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THAP1 is a nuclear proapoptotic factor that links

prostate-apoptosis-response-4 (Par-4) to PML nuclear bodies

Myriam Roussigne1, Corinne Cayrol1, Thomas Clouaire1, Francois Amalric1 and Jean-PhilippeGirardn,1

1Laboratoire de Biologie Vasculaire, Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne,31077 Toulouse, France

Promyelocytic leukemia (PML) nuclear bodies (PMLNBs) are discrete subnuclear domains organized by thepromyelocytic leukemia protein PML, a tumor suppressoressential for multiple apoptotic pathways. We haverecently described a novel family of cellular factors, theTHAP proteins, characterized by the presence at theiramino-terminus of an evolutionary conserved putativeDNA-binding motif, designated THAP domain. Here, wereport that THAP1 is a novel nuclear proapoptotic factorassociated with PML NBs, which potentiates both serumwithdrawal- and TNFa-induced apoptosis, and interactswith prostate-apoptosis-response-4 (Par-4), a well char-acterized proapoptotic factor, previously linked to pros-tate cancer and neurodegenerative diseases. We show thatendogenous Par-4 colocalizes with ectopic THAP1 withinPML NBs in primary endothelial cells and fibroblasts. Inaddition, we found that Par-4 is a component of PMLNBs in blood vessels, a major site of PML expression invivo. Finally, we investigated the role of the THAPdomain in THAP1 activities and found that this putativeDNA-binding domain is not required for Par-4 bindingand localization within PML NBs, but is essential forTHAP1 proapoptotic activity. Together, our resultsprovide an unexpected link between a nuclear factor ofthe THAP family, the proapoptotic protein Par-4 andPML nuclear bodies.Oncogene (2003) 22, 2432–2442. doi:10.1038/sj.onc.1206271

Keywords: PML nuclear bodies; Par-4; apoptosis;nuclear proteins; protein interaction

Introduction

Promyelocytic leukemia (PML) nuclear bodies (PMLNBs), also known as PODs (PML oncogenic domains),ND10 (nuclear domain 10) and Kr bodies, are discretesubnuclear domains that are specifically disrupted incells from acute promyelocytic leukemia (APL), adistinct subtype of human myeloid leukemia (Ruggeroet al., 2000; Zhong et al., 2000b; Negorev and Maul,

2001). Their name derives from their most intensivelystudied protein component, the PML protein, a RINGfinger IFN-inducible protein encoded by a gene origin-ally cloned as the t(15; 17) chromosomal translocationpartner of the retinoic acid receptor a (RARa) locus inAPL (de The et al., 1991). The analysis of mice, wherethe pml gene was disrupted by homologous recombina-tion, has revealed that PML functions as a tumorsuppressor in vivo (Wang et al., 1998a), which is essentialfor multiple apoptotic pathways (Wang et al., 1998b).PML �/� mice and cells are protected from Fas, tumornecrosis factor (TNFa), ceramide and IFN-inducedapoptosis as well as from DNA-damage-induced apop-tosis. However, the molecular mechanisms throughwhich PML modulates the response to proapoptoticstimuli are not well understood (Quignon et al., 1998;Wang et al., 1998b). Recent studies indicate that PMLmay act by recruiting into PML NBs, proapoptoticproteins such as p53 and Daxx (Ishov et al., 1999; Toriiet al., 1999; Ferbeyre et al., 2000; Fogal et al., 2000; Guoet al., 2000; Li et al., 2000;Pearson et al., 2000; Zhonget al., 2000c; Hofmann et al., 2001; D’Orazi et al., 2002).p53-Dependent DNA-damage induced apoptosis, tran-scriptional activation by p53 and induction of p53 targetgenes are all impaired in PML �/� primary cells (Guoet al., 2000; Pearson et al., 2000). PML physicallyinteracts with p53 and acts as a transcriptionalcoactivator for p53 (Fogal et al., 2000; Guo et al.,2000). This coactivatory role of PML is absolutelydependent on its ability to recruit p53 in the PML NBs(Fogal et al., 2000; Guo et al., 2000). PML may act bystimulating post-translational modifications of p53,since PML-mediated recruitment of p53 and the CBPacetyltransferase or the homeodomain-interacting pro-tein kinase-2 within PML NBs has been shown to induceformation of trimeric p53-PML-CBP or p53-PML-HIPK2 complexes, resulting in the stimulation of p53acetylation or phosphorylation, and increased p53transcriptional activity (Guo et al., 2000; Pearson et al.,2000; Hofmann et al., 2001; D’Orazi et al., 2002). Daxxcould be another important mediator of PML proa-poptotic activities (Ishov et al., 1999; Torii et al., 1999;Li et al., 2000; Zhong et al., 2000c). Daxx was initiallyidentified by its ability to enhance Fas-induced cell death(Yang et al., 1997). Daxx interacts with PML and

Received 21 August 2002; revised 22 November 2002; accepted 22November 2002

*Correspondence: Dr JP Girard;E-mail: [email protected]

Oncogene (2003) 22, 2432–2442& 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00

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accumulates in the PML NBs (Ishov et al., 1999; Toriiet al., 1999; Li et al., 2000; Zhong et al., 2000c).Inactivation of PML results in delocalization of Daxxfrom PML NBs and complete abrogation of Daxxproapoptotic activity (Zhong et al., 2000c). PML NBscontain several other proteins in addition to Daxx andp53, including the autoantigen Sp100 (Sternsdorf et al.,1999), the transcriptional coactivator CBP (LaMorteet al., 1998), the Bloom syndrome DNA helicase BLM(Zhong et al., 1999) and the small ubiquitin-likemodifier SUMO-1 (Ishov et al., 1999; Zhong et al.,2000a), but they are not known to be involved inapoptosis.

We have recently described a novel family of cellularfactors, the THAP proteins, characterized by thepresence of an evolutionary conserved protein motif attheir amino-terminus (Roussigne et al., 2003). Thismotif, designated THAP domain, exhibits strikingsimilarities with the DNA-binding domain of Drosophilamelanogaster P-element transposase, suggesting that theTHAP proteins may correspond to a novel family ofcellular DNA-binding proteins. In humans, the THAPfamily comprises at least 12 distinct members, includingthe death-associated protein DAP4/p52rIPK (Deisset al., 1995; Gale et al., 1998) as well as 11 novel humanproteins (THAP1 to THAP11) (Roussigne et al., 2003).

In this paper, we show that THAP1 is a novel nuclearproapoptotic factor associated with PML NBs, whichinteracts with the proapoptotic protein Par-4 andpotentiates both serum withdrawal- and TNFa-inducedapoptosis. Par-4 is a 38 kDa protein, initially identifiedas the product of a gene specifically upregulated inprostate tumor cells undergoing apoptosis (Sells et al.,1994), which has been shown to function as a transcrip-tional regulator with corepressor (Johnstone et al., 1996)or coactivator (Richard et al., 2001) properties fordistinct isoforms of transcription factor WT1, theWilms’ tumor suppressor protein. Consistent with animportant role of Par-4 in apoptosis, ectopic expressionof Par-4 in NIH-3T3 cells (Diaz-Meco et al., 1996,1999), neurons (Guo et al., 1998), prostate cancer andmelanoma cells (Sells et al., 1997) has been found tosensitize these cells to apoptotic stimuli. In addition,downregulation of Par-4 is critical for ras-inducedsurvival and tumor progression (Barradas et al., 1999)and suppression of Par-4 production by antisensetechnology prevents apoptosis in several systems (Sellset al., 1997), including different models of neurodegen-erative disorders (Guo et al., 1998; Mattson et al., 1999),further emphasizing the critical role of Par-4 inapoptosis. At the carboxy terminus, Par-4 containsboth a leucine zipper domain (aa 290–332), and apartially overlapping death domain (aa 258–332),deletion of which abrogates Par-4 proapoptotic function(Diaz-Meco et al., 1996; Sells et al., 1997; Guo et al.,1998). The Par-4 death domain (Par-4DD) mediatesPar-4 interaction with the three known Par-4-bindingpartners: WT1 (Johnstone et al., 1996), protein kinase Catypical isoforms (aPKCs) (Diaz-Meco et al., 1996) andDAP-like kinase Dlk/ZIP (Page et al., 1999). Here, weshow that Par-4DD also mediates Par-4 interaction with

THAP1 both in vitro and in vivo. Full-length endogen-ous Par-4 colocalizes with THAP1 within PML NBs inprimary endothelial cells or fibroblasts. Par-4 is alsofound to accumulate into PML NBs in blood vesselendothelial cells, a major site of PML expression in vivo(Flenghi et al., 1995; Terris et al., 1995). Altogether, ourresults provide an unexpected link between a nuclearfactor of the THAP family, the proapoptotic proteinPar-4 and PML nuclear bodies.

Results

THAP1 is a novel nuclear protein associated with PMLNBs

In the past few years, we have focused on the molecularcharacterization of novel genes expressed in the en-dothelial cells of postcapillary high endothelial venules(HEV), specialized blood vessels involved in lymphocytemigration (Girard and Springer, 1995b; Girard et al.,1999). We isolated the THAP1 cDNA fortuitously in atwo-hybrid screen of an HEV endothelial cell cDNAlibrary with a cytokine bait. It encodes a novel humanprotein of 213 amino acids that exhibits 93% identitywith its mouse orthologue (Figure 1a). The only

THAP1 PML Merge

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90 110 213146 162THAP1

hTHAP1 1 MVQSCSAYGCKNRYDKDKPVSFHKFPLTRPSLCKEWEAAVRRKNFKPTKYSSICSEHFTP

mTHAP1 1 MVQSCSAYGCKNRYDKDKPVSFHKFPLTRPSLCKQWEAAVKRKNFKPTKYSSICSEHFTP

********************************** ***** *******************

hTHAP1 61 DCFKRECNNKLLKENAVPTIFLCTEPHDKKEDLLEPQEQLPPPPLPPPVSQVDAAIGLLM

mTHAP1 61 DCFKRECNNKLLKENAVPTIFLYIEPHEKKEDL-ESQEQLPSPS--PPASQVDAAIGLLM

********************** *** ***** * ***** * ** ***********

hTHAP1 121 PPLQTPVNLSVFCDHNYTVEDTMHQRKRIHQLEQQVEKLRKKLKTAQQRCRRQERQLEKL

mTHAP1 118 PPLQTPDNLSVFCDHNYTVEDTMHQRKRILQLEQQVEKLRKKLKTAQQRCRRQERQLEKL

****** ********************** ******************************

hTHAP1 181 KEVVHFQKEKDDVSERGYVILPNDYFEIVEVPA

mTHAP1 178 KEVVHFQREKDDASERGYVILPNDYFEIVEVPA

******* **** ********************

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Figure 1 THAP1 is a novel nuclear protein associated with PMLNBs. (a) Alignment of human and mouse THAP1 orthologues.Identical residues (*) are indicated. (b) Primary structure of humanTHAP1. Positions of the THAP domain, the proline-rich region(PRO) and the bipartite nuclear localization sequence (NLS) areshown. Double immunofluorescence staining showing colocaliza-tion of ectopic GFP-THAP1 (green fluorescence) with endogenousPML NBs proteins (red fluorescence) PML (c) and Daxx (d) inhuman primary endothelial cells. Colocalization is indicated inyellow. Bar, 10 mm

THAP1, a link between Par-4 and PML nuclear bodiesM Roussigne et al

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noticeable motifs in the THAP1 predicted proteinsequence were a short proline-rich domain in the middlepart and a consensus nuclear localization sequence(NLS) in the carboxy terminal part (Figure 1b). How-ever, while performing database searches, we discoveredthat the first 89 amino-terminal residues of THAP1correspond to a novel evolutionary conserved proteinmotif, designated THAP domain (Figure 1b), whichdefines a novel family of cellular factors, with at least 12distinct members in the human genome (Roussigne et al.,2003). This domain presents striking similarities with thesite-specific DNA-binding domain of drosophila P-element transposase, including a similar size (B90residues), amino-terminal location and conservation ofthe residues, which define the THAP motif, such as theC2-CH signature (Cys-X2�4-Cys-X35�50-Cys-X2-His),indicating that the cellular THAP proteins may functionas DNA-binding factors.

To analyse the subcellular localization of the THAP1protein, a GFP-THAP1 expression construct wastransfected into primary human endothelial cells.Analysis by fluorescence microscopy revealed that theGFP-THAP1 fusion protein localizes exclusively in thenucleus with both a diffuse distribution and anaccumulation into nuclear dots in the majority oftransfected cells (Figure 1c, d). We used indirectimmunofluorescence microscopy to examine a possiblecolocalization of the nuclear dots containingGFP-THAP1 with known nuclear domains (replicationfactories, coiled bodies, nuclear bodies). This analysisrevealed that GFP-THAP1 staining exhibits a completeoverlap with the staining pattern obtained withantibodies directed against PML (Figure 1c) orDaxx, another well-characterized component of PMLNBs (Figure 1d). Together, these results reveal thatTHAP1 is a novel nuclear protein associated with PMLNBs.

THAP1 interacts with the proapoptotic protein Par-4

To identify cellular targets of THAP1, we performed ayeast two-hybrid screen of a human endothelial cellcDNA library using THAP1 as a bait. From 15� 106

yeast transformants, we identified three positive clonesthat exhibited a robust growth on selective medialacking histidine and adenine. Sequencing of theseclones revealed three identical library plasmids thatcorresponded to a partial cDNA coding for the last 147amino acids (positions 193–342) of the proapoptoticprotein Par-4. Interaction of THAP1 with Par-4 wasconfirmed using full-length Par-4 (Figure 2a). We thenexamined whether the death domain at the C-terminusof Par-4 (Par-4DD), previously shown to be involved inPar-4 binding to WT-1 and aPKC, was required for theinteraction between THAP1 and Par-4. We found thatdeletion of this domain (Par-4D) abrogates THAP1/Par-4 interaction. In contrast, positive interaction betweenTHAP1 and Par-4DD was observed, indicating that thePar-4DD is not only required but also sufficient for theinteraction with THAP1 (Figure 2a). Similar resultswere obtained when two-hybrid experiments were

performed in the opposite orientation using Par-4 orPar-4 mutants (Par-4D and Par-4DD) as baits instead ofTHAP1 (Figure 2a). To confirm the interaction ob-served in yeast, we performed in vitro GST pull-downassays. Par-4DD, expressed as a GST-tagged fusionprotein and immobilized on glutathione sepharose, wasincubated with radiolabelled in vitro translated THAP1.As expected, THAP1 interacted with GST/Par-4DD butnot with GST (Figure 2b, c).

We next addressed whether THAP1 is able toassociate with Par-4DD in vivo. For this purpose, we

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Figure 2 THAP1 interacts with the proapoptotic protein Par-4.(a) Interaction of THAP1 with Par-4 wild-type (Par-4) and Par-4death domain (Par-4DD) in yeast two-hybrid system. Yeast cellswere cotransformed with pGKT7(BD7)-THAP1 and pGAD-T7(AD7)-Par-4, AD7, AD7-Par-4DD or AD7-Par-4D expressionvectors. Transformants were selected on media lacking histidineand adenine. Identical results were obtained by cotransformationof AD7THAP1 with BD7-Par-4, BD7, BD7-Par-4DD or BD7-Par-4D. (b, c) In vitro binding of THAP1 to GST-Par-4DD. Par-4DDwas expressed as a GST fusion protein, purified on glutathionesepharose (b) and employed as an affinity matrix for binding of invitro translated 35S-methionine labelled THAP-1 (c). GST served asnegative control (b, c). The input represents 1/10 of the materialused in the binding assay


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used confocal immunofluorescence microscopy toanalyse the subcellular localization of epitope-taggedPar-4DD in primary human endothelial cells transientlycotransfected with myc-Par-4DD and GFP orGFP-THAP1 expression vectors. In cells transientlycotransfected with GFP expression vector, ectopicallyexpressed myc-Par-4DD was found to accumulateboth in the cytoplasm and in the nucleus of themajority of the cells (Figure 3a). In contrast,transient cotransfection of GFP-THAP1 andmyc-Par-4DD expression vectors dramatically shiftedthe latter to the nucleus with a preferential accumulationin PML NBs (Figure 3b). The effect of GFP-THAP1 onmyc-Par-4DD redistribution was specific since itwas not observed with GFP-APS kinase-1 (APSK1), anuclear enzyme unrelated to THAP1 (Figure 3c) (Bessetet al., 2000), nor with GFP-SUMO-1, another compo-nent of PML NBs (Figure 3d). These later resultssuggest specific recruitment of myc-Par-4DD withinPML NBs by GFP-THAP1 and strongly supportinteraction of the two proteins in vivo. Altogether, thesedata provide both in vitro and in vivo evidence for adirect interaction of THAP1 with the proapoptoticprotein Par-4, mediated through the Par-4 deathdomain.

Par-4 is a component of PML NBs in primary humanendothelial cells and fibroblasts

The localization of THAP1 within PML NBs and itsinteraction with Par-4 led us to investigate a possibleassociation of endogenous Par-4 with PML NBs.Confocal immunofluorescence microscopy using affi-nity-purified anti-Par-4 antibodies (Sells et al., 1997;Guo et al., 1998) was performed on primary humanendothelial cells (Figure 4a, c) or fibroblasts (Figure 4b,d) fixed with methanol/acetone, which makes PML NBscomponents accessible for antibodies (Sternsdorf et al.,1997). This analysis showed an association of Par-4immunoreactivity with nuclear dot-like structures, inaddition to diffuse nucleoplasmic and cytoplasmicstaining (Figure 4a, b). Double immunostaining withanti-PML antibodies revealed that the Par-4 focicolocalize perfectly with PML NBs in cell nuclei (Figure4a, b). In addition, the endogenous Par-4 foci were alsofound to colocalize with ectopic GFP-THAP1 (Figure4c, d).

Par4DD

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Figure 3 THAP1 interacts with Par-4DD in vivo. Myc-Par-4DDand GFP (a), GFP-THAP1 (b), GFP-APSK1 (c) or GFP-SUMO1(d) expression vectors were cotransfected in primary humanendothelial cells. Myc-Par-4DD was stained with monoclonalanti-myc antibody. Green fluorescence, GFP; red fluorescence, Par-4DD. Colocalization is indicated in yellow. Bar, 10 mm

MergePar4 PML

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Figure 4 Localization of Par-4 within PML NBs and colocaliza-tion with THAP1 (a, b) Par-4 is a novel component of PML NBs.Double immunofluorescence staining reveals colocalization of Par-4 and PML within PML NBs in primary human endothelial cells(a) or fibroblasts (b). Endogenous PAR-4 and PML were stainedwith polyclonal anti-PAR-4 (R334, red) and monoclonal anti-PML(green) antibodies, respectively. Bar, 10mm. (c, d) Par-4 colocalizeswith THAP1 in vivo. Double staining reveals colocalization of Par-4 and THAP1 in cells expressing ectopic GFP-THAP1. Primaryhuman endothelial cells (c) or fibroblasts (d) were transfected withGFP-THAP1 expression vector; endogenous Par-4 was stainedwith polyclonal anti-PAR-4 (R334, red) antibodies. Colocalizationis indicated in yellow. Bar, 10 mm


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Par-4 is a component of PML NBs in vivo

To further confirm the localization of endogenous Par-4within PML NBs, a possible association of Par-4 withPML NBs in normal tissue in vivo was analysed byimmunostaining of cryosections from human tonsils(Figure 5). It has previously been shown that, on frozensections, anti-PML antibodies detect the presence of oneto three individual dots in the nuclei of endothelial cellsfrom blood vessels, a preferential site of PML expressionin vivo (Flenghi et al., 1995; Terris et al., 1995). Weobserved a similar dot-like staining pattern with anti-Par-4 antibodies in endothelial cells from HEV bloodvessels (Figure 5a). Double labelling with anti-PMLantibodies revealed overlapping Par-4 and PML stainingpatterns (Figure 5b) indicating that Par-4 is a compo-nent of PML NBs in vivo in HEV blood vesselendothelial cells.

PML recruits THAP1 and Par-4 to PML NBs inimmortalized Hela cells

Since it has been shown that PML plays a critical role inthe assembly of PML NBs by recruiting other compo-nents (Ishov et al.,. 1999; Zhong et al., 2000a;Lallemand-Breitenbach et al., 2001), we next wantedto determine whether PML is involved in the recruit-ment of THAP1 and Par-4 to PML NBs. For thispurpose, we made use of the observation that bothendogenous Par-4 and ectopic GFP-THAP1 do notaccumulate in PML NBs in human Hela cells or mouse3T3 fibroblasts. Expression vectors for GFP-THAP1and HA-PML or HA-SP100 were cotransfected intoHela cells and the localization of endogenous Par-4,GFP-THAP1 and HA-PML or HA-SP100, was ana-lysed by triple staining confocal microscopy. In Helacells transfected with HA-PML (Figure 6a), endogenousPar-4 and GFP-THAP1 were recruited to PML NBs,whereas in cells transfected with HA-SP100 (Figure 6b),both Par-4 and GFP-THAP1 exhibited diffuse stainingwithout accumulation in PML NBs. These findingsindicate that recruitment of THAP1 and Par-4 to PMLNBs depends on PML but not on SP100.

THAP1 is a novel proapoptotic factor

Since PML and PML NBs have been linked toregulation of cell death and Par-4 is a well-establishedproapoptotic factor, we examined whether THAP1 canmodulate cell survival. To this end, we used mouse 3T3fibroblasts, which have previously been used to analysethe proapoptotic activity of Par-4 (Diaz-Meco et al.,1996; Berra et al., 1997), and can be easily transfected.We first analysed serum withdrawal-induced apoptosisin cells transiently transfected with expression vectorsfor GFP-THAP1, GFP-Par-4 or, as control, GFP-APSK1, a nuclear enzyme unrelated to THAP1 andapoptosis (Besset et al., 2000). Transfected cells weredeprived of serum for 24 h and apoptosis was analysedby in situ TUNEL assay (Figure 7a, b) or DAPI staining(Figure 7c). TUNEL assays revealed that overexpres-sion of GFP-THAP1, but not of control GFP-APSK1,

significantly increases vulnerability to apoptosis follow-ing serum withdrawal (Figure 7a, b). Analysis of nuclearcondensation upon DAPI staining gave similar results(Figure 7c). At 24 h after serum withdrawal, apoptosiswas found in 10–20% of untransfected 3T3 cells and in3T3 cells overexpressing GFP-APSK1. Levels of serumwithdrawal induced apoptosis were significantly in-creased to about 65–70% in cells overexpressing GFP-Par-4 or GFP-THAP1. These results demonstrate thatTHAP1, similarly to Par-4, does promote apoptosis in3T3 cells after serum withdrawal.

Since Par-4 has previously been shown to potentiate(TNFa)-induced apoptosis (Diaz-Meco et al., 1999) andPML is essential for apoptosis induction by TNFa

a

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Figure 5 Par-4 is a component of PML NBs in vivo in HEV bloodvessel endothelial cells. (a) Dot-like staining pattern of Par-4 inHEV blood vessel endothelial cells from human tonsil. EndogenousPAR-4 and DARC (an HEV-specific marker) were stained withpolyclonal anti-PAR-4 (R334, red) and monoclonal anti-DARC(green) antibodies, respectively. (b) Colocalization of Par-4 andPML within PML NBs in vivo. Endogenous PAR-4 and PML werestained with polyclonal anti-PAR-4 (R334, red) and monoclonalanti-PML (green) antibodies, respectively. Bar, 30mm

THAP1 MergePar4PML

THAP1 MergePar4SP100

a

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Figure 6 Role of PML in recruitment of THAP1 and Par-4 toPML NBs. (a, b) Triple immunofluorescence staining showingcolocalization of THAP1, Par-4 and PML in Hela cells over-expressing PML and absence of colocalization in Hela cellsexpressing ectopic Sp100. Hela cells were cotransfected withGFP-THAP1 (green) and HA-PML (a) or HA-SP100 (b) expres-sion vectors; HA-PML (a) or HA-SP100 (b) and endogenous Par-4were stained with monoclonal anti-HA (blue) and polyclonal anti-Par-4 (R334, red) antibodies, respectively. Colocalization is shownin Merge. Blue, green and red overlap results in white. Bar, 10mm


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(Wang et al., 1998b), we next examined the effectsof GFP-THAP1 overexpression on TNFa-inducedapoptosis. The 3T3 cells transiently transfected withexpression vectors for GFP-THAP1 or, as control,GFP-APSK1, were treated with TNFa in the presenceof serum, and apoptosis was scored 24 h later by DAPIstaining. We found that overexpression of GFP-THAP1enhances TNFa-induced apoptosis (Figure 7d). Thiseffect was specific since it was not observed in cellsoverexpressing GFP-APSK1 (Figure 7d). We concludedfrom these experiments that THAP1 is a proapoptoticpolypeptide that potentiates both serum-withdrawal andTNFa-induced apoptosis.

The THAP domain is essential for THAP1 proapoptoticactivity

To study the possible role of the amino-terminal THAPdomain in THAP1 proapoptotic activity and binding to

Par-4, we used a THAP1 mutant, THAP1-C1, that isdeleted of the THAP domain (Figure 8a). Confocalimmunofluorescence microscopy revealed that thisTHAP1 deletion mutant (GFP-THAP1-C1) colocalizeswith PML in PML NBs (Figure 8b), indicating that theTHAP domain is not required for THAP1 localizationwithin PML NBs.

We then performed in vitro GST pull-down assays toexamine the role of the THAP domain in Par-4 binding.We found that THAP1-C1 interacts with GST/Par-4DD(Figure 8c), indicating that the THAP domain is notrequired for THAP1/Par-4 interaction. To analyse therole of the THAP domain in THAP1 proapoptoticactivity, we performed cell death assays in mouse 3T3cells transiently transfected with GFP-THAP1-C1 or, ascontrols, GFP-APSK1 and GFP-THAP1 fusion pro-teins expression vectors. Levels of serum withdrawalinduced apoptosis were significantly increased from18% in cells overexpressing GFP-APSK1 (negativecontrol) to about 65% in cells overexpressing wild-typeGFP-THAP1 (positive control). Deletion of the THAPdomain abrogated most of this effect since serum-withdrawal-induced apoptosis was reduced to 28% incells overexpressing GFP-THAP1-C1 (Figure 8d). Theseresults indicate that the THAP domain, although notrequired for Par-4 binding and localization to PMLNBs, is essential for THAP1 proapoptotic activity.

Discussion

Although PML has emerged as a critical mediator ofmultiple apoptotic pathways (Wang et al., 1998b), themolecular mechanisms by which it exerts these effectsremain largely unknown (Ruggero et al., 2000). It hasbeen proposed that PML could exert its proapoptoticfunctions by actively recruiting to PML NBs, proapop-totic factors such as p53 and Daxx (Guo et al., 2000;Zhong et al., 2000c). Here, we report that the PML NBs-associated factor THAP1 is a novel nuclear proapopto-tic factor, which contains a putative DNA-bindingdomain (THAP domain) essential for its proapoptoticactivity. We show that THAP1 interacts and colocalizeswithin PML NBs with Par-4. Par-4 is a well-character-ized proapoptotic factor, which is upregulated in cellsundergoing apoptotic cell death and that seems to play akey role in prostate cancer and neurodegenerativediseases (Sells et al., 1994, 1997; Guo et al., 1998;Mattson et al., 1999). Cell fractionation experimentshave previously revealed that Par-4 is found in both thecytoplasm and the nucleus (Johnstone et al., 1996), andboth nuclear and cytoplasmic roles of Par-4 have beenproposed (Diaz-Meco et al., 1996; Johnstone et al.,1996; Richard et al., 2001). However, association ofPar-4 with PML NBs was not reported. Interestingly,another Par-4-binding partner, the DAP-like kinaseDlk/ZIP (Page et al., 1999), has also been foundassociated with PML NBs (Kogel et al., 1999) suggest-ing that Par-4 may associate within PML NBs withdistinct protein partners, including THAP1 and Dlk/

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Figure 7 Proapoptotic activity of THAP1. (a, b) TUNEL labellingof apoptotic nuclei in mouse 3T3 fibroblasts overexpressing GFP-THAP1 or GFP-APSK1 (as control) fusion proteins (a and b

respectively). TUNEL assays were performed 24 h after serumwithdrawal. Bar, 50mm. (c) THAP1 potentiates serum withdrawal-induced apoptosis. Comparison of apoptosis levels in cellstransfected with GFP-APSK1, GFP-Par-4 or GFP-THAP1 ex-pression vectors. Apoptosis was quantified by DAPI staining ofapoptotic nuclei, 24 h after serum withdrawal. Values are the meansof three independent experiments. (d) THAP1 potentiates TNFa-induced apoptosis. Comparison of apoptosis levels in cellstransfected with GFP-APSK1 or GFP-THAP1 expression vectors.Apoptosis was quantified by DAPI staining of apoptotic nuclei,24 h after addition of TNFa. Values are the means of threeindependent experiments


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ZIP kinase. Surprisingly, we discovered that Par-4 andTHAP1 are associated with PML NBs in primaryhuman endothelial cells and fibroblasts but not inimmortalized mouse 3T3 fibroblasts or human Helacells, which express endogenous PML. Although wefound that overexpression of PML in Hela cells, but notof Sp100, another major protein of PML NBs, issufficient to recruit Par-4 and THAP1 to PML NBs,PML does not appear to interact directly with Par-4 orTHAP1, at least in two-hybrid or GST pull-down assays(data not shown). Therefore, targeting of Par-4 andTHAP1 to PML NBs may be mediated by a PML-interacting adapter protein. This adapter protein couldbe expressed to lower levels within PML NBs fromimmortalized Hela cells compared to primary cells, andPML overexpression in Hela cells may therefore shiftPar4 and THAP1 to PML NBs by enhancing accumula-tion of this adapter protein within PML NBs.

Similarly to p53 and Daxx, THAP1 and Par-4 mayfunction in transcription regulation. Par-4 has pre-viously been shown to function as a transcriptionalregulator that exhibits corepressor (Johnstone et al.,1996) or coactivator activities (Richard et al., 2001) for

distinct isoforms of transcription factor WT1. Thepresence of the THAP domain in THAP1, a putativeDNA-binding motif homologous to the DNA-bindingdomain of Drosophila P-element transposase (Lee et al.,1998) supports the possibility that THAP1 is a DNA-binding transcription factor. As suggested for WT1,THAP1 may therefore recruit Par-4 to specific promo-ters to either stimulate or inhibit transcriptional activa-tion of genes involved in apoptosis, such as theantiapoptotic gene Bcl2, which is known to be down-regulated by Par-4 (Qiu et al., 1999).

Localization of THAP1 and Par4 to PML NBs isunlikely to be absolutely essential for their proapoptoticactivity since we found that both proteins exhibitsignificant proapoptotic activities in mouse 3T3 fibro-blasts, where they are not associated with PML NBs.PML and PML NBs may rather play a regulatory roleby modulating functions of Par-4 and THAP1 inapoptosis and/or transcriptional control with severaldistinct mechanisms. First, PML NBs could representcenters where Par-4 and THAP1 are assembled intomultiprotein complexes and/or post-translationallymodified, as it has been shown for p53 (Guo et al.,

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Figure 8 The THAP domain is required for THAP1 proapoptotic activity. (a) Primary structure of human THAP1 and deletionmutant THAP1-C1. Positions of the THAP domain, the proline-rich region (PRO) and the bipartite nuclear localization sequence(NLS) are shown. (b) The THAP domain (aa 1–90) is not required for localization to PML NBs. Double immunofluorescence stainingreveals colocalization of THAP1-C1 and PML within PML NBs in cells expressing ectopic GFP-THAP1-C1 (green). Primary humanendothelial cells were transfected with GFP-THAP1-C1 expression vector; endogenous PML was stained with monoclonal anti-PML(red) antibody. Colocalization is indicated in yellow. Bar, 5mm. (c) The THAP domain is not involved in Par-4 binding. In vitro bindingof THAP1 or THAP1-C1 to GST-Par-4DD. Par-4DD was expressed as a GST fusion protein, purified on glutathione sepharose andemployed as an affinity matrix for binding of in vitro translated 35S-methionine labelled THAP-1 (wt) or THAP1-C1 (C1). GST servedas negative control. The input represents 1/10 of the material used in the binding assay. (d) The THAP domain is essential for THAP1proapoptotic activity. The percentage of apoptotic cells in mouse 3T3 fibroblasts overexpressing GFP-APSK1, GFP-THAP1 or GFP-THAP1-C1 was determined by counting apoptotic nuclei after DAPI staining, 24 h after serum withdrawal. Values are the means ofthree independent experiments


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2000; Hofmann et al., 2001; Pearson et al., 2000;D’Orazi et al., 2002). Alternatively, the PML NBs couldrepresent nuclear degradation sites or nuclear storagedepots (Negorev and Maul, 2001), which sequester andthereby inactivate Par-4 and THAP1 by titrating themfrom the active pool in the nucleoplasm. This has beenshown for Daxx (Li et al., 2000; Lehembre et al., 2001).Daxx functions as a transcriptional repressor, whichinteracts with transcriptional regulators such as Pax3and Ets1, and inhibits gene activation. PML canregulate the repressor activity of Daxx by sequestratingit into the PML NBs (Li et al., 2000; Lehembre et al.,2001), thus allowing derepression of Daxx target genes.Whatever the effect of PML NBs localization, a role ofTHAP1 and Par-4 in transcriptional control would beconsistent with the observation that PML NBs containmany transcription factors and appear to play a key rolein the regulation of transcription (Zhong et al., 2000b).

We found that the THAP domain of human THAP1,which is not required for Par-4 binding and localizationto PML NBs, is essential for THAP1 proapoptoticactivity. This domain defines a novel evolutionaryconserved gene family with at least 12 distinct membersin the human genome (Roussigne et al., 2003). Althoughthe THAP proteins are likely to be involved in diversecellular processes, some of them could potentiallyfunction in apoptosis, similarly to THAP1. Theselater may include THAP4, which in GenBank(Accession No. AF258556) carried the intriguing anno-tation of ‘Novel cDNA clones with functions ininhibiting cancer cell growth’, and death-associatedprotein DAP4/p52rIPK which was previously identifiedin a genetic screen for genes involved in IFNg-inducedapoptosis in Hela cells (Deiss et al., 1995) and in a two-hybrid screen for indirect activators of the inter-feron-induced kinase PKR, an important mediator ofstress-induced apoptosis (Gale et al., 1998). In any case,future studies will be required to determine whetherother THAP proteins, in addition to THAP1, play a rolein apoptosis.

Materials and methods

Two-hybrid cDNA library construction and screening

For the yeast two-hybrid screening, a cDNA library wasgenerated from microvascular human high endothelial venulesendothelial cells (HEVEC). HEVEC were purified from humantonsils by immunomagnetic selection with monoclonal anti-body MECA-79 as previously described (Girard and Springer,1995a). The SMART PCR cDNA library Construction Kit(Clontech, Palo Alto, CA, USA) was first used to generate full-length cDNAs from 1mg HEVEC total RNA. Oligo-dT-primed HEVEC cDNA was then digested with SfiI anddirectionally cloned into pGAD424-Sfi, a two-hybrid vectorgenerated by inserting a SfiI linker (50-GAATTCGGCCAT-TATGGCCTGCAGGATCCGGCCGCCTCGGCCCAGGA-TCC-30) between EcoRI and BamHI cloning sites of pGAD424(Clontech). The resulting pGAD424-HEVEC cDNA two-hybrid library (mean insert size >1kb, B3� 106 independantclones) was amplified in E. coli. Screening of the two-hybridHEVEC cDNA library was performed in yeast strain AH109

(Clontech) using as a bait the human THAP1 full-lengthcDNA inserted into the MATCHMAKER two-hybrid system3 vector pGBKT7 (Clontech). Plasmid DNA was extractedfrom positive colonies and used to verify the specificity of theinteraction by cotransformation in AH109 withpGBKT7THAP1 or control baits pGBKT7, pGBKT7-laminand pGBKT7-hevin. Positive interaction between THAP1 andPar-4 was confirmed using full-length Par-4 bait (pGBKT-Par-4) and prey (pGADT7-Par-4).

Mammalian cell culture and transfection

Human primary endothelial cells from umbilical vein (HU-VEC, kindly provided by Dr A Bouloumie, Toulouse, France)were grown in complete ECGM medium (PromoCell,Heidelberg, Germany), plated on coverslips and transientlytransfected in RPMI medium using GeneJammer transfectionreagent according to the manufacturer’s instructions (Strata-gene, La Jolla, CA, USA). Human primary skin fibroblasts(kindly provided by Dr P Sassone-Corsi, Strasbourg, France)were grown in modified Eagle’s medium (MEM) supplementedwith 20% fetal calf serum and FGF-2 (all from LifeTechnologies, Grand Island, NY, USA), plated on coverslipsand transiently transfected using GeneJammer transfectionreagent (Stratagene). Human Hela cells and mouse 3T3-TOfibroblasts (Clontech) were grown in Dulbecco’s modifiedEagle’s medium supplemented with 10% fetal calf serum and1% penicillin–streptomycin (all from Life Technologies,Grand Island, NY, USA), plated on coverslips, and transientlytransfected with Lipofectamine reagent (Life Technologies)according to the supplier’s instructions or with calciumphosphate method using 2 mg plasmid DNA.

Plasmid constructions

The full-length coding region of THAP1 (GenBank #BC021721) was amplified by PCR from HEVEC cDNA withprimers 2HMR10 (50-CCGAATTCAGGATGGTGCAGTCCTGCTCCGCCT-30) and 2HMR9 (50-CGCGGATCCTGCTGGTACTTCAACTATTTCAAAGTAGTC-30), digested withEcoRI and BamHI, and cloned in frame downstream of theGal4-binding domain (Gal4-BD) or Gal-4 activation domain(Gal4-AD) in pGBKT7 and pGADT7 two-hybrid vectors(Clontech) to generate pGBKT7THAP1 and pGADT7-THAP1, or downstream of the enhanced green fluorescentprotein (EGFP) ORF in pEGFPC2 vector (Clontech) togenerate pEGFPC2THAP1. THAP1-C1 (aa 90–213) wasamplified by PCR, using pGBKT7THAP1 as template, withprimers 2HMR12 (50-GCGGAATTCAAAGAAGATCTTCTGGAGCCACAGGAAC-30) and 2HMR9, digested withEcoRI and BamHI, and cloned in pGBKT7 and pEGFP-C2vectors to generate pGBKT7THAP1-C1 and pEGFPC2THAP1-C1 expression vectors. Full-length human Par-4 wasamplified by PCR from human thymus cDNA (Clontech),with primers Par-4.8 (50-GCGGAATTCATGGCGACCGGTGGCTACCGGACC-30) and Par-4.5 (50-GCGGGATCCCTCTACCTGGTCAGCTGACCCACAAC-30), digested withEcoRI and BamHI, and cloned in pGBKT7, pGADT7 andpEGFP-C2 vectors, to generate pGBKT7-Par-4, pGADT7-Par-4 and pEGFPC2-Par-4. pGBKT7-Par-4D (aa 1–276) andpGADT7-Par-4D were constructed by subcloning a EcoRI–BglII fragment from pGADT7-Par-4 into the EcoRI andBamHI sites of pGBKT7 and pGADT7. Par-4DD (aa 250–342) was amplified by PCR, using pGBKT7-Par-4 as template,with primers Par-4.4 (50-CGCGAATTCGCCATCATGGGGTTCCCTAGATATAACAGGGATGCAA-30) and Par-4.5,and cloned into the EcoRI and BamHI sites of pGBKT7 and


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pGADT7 to obtain pGBKT7-Par-4DD and pGADT7-Par-4DD. Par-4DD (aa 250–342) was also amplified by PCR withprimers Par-4.10 50-GCCGGATCCGGGTTCCCTAGATA-TAACAGGGATGCAA-30 and Par-4.5, and cloned in framedownstream of the glutathion S-transferase ORF, into theBamHI site of the pGEX-2T prokaryotic expression vector(Amersham Pharmacia Biotech, Saclay, France). To generatepEF-mycPar-4DD eukaryotic expression vector, mycPar-4DD(aa 250–342) was amplified by PCR using pGBKT7-Par-4DDas template, with primers myc.BD7 (50-GCGCTCTAGAGC-CATCATGGAGGAGCAGAAGCTGATC-30) and Par-4.9(50-CTTGCGGCCGCCTCTACCTGGTCAGCTGACCCA-CAAC-30), and cloned into the XbaI and NotI sites of the pEF-BOS expression vector (Mizushima and Nagata, 1990).pEGFPC2-APSK1 was generated by subcloning a EcoRI–BamHI fragment from pGADT7-APSK1 (Besset et al., 2000)into the EcoRI and BamHI sites of pEGFPC2 expressionvector. pEGFPC2-SUMO1 was obtained by cloning theSUMO1 coding region, amplified by PCR with primersSumo-1 (50-GAACTGCAGCTAAACTGTTGAATGACCCCCCGTTTG-30) and Sumo-2 (50-GGCAGATCTTGTCT-GACCAGGAGGCAAAACCTTCAA-30) into the BglII andPstI sites of vector pEGFPC2. pcDNA.3-HA-PMLIII wasconstructed by subcloning a BglII–BamHI fragment frompGADT7-HA-PMLIII into the BamHI site of pcDNA3expression vector (Invitrogen, San Diego, CA, USA). Togenerate pGADT7-HA-PMLIII, PMLIII ORF was amplifiedby PCR, using pACT2-PMLIII (a gift from Dr De The, Paris,France) as template, with primers PML-1 (50-GCGGGATCCCTAAATTAGAAAGGGGTGGGGGTAGCC-30) andPML-2 (50-GCGGAATTCATGGAGCCTGCACCCGCCC-GATC-30) and cloned into the EcoRI and BamHI sites ofpGADT7. pSG5-HA-Sp100 was obtained from Dr Dejean(Institut Pasteur, Paris, France).

GST pull-down assays

GST-Par-4DD (250–342) fusion protein and control GSTprotein were expressed in E. coli. DH5a was purified byaffinity chromatography with glutathione sepharoseaccording to the supplier’s instructions (Amersham PharmaciaBiotech). The yield of proteins used in GST pull-downassays was determined by SDS–polyarylamide gel electrophor-esis (PAGE) and Coomassie blue staining analysis.In vitro-translated THAP1 and THAP1-C1 were generatedwith the TNT-coupled reticulocyte lysate system (Promega,Madison, WI, USA) using pGBKT7THAP1 andpGBKT7THAP1-C1 vectors as templates. A measure of25 ml of 35S-labelled wild-type THAP1 or THAP1-C1 wasincubated with immobilized GST-Par-4 or GST proteinsovernight at 41C in the following binding buffer: 10mm

NaPO4 pH 8.0, 140mm NaCl, 3mm MgCl2, 1mm dithio-threitol (DTT), 0.05% NP40, and 0.2mm phenylmethylsulphonyl fluoride (PMSF), 1mm Na Vanadate, 50mm bglycerophosphate, 25 mg/ml chymotrypsine, 5mg/ml aprotinin,10 mg/ml leupeptin. Beads were then washed five times in 1mlbinding buffer. Bound proteins were eluted with 2� LaemmliSDS–PAGE sample buffer, fractionated by 10% SDS–PAGEand visualized by fluorography using Amplify (AmershamPharmacia Biotech).

Immunofluorescence and confocal microscopy

Immunofluorescence staining of nontransfected cells andcells transfected with GFP-tagged and HA- or myc-taggedexpression constructs was performed as previously described(Besset et al., 2000). Briefly, cells were fixed for 15min

at room temperature in PBS containing 3.7% formaldehyde,and permeabilized 5min at room temperature in PBScontaining 0.1% Triton X-100. Alternatively, for Par-4immunostaining, cells were fixed in methanol for 5min at–201C, followed by incubation in cold acetone at –201C for30 s. Permeabilized cells were then blocked with PBS–BSA(PBS with 1% bovine serum albumin) and incubated2 h at room temperature with the following primaryantibodies: rabbit polyclonal antibodies against humanPar-4 (1/100, R-334, Santa Cruz Biotechnology, SantaCruz, CA, USA) or against human Daxx (1/50,M-112, Santa Cruz Biotechnology, Santa Cruz, CA, USA)and/or mouse monoclonal antibodies anti-PML (mouseIgG1, 1/100, mAb PG-M3 from Dako, Glostrup, Denmark),anti-HA tag (mouse IgG1, 1/1000, mAb 16B12 fromBabCO, Richmond, CA, USA) or anti-myc epitope(mouse IgG1, 1/200, Clontech). Cells were then washedthree times in PBS–BSA and incubated for 1 h withCy3 (red fluorescence)-conjugated goat anti-mouse oranti-rabbit IgG (1/1000, Amersham Pharmacia Biotech) orwith FITC-labelled goat anti-mouse-IgG (1/40, AmershamPharmacia Biotech) secondary antibodies, diluted inPBS–BSA. An Alexa Fluor-633 (blue fluorescence)goat anti-mouse IgG conjugate (1/100, Molecular Probes,Eugene, OR, USA) was used in triple immunofluorescencestaining. After extensive washing in PBS, samples were air-dried and mounted in Mowiol. Images were collected on aLeica confocal laser scanning microscope. The GFP (green),Cy3 (red) and Alexa 633 (blue) fluorescence signals wererecorded sequentially for identical image fields to avoidcrosstalk between the channels.

Immunohistochemistry

Tissue specimens of fresh palatine tonsils were embedded inOCT compound (TissueTek, Elkhart, IN, USA) and thensnap-frozen in liquid nitrogen. Cryosections (6 mm) were air-dried overnight and acetone fixed (10min, �201C). The tissuesections were first incubated with a mixture of rabbitpolyclonal antibodies against human Par-4 (1/100, R-334,Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mAbFy6 against the HEV blood vessel marker DARC (mouseIgG1, 1/30; kindly provided by R Horuk, Richmond, CA,USA) or mAb anti-PML (mouse IgG1, 1/10, mAb PG-M3from Dako, Glostrup, Denmark), followed by a mixture ofCy3-conjugated goat anti-rabbit IgG (1/1000, AmershamPharmacia Biotech) and Cy2-labelled goat anti-mouse-IgG(1/1000, Amersham Pharmacia Biotech) secondary antibodies,diluted in PBS–BSA. After extensive washing in PBS, sampleswere air-dried and mounted in Mowiol. Microscopy wasperformed with a Nikon Eclipse TE300 fluorescence micro-scope equipped with a Nikon digital camera DXM1200(Nikon Corp., Tokyo, Japan).

Cell death assays

Mouse 3T3-TO fibroblasts were seeded on coverslips in 12-wellplates at 50–80% confluency and transiently transfectedwith GFP or GFP-fusion protein expression vectorsusing Lipofectamine reagent (Life Technologies) according tothe supplier’s instructions or calcium phosphate method.Serum starvation of transiently transfected cells wasinduced 12–18 h later, by changing the medium to 0%serum, and the amount of GFP-positive apoptotic cells wasassessed 24 h after induction of serum starvation. Cells werefixed in PBS containing 3.7% formaldehyde, 30min at roomtemperature, and permeabilized with 0.1% Triton X-100, 0.1%


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sodium citrate, 2min on ice, and apoptosis was analysed by insitu terminal deoxynucleotidyl transferase-mediated dUTPnick end labelling (TUNEL) and/or 4,6-diamidino-2-pheny-lindole (DAPI) (0.2 mg/ml, 10min at room temperature)staining of apoptotic nuclei exhibiting nuclear condensation.The TUNEL reaction was performed for 1 h at 371Cusing the in situ cell death detection kit, TMR red(Roche Diagnostics, Meylan, France). At least 100 cells werescored for each experimental point using a fluorescencemicroscope.TNFa-induced apoptosis assays were performed by incubat-

ing transiently transfected cells in complete medium containing30 ng/ml of hTNFa (R&D, Minneapolis, MN, USA) for 24 h.Apoptosis was scored as described for serum withdrawal-induced apoptosis.

Acknowledgements

We thank Dr H De The (Paris, France) and Dr A Dejean(Paris, France) for the gifts of plasmids, and Dr A Bouloumie(Toulouse, France) and Dr P Sassone-Corsi (Strasbourg,France) for kindly providing human primary endothelial cellsand fibroblasts, respectively. We are grateful to Denis Jullien,Vincent Ecochard and Anne-Claire Lavigne for critical readingof the manuscript. Special thanks go to Stephanie Boireau andothers members of the Vascular Biology Laboratory (IPBS,Toulouse) for help in initial experiments and stimulatingdiscussions. This work was supported by grants fromFondation de France, ACI ‘Jeunes chercheurs’ Ministere dela Recherche, CNRS, Region Midi-Pyrenees, and Associationpour la Recherche sur le Cancer. This paper is dedicated to thememory of Jacques Feliu.

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