Signaling and Regulation Molecular Cancer Research TAF4b ... · ulation of integrin α6, an...

554

Signaling and Regulation Molecular
Cancer
Research

TAF4b and Jun/Activating Protein-1 Collaborate to Regulatethe Expression of Integrin α6 and Cancer CellMigration Properties

Margarita Kalogeropoulou1, Angeliki Voulgari1, Vassiliki Kostourou2, Raphael Sandaltzopoulos3, Rivka Dikstein4,Irwin Davidson5, Laszlo Tora4, and Alexander Pintzas1

Abstract

uthors' Astitute ofesearch Fenter “Axpressionf Moleculragana, Aeizmannunctionalellulaire, Clkirch Ced

ote: Supancer Res

orresponoundationhone: 30-

oi: 10.115

©2010 Am

Mol Canc

The TAF4b subunit of the transcription factor IID, which has a central role in transcription by polymerase II,is involved in promoter recognition by selective recruitment of activators. The activating protein-1 (AP-1) fam-ily members participate in oncogenic transformation via gene regulation. Utilizing immunoprecipitation of en-dogenous protein complexes, we documented specific interactions between Jun family members and TATA boxbinding protein–associated factors (TAF) in colon HT29 adenocarcinoma cells. Particularly, TAF4b and c-Junwere found to colocalize and interact in the nucleus of advanced carcinoma cells and in cells with epithelial-to-mesenchymal transition (EMT) characteristics. TAF4b was found to specifically regulate the AP-1 target geneinvolved in EMT integrin α6, thus altering related cellular properties such as migration potential. Using a chro-matin immunoprecipitation approach in colon adenocarcinoma cell lines, we further identified a synergistic rolefor TAF4b and c-Jun and other AP-1 family members on the promoter of integrin α6, underlining the existenceof a specific mechanism related to gene expression control. We show evidence for the first time of an interde-pendence of TAF4b and AP-1 family members in cell type–specific promoter recognition and initiation of tran-scription in the context of cancer progression and EMT. Mol Cancer Res; 8(4); 554–68. ©2010 AACR.

Introduction

The binding of the transcription factor IID (TFIID)complex, composed of the TATA box-binding protein(TBP) and 14 TBP-associated factors (TAF), to promoterDNA is responsive to cellular signals and constitutes thefirst step in transcription. A number of different TFIIDforms with functionally distinct properties exist, amongwhich the TAF10-free TFIID, the TAF4b-containingTFIID, the TAF6δ-containing TFIID, the TBP-freeTFIID, and the seven TAF complex have been described(1, 2). Notably, several studies suggest that TAFs are im-portant in specific events like cell cycle regulation (3, 4),

ffiliations: 1Laboratory of Signal Mediated Gene Expression,Biological Research and Biotechnology, National Hellenicoundation, Athens, Greece; 2Biomedical Sciences Researchlexander Fleming”, Vari, Greece; 3Laboratory of Gene, Molecular Diagnosis, and Modern Therapeutics, Departmentar Biology and Genetics, Democritus University of Thrace,lexandroupolis, Greece; 4Department of Biological Chemistry,Institute of Science, Rehovot, Israel; and 5Department ofGenomics, Institut de Génétique et de Biologie Moléculaire etNRS UMR 7104, INSERM U 964, Université de Strasbourg,

ex, France

plementary data for this article are available at Molecularearch Online (http://mcr.aacrjournals.org/).

ding Author: Alexander Pintzas, National Hellenic Research, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece.21072-73753; Fax: 30-21072-73755. E-mail: [email protected]

8/1541-7786.MCR-09-0159

AInRCEoDWFCIl

NC

CFP

d

erican Association for Cancer Research.

er Res; 8(4) April 2010

apoptosis (5, 6), cancer, and epithelial-to-mesenchymaltransition (EMT; ref. 7). TAF4b was first identified as atissue-specific TFIID subunit, present only in a limitednumber of complexes, and was later shown to be necessaryfor ovarian follicle development, proliferation, and function(8). TAF4b shares high homology with the COOH-termi-nal part of TAF4 in contrast to its coactivator NH2-terminaldomain (9). TAF4b contains a nuclear export signal allowingit to shuttle between the nucleus and the cytoplasm (10),although it displays DNA-binding capacity when incorpo-rated into the TFIID (11). Even though there is no evidencefor a direct sequence-specific contact between DNA andTAF4b, the involvement of TAF4b in direct promoter-selective recognition and subsequent recruitment of acti-vators in a cell type–specific manner has been suggested(12). Indeed, transcriptional induction of the activatingprotein-1 (AP-1) family member c-Jun by TAF4b in gran-ulosa cells has recently been proposed (13). As a memberof the AP-1 transcription factor, c-Jun participates in thecontrol of cellular responses, mainly by converting extra-cellular signals into specific gene expression profiles via thegeneral transcription machinery. Altering the transcrip-tion of target genes, c-Jun has been shown to interact withthe coactivator CBP (14) and with TAF7 in HEK293 andCOS cells (15).c-Jun follows a two-stage activation pattern including a

step of phosphorylation by mitogen-activated protein ki-nases (ERK, JNK, p38) and a subsequent selective forma-tion of dimers whose nature defines the activation of aspecific subset of AP-1 binding site containing target genes

TAF4b and c-Jun Regulate Cell Migration Properties

(16, 17). Notably, AP-1 activity is frequently elevated intransformed cell lines due to an oncogene-specific upregu-lation of the AP-1 family members c-Jun, JunB, Fra-1, andFra-2 (18, 19). Different types of tumors have been relatedto RAS-protein activation, which in turn, regulate the ac-tivity of AP-1 (20). For instance, c-Jun, which is frequentlyimplicated in the acquisition of invasive properties in ag-gressive forms of cancer (21), is required for in vitro cellulartransformation by oncogenic RAS partially via a phosphor-ylation mechanism (22, 23).Colorectal carcinogenesis occurs through the accumula-

tion of gene alterations in tumor suppressor genes and on-cogenes including RAS (24), leading to invasion/metastasis(25). EMT, occurring during the last steps of cancer pro-gression prior to metastasis, is controlled by a number ofregulators resulting in a loss of cell-cell adhesion, mediatedby repression of E-cadherin, whereas vimentin and othermesenchymal proteins like matrix metalloproteinases andfibronectin are upregulated (26). Importantly, activationand maintenance of EMT can be achieved by the signalingcascade of an oncogenic form of Harvey RAS (Ha-RAS;ref. 27). Even though the phenomenon of EMT reflectsa transient state in vivo, by constitutively expressing themutated Ha-RASV12 in the intermediate colon adenomaCaco-2 cell line, we have created a cell line (Caco-H)which adopts and maintains an EMT state (28).In this study, focusing on the investigation of Jun family

members and their interplay with TAFs in colon cancerand metastasis, we have identified an interaction betweenc-Jun and TAF4b and have evaluated its effect in the reg-ulation of integrin α6, an EMT-related AP-1 target gene.The implication of other AP-1 family members in thismechanism suggests a dynamic switch between these pro-teins and their interacting partners in the control oftranscription.

Materials and Methods

Propagation and Treatment of Cell LinesCaco-2, HT29, and HCT116 cells were obtained

from American Type Culture Collection and culturedin DMEM supplemented with 10% fetal bovine serum,antibiotics, and nonessential amino acids (all from Invi-trogen, Corp.). Caco-2 cells constitutively overexpressingHRASV12 (Caco-H) were cultured as mentioned above.For reasons of consistency, the name Caco-H will be usedthroughout the text when referring to Caco-2 cell linesoverexpressing Ha-RASV12 in the case of presenting re-sults of only one Caco-H cell line. Although in some ex-periments, two different Caco-H cell lines were presented(referred to as Caco-H1 and Caco-H2) for the validation ofresults.

Protein Extraction, Western Blotting, and AntibodiesNuclear, cytoplasmic, and whole cell lysate extracts were

prepared as described earlier (19, 29). Protein concentra-tions were determined by the Bradford method using aBio-Rad protein assay kit. Extracts were subjected to

www.aacrjournals.org

SDS-PAGE and transferred to a nitrocellulose membrane(Pall Corporation). The antibodies used for immunoblot-ting are described in Supplementary Data. Signals were vi-sualized using enhanced chemiluminescence (AmershamBiosciences) after exposure to Kodak Super RX film. Allexperiments were repeated at least three times. Representa-tive images are shown.

RNA Extraction and Reverse Transcription-PCRRNA was prepared from sampled cells by the TRIzol re-

agent (Invitrogen). Reverse transcription was carried outusing the SuperScript Reverse Transcriptase (Invitrogen)and oligo(15)-(dT), following the instructions of the manu-facturer. Primers are described in the Supplementary Data.Values were measured using the Image-Quant software(Amersham Biosciences). All experiments were repeated atleast three times. Representative images are shown.

Real-time PCRReal-time quantification was carried out using a Bio-Rad

iCycler and the iQ5 Multicolor Real-time PCR detectionsystem (Bio-Rad). Cycling conditions included a denatur-ing step of 3 min at 95°C followed by 40 cycles at 95°C for40 s and annealing/elongation at 60°C for 40 s. All geneswere tested in triplicate. Values were normalized to glycer-aldehyde-3-phosphate dehydrogenase (GAPDH). Resultswere analyzed on the iCycler software.

ImmunoprecipitationNuclear protein extracts (100 μg) were incubated over-

night at 4°C under rotation with 5 μg of c-Jun, JunB,JunD, and TAF4b (9) antibodies in a total volume of500 μL of 100 mmol/L NaCl immunoprecipitation buffer,adding 25 μL of dry Protein A-Sepharose matrix CL-4B(Amersham Biosciences) over a period of 2 h, followedby three washing steps with 500 mmol/L of KCl immuno-precipitation buffer and 100 mmol/L of KCl immunopre-cipitation buffer. To detect specific interactions with TAFs,Western blotting analysis of the immunoprecipitated com-plexes was done by immunoblotting with TAF antibodies.

Chromatin Immunoprecipitations (ChIP) andRe-ChIPsThe protocols used have been previously described (7).

Chromatin was incubated with 5 μg of anti-TAF4b (9) orany other antibody, as indicated overnight at 4°C. ForRe-ChIP experiments, complexes were eluted after thefirst round of immunoprecipitation by 30 min of incu-bation at 37°C in 10 mmol/L of DTT. The elutedchromatin was diluted 20 times in sonication bufferand again subjected to chromatin immunoprecipitationprocedures with the indicated antibodies. De–cross-linking of eluted chromatin was done by the additionof 200 mmol/L of NaCl plus RNase A overnight at65°C. The remaining proteins were digested withProteinase K for 2 h at 42°C. DNA fragments were re-covered by phenol/chloroform extraction.

Mol Cancer Res; 8(4) April 2010 555

Kalogeropoulou et al.

556

Confocal Laser Scanning MicroscopyCells on glass coverslips were fixed with 4% paraformal-

dehyde for 10 min at room temperature. Cell membraneswere permeabilized with 0.1% Triton X-100 in PBS for 15min. Blocking of cells was done with 5% fetal bovine se-rum in PBS for 1 h at room temperature. The antibodiesand dilutions used are listed in the Supplementary Data.Nuclei were stained with Hoechst 33258 (Sigma-Aldrich).The slides were viewed with a 40× objective, Leica TCSSPE confocal laser scanning microscope (Leica Leisertech-nik). The objective lens used was 63×. LAS AF softwarewas used for image acquisition.

DNA Transfection Short Interfering RNA TransfectionPlasmid DNA was transfected into cells by the calcium

phosphate method (30). For short interfering RNA (siRNA)treatment, cells were transfected with human TAF4b siRNA,c-Jun siRNA, TAF4 siRNA, or with siControl using theprotocols of the manufacturer (Dharmacon). After trans-fection (48 and 72 h), cells were harvested and extractionof proteins and RNA was done.

Luciferase Reporter AssayTo assess luciferase activity, the Promega Dual Luciferase

Reporter Assay (Promega Corporation) was used accordingto the instructions of the manufacturer. Luminescence wasmeasured using a Tecan Safire fluorescence plate reader(Tecan Group, Ltd.). The reporter plasmid used was5xcoll-TRE-tata-luciferase (31), whereas the expressionplasmids included TAF4b (9), c-Jun (32), and TAF4 (33).

Cellular Migration AssaysThe assays were done on transwell plates (Corning Co-

star, Co.). Twenty-four hours after transfection withsiRNA, treated cells (1 × 104) were trypsinized and migra-tion ability was measured as described previously (7). Cellswere visualized and counted by bright-field microscopywith a Nikon Eclipse TE200 inverted fluorescence/phasecontrast microscope equipped with a Sony charge-coupleddevice camera with a 40× objective. Data were obtainedfrom two independent experiments, each repeated twice.

Electrophoretic Mobility Shift AssayNuclear extracts (8 μg) were incubated for 60 min at

room temperature with a 32P-labeled double-strandedprobe ([γ-32P]ATP; Perkin-Elmer) in the presence of 1.5μg of poly(deoxyinosinic-deoxycytidylic acid; Sigma-Aldrich). The reaction was loaded onto native 6% poly-acrylamide gels containing 0.5× Tris-borate EDTA buffer.The gel was prerun for 30 min at 120 V, run at 250 V for2 h, dried for 1.5 h, and exposed for 12 h to a storagephosphor screen (Amersham Biosciences). Scanning wasdone with a Storm 860 scanner (Amersham Biosciences)and values were measured using ImageQuant software.For competition, nonlabeled probe (100 molar–fold excessof the labeled probe) was incubated with nuclear extractsfor 30min before the addition of the labeled probe. Double-

Mol Cancer Res; 8(4) April 2010

stranded oligos were blunt-ended (sequences of oligos inSupplementary Data).

Fluorescence-Activated Cell Sorting AnalysisForty-eight hours after the transfection of cells with

TAF4b siRNA or siRNA control, cells were harvested,washed with PBS, and 1 μg of integrin α6 antibody (SantaCruz Biotechnology) was added to 1 × 106 cells and kepton ice for 30 min. After washing twice with PBS, cells wereincubated for 30 min in secondary antibody (anti-mouseAlexa-488) and again washed twice with PBS before anal-ysis using FACScan CANTO II (Becton Dickinson).

Proliferation AssayForty-eight hours after TAF4b and TAF4 siRNA treat-

ment, cells in 12-well plates were fixed with methanol,stained with 0.5% crystal violet, and washed with PBS.Stained cells were extracted using 30% acetic acid. Absor-bance was measured at 595 nm.

Statistical AnalysisData are represented throughout the text with ±SD error

bars. Statistical significance was tested with unpaired Stu-dent's t test.

Results

In Human Colon Carcinoma Cells, c-Jun SpecificallyInteracts with TAF4bTo unveil any interactions between Jun family members

and human TAFs possibly playing a role in carcinogenesis,immunoprecipitations with anti–c-Jun, JunB, and JunDantibodies were done using nuclear extracts from the hu-man colon adenocarcinoma cell line HT29. Among the in-teractions identified (Table 1) by Western blot analysis, aspecific interaction between c-Jun and TAF4b was ob-served in the c-Jun immunoprecipitate, the specificity ofwhich was tested by siRNA against c-Jun and subsequentimmunoprecipitation with anti–c-Jun antibody (Fig. 1A).Verifying this interaction, in the inverse situation, c-Jun

Table 1. TAFs tested in immunoprecipitationswith anti–c-Jun, anti-JunB, and anti-JunDantibodies

TAFs

Interaction with
TAF1, TAF3, TAF4,TAF5, TAF6α, TAF6δ,TAF7, TAF9, TAF12
Molecular C

TAF4b

ancer R

TAF10

c-Jun
− + − JunB − − + JunD − − −
NOTE: TAF4b was shown to interact with c-Jun and TAF10with JunB.

esearch


was detected in the TAF4b immunoprecipitate (Fig. 1A,bottom). We focused on this interaction as they have beenshown to influence each other during the induction of spe-cific transcription programs (13). To identify a potentialrole in cancer-related mechanisms, the same experimentwas done using nuclear extracts from parental Caco-2and Caco-H cells with EMT characteristics. The sameinteraction was confirmed and was slightly enhanced inCaco-H cells (Fig. 1B). Together, these experiments notonly show a specific interaction between TAF4b andc-Jun but also stress out the possible significance of thisinteraction in EMT phenomena.

TAF4b Plays a Role in the Migration Ability of CellsTo further explore the two components involved in the

interaction and identify any possible role in cancer-related


mechanisms, we first analyzed the expression levels of thetwo proteins in Caco-2 and Caco-H cells. The levels ofTAF4b in the total extracts showed small differences be-tween the cell lines tested, whereas, as expected, c-Jun re-vealed an overexpression pattern in all cell lines ascompared with Caco-2 (Fig. 2A). The levels of EMTmar-kers, E-cadherin and vimentin, were also evaluated andshowed that these cell lines were at different stages of can-cer progression. Overexpression of the Ha-RAS protein inCaco-H cell lines (Caco-H1 and Caco-H2) was confirmed.In parallel, a quantification of the mRNA levels of c-Junshowed that they were highly increased in both Caco-Hcell lines as compared with Caco-2, whereas the mRNAlevels of TAF4b, in agreement with its protein expressionpattern, were slightly increased in Caco-H cells comparedwith the increase of c-Jun in the same cell lines (Fig. 2B).In an attempt to elucidate the specific role of TAF4b in

cancer, we reduced the levels of TAF4b and its homologueTAF4 by using specific siRNA either against TAF4b orTAF4. Seventy-two hours after transfection, the reductionof the mRNA levels of TAF4b and TAF4 was measuredby PCR analysis (Fig. 3A, left). Interestingly, the cellssubjected to TAF4b siRNA treatment, but not those trans-fected with TAF4 siRNAs, acquired up to a 76% differen-tiation in appearance, forming prolonged cell protrusions(Fig. 3A, right and bottom). In both cases, cell prolifera-tion was unaffected (Fig. 3B). Because this morphologicchange was predominant in cells with EMT, we investi-gated the role of TAF4b in related cell characteristics. Inter-estingly, the migration ability of Caco-H cells in responseto a chemoattractant gradient was increased by 80% afterTAF4b siRNA treatment (Fig. 3C). Similarly, Caco-2and colon cancer HCT116 cells with partial EMT charac-teristics in which c-Jun and TAF4n were also detected tointeract (Supplementary Data) showed increased migrationabilities of 50% and 30%, respectively, as compared withcontrol cells or cells treated with siControl. Together, theseresults indicate a negative role for TAF4b in cell migrationand metastasis-related phenomena.

TAF4b Regulates the Expression of IntegrinsBased on the proposed preferential regulation of AP-1 site–

containing genes by TAF4b and its implication in cell motil-ity, we focused our study on the search for a TAF4b-regulatedgene playing a role in cell migration while bearing an AP-1site in its promoter. Changes in the mRNA levels of somemetastatic/EMTmarkers after knockdown of TAF4b by siR-NA were analyzed revealing increased levels of only somegenes (e.g., MMP-2 and integrin α2; Supplementary Data).Interestingly, a gene important for cell migration—also bear-ing a putative AP-1 site in its promoter (34), i.e., integrin α6,after TAF4b siRNA treatment in Caco-2, Caco-H, andHCT116 cells—showed a decrease in its mRNA levels(Fig. 4A), surmising a regulatory function of TAF4b on itstranscriptional activation. Similarly, integrin α6 hemidesmo-some partners integrin β4 (Fig. 4C) and integrin β1 (Fig. 4D)showed lower mRNA levels after TAF4b siRNA treatment.

FIGURE 1. Specific interactions between Jun family members and TAFsin colon adenocarcinoma cells. A, top, HT29 nuclear extracts treated withsiRNA control (siControl) or c-Jun–specific siRNA (siRNA c-Jun) wereused for immunoprecipitation with c-Jun antibody. The samples wereanalyzed by Western blotting (WB) with anti–c-Jun and anti-TAF4bantibodies. Downregulation of c-Jun expression decreased theinteraction complex between TAFb and c-Jun as compared withimmunoprecipitation under siControl treatment. Western blotting withanti-actin antibody was used for the control of equal protein loading.Bottom, Western blotting analysis of immunoprecipitation withanti-TAF4b using HT29 nuclear extracts. B, Western blotting analysis ofimmunoprecipitation with anti–c-Jun using nuclear extracts from cellsexpressing mutant Ha-RAS (Caco-H) as compared with their parental cellline Caco-2.



558

Notably, integrin α6 showed higher mRNA levels in theEMT-like Caco-H cells, which have also shown a greaterextent of TAF4b/c-Jun colocalization (Fig. 2C) andin the advanced colon cancer HCT116 cells (Fig. 4A,left) as compared with Caco-2 cells. Interestingly, integrinα6 also showed higher protein levels in Caco-H andHCT116 cells as compared with Caco-2 cells, and thehighest overexpression rate as compared with integrin β4and β1 in the same cell lines (Supplementary Data). Im-munostaining of the integrin α6 protein confirmed the in-creased expression of integrin α6 in Caco-H cells, whereasvisualizing its accumulation in cell protrusions (Fig. 4D,right). At the same time, underlining the specificity ofTAF4b on the regulation of integrin α6, TAF4 siRNAtreatment in the same cells did not affect the mRNA levelsof integrin α6 or that of TAF4b (Supplementary Data).TAF4b-specific siRNA treatment, on the other hand, notonly reduced the levels of integrin α6 but also induced achange in its localization. As in cells under siControl treat-ment, the integrin α6 protein was localized mainly on thecell surface of Caco-H cells. In cells treated with siRNAagainst TAF4b, integrin α6 was found localized throughoutthe cell (Fig. 4E). The same was shown with fluorescence-activated cell sorting analysis comparing the cell surface ex-pression of integrin α6 in cells treated with siControl andcells treated with siRNA against TAF4b (Fig. 4F).The role of integrin α6 in cell migration properties was

verified in our cell system, as a reduction of integrin α6 le-vels through siRNA resulted in an increase of the migrationability of Caco-2 (an increase of 50%), Caco-H (70%), andHCT116 (90%) cells, as compared with cells treated withscrambled siRNA (siC; Fig. 5A). Investigating a possiblecoinvolvement of c-Jun in the regulation of integrin α6,we reduced c-Jun levels by siRNA and observed a decrease


of its expression in the cell lines tested, similarly to TAF4b(Fig. 5B). In addition, fluorescence-activated cell sortinganalysis showed that, similar to TAF4b (Fig. 5C), c-Junchanged the localization of integrinα6 in the cell surface aftertreatment of cells with c-Jun–specific siRNA. This dataindicates that integrin α6, β1, and β4 are transcriptionallyregulated byTAF4b, whereas a coinvolvement of AP-1 familymember and TAF4b-interacting partner c-Jun in the tran-scriptional regulation of integrin α6 has been proposed andwill be further studied.

Complex Formation of c-Jun and TAF4b on the AP-1Site of the Integrin α6 Promoter In vivo and a CellType–Specific, c-Jun–Binding Dependence of TAF4bTo understand the importance of an AP-1 site for gene

regulation by TAF4b, a luciferase reporter construct con-trolled by the AP-1 site (5xcoll-TRE-tata-luciferase) ofthe Collagenase gene promoter was activated after c-JunandTAF4b overexpression inCaco-2 cells. On the other hand,an overexpression of TAF4 did not produce the same effects,suggesting a specific involvement of TAF4b in AP-1–bindingcomplexes (Fig. 6A, left). Overexpression of the transfectedgenes was confirmed by Western blotting (Fig. 6A, right).To investigate the c-Jun/TAF4b interaction and eluci-

date its possible effect on AP-1 site–containing promotersin vivo, ChIP was done with primers encompassingthe AP-1 site of the integrin α6 promoter schematic-ally represented in Fig. 6B. Interestingly, both c-Junand TAF4b were found on the promoter of integrin α6in Caco-2 and Caco-H cells whereas, surprisingly inHCT116 cells, only TAF4b was bound (Fig. 6C and D).Re-ChIP analysis of the c-Jun immunoprecipitate revealedthat c-Jun and TAF4b simultaneously co-occupied the pro-moter of integrin α6 in Caco-2 as well as in Caco-H cells

FIGURE 2. Expression levels of TAF4b and c-Jun in Caco-2 and Caco-H cells. A, total extracts from cells expressing mutated Ha-RAS (Caco-H1 andCaco-H2) were subjected to Western blotting analysis. B, real-time PCR analysis of c-Jun and TAF4b mRNA levels in the same cell lines. All data werenormalized to GAPDH.

Molecular Cancer Research


FIGURE 3. TAF4b influences cell migration. A, left, reduction of TAF4b (siTAF4b) and TAF4 (siTAF4) mRNA levels in Caco-H cells compared withnontarget siRNA (siC) and untreated cells (Control). Right, changes in Caco-H cell morphology after 72 h of TAF4b and TAF4 siRNA treatment.Arrows, protrusions formed after TAF4b siRNA treatment. Bottom, the percentage of cells with changed morphologies after TAF4b siRNA treatmentcompared with the total number of transfected cells. B, graphs showing the proliferation rate of untreated cells (Control), cells with nontarget siRNA(siControl) treatment, and cells with TAF4b (left) and TAF4 (right) siRNA treatment. The results are an average of three independent experiments. C, graphexpressing the changes in the migratory ability of Caco-2, Caco-H, and HCT116 cells treated with siRNA, tested for their ability to migrate in responseto a serum gradient. The results are an average of two independent experiments (*, P < 0.05; **, P < 0.01, as determined by Student's t test).

Mol Cancer Res; 8(4) April 2010www.aacrjournals.org 559


560

FIGURE 4. TAF4b regulates the expression of integrins including integrin α6. A, B, and C, quantification of TAF4b, integrin α6 (ITGa6), integrin β4 (ITGb4)and integrin β1 (ITGb1) mRNA levels via real-time PCR analysis, after treatment of Caco-2, Caco-H, and HCT116 cells with TAF4b siRNA (siTAF4b) comparedwith control nontarget siRNA-treated (siC) cells. D, left, real-time PCR analysis of integrin α6 mRNA levels in Caco-2, Caco-H (H1 and H2), and HCT116 cells.Right, immunofluorescence confocal imaging visualizing integrin α6 localization (green) in Caco-2 and Caco-H cells. Nuclei were stained with Hoechst dye. Mergedimages of nuclei and integrin α6 antibody are shown. E, immunofluorescence confocal imaging visualizing changes in signal intensity and localization of TAF4band integrin α6 after treatment of cells with TAF4b-specific siRNA as compared with cells treated with control nontarget siRNA (siC). F, fluorescence-activated cellsorting analysis of integrin α6 expression levels in live Caco-H cells 48 h after transfection with control nontarget siRNA and TAF4b siRNA.

Mol Cancer Res; 8(4) April 2010 Molecular Cancer Research


(Fig. 6E). In summary, TAF4b was found on the regula-tory AP-1 site of the integrin α6 promoter whereas its in-terplay with c-Jun on this particular site, and only under aspecific cellular context, suggests a differential regulationpattern.To understand the importance of the cell context on the

regulation pattern of integrin α6 by c-Jun and TAF4b, weknocked down c-Jun by siRNA in Caco-2 and Caco-Hcells (Fig. 7A), and followed the integrin α6 promoter oc-cupancy by TAF4b with ChIP analysis. Interestingly, thebinding of TAF4b on this promoter was dependent on


the binding of c-Jun on the AP-1 site in Caco-H cells;the anti-TAF4b antibody could not precipitate promoterchromatin when c-Jun was knocked down (Fig. 7A, bot-tom). On the contrary, in Caco-2 cells, TAF4b remainedbound on the promoter even after the reduction of c-Jun(siRNA c-Jun) protein levels. In the inverse situation inwhich TAF4b levels were reduced by siRNA (Fig. 7B,top), ChIP experiments showed that the binding of c-Junon the promoter of integrin α6 was independent of the pres-ence (siControl) or absence (siTAF4b) of TAF4b (bottom).This enforces the assumption that c-Jun controls the

FIGURE 5. Reduced levels of integrin α6 leads to higher rates of cell migration. A, left, graph expressing the changes in the migratory ability of each cellline after integrin α6 siRNA treatment, presented as an average of two independent experiments. Caco-2, Caco-H, and HCT116 cells were treated withcontrol nontarget siRNA (siC) or integrin α6 siRNA. Right, reduction of integrin α6 (si-integrin α6) mRNA levels are shown compared with nontarget siRNA(siC; *, P < 0.05; **, P < 0.01, as determined by Student's t test). B, quantification of c-Jun and integrin α6 (ITGa6) mRNA levels in Caco-2, Caco-H,and HCT116 cells, after c-Jun siRNA (si-c-Jun) treatment. All data were normalized to GAPDH. C, fluorescence-activated cell sorting analysis of integrin α6expression levels in live Caco-H cells 48 h after transfection with control nontarget siRNA and c-Jun siRNA.



562

binding of TAF4b on the promoter of integrin α6 inCaco-H cells with an EMT-like phenotype, whereas inCaco-2, this binding is independent of c-Jun. Therefore,a partial, cell type–specific c-Jun dependency of TAF4b-binding on this specific promoter is proposed.


Cell Type–Specific AP-1 Complex Formation at the AP-1Site of the Integrin α6 Promoter Together with TAF4bTo further clarify the AP-1 composition on the promoter

of integrin α6 in relation with TAF4b, we did Re-ChIPexperiments in Caco-H cells. Interestingly, the integrin

FIGURE 6. c-Jun and TAF4b bind on the AP-1 site of the promoter of integrin α6. A, left, chart showing the luciferase activity in Caco-2 cells transfected with5xcoll-TRE-tata-luciferase plasmid or cotransfected with c-Jun (c-Jun-TRE-luc), TAF4b (TAF4b-TRE-luc), and TAF4 (TAF4-TRE-luc). Samples wereprepared in triplicate and data are shown as an average of three independent experiments. All data are presented compared with control cells cotransfectedwith an empty luciferase vector (*, P < 0.05; **, P < 0.01, as determined by Student's t test). Right, total extracts analyzed by Western blotting, showingprotein overexpression levels in Caco-2 cells. For the detection of overexpressed proteins, anti-Ha (TAF4b) and anti-His (c-Jun) antibodies were used,whereas for TAF4, an antibody against its protein was used. B, schema presenting the integrin α6 promoter close to its +1 site indicating the AP-1 site plusthe region encompassed by the primers designed for the ChIP experiments; C, PCR end point; D, real-time PCR analysis of ChIP experiments on theAP-1 site of the integrin α6 promoter with chromatin extracted from Caco-2, Caco-H, and HCT116 cells. Immunoprecipitation with the indicated antibodiesor, as a negative control, in the absence of any antibody (NoAb). D, results are normalized to inputs. E, Re-ChIP experiments with cross-linkedchromatin from Caco-2 and Caco-H cells, using antibodies as indicated.



α6 promoter was found to be occupied by FRA-2 andc-Fos in addition to c-Jun and TAF4b (Fig. 8A, i), indicat-ing that c-Jun might dimerize with any of these proteinswhile in complex with TAF4b. To clarify the specificityof the complex formation between TAF4b and AP-1 fam-ily members on specific promoters regulated by TAF4b, wefollowed with the same experiment on another promoter,that of vimentin. Vimentin was not found to be regulatedby TAF4b in our system (Fig. 4E), and interestingly,TAF4b was also not found on its promoter together withany AP-1 family member (Fig. 8A, ii).On the other hand, in the Caco-2 cell line, in which

TAF4b exhibited a c-Jun–independent binding, only


FRA-1 was detected on the integrin α6 promoter afterChIP (Fig. 8B, top). Re-ChIP analysis (Fig. 8B, bottom)showed that in Caco-2 cells, in addition to c-Jun, FRA-1was also present on the promoter. In the case of HCT116cells, in which a c-Jun–independent binding of TAF4b wasobserved, FRA-1, ATF2, and c-Fos were identified on thepromoter (Fig. 8C, top). Re-ChIP experiments furthershowed that TAF4b was able to interfere with FRA-1,ATF2, and c-Fos, but not FRA-2 (Fig. 8C, bottom). Be-cause c-Jun was not detected within the TAF4b/AP-1 fam-ily complex formed in HCT116 cells, we examinedwhether it could be substituted by another Jun familymember on the integrin α6 promoter. Re-ChIP analysisshowed that in HCT116 cells, JunB was detected inTAF4b-immunoprecipitated chromatin whereas in Caco-2 and Caco-H cells, neither JunB nor JunD were present(Fig. 9A, top). Interestingly, JunB was not able to inter-act with TAF4b in the same cell line (Fig. 9A, bottom),indicating that the presence of both proteins, TAF4band JunB, on the promoter of integrin α6 was promoterDNA–dependent. JunB siRNA treatment in HCT116

FIGURE 7. Binding of TAF4b on the promoter of target gene integrin α6 ismerely dependent on c-Jun. A and B, top, total protein extracts ofCaco-H and Caco-2 cells analyzed by Western blotting, showingchanges in protein levels of c-Jun, TAF4b, and integrin α6 after siRNAknockdown of c-Jun and TAF4b, respectively, Bottom, ChIP experimentdone with the indicated antibodies under siControl treatment and c-JunsiRNA or TAF4b siRNA treatment, respectively, for 48 h.

FIGURE 8. TAF4b forms a complex with different AP-1 familymembers around the promoter of integrin α6, depending on the cell line.A, Re-ChIP experiments with Caco-H cells chromatin on the promoterof integrin α6 (i) and vimentin (ii). The first round of immunoprecipitationwas done with anti-TAF4b, whereas the second immunoprecipitationwas done with the indicated antibodies. B, top, ChIP experiment withCaco-2 cells. Immunoprecipitation with FRA-1 antibody. Bottom,Re-ChIP analysis in Caco-2 cells with antibodies as indicated. C, top,ChIP experiment in HCT116 cells. Binding of FRA-1, ATF2, and c-Foson the AP-1 site of integrin α6 was tested. Bottom, Re-ChiP experimentwith antibodies as indicated.



564

FIGURE 9. JunB recruits TAF4b on the promoter of integrin α6 in HCT116 cells, replacing c-Jun. A, Re-ChIP experiments with cross-linked chromatinfrom Caco-2, Caco-H and HCT116 cells. The first round of immunoprecipitation was done with anti-TAF4b, whereas the second immunoprecipitation wasdone with the antibodies indicated (top). Western blotting analysis of immunoprecipitation with anti-JunB antibodies, using nuclear extracts fromHCT116 cells. The samples were analyzed by Western blot (WB) with anti-JunB and anti-TAF4b antibodies (bottom). B, quantification of JunB and integrinα6 (ITGa6) mRNA levels in HCT116 (top) and Caco-H (bottom) cells, after JunB siRNA (siJunB) treatment as compared with cells treated withscrambled siRNA (siC). All data were normalized to GAPDH. C, top, total protein extracts analyzed by Western blotting, showing the change in protein levelsof JunB and integrin α6 after JunB siRNA knockdown, in HCT116 and Caco-H cells. Bottom, ChIP experiment done with the indicated antibodiesunder siControl treatment and JunB siRNA treatment for 48 h in HCT116 and Caco-H cells. D, total extracts from Caco-2, Caco-H, and HCT116 cellssubjected to Western blotting analysis using the antibodies indicated.

Mol Cancer Res; 8(4) April 2010 Molecular Cancer Research


cells showed decreased levels of integrin α6 expression,whereas in Caco-H cells, under the same conditions,the expression of integrin α6 presented no significantchanges (Fig. 9B). Therefore, to determine a possibleJunB-dependence in the binding of TAF4b on the integrinα6-promoter in this particular cell line, HCT116, wefollowed with ChIP experiments after the knockdown ofJunB by specific siRNA treatment (Fig. 9C, top). As acontrol, the same experiment was done in Caco-H cellsin which JunB was not found on the promoter of integrinα6 together with TAF4b. Interestingly, the integrin α6promoter occupancy in HCT116 cells by TAF4b showeda dependency on the presence of JunB because the anti-TAF4b antibody could not precipitate promoter chromatinafter the exclusion of JunB (Fig. 9C, bottom). On the con-trary, in Caco-H cells, TAF4b remained on the promoterunaffected by the reduced JunB protein levels from siRNA.To assess both the preferential binding of specific AP-1

family members on the promoter of integrin α6 and theircell type–specific interplay with TAF4b, we analyzed theexpression levels of particular AP-1 family members in to-tal extracts of Caco-2, Caco-H, and HCT116 cells. Asjudged by Western blot analysis (Fig. 9D), the levels ofthese proteins were cell type–dependent. Interestingly,the expression levels of AP-1 factors c-Jun, FRA-2, andATF2 correlated with the occupancy of integrin α6 pro-moter; whereas some of them, e.g., FRA-1, did not. Theformation of complexes between AP-1 family members,together with TAF4b, might not be explained solely bythe respective expression levels of each AP-1 member.Validating the proposed preferential binding of Jun fam-

ily members, together with TAF4b on the promoter of in-tegrin α6, we analyzed the actual binding of proteincomplexes on this exact DNA segment (AP-1 site) by gelshift assays using nuclear extracts from Caco-2, Caco-H,and HCT116 cells. Indeed, the presence of the integrinα6 probe induced the specific binding of a protein com-plex in all cell lines (Fig. 10; lanes 5, 6, and 7). Interest-ingly, the DNA-binding activity in each cell line wasrelevant to the expression levels of integrin α6 in the re-spective cell line, as shown in Fig. 5C. Caco-H andHCT116 cell extracts (lanes 6 and 7) showed stronger pro-tein complex signals as compared with Caco-2 (lane 5). Toexclude any off-target effects, we added an excess of non-radiolabeled integrin α6 probe (lane 4), which almost abol-ished the protein complex formation on the AP-1 site ofintegrin α6, whereas with other nonspecific competitors(lanes 2 and 3), the complex formations on these sites re-mained intact. In agreement, a nonspecific probe (coldETS 1.3) did not affect the intensity of the signal (lane2) whereas a cold vimentin probe (Vim) containing anAP-1 site competed with the integrin α6 probe (lane 3).Moreover, an integrin α6–mutated (iα6 mut, lane 8)probe, bearing three point mutations, failed to build thesame protein complex as the wild-type AP-1 site of integrinα6. These results indeed validate the protein complex for-mation around the AP-1 binding site of the integrin α6promoter.


Discussion

For many years, it had been proposed that TAFs weretransmitters of information between activators and the coretranscriptional machinery. Notably, it has been shown thatindividual TAFs are required for the expression of only aspecific subset of genes (35-38), and that the TFIID, orany of its other forms, was recruited in core promotersby a direct interaction between TAFs and their gene-specific activators (39). Given that TAF4b target pro-moter selectivity is enhanced by activators such as c-Junand Sp-1 (12), we propose a synergistic function of thetwo factors, c-Jun and TAF4b, through their interactionin which TAF4b is the coactivator of AP-1 during regu-lation of AP-1 target genes (Fig. 11). Importantly, becauseamong all the human TAFs, only TAF4b was found tointeract with c-Jun, we cannot exclude the possibility ofthis interaction taking place independently of the TFIIDcomplex.

FIGURE 10. Cell type–specific complex formation around the AP-1 site ofintegrin α6 evaluated by electrophoretic mobility shift assay analysis.Electrophoretic mobility shift assay was done using labeled probescontaining the AP-1 binding site of integrin α6. Probes were incubatedin the presence (+) of Caco-2, Caco-H, or HCT116 nuclear extracts or inthe absence of any cell extract (−). For competition experiments, anexcess of the indicated unlabeled probe (competitor) was added. ETS 1.3binding site was used as a nonspecific competitor. Free probe (F) andsite-specific protein complexes (C) are indicated.



566

Interestingly, TAF12, which heterodimerizes with TAF4or TAF4b, has also been shown to directly interact withAP-1 family member ATF7, controlling transcription(40). In the present study, we suggest that TAF4b func-tions as a coactivator of integrin α6 transcription in con-cert with activator c-Jun or, depending on the cell type,with other AP-1 family members. We propose that TAF4btogether with AP-1 factors induces preinitiation complexformation and transcriptional regulation of certain genescontaining an AP-1 binding site (Fig. 8).The coactivator domain of TAF4 and TAF4b is not con-

served, leading to the hypothesis of an independent se-quence-specific coactivator function. Our analysis, usingluciferase reporter constructs transfected to colorectal can-cer cells, supports that TAF4b, but not TAF4, acts as acoactivator on specific AP-1 targets. Furthermore, in ourstudy, we observed that an intracellular reduction ofTAF4b might be compensated by induced expression levelsof TAF4 (Supplementary Data), reinforcing the previousassumption that TAF4 was limiting the TAF4b-containingTFIID complexes present in cells (36). Interestingly, TAF4and TAF4b, through their competitive equilibrium inTFIID, have been shown to regulate the expression ofgenes involved in cell proliferation and transforminggrowth factor-β signaling, known to drive tumor cells toEMT transformation (41, 42). This underlines the impor-tance of further analyzing the gene- and signal-specificcoactivator functions of both factors.


AP-1 Family Members Share Transcriptional Regulationof Target Genes through Interaction with TAF4bBased on our observations, the modulation of AP-1 com-

position in response to external signals, possibly by exchang-ing the most abundant AP-1 family member in each celltype, serves as a regulatory mechanism triggering the expres-sion of specific genes. Indeed, we have shown that in theintegrinα6 promoter, c-Jun is not the only Jun family mem-ber occupying the AP-1 site together with TAF4b but mightas well be replaced by JunB, for example, within HCT116cells. Other AP-1 family members were found on this site aswell, probably building dimers in the absence or presence ofc-Jun and in a cell type–dependent mechanism, to some ex-tent, in an expression level–proportional manner (Fig. 8).Because the protein levels of transcription factors do not per-fectly correlate with the pattern of occupancy of the integrinα6 promoter, other mechanisms that fine-tune the affinityof particular AP-1 family members on the cognate cis ele-ment could be considered. For instance, the interaction withTAF4b or the existence of particular posttranslational mod-ifications could result in DNA-affinity alteration. In thisvein, it is well known that the mutational profile of each cellline influences signaling pathways such as JNK and mito-gen-activated protein kinase, which are crucial for AP-1 reg-ulation. Notably, theHCT116 cell line bears, among others,endogenous mutated forms of K-RAS and PIK3CA onco-genes, whereas the Caco-H cell line overexpresses a mutatedHa-RAS oncogene leading to high levels of JNK activity

FIGURE 11. Model of TAF4b/AP-1 complexfunction on target genes, depending on theircellular environment. According to its cellularenvironment, TAF4b forms distinct complexeswith AP-1 family members to drivetranscriptional activation. A, activator (c-Jun)plus coactivator (TAF4b)–dependenttranscriptional activation on the promoter ofintegrin α6 in the model of Caco-H cells. TAF4brecruitment to the AP-1 site of the promoter andsubsequent activation of transcription isdependent on c-Jun. B, activation oftranscription by TAF4b and FRA-1. c-Jun is alsopresent in the promoter but recruitment ofTAF4b on the AP-1 site is not solely dependenton c-Jun itself. C, transcriptional activation oftarget gene by TAF4b and Jun family memberJunB in HCT116 cells. JunB, which isoverexpressed in these cells, takes over thetransactivation role of c-Jun.



(19). Our study illustrates the significance of screening dif-ferent cell types as a way to link genetic alterations to partic-ular patterns of gene regulation.Jun proteins have diverse expression profiles and biolog-

ical functions, even though they share high sequence ho-mologies. Although, c-Jun and JunB may induce oppositeeffects (43), they also have gene targets in common (44).Interestingly, JunB has been suggested to substitute c-Junduring mouse development and cell proliferation whenc-Jun is depleted (45), further supporting our suggestionthat AP-1 family members take their promoter-specificinteraction with TAF4b into their own hands and followwith regulation of transcription.

TAF4b is Linked toCellMigrationMechanisms and EMTThe involvement of other TAFs in cancer and/or metas-

tasis has already been brought into light (7, 46), however,this is the first report linking TAF4b itself with the regula-tion of tumor cell migration. Herewith, we provide evidencethat TAF4b is a suppressor of cell migration regulating, inconcert with c-Jun, the expression of genes playing a pivotalrole in EMT. Accordingly, in a microarray analysis done ingranulosa cells stably overexpressing TAF4b, a number ofAP-1 target genes were upregulated (13), including theEMTmarker vimentin. In our hands, the expression of thisgene was unaffected by the alteration of TAF4b levels inCaco-H cells, possibly due to an already highly activatedstate of this promoter in the EMT cells. Indeed, in theseparticular cells, in contrast with the epithelial Caco-2 cells,the vimentin gene was found to be predominantly regulatedby FRA-1 (19), which did not interact with TAF4b in ourexperiments, suggesting a cell type–specific and TAF4b-independent mechanism regulated by AP-1 family memberselectivity. On the other hand, TAF4bwas shown to regulateintegrin α6 in different cell lines: a colon adenocarcinomacell line, its derivative Caco-H cell line constitutively over-expressing Ha-RAS, and in parallel, in an established coloncell line that has gained some EMT characteristics. Interest-ingly, in another study (36), integrin α6 was found to beupregulated in mouse fibroblasts after knockdown ofTAF4 and its proven replacement by TAF4b.The integrin subunits α6 and β4 together form the

hemidesmosomal α6β4 integrin, a transmembrane receptor


known to play a critical role in a number of cellular func-tions including cell migration and differentiation (47).Both integrins have been found elevated in several typesof carcinomas, whereas increased expression levels correlatewith invasive phenotypes and EMT (48, 49). Integrin α6 isa controversial member of the integrin family because it hasbeen shown to have a dual role depending on the mutation-al oncogenic profile of the tumor tissue and its stage (50).Interestingly, in human mammary cancer and in mousekeratinocytes, reduced α6 expression has been linked to in-creased cell migration and metastasis (51, 52). On the onsetof cell migration, cells must detach from the basal mem-brane, in part by reducing the expression of integrin α6,which is responsible for detachment and migration. Onthe other hand, when gaining a more mesenchymal pheno-type, integrin α6 expression must again be upregulated toinvade the basal membrane. Nevertheless, under conditionsof induced migration ability (e.g., after TAF4b knock-down), integrin α6 expression must be downregulated sothat migration could begin. Accordingly, integrin α6 ex-pression was reduced in tumors formed after the injectionof Caco-H cells in severe combined immunodeficiencymice, in comparison with cultured cells, supporting itsrole in invasion mechanisms (28).In conclusion, we report a cell type–specific initiation of

transcription controlled by TAF4b together with variantAP-1 family members in a promoter-dependent manner,emphasizing the importance of deciphering the indepen-dent roles of specific TFIID subunits as cofactors that gov-ern transcription.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

Marie Curie Fellowship and EU Marie Curie Research Training Network “TAF-Chromatin” grant MRTN-CT-2004 504228 (L. Tora, A. Pintzas, and R. Dikstein).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 04/15/2009; revised 03/02/2010; accepted 03/03/2010; publishedOnlineFirst 03/30/2010.

References
1. Bell B, Tora L. Regulation of gene expression by multiple forms of
TFIID and other novel TAFII-containing complexes. Exp Cell Res1999;246:11–9.

2. Demeny MA, Soutoglou E, Nagy Z, et al. Identification of a small TAFcomplex and its role in the assembly of TAF-containing complexes.PLoS ONE 2007;2:e316.

3. Wassarman DA, Aoyagi N, Pile LA, Schlag EM. TAF250 is requiredfor multiple developmental events in Drosophila. Proc Natl Acad SciU S A 2000;97:1154–9.

4. Metzger D, Scheer E, Soldatov A, Tora L. Mammalian TAF(II)30 isrequired for cell cycle progression and specific cellular differentiationprogrammes. EMBO J 1999;18:4823–34.

5. Silkov A, Wolstein O, Shachar I, Dikstein R. Enhanced apoptosis of B

and T lymphocytes in TAFII105 dominant-negative transgenic mice islinked to nuclear factor-κB. J Biol Chem 2002;277:17821–9.

6. Lu H, Levine AJ. Human TAFII31 protein is a transcriptionalcoactivator of the p53 protein. Proc Natl Acad Sci U S A 1995;92:5154–8.

7. Voulgari A, Voskou S, Tora L, et al. TATA box-binding protein-associated factor 12 is important for RAS-induced transformationproperties of colorectal cancer cells. Mol Cancer Res 2008;6:1071–83.

8. Voronina E, Lovasco LA, Gyuris A, Baumgartner RA, Parlow AF,Freiman RN. Ovarian granulosa cell survival and proliferation re-quires the gonad-selective TFIID subunit TAF4b. Dev Biol 2007;303:715–26.



568

9. Dikstein R, Zhou S, Tjian R. Human TAFII 105 is a cell type-specificTFIID subunit related to hTAFII130. Cell 1996;87:137–46.

10. Rashevsky-Finkel A, Silkov A, Dikstein R. A composite nuclearexport signal in the TBP-associated factor TAFII105. J Biol Chem2001;276:44963–9.

11. Shao H, Revach M, Moshonov S, et al. Core promoter binding byhistone-like TAF complexes. Mol Cell Biol 2005;25:206–19.

12. Liu WL, Coleman RA, Grob P, et al. Structural changes in TAF4b-TFIID correlate with promoter selectivity. Mol Cell 2008;29:81–91.

13. Geles KG, Freiman RN, Liu WL, Zheng S, Voronina E, Tjian R. Cell-type-selective induction of c-jun by TAF4b directs ovarian-specifictranscription networks. Proc Natl Acad Sci U S A 2006;103:2594–9.

14. Bannister AJ, Oehler T, Wilhelm D, Angel P, Kouzarides T.Stimulation of c-Jun activity by CBP: c-Jun residues Ser63/73are required for CBP induced stimulation in vivo and CBP bindingin vitro. Oncogene 1995;11:2509–14.

15. Munz C, Psichari E, Mandilis D, et al. TAF7 (TAFII55) plays a role inthe transcription activation by c-Jun. J Biol Chem 2003;278:21510–6.

16. Halazonetis TD, Georgopoulos K, Greenberg ME, Leder P. c-Jundimerizes with itself and with c-Fos, forming complexes of differentDNA binding affinities. Cell 1988;55:917–24.

17. Hess J, Angel P, Schorpp-Kistner M. AP-1 subunits: quarrel andharmony among siblings. J Cell Sci 2004;117:5965–73.

18. Zoumpourlis V, Papassava P, Linardopoulos S, Gillespie D,Balmain A, Pintzas A. High levels of phosphorylated c-Jun, Fra-1,Fra-2 and ATF-2 proteins correlate with malignant phenotypes inthe multistage mouse skin carcinogenesis model. Oncogene 2000;19:4011–21.

19. Andreolas C, Kalogeropoulou M, Voulgari A, Pintzas A. Fra-1regulates vimentin during Ha-RAS-induced epithelial mesenchymaltransition in human colon carcinoma cells. Int J Cancer 2008;122:1745–56.

20. Mechta F, Lallemand D, Pfarr CM, Yaniv M. Transformation byras modifies AP1 composition and activity. Oncogene 1997;14:837–47.

21. Bos TJ, Margiotta P, Bush L, Wasilenko W. Enhanced cell motilityand invasion of chicken embryo fibroblasts in response to Junover-expression. Int J Cancer 1999;81:404–10.

22. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M. Oncogenic andtranscriptional cooperation with Ha-Ras requires phosphorylation ofc-Jun on serines 63 and 73. Nature 1991;354:494–6.

23. Johnson R, Spiegelman B, Hanahan D, Wisdom R. Cellular transfor-mation and malignancy induced by ras require c-jun. Mol Cell Biol1996;16:4504–11.

24. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigen-esis. Cell 1990;61:759–67.

25. Grunert S, Jechlinger M, Beug H. Diverse cellular and molecularmechanisms contribute to epithelial plasticity and metastasis. NatRev Mol Cell Biol 2003;4:657–65.

26. Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin CellBiol 2005;17:548–58.

27. Janda E, Lehmann K, Killisch I, et al. Ras and TGFβ cooperativelyregulate epithelial cell plasticity and metastasis: dissection of Rassignaling pathways. J Cell Biol 2002;156:299–313.

28. Roberts ML, Drosopoulos KG, Vasileiou I, et al. Microarray analysisof the differential transformation mediated by Kirsten and HarveyRas oncogenes in a human colorectal adenocarcinoma cell line.Int J Cancer 2006;118:616–27.

29. Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detectionof octamer binding proteins with ‘mini-extracts’, prepared from asmall number of cells. Nucleic Acids Res 1989;17:6419.

30. Chen C, Okayama H. High-efficiency transformation of mammaliancells by plasmid DNA. Mol Cell Biol 1987;7:2745–52.

31. van Dam H, Huguier S, Kooistra K, et al. Autocrine growth andanchorage independence: two complementing Jun-controlled


genetic programs of cellular transformation. Genes Dev 1998;12:1227–39.

32. Angel P, Hattori K, Smeal T, Karin M. Oncogene jun encodes asequence-specific trans-activator similar to AP-1. Cell 1988;55:875–85.

33. Gangloff YG, Werten S, Romier C, et al. The human TFIID compo-nents TAF(II)135 and TAF(II)20 and the yeast SAGA componentsADA1 and TAF(II)68 heterodimerize to form histone-like pairs. MolCell Biol 2000;20:340–51.

34. Nishida K, Kitazawa R, Mizuno K, Maeda S, Kitazawa S. Identi-fication of regulatory elements of human α6 integrin subunit gene.Biochem Biophys Res Commun 1997;241:258–63.

35. Lee TI, Causton HC, Holstege FC, et al. Redundant roles for theTFIID and SAGA complexes in global transcription. Nature 2000;405:701–4.

36. Mengus G, Fadloun A, Kobi D, et al. TAF4 inactivation in embryonicfibroblasts activates TGF β signalling and autocrine growth. EMBO J2005;24:2753–67.

37. Fadloun A, Kobi D, Pointud JC, et al. The TFIID subunit TAF4regulates keratinocyte proliferation and has cell-autonomousand non-cell-autonomous tumour suppressor activity in mouseepidermis. Development 2007;134:2947–58.

38. Tatarakis A, Margaritis T, Martinez-Jimenez CP, et al. Dominant andredundant functions of TFIID involved in the regulation of hepaticgenes. Mol Cell 2008;31:531–43.

39. Mencia M, Moqtaderi Z, Geisberg JV, Kuras L, Struhl K. Activator-specific recruitment of TFIID and regulation of ribosomal proteingenes in yeast. Mol Cell 2002;9:823–33.

40. Hamard PJ, Dalbies-Tran R, Hauss C, Davidson I, Kedinger C,Chatton B. A functional interaction between ATF7 and TAF12 thatis modulated by TAF4. Oncogene 2005;24:3472–83.

41. Davidson I, Kobi D, Fadloun A, Mengus G. New insights into TAFsas regulators of cell cycle and signaling pathways. Cell Cycle 2005;4:1486–90.

42. Pardali K, Moustakas A. Actions of TGF-β as tumor suppressor andpro-metastatic factor in human cancer. Biochim Biophys Acta 2007;1775:21–62.

43. Shaulian E, Karin M. AP-1 as a regulator of cell life and death. NatCell Biol 2002;4:E131–6.

44. Leaner VD, Kinoshita I, Birrer MJ. AP-1 complexes containing cJunand JunB cause cellular transformation of Rat1a fibroblasts andshare transcriptional targets. Oncogene 2003;22:5619–29.

45. Passegue E, Jochum W, Behrens A, Ricci R, Wagner EF. JunBcan substitute for Jun in mouse development and cell proliferation.Nat Genet 2002;30:158–66.

46. Nagy Z, Tora L. Distinct GCN5/PCAF-containing complexes functionas co-activators and are involved in transcription factor and globalhistone acetylation. Oncogene 2007;26:5341–57.

47. Watt FM. Role of integrins in regulating epidermal adhesion, growthand differentiation. EMBO J 2002;21:3919–26.

48. Dajee M, Tarutani M, Deng H, Cai T, Khavari PA. Epidermal Rasblockade demonstrates spatially localized Ras promotion of prolifer-ation and inhibition of differentiation. Oncogene 2002;21:1527–38.

49. Cruz-Monserrate Z, O'Connor KL. Integrin α6β4 promotes migration,invasion through Tiam1 upregulation, and subsequent Rac activa-tion. Neoplasia 2008;10:408–17.

50. Raymond K, Kreft M, Song JY, Janssen H, Sonnenberg A. Dualrole of α6β4 integrin in epidermal tumor growth: tumor-suppressiveversus tumor-promoting function. Mol Biol Cell 2007;18:4210–21.

51. Natali PG, Nicotra MR, Botti C, Mottolese M, Bigotti A, Segatto O.Changes in expression of α6/β4 integrin heterodimer in primary andmetastatic breast cancer. Br J Cancer 1992;66:318–22.

52. Rodius S, Indra G, Thibault C, Pfister V, Georges-Labouesse E.Loss of α6 integrins in keratinocytes leads to an increase in TGFβand AP1 signaling and in expression of differentiation genes. J CellPhysiol 2007;212:439–49.


Signaling and Regulation Molecular Cancer Research TAF4b ... · ulation of integrin α6, an...

Documents

Transcript of Signaling and Regulation Molecular Cancer Research TAF4b ... · ulation of integrin α6, an...