loading control. B, ⌬Np63 reintroduction in TAZ-overexpressing cells partially rescues TAZ-induced increased cell migration. MCF10A-WPI, MCF10A-TAZ, and MCF10A-TAZ-⌬Np63 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-⌬Np63. 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.



deacetylation complex (Fig. 6D) and that inhibition of histone deacetylase partially releases TAZ-induced suppression of ⌬Np63 transcription by directly interacting with histone deacetylation complex (Fig. 6C), suggesting that TAZ may suppress gene transcription by activating histone deacetylation and histone deacetylase-mediated chromatin tightening. Besides TEAD-dependent transcriptional suppression by TAZ, we also observed TEAD-independent suppression of some downstream genes such as TAp63 and GJA1 (Fig. 3D). In addition, a recent study reported a ZEB2-dependent suppression of ⌬Np63 promoter by YAP (TAZ paralog) during squamous transdifferentiation of lung epithelial cells (60). These studies suggest that TAZ, like YAP, may also suppress cellular gene transcription through interacting with other transcription factors. Nevertheless, our findings have shed light on the unanticipated complexity of the mechanisms underlying TAZ and TEAD interaction for gene transcriptional regulation and provide convincing evidence that TAZ can exert its function by negatively regulating transcription of downstream cellular genes such as ⌬Np63 through interaction with TEAD transcription factor. Acknowledgments—We thank Dr. Wanji Hong for providing the TAZ-WWm plasmid, Dr. David Berman for using the Applied Biosystems ViiA 7 Real-Time PCR System, and Dr. Robin Hallett for assistance in heat map design. References 1. Yang, X., and Xu, T. (2011) Molecular mechanism of size control in development and human diseases. Cell Res. 21, 715–729 2. Pan, D. (2010) The hippo signaling pathway in development and cancer. Dev. Cell 19, 491–505 3. Badouel, C., and McNeill, H. (2011) SnapShot: the hippo signaling pathway. Cell 145, 484 – 484.e1 4. Barron, D. A., and Kagey, J. D. (2014) The role of the Hippo pathway in human disease and tumorigenesis. Clin. Transl. Med. 3, 25 5. Gomez, M., Gomez, V., and Hergovich, A. (2014) The Hippo pathway in disease and therapy: cancer and beyond. Clin. Transl. Med. 3, 22 6. Halder, G., and Johnson, R. L. (2011) Hippo signaling: growth control and beyond. Development 138, 9 –22 7. Johnson, R., and Halder, G. (2014) The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment. Nat. Rev. Drug Discov. 13, 63–79 8. Mo, J. S., Park, H. W., and Guan, K. L. (2014) The Hippo signaling pathway in stem cell biology and cancer. EMBO Rep. 15, 642– 656 9. Zhao, B., Tumaneng, K., and Guan, K. L. (2011) The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal. Nat. Cell Biol. 13, 877– 883 10. Liu, H., Jiang, D., Chi, F., and Zhao, B. (2012) The Hippo pathway regulates stem cell proliferation, self-renewal, and differentiation. Protein Cell 3, 291–304 11. Hao, Y., Chun, A., Cheung, K., Rashidi, B., and Yang, X. (2008) Tumor suppressor LATS1 is a negative regulator of oncogene YAP. J. Biol. Chem. 283, 5496 –5509 12. Zhao, B., Wei, X., Li, W., Udan, R. S., Yang, Q., Kim, J., Xie, J., Ikenoue, T., Yu, J., Li, L., Zheng, P., Ye, K., Chinnaiyan, A., Halder, G., Lai, Z. C., and Guan, K. L. (2007) Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 21, 2747–2761 13. Chan, E. H., Nousiainen, M., Chalamalasetty, R. B., Schäfer, A., Nigg, E. A., and Silljé, H. H. (2005) The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene 24, 2076 –2086


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tially reverse TAZ-mediated cell migration (Fig. 7, A–C). Specifically, assessment of the migration distance of MCF10A cells expressing TAZ plus ⌬Np63 showed a significantly slower wound closure rate that was more similar to that displayed by MCF10A-WPI control than by MCF10A-TAZ cells. Because multiple genes may regulate breast cancer cell migration, ⌬Np63 re-expression could not completely reverse this TAZmediated phenotype. Nonetheless, our studies strongly suggest that ⌬Np63 is one of the genes involved in TAZ-induced cell migration. These results are consistent with studies showing ⌬Np63 as an inhibitor of cell migration, invasion, and metastatic progression in breast cells (45, 46). Moreover, p63 and particularly ⌬Np63 expression has been specifically observed in normal myoepithelial breast cells, has been proposed as a marker for cell differentiation, and is shown to be down-regulated in non-metaplastic invasive breast carcinomas (33, 53–55). Importantly, TAZ overexpression is highly correlated with breast cancer invasiveness and dissemination (32). Thus, it is likely that TAZ-induced suppression of ⌬Np63 enhances its metastatic potential by promoting EMT and breast cell migration. It will be interesting to further investigate the correlation between the levels of TAZ and ⌬Np63 and whether these could be used as prognostic biomarkers for clinical metastatic breast cancer. Until then, our results have provided a better understanding of the molecular mechanisms of TAZ-induced metastatic dissemination that might be critical for future development of effective targeted therapies for breast and lung cancer patients. TEADs Are Critical Binding Partners of TAZ, Which Suppresses ⌬Np63 Transcription—Our results have identified the members of the TEAD/transcriptional enhancer factor family as major transcription factors mediating TAZ-induced transcriptional repression of downstream genes, particularly ⌬Np63. We showed that TAZ could no longer suppress ⌬Np63 promoter activity in TEAD-null SK-luci6 cells or MCF10A cells with TEAD knockdown (Fig. 4, D and E). This TAZ-TEADmediated suppression seems to be direct because our results have shown that TEAD itself can suppress ⌬Np63 promoter activity and that TAZ can suppress ⌬Np63 promoter activity by directly interacting with two TREs on the ⌬Np63 promoter (Fig. 5, B and C). Consistent with our findings, through ChIP sequencing, a recent study also suggests that TEAD2 can regulate EMT-relevant genes by acting as a transcriptional activator or repressor mainly by directly binding to the promoters containing TREs (56). It is still unclear why the same TAZ-TEAD complex activates transcription of some cellular genes such as CTGF and Cyr61 but suppresses transcription of other genes such as ⌬Np63. Most interestingly, several recent studies have identified components of the chromatin/chromatin-remodeling complexes (BRM, MED mediator complexes, and SWI/SNF complexes) and histone methyltransferase (Ncoa6) complexes as binding partners of TAZ or its Drosophila homolog Yki (41, 57–59). Because these complexes control transcriptional status (activation or inactivation) of a specific gene, the methylation or acetylation status of chromatin on the promoter regions of a specific gene may determine the transcriptional activation or suppression functions of the TAZ-TEAD complex. Indeed, our data further showed that TAZ can directly interact with histone



36.

37.

38.

39.

40.

41. 42.

43.

44.

45.

46.

47.

48.

49.

50. 51. 52.

53.

54.

55.

apoptosis in the SGC7901 gastric cancer cell line. Mol. Med. Rep. 4, 1075–1082 Murakami, M., Nakagawa, M., Olson, E. N., and Nakagawa, O. (2005) A WW domain protein TAZ is a critical coactivator for TBX5, a transcription factor implicated in Holt-Oram syndrome. Proc. Natl. Acad. Sci. U.S.A. 102, 18034 –18039 Di Palma, T., D’Andrea, B., Liguori, G. L., Liguoro, A., de Cristofaro, T., Del Prete, D., Pappalardo, A., Mascia, A., and Zannini, M. (2009) TAZ is a coactivator for Pax8 and TTF-1, two transcription factors involved in thyroid differentiation. Exp. Cell Res. 315, 162–175 Murakami, M., Tominaga, J., Makita, R., Uchijima, Y., Kurihara, Y., Nakagawa, O., Asano, T., and Kurihara, H. (2006) Transcriptional activity of Pax3 is co-activated by TAZ. Biochem. Biophys. Res. Commun. 339, 533–539 Koontz, L. M., Liu-Chittenden, Y., Yin, F., Zheng, Y., Yu, J., Huang, B., Chen, Q., Wu, S., and Pan, D. (2013) The Hippo effector Yorkie controls normal tissue growth by antagonizing scalloped-mediated default repression. Dev. Cell 25, 388 – 401 Zhang, W., Gao, Y., Li, P., Shi, Z., Guo, T., Li, F., Han, X., Feng, Y., Zheng, C., Wang, Z., Li, F., Chen, H., Zhou, Z., Zhang, L., and Ji, H. (2014) VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex. Cell Res. 24, 331–343 Basu, D., Reyes-Múgica, M., and Rebbaa, A. (2013) Histone acetylationmediated regulation of the Hippo pathway. PLoS One 8, e62478 Hildmann, C., Riester, D., and Schwienhorst, A. (2007) Histone deacetylases—an important class of cellular regulators with a variety of functions. Appl. Microbiol. Biotechnol. 75, 487– 497 Hublitz, P., Albert, M., and Peters, A. H. (2009) Mechanisms of transcriptional repression by histone lysine methylation. Int. J. Dev. Biol. 53, 335–354 Lai, D., and Yang, X. (2013) BMP4 is a novel transcriptional target and mediator of mammary cell migration downstream of the Hippo pathway component TAZ. Cell. Signal. 25, 1720 –1728 Lindsay, J., McDade, S. S., Pickard, A., McCloskey, K. D., and McCance, D. J. (2011) Role of ⌬Np63␥ in epithelial to mesenchymal transition. J. Biol. Chem. 286, 3915–3924 Bergholz, J., Zhang, Y., Wu, J., Meng, L., Walsh, E. M., Rai, A., Sherman, M. Y., and Xiao, Z. X. (2014) ⌬Np63␣ regulates Erk signaling via MKP3 to inhibit cancer metastasis. Oncogene 33, 212–224 Wu, J., Liang, S., Bergholz, J., He, H., Walsh, E. M., Zhang, Y., and Xiao, Z. X. (2014) ⌬Np63␣ activates CD82 metastasis suppressor to inhibit cancer cell invasion. Cell Death Dis. 5, e1280 Hong, J. H., Hwang, E. S., McManus, M. T., Amsterdam, A., Tian, Y., Kalmukova, R., Mueller, E., Benjamin, T., Spiegelman, B. M., Sharp, P. A., Hopkins, N., and Yaffe, M. B. (2005) TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Science 309, 1074 –1078 Jang, E. J., Jeong, H., Han, K. H., Kwon, H. M., Hong, J. H., and Hwang, E. S. (2012) TAZ suppresses NFAT5 activity through tyrosine phosphorylation. Mol. Cell. Biol. 32, 4925– 4932 Vo, N., and Goodman, R. H. (2001) CREB-binding protein and p300 in transcriptional regulation. J. Biol. Chem. 276, 13505–13508 Xu, J., Kawai, Y., and Arinze, I. J. (2013) Dual role of C/EBP␣ as an activator and repressor of G␣i2 gene transcription. Genes Cells 18, 1082–1094 Bartucci, M., Dattilo, R., Moriconi, C., Pagliuca, A., Mottolese, M., Federici, G., Benedetto, A. D., Todaro, M., Stassi, G., Sperati, F., Amabile, M. I., Pilozzi, E., Patrizii, M., Biffoni, M., Maugeri-Saccà, M., Piccolo, S., and De Maria, R. (2015) TAZ is required for metastatic activity and chemoresistance of breast cancer stem cells. Oncogene 34, 681– 690 Reis-Filho, J. S., Milanezi, F., Amendoeira, I., Albergaria, A., and Schmitt, F. C. (2003) Distribution of p63, a novel myoepithelial marker, in fineneedle aspiration biopsies of the breast: an analysis of 82 samples. Cancer 99, 172–179 Wang, X., Mori, I., Tang, W., Nakamura, M., Nakamura, Y., Sato, M., Sakurai, T., and Kakudo, K. (2002) p63 expression in normal, hyperplastic and malignant breast tissues. Breast Cancer 9, 216 –219 Stefanou, D., Batistatou, A., Nonni, A., Arkoumani, E., and Agnantis, N. J. (2004) p63 expression in benign and malignant breast lesions. Histol. Histopathol. 19, 465– 471



14. Lei, Q. Y., Zhang, H., Zhao, B., Zha, Z. Y., Bai, F., Pei, X. H., Zhao, S., Xiong, Y., and Guan, K. L. (2008) TAZ promotes cell proliferation and epithelialmesenchymal transition and is inhibited by the hippo pathway. Mol. Cell. Biol. 28, 2426 –2436 15. Chan, S. W., Lim, C. J., Guo, K., Ng, C. P., Lee, I., Hunziker, W., Zeng, Q., and Hong, W. (2008) A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells. Cancer Res. 68, 2592–2598 16. Cordenonsi, M., Zanconato, F., Azzolin, L., Forcato, M., Rosato, A., Frasson, C., Inui, M., Montagner, M., Parenti, A. R., Poletti, A., Daidone, M. G., Dupont, S., Basso, G., Bicciato, S., and Piccolo, S. (2011) The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells. Cell 147, 759 –772 17. Zhou, Z., Hao, Y., Liu, N., Raptis, L., Tsao, M. S., and Yang, X. (2011) TAZ is a novel oncogene in non-small cell lung cancer. Oncogene 30, 2181–2186 18. Xie, M., Zhang, L., He, C. S., Hou, J. H., Lin, S. X., Hu, Z. H., Xu, F., and Zhao, H. Y. (2012) Prognostic significance of TAZ expression in resected non-small cell lung cancer. J. Thorac. Oncol. 7, 799 – 807 19. Yuen, H. F., McCrudden, C. M., Huang, Y. H., Tham, J. M., Zhang, X., Zeng, Q., Zhang, S. D., and Hong, W. (2013) TAZ expression as a prognostic indicator in colorectal cancer. PLoS One 8, e54211 20. de Cristofaro, T., Di Palma, T., Ferraro, A., Corrado, A., Lucci, V., Franco, R., Fusco, A., and Zannini, M. (2011) TAZ/WWTR1 is overexpressed in papillary thyroid carcinoma. Eur. J. Cancer 47, 926 –933 21. Avruch, J., Zhou, D., Fitamant, J., Bardeesy, N., Mou, F., and Barrufet, L. R. (2012) Protein kinases of the Hippo pathway: regulation and substrates. Semin. Cell Dev. Biol. 23, 770 –784 22. Dupont, S., Morsut, L., Aragona, M., Enzo, E., Giulitti, S., Cordenonsi, M., Zanconato, F., Le Digabel, J., Forcato, M., Bicciato, S., Elvassore, N., and Piccolo, S. (2011) Role of YAP/TAZ in mechanotransduction. Nature 474, 179 –183 23. Hong, W., and Guan, K. L. (2012) The YAP and TAZ transcription coactivators: key downstream effectors of the mammalian Hippo pathway. Semin. Cell Dev. Biol. 23, 785–793 24. Varelas, X. (2014) The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease. Development 141, 1614 –1626 25. Liu, C., Huang, W., and Lei, Q. (2011) Regulation and function of the TAZ transcription co-activator. Int. J. Biochem. Mol. Biol. 2, 247–256 26. Lai, D., Visser-Grieve, S., and Yang, X. (2012) Tumour suppressor genes in chemotherapeutic drug response. Biosci. Rep. 32, 361–374 27. Zhao, Y., and Yang, X. (2014) The Hippo pathway in chemotherapeutic drug resistance. Int. J. Cancer 10.1002/ijc.29293 28. Chan, S. W., Lim, C. J., Loo, L. S., Chong, Y. F., Huang, C., and Hong, W. (2009) TEADs mediate nuclear retention of TAZ to promote oncogenic transformation. J. Biol. Chem. 284, 14347–14358 29. Zhang, H., Liu, C. Y., Zha, Z. Y., Zhao, B., Yao, J., Zhao, S., Xiong, Y., Lei, Q. Y., and Guan, K. L. (2009) TEAD transcription factors mediate the function of TAZ in cell growth and epithelial-mesenchymal transition. J. Biol. Chem. 284, 13355–13362 30. Zhao, B., Kim, J., Ye, X., Lai, Z. C., and Guan, K. L. (2009) Both TEADbinding and WW domains are required for the growth stimulation and oncogenic transformation activity of yes-associated protein. Cancer Res. 69, 1089 –1098 31. Zhao, B., Ye, X., Yu, J., Li, L., Li, W., Li, S., Yu, J., Lin, J. D., Wang, C. Y., Chinnaiyan, A. M., Lai, Z. C., and Guan, K. L. (2008) TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 22, 1962–1971 32. Lai, D., Ho, K. C., Hao, Y., and Yang, X. (2011) Taxol resistance in breast cancer cells is mediated by the hippo pathway component TAZ and its downstream transcriptional targets Cyr61 and CTGF. Cancer Res. 71, 2728 –2738 33. Koker, M. M., and Kleer, C. G. (2004) p63 expression in breast cancer: a highly sensitive and specific marker of metaplastic carcinoma. Am. J. Surg. Pathol. 28, 1506 –1512 34. Deyoung, M. P., and Ellisen, L. W. (2007) p63 and p73 in human cancer: defining the network. Oncogene 26, 5169 –5183 35. Zhou, Z., Zhu, J. S., Xu, Z. P., and Zhang, Q. (2011) Lentiviral vectormediated siRNA knockdown of the YAP gene inhibits growth and induces

TAZ Suppresses ⌬Np63 Transcription as a Co-repressor 56. Diepenbruck, M., Waldmeier, L., Ivanek, R., Berninger, P., Arnold, P., van Nimwegen, E., and Christofori, G. (2014) Tead2 expression levels control the subcellular distribution of Yap and Taz, zyxin expression and epithelial-mesenchymal transition. J. Cell Sci. 127, 1523–1536 57. Qing, Y., Yin, F., Wang, W., Zheng, Y., Guo, P., Schozer, F., Deng, H., and Pan, D. (2014) The Hippo effector Yorkie activates transcription by interacting with a histone methyltransferase complex through Ncoa6. Elife 3, e02564 58. Skibinski, A., Breindel, J. L., Prat, A., Galván, P., Smith, E., Rolfs, A., Gupta, P. B., Labaer, J., and Kuperwasser, C. (2014) The Hippo transducer TAZ

interacts with the SWI/SNF complex to regulate breast epithelial lineage commitment. Cell Rep. 6, 1059 –1072 59. Oh, H., Slattery, M., Ma, L., Crofts, A., White, K. P., Mann, R. S., and Irvine, K. D. (2013) Genome-wide association of Yorkie with chromatin and chromatin-remodeling complexes. Cell Rep. 3, 309 –318 60. Gao, Y., Zhang, W., Han, X., Li, F., Wang, X., Wang, R., Fang, Z., Tong, X., Yao, S., Li, F., Feng, Y., Sun, Y., Hou, Y., Yang, Z., Guan, K., Chen, H., Zhang, L., and Ji, H. (2014) YAP inhibits squamous transdifferentiation of Lkb1-deficient lung adenocarcinoma through ZEB2-dependent DNp63 repression. Nat. Commun. 5, 4629




16917

Table S1. Cellular genes down-‐regulated by TAZ Gene Name

Description (from microarray 1, last version)

Average ratio

Number of spots

Standard deviation

P value (t-test)

A_23_P103951

Unknown

0,4448

3

0,08025

0,0084508

A_23_P15226

Unknown

0,3809

3

0,05727

0,0030817

A_23_P170719

Unknown

0,3797

3

0,10059

0,0092713

A_23_P213350

Unknown

0,2544

3

0,23367

0,0239235

A_24_P152845

Unknown

0,3215

3

0,18560

0,0220853

A_24_P233560

Unknown

0,4349

3

0,19071

0,040777

A_24_P398370

Unknown

0,2408

3

0,07339

0,0023317

A_24_P524164

Unknown

0,5046

3

0,24555

0,0855687

A_24_P918926

Unknown

0,5079

3

0,24384

0,08589

A_24_P934975

Unknown

0,4692

3

0,11627

0,0196395

A_32_P109876

Unknown

0,4641

3

0,16408

0,036051

A_32_P132169

Unknown

0,4041

3

0,16749

0,0277052

A_32_P135767

Unknown

0,4171

3

0,01964

0,0004452

A_32_P208076

Unknown

0,4205

3

0,08332

0,007978

A_32_P227158

0,4760

3

0,11008

0,0183682

0,4566

3

0,15222

0,0303147

0,2878

3

0,06484

0,0023776

0,4287

6

0,13216

0,0005177

0,4668

3

0,09736

0,0138368

ADAMTS9

Unknown Homo sapiens acetyl-Coenzyme A carboxylase beta (ACACB), mRNA [NM_001093] Homo sapiens actin, gamma 2, smooth muscle, enteric (ACTG2), mRNA [NM_001615] Homo sapiens ADAM metallopeptidase domain 12 (meltrin alpha) (ADAM12), transcript variant 2, mRNA [NM_021641] Homo sapiens ADAM metallopeptidase with thrombospondin type 1 motif, 5 (aggrecanase-2) (ADAMTS5), mRNA [NM_007038] Homo sapiens ADAM metallopeptidase with thrombospondin type 1 motif, 9 (ADAMTS9), mRNA [NM_182920]

0,2769

6

0,15370

0,00015

AF074982

Homo sapiens full length insert cDNA YH65B08. [AF074982]

0,2841

3

0,14773

0,0117498

AF085846

Homo sapiens full length insert cDNA clone YI46F09. [AF085846]

0,2707

3

0,26736

0,0333408

AF086216

Homo sapiens full length insert cDNA clone ZC65B11. [AF086216]

0,4447

3

0,06478

0,0055535

AF086511

Homo sapiens full length insert cDNA clone ZE03A08. [AF086511]

0,4155

3

0,03590

0,0014698

AF088076

0,2812

3

0,08024

0,0034979

0,3132

3

0,18081

0,020148

0,4383

3

0,07765

0,0076509

0,3615

3

0,04438

0,0016725

0,4007

3

0,00958

9,697E-05

AK021785

Homo sapiens full length insert cDNA clone ZE01A04. [AF088076] Homo sapiens uncharacterized gastric protein ZG33P mRNA, partial cds. [AF264623] AI827481 wf29h01.x1 Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone IMAGE:2357041 3', mRNA sequence [AI827481] Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 1090207. [AJ420543] Homo sapiens cDNA FLJ11104 fis, clone PLACE1005730. [AK001966] Homo sapiens cDNA FLJ11723 fis, clone HEMBA1005314. [AK021785]

0,3339

3

0,12841

0,0116841

AK026517

Homo sapiens cDNA: FLJ22864 fis, clone KAT02164. [AK026517]

0,3646

3

0,07158

0,0043888

AK057923

Homo sapiens cDNA FLJ25194 fis, clone REC04095. [AK057923] Homo sapiens cDNA FLJ37467 fis, clone BRAWH2011920. [AK094786] Homo sapiens cDNA FLJ37480 fis, clone BRAWH2013866, highly similar to Homo sapiens stonin 2 mRNA. [AK094799] Homo sapiens cDNA FLJ38645 fis, clone HHDPC2005794. [AK095964] Homo sapiens cDNA FLJ42287 fis, clone TLIVE2005866. [AK124281] Homo sapiens cDNA FLJ42851 fis, clone BRHIP2005719. [AK124841] Homo sapiens cDNA FLJ43172 fis, clone FCBBF3007242. [AK125162]

0,3730

3

0,06895

0,0042646

0,3839

3

0,13151

0,0158839

0,4002

3

0,06314

0,0041483

0,4757

3

0,06589

0,0068097

0,4857

3

0,13845

0,0296278

0,4684

3

0,04603

0,0032227

0,0915

3

0,05918

0,0005405

ACACB ACTG2 ADAM12 ADAMTS5

AF264623 AI827481 AJ420543 AK001966

AK094786 AK094799 AK095964 AK124281 AK124841 AK125162

AK130118

Homo sapiens cDNA FLJ26608 fis, clone LVR00914. [AK130118] Homo sapiens aldo-keto reductase family 1, member B10 (aldose reductase) (AKR1B10), mRNA [NM_020299] Homo sapiens aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1), mRNA [NM_001353] Homo sapiens aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II) (AKR1C3), mRNA [NM_003739] Homo sapiens mRNA; cDNA DKFZp586N0121 (from clone DKFZp586N0121). [AL133118]

0,2352

3

0,10120

0,004265

0,3264

6

0,04360

6,144E-07

0,2330

12

0,17122

2,99E-08

0,2879

3

0,09611

0,0051829

0,0727

3

0,19885

0,0050098

Homo sapiens EST from clone 24355, full insert. [AL355687] AL533377 Homo sapiens ADULT BRAIN Homo sapiens cDNA clone CS0DN004YJ04 3-PRIME, mRNA sequence [AL533377] Homo sapiens angiopoietin-like 4 (ANGPTL4), transcript variant 1, mRNA [NM_139314]

0,2183

3

0,19161

0,0134428

0,2628

3

0,61793

0,1226702

0,4098

3

0,04632

0,0023653

Homo sapiens annexin A10 (ANXA10), mRNA [NM_007193] Homo sapiens adenomatosis polyposis coli down-regulated 1-like (APCDD1L), mRNA [NM_153360]

0,4746

3

0,24148

0,0729968

0,3559

3

0,09492

0,0072976

Homo sapiens aquaporin 9 (AQP9), mRNA [NM_020980] Homo sapiens ankyrin repeat and SOCS box-containing 9 (ASB9), transcript variant 2, mRNA [NM_024087]

0,0975

3

0,21430

0,007326

0,5046

3

0,01303

0,0003202

0,1900

3

0,18334

0,0104325

0,3793

3

0,19566

0,0325265

0,4130

3

0,49405

0,1552374

0,2939

3

0,24289

0,0315183

0,4044

3

0,13957

0,0197671

BC024289

Homo sapiens asporin (LRR class 1) (ASPN), mRNA [NM_017680] Homo sapiens ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide (ATP1A2), mRNA [NM_000702] AW076051 xa83g01.x1 NCI_CGAP_CML1 Homo sapiens cDNA clone IMAGE:2573424 3' similar to TR:O75594 O75594 PEPTIDOGLYCAN RECOGNITION PROTEIN PRECURSOR. ;, mRNA sequence [AW076051] Homo sapiens clone DNA108923 SFVP2550 (UNQ2550) mRNA, complete cds. [AY358815] Homo sapiens BTB and CNC homology 1, basic leucine zipper transcription factor 2 (BACH2), mRNA [NM_021813] Homo sapiens cDNA clone MGC:39273 IMAGE:5440834, complete cds. [BC024289]

0,4827

3

0,13490

0,0278059

BC032027

Homo sapiens cDNA clone IMAGE:4825733. [BC032027]

0,2046

3

0,18595

0,011713

BC038556

Homo sapiens, clone IMAGE:3446976, mRNA. [BC038556] Homo sapiens bradykinin receptor B1 (BDKRB1), mRNA [NM_000710] BE749146 601123310F1 NIH_MGC_5 Homo sapiens cDNA clone IMAGE:3348051 5', mRNA sequence [BE749146] BF213738 601847628F1 NIH_MGC_55 Homo sapiens cDNA clone IMAGE:4078519 5', mRNA sequence [BF213738] Homo sapiens bone morphogenetic protein 2 (BMP2), mRNA [NM_001200] Homo sapiens bone morphogenetic protein 6 (BMP6), mRNA [NM_001718] Homo sapiens BTB (POZ) domain containing 16 (BTBD16), mRNA [NM_144587] Homo sapiens butyrophilin-like 8 (BTNL8), transcript variant 1, mRNA [NM_024850] Homo sapiens mRNA; cDNA DKFZp686B14224 (from clone DKFZp686B14224). [BX640843] Homo sapiens chromosome 10 open reading frame 90 (C10orf90), mRNA [NM_001004298] Homo sapiens chromosome 19 open reading frame 21 (C19orf21), mRNA [NM_173481] Homo sapiens chromosome 1 open reading frame 168 (C1orf168), mRNA [NM_001004303] Homo sapiens chromosome 1 open reading frame 87 (C1orf87), mRNA [NM_152377]

0,5088

3

0,13720

0,032882

0,2722

6

0,19750

0,0004559

0,4767

3

0,11364

0,0195749

0,1551

3

0,17718

0,0077989

0,1708

3

0,15453

0,0066217

0,1629

3

0,24396

0,0152466

0,3979

3

0,07982

0,0064995

0,3040

3

0,15798

0,0148617

0,3495

6

0,13440

0,0002048

0,4640

3

0,05567

0,0045804

0,3848

3

0,08183

0,0063621

0,2190

6

0,72364

0,0379457

0,1661

3

0,07859

0,0016856

0,3336

9

0,11591

8,669E-07

C8orf57

Homo sapiens mRNA for KIAA0386 gene, partial cds. [AB002384] Homo sapiens mRNA; cDNA DKFZp761D112 (from clone DKFZp761D112). [AL136588]

0,3768

3

0,06665

0,0040706

C9orf26

Homo sapiens chromosome 9 open reading frame 26 (NF-HEV)

0,2295

3

0,03362

0,0004603

AKR1B10 AKR1C1 AKR1C3 AL133118 AL355687 AL533377 ANGPTL4 ANXA10 APCDD1L AQP9 ASB9 ASPN ATP1A2

AW076051 AY358815 BACH2

BDKRB1 BE749146 BF213738 BMP2 BMP6 BTBD16 BTNL8 BX640843 C10orf90 C19orf21 C1orf168 C1orf87 C6orf32

(C9orf26), mRNA [NM_033439]

CD36

AGENCOURT_13543108 NIH_MGC_148 Homo sapiens cDNA clone IMAGE:30330694 5', mRNA sequence [CB995946] Homo sapiens coiled-coil domain containing 3 (CCDC3), mRNA [NM_031455] CD36=collagen type I/thrombospondin receptor {one exon} [human, mRNA Partial, 369 nt]. [S67044]

0,0905

6

0,12444

2,533E-06

CD52

Homo sapiens CD52 molecule (CD52), mRNA [NM_001803]

0,1872

3

0,11548

0,0041437

CDH19

Homo sapiens cadherin 19, type 2 (CDH19), mRNA [NM_021153] Homo sapiens cholinergic receptor, nicotinic, alpha 9 (CHRNA9), mRNA [NM_017581] Homo sapiens C-type lectin domain family 1, member B (CLEC1B), mRNA [NM_016509] Homo sapiens C-type lectin domain family 2, member B (CLEC2B), mRNA [NM_005127] Homo sapiens chemokine orphan receptor 1 (CMKOR1), mRNA [NM_020311] Homo sapiens contactin 3 (plasmacytoma associated) (CNTN3), mRNA [NM_020872] Homo sapiens collagen, type XXI, alpha 1 (COL21A1), mRNA [NM_030820] Homo sapiens collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive) (COL7A1), mRNA [NM_000094] full-length cDNA clone CS0DI072YG17 of Placenta Cot 25normalized of Homo sapiens (human). [CR600419] full-length cDNA clone CS0DJ001YJ05 of T cells (Jurkat cell line) Cot 10-normalized of Homo sapiens (human). [CR601458] full-length cDNA clone CS0DF021YL03 of Fetal brain of Homo sapiens (human). [CR603982] Homo sapiens mRNA; cDNA DKFZp686J17110 (from clone DKFZp686J17110) [CR749547] Homo sapiens cytokine receptor-like factor 1 (CRLF1), mRNA [NM_004750] Homo sapiens colony stimulating factor 2 (granulocyte-macrophage) (CSF2), mRNA [NM_000758] Homo sapiens catenin (cadherin-associated protein), delta 2 (neural plakophilin-related arm-repeat protein) (CTNND2), mRNA [NM_001332] Homo sapiens chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1), mRNA [NM_001511] Homo sapiens chemokine (C-X-C motif) ligand 14 (CXCL14), mRNA [NM_004887] Homo sapiens chemokine (C-X-C motif) ligand 2 (CXCL2), mRNA [NM_002089] Homo sapiens chemokine (C-X-C motif) ligand 3 (CXCL3), mRNA [NM_002090] Homo sapiens chromosome X open reading frame 57 (CXorf57), mRNA [NM_018015] Homo sapiens cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1), nuclear gene encoding mitochondrial protein, mRNA [NM_000781] Homo sapiens cytochrome P450, family 2, subfamily J, polypeptide 2 (CYP2J2), mRNA [NM_000775] Homo sapiens cytochrome P450, family 4, subfamily B, polypeptide 1 (CYP4B1), mRNA [NM_000779] Homo sapiens cytochrome P450, family 4, subfamily F, polypeptide 11 (CYP4F11), mRNA [NM_021187] Homo sapiens cytochrome P450, family 4, subfamily F, polypeptide 12 (CYP4F12), mRNA [NM_023944] Homo sapiens cytochrome P450, family 4, subfamily F, polypeptide 8 (CYP4F8), mRNA [NM_007253] Homo sapiens DNA segment on chromosome 4 (unique) 234 expressed sequence (D4S234E), transcript variant 1, mRNA [NM_014392] Human monocyte PABL (pseudoautosomal boundary-like sequence) mRNA, clone Mo1. [D55639] DB097529 TESTI4 Homo sapiens cDNA clone TESTI4048830 5', mRNA sequence [DB097529]

0,3930

6

0,47733

0,0459515

0,0483

3

0,28674

0,0077244

0,2497

3

0,08404

0,0032098

0,1346

3

0,07597

0,0012633

0,3871

3

0,07229

0,0050507

0,2528

9

0,22795

2,477E-05

0,0242

3

0,50871

0,0157244

0,4653

3

0,11658

0,0193314

0,3862

3

0,06691

0,0043131

0,3754

3

0,06214

0,0035178

0,1817

3

0,15369

0,0070275

0,3491

3

0,39887

0,0928166

0,2705

3

0,07569

0,0029353

0,2187

3

0,05488

0,0011476

0,4857

6

0,06727

4,505E-05

0,2079

3

0,06257

0,0013965

0,3773

3

0,07254

0,0048237

0,3643

6

0,08317

2,478E-05

0,2459

6

0,17136

0,0001653

0,4689

6

0,12735

0,0007271

0,3238

3

0,27994

0,0469163

0,4179

3

0,06818

0,0053082

0,2604

3

0,12233

0,0071486

0,3274

6

0,20212

0,0010129

0,4098

3

0,10813

0,0125001

0,3475

3

0,12731

0,0123463

0,4972

3

0,01854

0,000621

0,3021

3

0,14485

0,0124612

0,4026

3

0,28732

0,07007

CB995946 CCDC3

CHRNA9 CLEC1B CLEC2B CMKOR1 CNTN3 COL21A1 COL7A1 CR600419 CR601458 CR603982 CR749547 CRLF1 CSF2 CTNND2 CXCL1 CXCL14 CXCL2 CXCL3 CXorf57 CYP11A1 CYP2J2 CYP4B1 CYP4F11 CYP4F12 CYP4F8 D4S234E D55639 DB097529

0,1994

3

0,25673

0,0210016

0,2473

3

0,05408

0,0013187

DB381305 DCAMKL1 DDC

DB381305 PLACE3 Homo sapiens cDNA clone PLACE3000400 3', mRNA sequence [DB381305] Homo sapiens doublecortin and CaM kinase-like 1 (DCAMKL1), mRNA [NM_004734] Homo sapiens dopa decarboxylase (aromatic L-amino acid decarboxylase) (DDC), mRNA [NM_000790]

0,2940

3

0,14489

0,0119391

0,4117

6

0,43241

0,0403959

0,1905

3

0,08734

0,0024335

Homo sapiens defensin, beta 126 (DEFB126), mRNA [NM_030931] Homo sapiens dehydrogenase/reductase (SDR family) member 2 (DHRS2), transcript variant 1, mRNA [NM_182908] Homo sapiens dynein, axonemal, light intermediate polypeptide 1 (DNALI1), mRNA [NM_003462] Homo sapiens dedicator of cytokinesis 2 (DOCK2), mRNA [NM_004946] Homo sapiens desmocollin 2 (DSC2), transcript variant Dsc2a, mRNA [NM_024422] Homo sapiens Down syndrome critical region gene 1-like 1 (DSCR1L1), mRNA [NM_005822] Homo sapiens dynein, light chain, roadblock-type 2 (DYNLRB2), mRNA [NM_130897] Homo sapiens endothelial differentiation, lysophosphatidic acid Gprotein-coupled receptor, 2 (EDG2), transcript variant 2, mRNA [NM_057159] Homo sapiens early growth response 2 (Krox-20 homolog, Drosophila) (EGR2), mRNA [NM_000399]

0,5034

6

0,13185

0,0013173

0,4778

6

0,03908

2,815E-06

0,0809

3

0,09321

0,0012097

0,4540

6

0,11261

0,0003413

0,4204

3

0,22821

0,0519048

0,2782

3

0,07951

0,0033778

0,2468

3

0,30538

0,0374234

0,0990

3

0,22167

0,0079258

0,3757

3

0,07074

0,0045505

Homo sapiens early growth response 4 (EGR4), mRNA [NM_001965] Homo sapiens EGF, latrophilin and seven transmembrane domain containing 1, mRNA (cDNA clone MGC:34204 IMAGE:5229055), complete cds. [BC025721]

0,2556

3

0,23481

0,024291

0,2374

3

0,15344

0,0097658

Homo sapiens endomucin (EMCN), mRNA [NM_016242] H.sapiens mRNA for immunoglobulin kappa variable region (clone 8F1C-06). [X85155] Homo sapiens clone JM6 immunoglobulin light chain variable region mRNA, partial cds. [AF078945]

0,0403

3

0,10027

0,0008587

0,2952

3

0,11537

0,0077199

0,4695

3

0,01197

0,0002212

Unknown Homo sapiens clone SC4132 anti-rabies virus immunoglobulin light chain variable region mRNA, complete cds. [AY942106] Homo sapiens fibroblast growth factor homologous factor 2 isoform 1Z+1Y (FHF-2) mRNA, partial cds. [AF199613]

0,3477

3

0,14556

0,0159725

0,3847

3

0,25435

0,0529013

0,4704

3

0,11507

0,0193858

0,4469

3

0,24883

0,0676289

ENST00000376752

Homo sapiens OVC10-2 mRNA, complete cds. [AF230201] Homo sapiens cDNA FLJ13034 fis, clone NT2RP3001232. [AK023096]

0,3756

3

0,08289

0,0062142

ENST00000376881

Unknown

0,4426

3

0,03835

0,0019444

ENST00000379895

Human IG rearranged H-chain mRNA V-region, partial cds. [L03822] Homo sapiens disintegrin metalloproteinase with thrombospondin repeats (ADAMTS9) mRNA, complete cds. [AF261918] Homo sapiens endothelial cell-specific molecule 1 (ESM1), mRNA [NM_007036]

0,4930

3

0,07469

0,0095734

0,2358

3

0,15569

0,0099579

0,1549

6

0,21921

0,0001376

Homo sapiens ets variant gene 1 (ETV1), mRNA [NM_004956] Homo sapiens eyes absent homolog 2 (Drosophila) (EYA2), transcript variant 2, mRNA [NM_172113] Homo sapiens fatty acid binding protein 4, adipocyte (FABP4), mRNA [NM_001442] Homo sapiens family with sequence similarity 26, member A (FAM26A), mRNA [NM_182494] Homo sapiens family with sequence similarity 47, member B (FAM47B), mRNA [NM_152631] Homo sapiens family with sequence similarity 47, member C (FAM47C), mRNA [NM_001013736] Homo sapiens family with sequence similarity 5, member C (FAM5C), mRNA [NM_199051]

0,4323

6

0,15444

0,0010934

0,3645

6

0,10649

8,222E-05

0,1511

3

0,21183

0,0107494

0,4159

3

0,15347

0,0250351

0,0009

3

0,09420

0,0001578

0,0672

3

0,06925

0,0005804

0,1862

3

0,26970

0,0213096

Homo sapiens fibulin 5 (FBLN5), mRNA [NM_006329] Homo sapiens fibroblast growth factor 13 (FGF13), transcript variant 1A, mRNA [NM_004114] Homo sapiens fibroblast growth factor binding protein 1 (FGFBP1), mRNA [NM_005130]

0,3690

6

0,10579

8,448E-05

0,3070

3

0,13031

0,010426

0,3899

3

0,12821

0,0156085

DEFB126 DHRS2 DNALI1 DOCK2 DSC2 DSCR1L1 DYNLRB2 EDG2 EGR2 EGR4 ELTD1 EMCN ENST00000295339 ENST00000295410 ENST00000339363 ENST00000359488 ENST00000370599 ENST00000371497

ENST00000383706 ESM1 ETV1 EYA2 FABP4 FAM26A FAM47B FAM47C FAM5C FBLN5 FGF13 FGFBP1

FLJ14167 FLJ20701 FOSB

Homo sapiens cDNA FLJ14167 fis, clone NT2RP2001214. [AK024229] Homo sapiens hypothetical protein FLJ20701 (FLJ20701), mRNA [NM_017933] Homo sapiens FBJ murine osteosarcoma viral oncogene homolog B (FOSB), mRNA [NM_006732]

0,1672

3

0,25446

0,0169754

0,4378

3

0,03944

0,0020034

0,3414

3

0,02412

0,0004444

0,4628

6

0,14008

0,0010332

FZD10

Homo sapiens Fraser syndrome 1 (FRAS1), mRNA [NM_025074] Homo sapiens frizzled homolog 10 (Drosophila) (FZD10), mRNA [NM_007197]

0,3228

3

0,16868

0,01858

GAGE7

Homo sapiens G antigen 7 (GAGE7), mRNA [NM_021123]

0,3172

3

0,33802

0,0625247

GAL

Homo sapiens galanin (GAL), mRNA [NM_015973] Homo sapiens UDP-N-acetyl-alpha-D-galactosamine:polypeptide Nacetylgalactosaminyltransferase 14 (GalNAc-T14) (GALNT14), mRNA [NM_024572] Homo sapiens grancalcin, EF-hand calcium binding protein (GCA), mRNA [NM_012198] Homo sapiens granulysin (GNLY), transcript variant NKG5, mRNA [NM_006433] Homo sapiens G protein-coupled receptor 143 (GPR143), mRNA [NM_000273] Homo sapiens G protein-coupled receptor 64 (GPR64), mRNA [NM_005756] Homo sapiens G protein-coupled receptor 85 (GPR85), mRNA [NM_018970] Homo sapiens G protein-coupled receptor 87 (GPR87), mRNA [NM_023915]

0,3672

3

0,17228

0,024242

0,2537

3

0,10595

0,0051915

0,3531

3

0,03214

0,0008404

0,3150

3

0,10508

0,0071553

0,4487

3

0,08295

0,0092058

0,4888

6

0,19751

0,0059708

0,4530

6

0,10572

0,0002511

0,5070

3

0,04704

0,0041846

Homo sapiens mRNA for KIAA1943 protein. [AB075823] Homo sapiens growth factor receptor-bound protein 10 (GRB10), transcript variant 4, mRNA [NM_001001555] Homo sapiens growth factor receptor-bound protein 14 (GRB14), mRNA [NM_004490] Homo sapiens glutamate receptor, metabotropic 8 (GRM8), mRNA [NM_000845] Homo sapiens major histocompatibility complex, class II, DR beta 4 (HLA-DRB4), mRNA [NM_021983] Homo sapiens histamine N-methyltransferase (HNMT), transcript variant 1, mRNA [NM_006895] Homo sapiens 4-hydroxyphenylpyruvate dioxygenase (HPD), mRNA [NM_002150] Homo sapiens hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), transcript variant 2, mRNA [NM_181755] Homo sapiens inhibitor of DNA binding 1, dominant negative helixloop-helix protein (ID1), transcript variant 1, mRNA [NM_002165] Homo sapiens inhibitor of DNA binding 2, dominant negative helixloop-helix protein (ID2), mRNA [NM_002166] Homo sapiens inhibitor of DNA binding 3, dominant negative helixloop-helix protein (ID3), mRNA [NM_002167] Homo sapiens interferon-induced protein 44 (IFI44), mRNA [NM_006417]

0,4165

9

0,54815

0,0354505

0,4809

6

0,11482

0,0005299

0,2697

3

0,06024

0,0018566

0,3865

3

0,06213

0,0037329

0,3300

3

0,13165

0,0120143

0,4927

6

0,27651

0,0207989

0,3429

3

0,11757

0,0103345

0,4231

3

0,09103

0,0096113

0,4266

3

0,04978

0,0029896

0,3791

6

0,13104

0,0002653

0,3779

3

0,06198

0,0035462

0,3944

3

0,02084

0,0004428

0,3859

3

0,07722

0,0057117

0,3563

3

0,07201

0,004247

IL18RAP

Homo sapiens interferon epsilon 1 (IFNE1), mRNA [NM_176891] Homo sapiens insulin growth factor-like family member 2 (IGFL2), mRNA [NM_001002915] Homo sapiens interleukin 18 receptor accessory protein (IL18RAP), mRNA [NM_003853]

0,4853

3

0,02471

0,0010292

IL1A

Homo sapiens interleukin 1, alpha (IL1A), mRNA [NM_000575]

0,2544

30

0,20554

4,069E-16

IL1B

Homo sapiens interleukin 1, beta (IL1B), mRNA [NM_000576] Homo sapiens interleukin 1 receptor-like 1 (IL1RL1), transcript variant 1, mRNA [NM_016232] Homo sapiens interleukin 32 (IL32), transcript variant 1, mRNA [NM_001012631] Homo sapiens interleukin 6 (interferon, beta 2) (IL6), mRNA [NM_000600]

0,3229

30

0,07957

3,825E-25

0,2326

3

0,08399

0,0029053

0,4299

3

0,05527

0,0037444

0,1256

30

0,08401

5,232E-32

Homo sapiens interleukin 8 (IL8), mRNA [NM_000584] Homo sapiens inhibitory caspase recruitment domain (CARD) protein (INCA), mRNA [NM_001007232]

0,1318

3

0,10009

0,0021415

0,4096

3

0,01650

0,0003016

FRAS1

GALNT14 GCA GNLY GPR143 GPR64 GPR85 GPR87 GPR98 GRB10 GRB14 GRM8 HLA-DRB4 HNMT HPD HSD11B1 ID1 ID2 ID3 IFI44 IFNE1 IGFL2

IL1RL1 IL32 IL6 IL8 INCA

KRT16

Homo sapiens integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) (ITGA2), mRNA [NM_002203] Homo sapiens junctional adhesion molecule 2 (JAM2), mRNA [NM_021219] Homo sapiens potassium inwardly-rectifying channel, subfamily J, member 15 (KCNJ15), transcript variant 1, mRNA [NM_170736] Homo sapiens potassium inwardly-rectifying channel, subfamily J, member 6 (KCNJ6), mRNA [NM_002240] Homo sapiens potassium large conductance calcium-activated channel, subfamily M, beta member 4 (KCNMB4), mRNA [NM_014505] Homo sapiens killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 2 (KIR2DL2), mRNA [NM_014219] Homo sapiens killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 4 (KIR2DL4), mRNA [NM_002255] Homo sapiens killer cell immunoglobulin-like receptor, two domains, long cytoplasmic tail, 5A (KIR2DL5A), mRNA [NM_020535] Homo sapiens killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 1 (KIR2DS1), mRNA [NM_014512] Homo sapiens killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 2 (KIR2DS2), mRNA [NM_012312] Homo sapiens killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 4 (KIR2DS4), transcript variant 2, mRNA [NM_178228] Homo sapiens killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), mRNA [NM_013289] Homo sapiens keratin 16 (focal non-epidermolytic palmoplantar keratoderma) (KRT16), mRNA [NM_005557]

KRT6B

Homo sapiens keratin 6B (KRT6B), mRNA [NM_005555]

0,2246

3

0,27081

0,0267502

KRT6C

Homo sapiens keratin 6C (KRT6C), mRNA [NM_058242]

0,3323

3

0,21991

0,0318755

KRT6E

Homo sapiens keratin 6E (KRT6E), mRNA [NM_173086] Homo sapiens kynureninase (L-kynurenine hydrolase) (KYNU), transcript variant 1, mRNA [NM_003937] Homo sapiens laminin, beta 3 (LAMB3), transcript variant 2, mRNA [NM_001017402] Homo sapiens laminin, gamma 2 (LAMC2), transcript variant 2, mRNA [NM_018891] Homo sapiens late cornified envelope 3D (LCE3D), mRNA [NM_032563] PREDICTED: Homo sapiens similar to Ig heavy chain V-III region VH26 precursor (LOC390712), mRNA [XM_372630] PREDICTED: Homo sapiens similar to Ig heavy chain V-III region VH26 precursor (LOC652159), mRNA [XM_941503] PREDICTED: Homo sapiens similar to Ig heavy chain V-III region VH26 precursor (LOC652791), mRNA [XM_942451] Homo sapiens loss of heterozygosity, 11, chromosomal region 2, gene A (LOH11CR2A), transcript variant 1, mRNA [NM_014622]

0,2969

3

0,24002

0,0313141

0,4605

6

0,06232

2,199E-05

0,4431

3

0,08631

0,00965

0,2787

6

0,05752

1,265E-06

0,2906

3

0,13254

0,0098636

0,4751

3

0,08070

0,0100774

0,4995

3

0,13014

0,0284401

0,5088

3

0,06418

0,0077839

0,2777

6

0,10014

1,919E-05

Homo sapiens lipoprotein lipase (LPL), mRNA [NM_000237] Homo sapiens leucine rich repeat containing 49 (LRRC49), mRNA [NM_017691] Homo sapiens lymphotoxin beta (TNF superfamily, member 3) (LTB), transcript variant 1, mRNA [NM_002341] Homo sapiens LY6/PLAUR domain containing 3 (LYPD3), mRNA [NM_014400] Homo sapiens mal, T-cell differentiation protein 2 (MAL2), mRNA [NM_052886] Homo sapiens mannosidase, alpha, class 1A, member 1 (MAN1A1), mRNA [NM_005907] Homo sapiens macrophage receptor with collagenous structure (MARCO), mRNA [NM_006770] Homo sapiens MCF.2 cell line derived transforming sequence (MCF2), mRNA [NM_005369] Homo sapiens mannosyl (alpha-1,3-)-glycoprotein beta-1,4-Nacetylglucosaminyltransferase, isozyme C (putative) (MGAT4C), mRNA [NM_013244] Homo sapiens similar to Keratin, type I cytoskeletal 16 (Cytokeratin16) (CK-16) (Keratin-16) (K16), mRNA (cDNA clone MGC:102966 IMAGE:4752428), complete cds. [BC110641]

0,1359

3

0,12924

0,0036657

0,4881

3

0,05109

0,0044237

0,4762

3

0,03627

0,0020982

0,4104

6

0,04649

2,63E-06

0,2964

3

0,25097

0,033855

0,1815

6

0,34857

0,0017221

0,2808

3

0,19624

0,019843

0,3768

3

0,14274

0,017895

0,0684

3

0,50485

0,0286513

0,5032

6

0,22874

0,01208

ITGA2 JAM2 KCNJ15 KCNJ6 KCNMB4 KIR2DL2 KIR2DL4 KIR2DL5A KIR2DS1 KIR2DS2 KIR2DS4 KIR3DL1

KYNU LAMB3 LAMC2 LCE3D LOC390712 LOC652159 LOC652791 LOH11CR2A LPL LRRC49 LTB LYPD3 MAL2 MAN1A1 MARCO MCF2 MGAT4C MGC102966

0,3342

6

0,13207

0,000155

0,3240

3

0,28252

0,0477128

0,4217

3

0,02909

0,0010003

0,4303

3

0,20255

0,0443077

0,4404

3

0,04886

0,0031071

0,2152

3

0,10564

0,0041271

0,2200

6

0,18181

0,0001524

0,2597

3

0,10528

0,0053017

0,2571

3

0,07024

0,0023459

0,2326

3

0,04961

0,0010191

0,1682

9

0,26396

1,094E-05

0,3202

3

0,11622

0,0089577

0,4513

6

0,07778

5,689E-05

MGC23985

Homo sapiens similar to AVLV472 (MGC23985), mRNA [NM_206966]

0,2438

3

0,05643

0,0014065

Homo sapiens matrix Gla protein (MGP), mRNA [NM_000900] Homo sapiens matrix metallopeptidase 1 (interstitial collagenase) (MMP1), mRNA [NM_002421] Homo sapiens matrix metallopeptidase 13 (collagenase 3) (MMP13), mRNA [NM_002427] Homo sapiens matrix metallopeptidase 16 (membrane-inserted) (MMP16), transcript variant 1, mRNA [NM_005941] Homo sapiens matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3), mRNA [NM_002422] Homo sapiens melanoma associated antigen (mutated) 1-like 1 (MUM1L1), mRNA [NM_152423] Homo sapiens myosin, light polypeptide 1, alkali; skeletal, fast (MYL1), transcript variant 3f, mRNA [NM_079422] Homo sapiens neutrophil cytosolic factor 2 (65kDa, chronic granulomatous disease, autosomal 2) (NCF2), mRNA [NM_000433] Homo sapiens nebulette (NEBL), transcript variant 1, mRNA [NM_006393] Homo sapiens Nance-Horan syndrome (congenital cataracts and dental anomalies) (NHS), mRNA [NM_198270] Homo sapiens nephroblastoma overexpressed gene (NOV), mRNA [NM_002514] Homo sapiens nuclear receptor subfamily 4, group A, member 2 (NR4A2), transcript variant 1, mRNA [NM_006186]

0,3972

3

0,34728

0,0917536

0,4519

33

0,17435

4,51E-13

0,4304

3

0,05938

0,0043275

0,4159

6

0,16047

0,001061

0,0924

3

0,20514

0,0064272

0,2432

3

0,06643

0,0019392

0,1521

3

0,03657

0,0003329

0,2924

3

0,13048

0,0096612

0,4530

6

0,07724

5,629E-05

0,4026

3

0,04740

0,0023811

0,3833

6

0,07439

1,851E-05

0,3824

3

0,05412

0,0027779

0,2207

3

0,19820

0,0145434

0,4738

3

0,14995

0,0322128

0,3181

6

0,07462

7,85E-06

0,4049

6

0,08418

4,499E-05

0,4509

3

0,08486

0,0097399

0,4138

6

0,37138

0,0263475

0,2438

3

0,34360

0,0453493

0,1310

3

0,04948

0,0005228

0,5064

9

0,12951

6,588E-05

0,3202

3

0,09820

0,0064445

0,2602

3

0,05237

0,001332

0,4100

3

0,08554

0,007939

0,3806

3

0,07989

0,0059361

0,4476

3

0,03050

0,0012675

0,0951

3

0,19939

0,0062298

PPEF1

Homo sapiens osteomodulin (OMD), mRNA [NM_005014] Homo sapiens olfactory receptor, family 10, subfamily P, member 1 (OR10P1), mRNA [NM_206899] Homo sapiens procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide III (P4HA3), mRNA [NM_182904] Homo sapiens proprotein convertase subtilisin/kexin type 1 (PCSK1), mRNA [NM_000439] Homo sapiens proprotein convertase subtilisin/kexin type 5 (PCSK5), mRNA [NM_006200] Homo sapiens phosphate cytidylyltransferase 1, choline, beta (PCYT1B), mRNA [NM_004845] Homo sapiens PDZ and LIM domain 3 (PDLIM3), mRNA [NM_014476] Homo sapiens platelet/endothelial cell adhesion molecule (CD31 antigen) (PECAM1), mRNA [NM_000442] Homo sapiens cDNA FLJ37405 fis, clone BRAMY2028269. [AK094724] Homo sapiens phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma) (PLA2G7), mRNA [NM_005084] Homo sapiens phospholipase C, epsilon 1 (PLCE1), mRNA [NM_016341] Homo sapiens pleckstrin homology domain containing, family G (with RhoGef domain) member 1 (PLEKHG1), mRNA [NM_001029884] Homo sapiens plexin domain containing 2 (PLXDC2), mRNA [NM_032812] Homo sapiens podocalyxin-like (PODXL), transcript variant 2, mRNA [NM_005397] Homo sapiens periostin, osteoblast specific factor (POSTN), mRNA [NM_006475] Homo sapiens protein phosphatase, EF-hand calcium binding domain 1 (PPEF1), transcript variant 1, mRNA [NM_006240]

0,2970

3

0,06917

0,0028442

PRG4

Homo sapiens proteoglycan 4 (PRG4), mRNA [NM_005807]

0,2331

3

0,09367

0,0036168

PRL

Homo sapiens prolactin (PRL), mRNA [NM_000948] Homo sapiens prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) (PTGS1), transcript variant 1, mRNA [NM_000962] Homo sapiens pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1) (PTN), mRNA [NM_002825] Homo sapiens retinoic acid early transcript 1E (RAET1E), mRNA [NM_139165]

0,0532

3

0,04751

0,0002317

0,3398

6

0,12396

0,0001232

0,3675

6

0,07989

2,125E-05

0,4053

3

0,01559

0,0002631

MGP MMP1 MMP13 MMP16 MMP3 MUM1L1 MYL1 NCF2 NEBL NHS NOV NR4A2 OMD OR10P1 P4HA3 PCSK1 PCSK5 PCYT1B PDLIM3 PECAM1 PITPNC1 PLA2G7 PLCE1 PLEKHG1 PLXDC2 PODXL POSTN

PTGS1 PTN RAET1E

RAPGEF6

Homo sapiens Rap guanine nucleotide exchange factor (GEF) 6 (RAPGEF6), mRNA [NM_016340] Homo sapiens RAS-like, estrogen-regulated, growth inhibitor (RERG), mRNA [NM_032918] Homo sapiens roundabout, axon guidance receptor, homolog 1 (Drosophila) (ROBO1), transcript variant 2, mRNA [NM_133631] Homo sapiens SAM domain, SH3 domain and nuclear localisation signals, 1 (SAMSN1), mRNA [NM_022136] Homo sapiens serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1), transcript variant 2, mRNA [NM_001002236] Homo sapiens serpin peptidase inhibitor, clade D (heparin cofactor), member 1 (SERPIND1), mRNA [NM_000185] Homo sapiens serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 (SERPINE2), mRNA [NM_006216]

0,2747

3

0,14933

0,0114028

0,4873

6

0,07378

7,198E-05

0,4708

6

0,09590

0,0002008

0,4557

3

0,22528

0,0598519

0,4137

3

0,00888

8,945E-05

0,1896

33

0,22924

1,139E-18

0,3119

3

0,16876

0,017568

Homo sapiens sestrin 3 (SESN3), mRNA [NM_144665] Homo sapiens secreted frizzled-related protein 1 (SFRP1), mRNA [NM_003012] Homo sapiens SLAM family member 8 (SLAMF8), mRNA [NM_020125] Homo sapiens solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1 (SLC1A1), mRNA [NM_004170] Homo sapiens solute carrier family 1 (glial high affinity glutamate transporter), member 3 (SLC1A3), mRNA [NM_004172] Homo sapiens solute carrier family 7, (cationic amino acid transporter, y+ system) member 11 (SLC7A11), mRNA [NM_014331] Homo sapiens solute carrier family 9 (sodium/hydrogen exchanger), member 2 (SLC9A2), mRNA [NM_003048] Homo sapiens SLIT and NTRK-like family, member 6 (SLITRK6), mRNA [NM_032229] Homo sapiens SPARC related modular calcium binding 1 (SMOC1), transcript variant 1, mRNA [NM_001034852]

0,4643

3

0,28930

0,0920945

0,4595

6

0,14983

0,0013357

0,1564

3

0,04037

0,0004178

0,3365

6

0,24210

0,0024731

0,2670

6

0,19971

0,0004481

0,4288

9

0,12134

8,594E-06

0,4752

3

0,22961

0,0675125

0,1548

3

0,12549

0,0039508

0,1433

6

0,19502

6,466E-05

Homo sapiens mRNA for FLJ00133 protein. [AK074062] Homo sapiens Sp8 transcription factor (SP8), transcript variant 2, mRNA [NM_198956] Homo sapiens sushi-repeat-containing protein, X-linked (SRPX), mRNA [NM_006307] Homo sapiens ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 3 (ST6GALNAC3), mRNA [NM_152996] Homo sapiens signal transducer and activator of transcription 4 (STAT4), mRNA [NM_003151] Homo sapiens serine/threonine kinase 32A (STK32A), mRNA [NM_145001] Homo sapiens sulfatase 2 (SULF2), transcript variant 1, mRNA [NM_018837] Homo sapiens sulfotransferase family 1E, estrogen-preferring, member 1 (SULT1E1), mRNA [NM_005420] Homo sapiens synaptonemal complex protein 2 (SYCP2), mRNA [NM_014258] Homo sapiens transgelin (TAGLN), transcript variant 1, mRNA [NM_001001522] Homo sapiens trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) (TFF1), mRNA [NM_003225]

0,4959

6

0,04357

6,209E-06

0,3241

3

0,06208

0,0026616

0,4473

3

0,01722

0,0004045

0,3941

3

0,03229

0,0010592

0,4703

6

0,36768

0,0404056

0,1523

3

0,14180

0,0049427

0,4473

3

0,08230

0,0089969

0,2254

3

0,18334

0,0128678

0,3475

6

0,14589

0,0002933

0,3354

6

0,07492

1,011E-05

0,4884

6

0,20051

0,0062989

Unknown A40201 artifact-warning sequence (translated ALU class A) - human {Homo sapiens;} , partial (10%) [THC2279377] ALU7_HUMAN (P39194) Alu subfamily SQ sequence contamination warning entry, partial (10%) [THC2279402]

0,3438

3

0,17183

0,0214238

0,4904

3

0,13321

0,0282899

0,1468

3

0,06506

0,0010129

0,1601

3

0,22173

0,0124658

THC2283371

Unknown Q9BXX3 (Q9BXX3) Breast cancer antigen NY-BR-1, partial (4%) [THC2283371]

0,1438

3

0,39168

0,0325598

THC2290220

Unknown

0,4587

3

0,15416

0,0313514

THC2301901

RL2A_HUMAN (P46776) 60S ribosomal protein L27a, partial (20%)

0,2167

3

0,22683

0,0183785

RERG ROBO1 SAMSN1 SERPINA1 SERPIND1 SERPINE2 SESN3 SFRP1 SLAMF8 SLC1A1 SLC1A3 SLC7A11 SLC9A2 SLITRK6 SMOC1 SNED1 SP8 SRPX ST6GALNAC3 STAT4 STK32A SULF2 SULT1E1 SYCP2 TAGLN TFF1 THC2278737 THC2279377 THC2279402 THC2281837

[THC2301901]

THC2308403

CA307538 UI-H-FT1-bic-o-24-0-UI.s1 NCI_CGAP_FT1 Homo sapiens cDNA clone UI-H-FT1-bic-o-24-0-UI 3', mRNA sequence [CA307538]

0,5016

3

0,09020

0,0144531

THC2320128

Unknown

0,3430

3

0,20224

0,0288568

THC2321585

Unknown

0,3905

3

0,18903

0,0322937

THC2341129

Unknown

0,1506

3

0,03377

0,0002808

THC2343678

Q6E5T4 (Q6E5T4) Claudin 2, partial (5%) [THC2343678]

0,4360

3

0,06269

0,0049663

THC2346121

Unknown

0,4780

3

0,01996

0,0006449

THC2350949

Unknown AE017295 pyridoxal phosphate biosynthetic protein {Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130;} , partial (9%) [THC2376915]

0,4768

3

0,03968

0,0025168

0,4428

3

0,18855

0,0415312

0,2615

3

0,47609

0,0841018

THC2380750

Q93NK8 (Q93NK8) YsaW, partial (7%) [THC2379106] COAT_BPHK7 (P49861) Major capsid protein precursor (Gp5) (Head protein), partial (5%) [THC2380750]

0,3938

3

0,15380

0,0224633

THC2384167

Unknown

0,2763

3

0,40343

0,0693034

THC2391454

Unknown

0,4677

3

0,20287

0,053131

THC2402993

0,4646

3

0,05098

0,0038616

THC2406944

Unknown Q9F8M7 (Q9F8M7) DTDP-glucose 4,6-dehydratase (Fragment), partial (11%) [THC2406944]

0,3775

3

0,21895

0,0394318

THC2407272

Unknown

0,3718

3

0,09081

0,0072812

THC2408099

Q6IQD8 (Q6IQD8) Zgc:56665 protein, partial (12%) [THC2408099]

0,1896

3

0,40951

0,0462661

THC2411757

Unknown

0,4971

3

0,17129

0,0458845

THC2425047

0,4378

3

0,18100

0,0376909

0,3258

3

0,17908

0,0211176

THC2440554

Unknown O60448 (O60448) Neuronal thread protein AD7c-NTP, partial (15%) [THC2431967] Q60501 (Q60501) Hamster p7 preinsertion DNA, partial (9%) [THC2440554]

0,4451

3

0,29193

0,0861702

THC2443836

Unknown

0,2355

3

0,22986

0,0209389

THC2446811

Unknown

0,2537

3

0,23564

0,0242066

TIMP3

Homo sapiens mRNA for KIAA1657 protein, partial cds. [AB051444]

0,4577

6

0,37354

0,038299

TLR2

0,0807

30

0,22354

2,619E-22

0,5099

9

0,23482

0,0028659

0,3966

6

0,13966

0,0004449

TMEPAI

Homo sapiens toll-like receptor 2 (TLR2), mRNA [NM_003264] Homo sapiens cDNA FLJ40300 fis, clone TESTI2029131. [AK097619] Homo sapiens transmembrane protein 92 (TMEM92), mRNA [NM_153229] Homo sapiens transmembrane, prostate androgen induced RNA (TMEPAI), transcript variant 1, mRNA [NM_020182]

0,3947

6

0,08342

3,757E-05

TMSL8

Homo sapiens thymosin-like 8 (TMSL8), mRNA [NM_021992]

0,3063

3

0,20965

0,0256316

TNC

Homo sapiens tenascin C (hexabrachion) (TNC), mRNA [NM_002160] Homo sapiens tumor necrosis factor, alpha-induced protein 2 (TNFAIP2), mRNA [NM_006291] Homo sapiens tumor necrosis factor, alpha-induced protein 6 (TNFAIP6), mRNA [NM_007115] Homo sapiens tumor necrosis factor receptor superfamily, member 19 (TNFRSF19), transcript variant 2, mRNA [NM_148957] Homo sapiens troponin I type 2 (skeletal, fast) (TNNI2), mRNA [NM_003282]

0,1855

3

0,03181

0,0003148

0,2248

3

0,18981

0,0137042

0,2189

3

0,13012

0,0063589

0,5055

6

0,35197

0,0471048

0,3583

3

0,10594

0,0091547

0,2524

6

0,67090

0,0010101

0,3512

3

0,13620

0,0143336

0,3669

3

0,12711

0,0136266

0,4255

6

0,10576

0,0001758

TSC22D1

Homo sapiens tumor protein p73-like (TP73L), mRNA [NM_003722] Homo sapiens thyroid peroxidase (TPO), transcript variant 1, mRNA [NM_000547] Homo sapiens triggering receptor expressed on myeloid cells 1 (TREM1), mRNA [NM_018643] Homo sapiens tripartite motif-containing 29 (TRIM29), mRNA [NM_012101] Homo sapiens TSC22 domain family, member 1 (TSC22D1), transcript variant 1, mRNA [NM_183422]

0,4805

3

0,01116

0,0002048

TSPAN7

Homo sapiens tetraspanin 7 (TSPAN7), mRNA [NM_004615]

0,2234

3

0,04555

0,0008142

THC2376915 THC2379106

THC2431967

TMEM16A TMEM92

TNFAIP2 TNFAIP6 TNFRSF19 TNNI2 TP73L (TP63) TPO TREM1 TRIM29

TUBAL3 TYRP1 U21252 UNQ3072

W60781 ZBED2 ZNF521 ZNF683 ZPLD1

Homo sapiens tubulin, alpha-like 3 (TUBAL3), mRNA [NM_024803] Homo sapiens tyrosinase-related protein 1 (TYRP1), mRNA [NM_000550] Human rearranged Ig gamma heavy chain V region (VH3-JH4b) mRNA, partial cds, clone JGpFv3-06 VH. [U21252]

0,4457

3

0,19918

0,0463325

0,2660

3

0,08631

0,0037114

0,4957

3

0,19718

0,057893

Homo sapiens epithelial mitogen (EPGN), mRNA [NM_001013442] W60781 zd26f05.r1 Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone IMAGE:341793 5' similar to gb:J02874 FATTY ACIDBINDING PROTEIN, ADIPOCYTE (HUMAN);, mRNA sequence [W60781] Homo sapiens zinc finger, BED-type containing 2 (ZBED2), mRNA [NM_024508] Homo sapiens zinc finger protein 521 (ZNF521), mRNA [NM_015461] Homo sapiens zinc finger protein 683 (ZNF683), mRNA [NM_173574] Homo sapiens zona pellucida-like domain containing 1 (ZPLD1), mRNA [NM_175056]

0,4148

3

0,08501

0,0080476

0,0123

3

0,37607

0,0063332

0,3488

3

0,25569

0,0451449

0,3266

3

0,14874

0,0149137

0,2654

3

0,17381

0,0145119

0,3434

3

0,76836

0,2026115

Signal Transduction: Hippo Component TAZ Functions as a Co-repressor and Negatively Regulates ∆ Np63 Transcription through TEA Domain (TEAD) Transcription Factor Ivette Valencia-Sama, Yulei Zhao, Dulcie Lai, Helena J. Janse van Rensburg, Yawei Hao and Xiaolong Yang

Access the most updated version of this article at doi: 10.1074/jbc.M115.642363 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts

Supplemental material: http://www.jbc.org/content/suppl/2015/05/20/M115.642363.DC1.html This article cites 60 references, 20 of which can be accessed free at http://www.jbc.org/content/290/27/16906.full.html#ref-list-1


J. Biol. Chem. 2015, 290:16906-16917. doi: 10.1074/jbc.M115.642363 originally published online May 20, 2015

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