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Dépôt Institutionnel de l’Université libre de Bruxelles / Université libre de Bruxelles Institutional Repository

Thèse de doctorat/ PhD Thesis Citation APA:

Francoz, S. (2006). Mdm4 and Mdm2 cooperate to inhibit p53 activity in proliferating and quiescent cells in vivo (Unpublished doctoral dissertation).

Université libre de Bruxelles, Faculté des Sciences – Sciences biologiques, Bruxelles.

Disponible à / Available at permalink : https://dipot.ulb.ac.be/dspace/bitstream/2013/210858/4/7b2a5a4d-4b67-44a6-97c1-7b7b26fd5cd3.txt

(English version below)

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DBM 00664 Sarah FRANCOZ

Mdm4 and Mdm2 cooperate to inhibit p53 activity in proliferating

and quiescent cells in vivo

Promoter: Dr. Jean-Christophe MARINE Co-promoter: Dr. Eric BELLE FROID Laboratory of Molecular E mbryology Institute of Molecular Biology and Medicine

Université Libre de Bruxelles Belgium

Academie year 2005-2006

Thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR IN SCIENCES

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Mdm4 and Mdm2 cooperate to inhibit p53 activity in proliferating

and quiescent cells in vivo

Promoter: Dr. Jean-Christophe MARINE Co-promoter: Dr. Eric BELLEFROID Laboratory of Molecular Embryology Institute of Molecular Biology and Medicine

Université Libre de Bruxelles Belgium

Academie year 2005-2006

Thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR IN SCIENCES

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[1 ©(i [1 [M] 1 JfblË)

I would like to thank my promoter Jean-Christophe Marine who supervisée! my PhD during ail these exciting years ... almost 5 in Gosselies and Ghent! I really appreciated to work with you because of your enthusiasm that is aiways présent. You were aiways there to motivate me and make me do the best I can! I was and still am very impressed with your great ideas (sometimes too much!) concerning the projects that we conducted together! I aiso appreciate that you are aiways ready to push us to perform and sometimes succeed new experiments with lots of support. I aIso appreciate your dedication for our projects (1 will aiways remember the “nice” evenings at the microscope!) and ail opportunities you gave me for new collaborations, l’d aiso like to thank you for your nice mood ... almost aiways présent.

I was aiso happy that, during the “crisis times” ... and there were several ... we could discuss and “calm down” without problems ... at least I hope! l’d aiso like to thank you for helping me finding a post- doc lab ... l’Il never forget the Mdm2 workshoplll Thus I think ... or hope ... that we were forming a “good team” and I was really happy to work with you. I therefore hope that we’ll maybe hâve the opportunity to work again together in the future ... who knows?!

l’m gratefui to hâve worked with Eric Bellefroid who was my co-promoter and who gave me the opportunity to begin my project in Gosselies. I would aiso like to thank you for your help and support to obtain my F.N.R.S.-Télévie Grant.

l’d like to thank the F.N.R.S. (Fond National pour la Recherche Scientifique) for giving me a Télévie Grant for 5 years. I really appreciate that this organization continued to support my work even after the moving of the lab to Flanders. It’s aiso fun to go to TV shows ... especially as a background for Frédéric François!

I would like to thank our external collaborators. First of ali, Aart Jochemsen who cloned Mdm4 ... Mdmx sorry! ... and so without whom ail this research would not hâve been possible. Thanks aiso for the nice ideas and discussions about my project. Spécial thanks goes out to Gigi Lozano for the Mdm2 and Mdm4 conditional mice, but aiso and especially for the cosubmission ... and acceptance after several attempts of the work of our 2 labs. I thank aiso ali the people who provided us with the mice we used: Tyler Jacks for the p53LSL, Klaus-Armin Nave for the NEX-Cre and Stéphane Schurmans for the Nestin-Cre.

I would like to thank ail LMCB members for the good work we did ail together, but aiso of course for the nice ambiance (“lunch time”.

IBMM-ULB PhD thesis Sarah Francoz - 2006

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énergie et ta disponibilité à “tchatcher” avec moi! Tu vas aussi beaucoup me manquer à Madrid!!! Je crois en toi ma p’tite poulette ...

je ne sais pas de quoi ton futur sera fait, mais il sera magnifique!

Dieter, I found in you a nice colleague aiways motivated by the work.

Thanks a lot for ail the experiments we conducted together, it’s really nice and productive! I aiso found in you a great friend and I really appreciated ail our discussions about “everything and nothing”

(especially at the microtome!), the swimming ... and running (you're a good coach!). You’Il aiways be welcome in Spain my friend! Sven, thanks for the MEFs experiments and your availability in the lab ... and printing of my PhD! Thanks aiso for the cakes and the bets! It’s fun anywayü! Sarah (2! ou l’apprentie!), j’ai été vraiment contente de travailler avec toi sur le projet SM22. Nous avons formé une bonne équipe ... très agréable je pense! J’ai été impressionnée par ta capacité à devenir rapidement indépendante. Bon courage pour la suite! Gilles, merci pour ton sens de l’humour toujours très juste ... et tes imitations mémorables! Merci aussi pour ton aide précieuse au cours du repiquage des clones ES. Ce travail laborieux et devenu

“fun” grâce à toi! Merci aussi pour le ChIP. Alinounet Hiroshima, j’ai été aussi contente de travailler à tes côtés à Gosselies dans l’ambiance “demerdum”! Ludivine, merci merci et merci pour ton aide pour la gestion des colonies de souris à Gosselies ... et les phenol- chloro! Je pense que nous avons tous vraiment apprécié ta motivation et ton énergie ... à déplacer des montagnes! Fred, my dear Fred, I was really happy that you arrived to the lab and glad to discuss with you about your project! l’m sure you’Il be one of the best technician ...

of the DMBR?! l’m Crossing my fingers for you! Thanks aiso for your help with my PhD! Alex, l’m aiways impressed by your energy and motivation ... l’m sure you’Il be a great scientist! Thanks for the lucky owl! Inès, thanks for the ordering which is really efficient and for the organization of the “lab’s life”! Chantal, fortunately you’re here to help me to look for a fiat when I moved to Ghent ... my Dutch is still really bad today! Davide, I just met you few times in Milan, but I directiy appreciated your nice mood ... thanks for the MCB paper! Mimmo ...

crazy Mimmo ... it’s fun to meet you! Thanks for the characterization of the Mdm4 KO. Your work was the starting point of mine! Riet, thanks for the EM pictures. Joris, thank you so much for ALL the work with the mice at the Ledeganck. Fortunately you’re here ... complaining but aiways working like crazy!

l’d like to thank Lianne and Suzanne for the SM22 project. We built ail together with Sarah a nice story! Philip, the “fat guy” as you call you! I was happy to collaborate with you and learn everything about fat!

Thanks to Geert Berx and Jody Haigh for their commenta on

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the PNAS paper. Thanks aiso to ail the DMBR members!

J’aimerais remercier tous les membres de l’IBMM pour leur aide and sympathie au cours de mon travail à Gosselies. J’ai particulièrement apprécié la bonne ambiance du labo d’Embryologie Moléculaire. Merci à: Vincent, Massimo, Kaki, Sadia, Virginie, Coralie, Aiine et Fred.

J’aimerais maintenant remercier tous mes amis de France, de Belgique et d’ailleurs...

Tout d’abord mes amis de BXL! Un grand merci à Claude et Julien ... et à leur canapé qui m’ont accueillie si souvent! J’ai vraiment apprécié les nombreux moments que nous avons passé ensemble ... surtout à la “Petite Planète” et au Marché du Midi à manger des crêpes! Chiat and Ivo, you’re really nice neighbors!

Thanks for your friendship, the mosaic projects and the discovery of the Dutch and Chinese cultures! Fer y Gaby, muchas gracias para todo! Creo que compartimos un amistad muy muy especial y espero que va a quedar para todavia. Vos deseo todo el mejor para vuestra vida. Un besito enorme para vosotros 2! Grazie mille per ti Marco. I was really to hâve you close to me in BXL and to visit Italy with the best guide! I hope we’ll stay good friend even with the distance ... till now we’re dealing really well with it! Dear Enni, l’m so happy that you came back to Belgium! I wish you ail the best my friend. J’aimerais aussi remercier Delphine, Eric et Fred pour tous les bons moments passes à BXL et à Bordeaux. Gracias tambien para ti Angelita! J’ai vraiment apprécié ton amitié ... même si nos chemins se sont séparés depuis ... Que penal Je voudrais remercier du fond du coeur Claude, Fabienne, Laura, Nico et Charlie Van Veerdegem pour leur accueil si chaleureux à BXL. J’ai trouvé en vous une nouvelle famille d’adoption ... belge et je ne suis pas prête de l’oublier! J’espère que malgré les kilomètres qui vont nous séparer à présent nous resteront toujours aussi proches!

A Gand, Gent, Ghent ... j’aimerais surtout remercier ma chère amie Delphine pour toutes les chouette bouffes et bons moments passé ensemble à parler de nos “vies”! Merci pour ton soutien et ton amitié durant cette année à Gand. Je te souhaite plein de bonnes choses pour ta nouvelle vie australienne! Trui, thanks for your friendship. Since we hâve been friends I really learned to appreciate my life in Ghent ... and I felt much more integrated in the DMBR!

Thanks aIso to Rebecca and Sylvester for the fun time we spent together! Debby and Tom, l’d like to thank you so much cause you’re the first ones in the department ... to talk to me and be so nice!

Thanks aiso to Unit 3 and 8 for the Friday evening drinks!

Je voudrais évidemment remercier tous mes amis de Paris pour leur amitié de toujours, leur soutien dans les moments de “coup de mou” et leur patience ... à attendre mes coups de fils et mes réponses aux mails qui tardent trop ... et à supporter mes week-ends pas toujours très bien organisés! Merci aussi pour toutes les visites en

IBMM-ULB PhD thesis Sarah Francoz - 2006

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Ronan. J’espère que mon passage de l'autre côté des Pyrénées ne changera rien à notre longue amitié et ne fera que la renforcer encore plus!

Je tiens à remercier plus que tout mes parents, ainsi que toute ma famille, qui m’ont toujours soutenue et apporté tout leur amour au cours de ma thèse. Maman et Papa, merci beaucoup pour avoir toujours été là tout au long de ces “années belges” et pour être devenu tout comme moi un peu “belches” aussi! Merci aussi pour les déménagements et les découvertes de BXL et Gand en votre compagnie. Merci aussi à toi Raphaël pour avoir toujours été mon p’tit frérot adoré! Et merci aussi pour nous avoir donné avec Florence, notre petit Sacha qui est le rayon de soleil de toute la famille! Belle vie à tous les 3! Je remercie aussi de tout mon coeur mes grands- parents pour avoir été si formidables depuis mon enfance. Un merci très spécial à mon Papl qui est bien le seul à penser ... que je suis une nouvelle Einstein!!!

Je voudrais également remercier tous les amis qui font aussi partie de ma famille ... vous comprendrez je crois! Merci aux Krejcl, aux Rlgois, aux Motsch, aux Lunel et aux Morin.

Pour finir j’aimerais remercier la Belgique, ce pays qui m’a

vraiment bien accueillie tant à BXL, qu’à Gosselies et qu’à Gand! J’ai

vraiment apprécié tous les gens que j’ai pu y rencontrer. Merci à

tous!!!

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President Pr. Jacques Urbain

Secretary Pr. Josiane Roscam

Promoter Dr. Jean-Christophe Marine

Co- Promoter Dr. Eric Bellefroid

Internai expert Pr. Claude Szpirer Dr. Stéphane Schurmans

External expert

Pr. Aart G. Jochemsen Leiden University The Netherlands

Private defense: 12"^ of May 2006 Public defense: 2"*^ of June 2006

IBMM-ULB PhD thesis Sarah Francoz - 2006

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aMT[^®®[ü)©?[i©M

A. p 53. the aatekeeper for qrowth and death...1

1. p53 structure and function... 1

2. p53 régulation... 5

a) Keeping o53 in check in unstressed conditions... 5

(1) Ubiquitination and dégradation... 5

(2) Mdm family...6

(a) Mdm2:...6

(i) in vitro...6

(ii) in vivo... 10

(b) Mdm4:... 11

(i) in vitro ... 11

(ii) in vivo... 15

(3) Additional négative reguiators...18

b) Activation followina DNA damage / stress... 19

(1) Phosphorylation on Ser and Thr residues...19

(2) Acetyiation on Lys...21

(3) Sumoylation on Lys... 22

(4) Deubiquitination by HAUSP... 22

(5) ARF régulation...23

(6) PML régulation... 24

(7) ASPP régulation... 24

3. p53 pathway and tumorigenesis...25

B. Mouse development...29

1. Development of the mouse embryo...29

2. Central nervous System...30

a) Organization of the brain... 30

b) Development of the central nervous svstem...30

(1) Development of the cérébral cortex...30

(2) Development of the cerebellum... 31

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Table of contents

A.

B. o53 expression in Mdm2 or/and Mdm4-deficient

MEFs... ... 36

1. p53 protein levels...36

2. p53 transcriptional activity... 36

3. p53 biological response... 38

C. Tissue spécifie conditional expression of p53 in Mdm2 or/and Md/n4-deficient mice... 38

D. p 53 expression in neuronal oroaenltor cells In Mdm2 or/and /Vtd/n4-deficient mice... 39

1. Mdm4 is critical for cell prolifération of the neuronal progenitor ceils and prévention of ataxia...39

2. Mdm2 is essential for neuronai progenitor celi survivai ... 42

3. Mdm2 and Mdm4 cooperate to prevent apoptosis in neuronal progenitors... 43

E. p 53 expression in postmitotic neurons in Mdm2 or/and Md/n4-deficient mice... 43

F. p 53 activation without DNA-damaae in vivo...45

G. Studv of the p53-independent function of Mdm2 and Mdm4... 46

Mdm2 and Mdm4 are reauired to control o53 in neuronai celis... .49

Mcfm4-null ohenotvoe is non-celi autonomous... 50

Mdm2 is reauired to maintain o53 at low ieveis and to orevent ceii death in neuronal oroaenitors and postmitotic neurons... 51

IBMM-ULB PhD thesis Sarah Francoz - 2006

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D. Mdm4 is essential for restralnina p 53 activitv in

neuronal proqenitors and postmitotic neurons... 53

E. Mdm4 contributes to the overall inhibition of o53 function in an Mdm2-independent manner...55

F. Mdm4 does not modulate p 53 levels independentiv of Mdm2...56

G. p 53 fonctions in absence of DNA damage in vivo . 58 H. The Mdm2-Mdm4-p53 interolav as a taroet for therapeutic intervention...59

I. Lack of qenetic evidence for a p53-independent rôle of Mdm2 and Mdm4 under phvsioloaical expression levels ..61

w. ...J A. Mouse strains... 1. p53... 2. LSL p53... 3. Mdm2... 4. floxed Mdm2... i

5. Mdm4... i

6. floxed Mdm4... Il 7. Nes-Cre...Il 8. NEX-Cre...il B. DNA préparation and aenotvoina... iv

C. Histoloqy... v

1. Time mating... v

2. BrdU injection...v

3. Samples collection and fixation... vi

4. Embedding and sectioning... vi

5. Hematoxyiin & eosin (H&E) staining...vi

6. Immunohistochemistry (IHC)... vii

7. in situ end labeling (ISEL) staining with Klenow

FragEL™ DNA Fragmentation Détection Kit (Calbiochem

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Table of contents

#QIA21)... ix

D. Mouse Embrvonic Fibroblasts (MEFs) culture... ix

1. MEFs préparation... ix

2. Adenovirai infection... x

3. Growth curve...x

4. Colony formation assay... x

5. S-phase entry following sérum starvation and y- irradiation assay...x

6. BrdU incorporation assay...xi

7. Cycloheximide treatment assay... xi

E. Western blottina... xi

F. Real-time quantitative RT-PCR assavs... xiii

1. RNA extraction and cDNA amplification...xiii

2. Real-time quantitative RT-PCR assays...xiii

G. Chromatin Immuno-Precipitation Assay...xiv

mL

wa waa

IBMM-ULB PhD thesis Sarah Francoz - 2006

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aa Ad APAF-1 ARF ARF-BP1 ASPP ATM ATR bp BrdU BSA casp-3*

CBP Cdk cDNA CHAPS Chk1/2 CMV CNS COP1 et Da DAB DAPI DNA dNTP EDTA EGL EGTA En EtOH FITC

LiST ©F AiiREVIATlONS

amino acid Adenovirus

Apoptosis-Protease Activating Factor 1 Alternative Reading Frame protein ARF Binding Protein 1

Apoptosis-Stimulating Protein of p53 Ataxia Telangiectasia Mutated

Ataxia Telangiectasia and Rad3 related protein base pair

5-Bromo-2’-deoxyUridine Bovine Sérum Albumin caspase 3-cleaved CREB Binding Protein Cyclin dépendent kinase complementary DNA

sulfonic acid 3[(3-cholamidopropyl)dimethylammonio]-propane Checkpoint kinase 1/2

CytoMegaloVirus Central Nervous System

COnstitutively Photomorphogenic 1 Carboxy terminus

Dalton

3,3’-DiAminoBenzidine DiAmidoPhenylIndole DeoxyriboNucleic Acid

deoxyriboNucleotid TriPhosphate EthyleneDiamine-Tetraacetic Acid External Granular Layer

Ethylene Glycol bis(2-aminoethyl ether)-N,N,N'N'-Tetraacetic Acid Embryonic day n

Ethanol

Fluorescein IsoThioCyanate

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Abbreviations

gapdh glyceraldehydes-3-phosphate dehydrogenase GFP Green Fluorescent Protein

Gl Gastro-Intestinal

Gy gray

H&E Hematoxylin and Eosin

H

2

O water

HAT Histone AcetylTransferases

HAUSP Herpes virus-Associated Ubiquitin-Specific Protease HDAC Histone DeACetylase

Human Mdm2 Human ortholog of Mdm2 Human Mdm4 Human ortholog of Mdm4

HEPES 4-(2-HydroxyEthyl)Piperazine-1-EthaneSulfonic acid H RP HorseRadish Peroxidase

IgG Immunoglobulin G IGL Internai Granular Layer IHC ImmunoHistoChemistry IP Immunoprécipitation ISEL In Situ End Labeling

kb kilobase

kDa kiloDalton

L LoxP

LSL LoxP-STOP-LoxP LTR Long Terminal Repeat Lys Lysine residue

Mdm2 Mouse double minute 2 Mdm4 Mouse double minute 4 MEFs Mouse Embryonic Fibroblasts NBS1 Nijmegen Breakage Syndrome 1

NEDD8 NEural precursor cell expressed, Developmentally Down-regulated 8

Neo Neomycine

NES Nuclear Export Signal

Nés Nestin

NEX NEuronal helix-loop-heliX protein

IBMM-ULB PhD thesis Sarah Francoz - 2006

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NLS Nuclear Localisation Signal NoLS Nucleolar Localisation Signal NP-40 Nonidet-P40

NPC Neural Progenitor Cells

Nt Amino terminus

nt nucléotide

PAGE PolyAcrylamide Gel Electrophoresis

Parc p53-associated, parkin-like cytoplasmic protein PBS Phosphate Buffered Saline

PCAF p300/CBP Associated Factor PCR Polymerase Chain Reaction pHH3 phosphorylated Histone H3

Pirh-2 p53-induced protein with a RING-H2 domain PML Promyelocytic Leukemia Protein

PMSF PhenylMethylSulphonyl Fluoride Pn Postnatal day n

prom promoter

qRT-PCR quantitative Reverse Transcription-Polymerase Chain Reaction

Rb Retinoblastoma

RING Really Interesting New Gene RNA RiboNucleic Acid

rRNA ribosomal RiboNucleic Acid RT Reverse Transcription SDS Sodium Dodecyl Sulphate

Ser Serine residue

SMC Smooth Muscle Cells

SUMO Small Ubiquitin-related Modifier TBS T ris Buffered Saline

Thr Threonine residue

Triton X100 4-(1,1,3,3-Tetraethylbutyl) phenyl-polyethylene glycol

TUNEL Terminal deoxynucleotidyltransferase-mediated dUTP-blotIn Nick End Labelling

Tween 20 monolaurate of polyethylene glycol sorbitan

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Abbreviations

U Unit

uv Ultra Violet light

WT Wild-Type

pTublII p-TubulinlII

A délétion

IBMM-ULB PhD thesis Sarah Francoz - 2006

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NLS NES

1 50 58 98102 292 325 363 393

Figure 1: Schematic représentation of the primary structure of the p53 protein. The different

functionai domains of the protein are represented; the transactivation domain (red), the

Proline-rich domain (orange), the DNA binding domain (blue), the oiigomerisation domain

(yellow) and the reguiatory domain (green). NLS, Nuclear Localization Signal and NES,

Nuclear Export Signal.

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I. Introduction

L OKnTM©[y){êTQ©M

p53 is possibly the most studied protein in cancer research.

Indeed, more than 30.000 papers hâve been published on this molécule. This section is a short and not exhaustive summary and focuses on the introduction of general concepts concerning this field.

A. p 53. the qatekeeper for arowth and death

The p53 tumor suppressor gene is frequently mutated during tumorigenesis. Indeed, more than half of human tumors is associated with mutations that either directiy affect the p53 protein or affect components of pathways that regulate p53. Moreover, mice inactivated for p53 are tumor prone (Donehower étal., 1992; Jacks étal., 1994).

Originally, the p53 protein was identified because of its association with the SV40 DNA large T antigen (Lane and Crawford, 1979; Linzer and Levine, 1979). It was later discovered that this experiment was conducted with a mutated p53 (Finlay et al., 1989).

Moreover, the mutated Ras combined with mutated p53 overexpression leads to transformation of primary rat fibroblasts (Eliyahu et al., 1984; Hicks et al., 1991; Hinds et al., 1989; Parada et al., 1984). Subséquent research with the wild-type protein demonstrated its tumor suppressive rôle. Indeed, p53 expression inhibits the growth of tumor cells In vitro (Baker et al., 1990) and decreases their tumorigenicity (Chen et al., 1990).

1. p53 structure and function

p53 is a protein of 393 aa (amino-acids) in human and 390 aa in mouse. The primary structure of the protein is conserved throughout évolution, particularly in its five characteristic domains (Figure 1):

O the Nt (amino-terminal) transactivation domain (50 aa) is composed of two independently functioning subdomains. These domains interact with several transcription factors (Candau et al., 1997; Lu

IBMM-ULB PhD thesis Sarah Francoz - 2006 1

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and Levine, 1995; Thut et al., 1995; Zhu et al., 1998).

O the proline-rich domain (residues 58-98 in human) contains 15 proline residues of which five PXXP repeats (where P désignâtes proline and X any aa). This domain is implicated in p53 stability control by binding to the corepressor mSinSa. Indeed, mSin3a-p53 binding stabilizes p53 by inhibiting proteasome-mediated dégradation of p53 (Zilfou et al., 2001). Moreover, this domain médiates p53 transactivation by binding to p300 (Dornan et al., 2003; Toledo et al., 2006).

O the DNA binding domain (residues 102-292 in human) is required for sequence-specific DNA binding of the p53 response element defined as: 5’(A/G)3

x

C(A/T)2

x

G(T/C)3

x

[ variable part: 0-13 nt](A/G)3xC(A/T)2xG(T/C)3x3’ (el-Deiry et al., 1992). This domain is often mutated in human tumors (Hollstein étal., 1994).

O Residues 325-363 in human form the tetramerization domain. As a tetramer, p53 binds DNA in a sequence-specific manner more efficiently (Hupp and Lane, 1994). An NLS (Nuclear Localization Signal) is located between the DNA binding domain and the tetramerization domain on residues 316-322 (Liang and Clarke, 2001) and a NES (Nuclear Export Signal) is présent in the tetramerization domain on residues 340-351 (Stommel étal., 1999).

O the et (carboxy-terminal) regulatory domain (residues 363-393 in human) is implicated in the régulation of the activity and the stability of the protein. This région is the target of a number of spécifie posttranslational modifications (Ryan et al., 2001).

p53 is a transcription factor, orchestring the expression of

spécifie target genes implicated in either cell cycle arrest (to prevent

prolifération of damaged cells) or apoptosis (to remove damaged cells

from the organism). However, new data suggest that p53 itself aiso

has a direct rôle in accomplishing cell death, at the mitochondria

(Murphy et al., 2004). During p53-dependent apoptosis, a fraction of

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I. Introduction

p53 protein localizes to mitochondria and induces cytochrome c release, ieading to activation of caspase-3, the main effector caspase (Marchenko et al., 2000). These data suggest that p53 has both transcription-dependent and -indépendant fonctions.

To ciarify this point, based on in vitro transfection and biochemical anaiyses, two knock-in mouse in which the p53 protein carries mutations of two residues that are crucial for transactivation (L25Q, W26S) hâve been generated (Jimenez étal., 2000; Johnson et al., 2005). In the first mouse model (Jimenez et al., 2000), the p53- mutant ailele behaves indistinguishably from a p53-nuil allele. These data suggest therefore that the transactivation fonction of p53 is essential for its fonction. However, it was iater found that these mice aiso carry a mutation in its DNA binding domain, which severely compromises p53 fonction. The second knock-in aileie generated harbors an inducibie mutated p53 (L25Q, W26S) (Johnson et al., 2005). The mice generated retain partiai transactivation activity and therefore apoptotic and growth suppressive activation. Therefore, no clear conciusions can be draw about the invoivement of p53 transactivation fonction in p53 proapoptotic activity.

Despite its transactivation-independent fonction, p53’s major contribution in maintaining the genomic integrity is through transcriptionai régulation of genes required for ceil cycle arrest and apoptosis. Numerous p53 target genes hâve been identified that play a fonction as downstream effectors of p53 fonction.

Many proapoptotic genes are transcriptionai targets of p53 (ei- Deiry, 1998). Most of these genes are coding for:

O Mitochondrial proteins: Bax (Miyashita et al., 1994; Miyashita and Reed, 1995), Bid (Sax et al., 2002), NoxA (Oda et al., 2000a), PUMA (Nakano and Vousden, 2001; Yu et al., 1999) and p53AIP (Oda et al., 2000b).

O Effectors of the apoptotic machinery: Apaf-1 (Moroni et al., 2001).

O Death domain proteins: Fas/APOl (Munsch et al., 2000; Owen-

IBMM-ULB PhD thesis Sarah Francoz - 2006 3

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Schaub et al., 1995), KILLER/DR5 (Takimoto and El-Deiry, 2000;

Wu et al., 1997) and Pidd (Lin et al., 2000).

Therefore, p53 is abie to induce apoptosis In vitro, through the mitochondrial pathway and the release of cytochrome C or through the induction of death domain receptors. Interestingly, oniy few of the p53 pro-apoptotic target genes were shown to be indispensable to médiate p53-dependent apoptosis in vivo (Fridman and Lowe, 2003; Vousden, 2000 ).

p53 aiso induces the expression of target genes involved in G1/S or in G2/M cell cycle checkpoints, controlling in this way the cell cycle progression.

O The main target gene implicated in cell cycle arrest at G1 is WAF1/Cip1 coding for p21 or Cdknia. This protein is a cyclin- dependent kinase inhibitor and therefore negatively régulâtes the cell cycle progression (el-Deiry et al., 1993; Harper ef al., 1993;

Xiong et al., 1993). Ptprv/ESP is another p53 target gene implicated in the régulation of the G1 cell cycle checkpoint (Doumont et al., 2005b).

O Several target genes of p53 that play a rôle in the G2/M cell cycle checkpoint hâve been identified: f4-3-3a (Hermeking et al., 1997), Gadd45 (Kastan et al., 1992), cyclln G1 (Okamoto and Beach, 1994; Zauberman et al., 1995) and Gtse1 (Utrera et al., 1998).

Cell cycle arrest is thought to allow time for repair of DNA damage. In addition, some controversial data suggest that p53 modulâtes DNA repair processes (Dasika et al., 1999; Smith et al., 2000 ).

Whether p53 directiy influence the sélection of the anti­

proliférative response (either cell cycle arrest or apoptosis) is still a matter of debate. Two alternative models hâve been proposed (Vousden, 2000):

O The Dumb model: p53 does not play any rôle in determining the

cellular response. Thus p53 activâtes the expression of ail of its

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I. Introduction

target genes, implicated in cell cycle arrest or apoptosis, in the same manner. Indépendant transcription factors play, in this case, a rôle in differentially regulating expression.

O The Smart model: The choice of the response is governed by p53 itself. Indeed, the abundance of p53 affects the response: low amounts of p53 lead to cell cycle arrest and high amounts of p53 lead to apoptosis. This could be due to the fact that the promoters of apoptotic target genes bind p53 with lower affinity than promoters of cell cycle target genes (el-Deiry, 1998). Alternatively, promoter binding could aiso be regulated by p53-modifications.

Finally modifications of p53 could aIso affect its ability to interact with coactivators.

The exact outcome of p53 induction may be cell-specific and dépend on both the type and strength of the stimulus.

2. p53 régulation

a) Keeping o53 in check in unstressed conditions

Since activation of p53 is detrimental to cell survival, the mechanisms controlling the activity of p53 must be exquisitely balanced. Thus, under normal cell proliférative conditions (such as normal growth and development) and/or maintenance of cell viability, p53 has to be kept in check. Indeed, p53 is undetectable in most embryonic and adult tissue (MacCallum et al., 1996), despite that p53 mRNA can be detected by qRT-PCR (Jean-Christophe Marine, Personal communication). These data suggest that either p53 protein is expressed at low levels or undergoes constitutive dégradation in vivo.

(1) Ubiquitination and dégradation

In vitro studies hâve shown that p53 protein stability is regulated

IBMM-ULB PhD thesis Sarah Francoz - 2006 5

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Figure 2: The ubiquitin System and proteasomal-dependent dégradation of cellular

proteins. Ubiquitin is activated by the ubiquitin-activating enzyme, E1 (1) foliowed by

its transfer to an E2-ubiquitin-carrier protein (2). E2 transfers the activated ubiquitin

moieties to the protein substrate that is bound specificaily to a unique ubiquitin

ligase E3 (3). Successive conjugation of ubiquitin moieties to one another generates

a poiyubiquitin Chain that serves as the dégradation signal for the downstream 26S

protéasome (4). The substrate is degraded to short peptides, and free and reusabie

ubiquitin is released by de-ubiquitinating enzymes (DUBs)(5). (Adapted from

Ciechanover et al., 2004)

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I. Introduction

by the ubiquitin-dependent 26S protéasome pathway (Chowdary et al., 1994; Maki et al., 1996). Ubiquitin is a 76-residue polypeptide that can be connected to the substrate protein by a covalent link. This modification targets the modified protein to the 26S protéasome, a large multicatalytic protease complex for dégradation (Ciechanover and Iwai, 2004). The binding of ubiquitin on the substrate protein involves 3 spécifie proteins: the ubiquitin-activating enzyme El which is linked to ubiquitin, the ubiquitin-conjugating enzyme E2 which transfers ubiquitin from El to the substrate and the ubiquitin protein ligase E3 which recruits the substrate (Figure 2). The E3-ligase catalyzes the covalent attachment of ubiquitin to the substrate. In most of the cases, the addition of a single ubiquitin residue to a substrate protein (monoubiquitination) is not sufficient to cause its dégradation.

Indeed, the dégradation of the substrate occurs after polyubiquitination (chain of ubiquitins) of a monoubiquitinated substrate (Sun, 2003). The E3 ligase plays a key rôle in the ubiquitin- mediated proteolytic cascade since it serves as the specific- recognition element of the System.

(2) Mdm family (a) Mdm2;

(i) in vitro

The murine double minute 2 (Mdm2) gene was originally identified by virtue of its amplification in a spontaneousiy transformed mouse BALB/c cell line (3T3-DM) (Cahilly-Snyder et al., 1987;

Fakharzadeh étal., 1991).

Mdm2 is composed of 489 aa and human Mdm2, the human ortholog of Mdm2, of 491 aa. The primary structure of these proteins is characterized by the following conserved domains (Figure 3):

O the p53-binding domain localized in the Nt part of the protein (residues 18-101). Mdm2 is a p53-binding partner (Oliner et al..

IBMM-ULB PhD thesis Sarah Francoz - 2006 6

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Mdm2

18 101 178192 237 288289 331 436 482

Figure 3: Schematic représentation of the primary structure of the Mdm2 protein. . Mdm2 is

composed of a p53 binding domain (red), an acidic domain (blue), a Zinc finger domain

(yellow) and a RING finger domain (green). NLS, Nuclear Localization Signal and NES,

Nuclear Export Signal. (Adapted from Marine et al., 2004)

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I. Introduction

1992) and inhibits its transcription activation fonction upon overexpression (Momand étal., 1992).

O the acidic région iocaiized in the central région. This domain is the target for phosphoryiation on serine residues and appears involved in p53 dégradation (Kawai et al., 2003b; Meulmeester ef a/., 2003).

O the zinc-finger domain Iocaiized in the centrai région. The function of this domain is stili unknown.

O the RING (Really Interesting New Gene) finger domain composed of four pairs of metai binding residues. Aimost ali RING-containing proteins hâve E3 ubiquitin ligase activity towards themseives as weii as other protein substrates (Sun, 2003). Indeed, the RING finger domain of Mdm2 confers the E3 ligase activity to the protein, leading to p53-degradation and its autodegradation (see below) (Fang et al., 2000; Haupt et al., 1997; Honda et al., 1997; Kubbutat étal., 1997).

O a nucieolar location signal (NoLS) is iocated within the RING finger domain of Mdm2 (Lohrum et al., 2000; Weber et al., 2000). Mdm2 can be acetyiated, within its RING finger domain, in particuiar on two key iysines (Lys466 and Lys467) situated within the NoLS (Wang et al., 2001). This modification appears to modulate its activity and might aiso affect its cellular distribution.

O an NLS (nuciear iocation signal) and a NES (nuclear export signal) are Iocaiized in between the p53-binding domain and the acidic domain of the protein. These signais are implicated in the shuttling of Mdm2 between the cytoplasm and the nucléus (Roth et al., 1998).

Several studies hâve shown that Mdm2 is one of the major négative regulators of p53. It was first shown that Mdm2 directiy binds p53 in its transactivation domain (Oliner et al., 1992), thereby blocking the transactivation function of p53 (Momand et al., 1992). This binding induces the disruption of p53-interaction with the basal transcriptional machinery and/or essential coactivators, such as p300/CBP (Thut et

IBMM-ULB PhD thesis Sarah Francoz - 2006 7

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al., 1997). A recent study has shown that Mdm2 inhibits the p53 response in normal growing celle by binding to chromatin-associated p53 (White et al., 2006). Mdm2 is recruited, in a p53-dependent manner, to p53-responsive éléments of the target genes Mdm2 and p21. Moreover, after DNA damage, a decrease of Mdm2 bound to chromatin-associated p53 was observed.

Direct binding of Mdm2 with p53 is also required for the export of p53 from the nucléus to the cytoplasm, where p53 accumulâtes (Geyer et al., 2000; Roth et al., 1998; Tao and Levine, 1999a) and is efficiently degraded by the protéasome.

Moreover, Mdm2 is a E3-ubiquitin ligase implicated in the régulation of p53 dégradation by the 26S protéasome (Fang et al., 2000; Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997).

This activity, due to its RING finger domain. Mdm2 can also regulate its own stability through autoubiquitination, thereby targeting itself for proteasomal dégradation. In vitro studies hâve shown that Mdm2 catalyzes p53 monoubiquitination on six Lys residues localized in the et part of p53 (Lai et al., 2001) and p300 catalyses its polyubiquitination (Grossman et al., 2003). Indeed, p300 acts as a

“E4” ligase towards p53 and is needed for its polyubiquitination (chain of ubiquitins) and subséquent dégradation by the protéasome. More recent data indicate that Mdm2 differentially catalyzes either monoubiquitination or polyubiquitination of p53 in a dose-dependent manner (Li et al., 2003). As a conséquence, low levels of Mdm2 activity induce monoubiquitination and nuclear export of p53, whereas high levels promote polyubiquitination and nuclear dégradation of p53.

It seems likely that these distinct mechanisms are exploited under

different physiological settings (Brooks and Gu, 2006). Mdm2-

mediated polyubiquitination and nuclear dégradation may play a

critical rôle in suppressing p53 fonction during the later stages of a

DNA damage response or when Mdm2 is malignantly overexpressed

(Shirangi et al., 2002; Xirodimas et al., 2001). On the other hand.

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Mdm2 locus

1 23 4 5 6 78 9 10 11 12

^- - - - - - - - - -1- - - - - -H- - - - - - - - - - -HH—hH^

PI P2 ATG

Figure 4; Schematic représentation of the Mdm2 locus. Promoters are shown by blue

arrows. The red diamond represent two p53 response éléments and the star is the STOP

codon. The ATG start codon is indicated in green.

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Mdm2-mediated monoubiquitination and subséquent cytoplasmic translocation of p53 may represent an important means of p53 régulation in unstressed cells, where Mdm2 is maintained at low levels (Boyd et al., 2000; Freedman et al., 1999; Geyer et al., 2000; Stommel et al., 1999). Moreover, movement of p53 into the cytoplasm may be important for transcription-independent fonctions of p53 such as interactions with mitochondria proteins in the apoptotic response (Brooks and Gu, 2006).

Mdm2 aiso induces the monoubiquitination of histones surrounding p53-response éléments resulting in transcriptional repression (Minsky and Oren, 2004).

In contrast of p300 polyubiquitination activity, p53 is stabilized by acétylation by p300 and CBP in cells exposed to stress. Mdm2 is implicated in p53 down-regulation by inhibiting its acétylation by p300 in vivo (Ito et al., 2001; Kobet et al., 2000). Moreover, Mdm2 is promoting p53 deacetylation by recruiting a complex containing HDAC1 (Histone deacetylase 1) (Ito et al., 2002).

Mdm2 promotes aIso conjugation of NEDD8 (neural precursor cell expressed, developmentally down-regulated 8), a ubiquitin-like protein, to p53 and thereby inhibits its transcriptional activity (Harper, 2004; Xirodimas ef a/., 2004). NEDD8 conjugation is mechanistically similar to ubiquitination and Mdm2 has a spécifie neddylation E3 ligase activity for p53, due to its RING finger domain. While ubiquitination is known to occur on as many as six Lys residues in the et part of p53, only three of these are required for efficient neddylation. Mdm2 is aiso able to induce its own neddylation.

Mdm2 is a transcriptional target of p53, creating a négative

feedback loop where p53 activâtes Mdm2 expression, which keeps

p53 levels low during normal growth and development (Barak et al.,

1993; Wu et al., 1993). The transcription of Mdm2 is regulated by two

promoters localized in the first and the second noncoding exons of the

gene (Figure 4). Two p53-responsive éléments are présent in the first

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A

Mdni2 WT allele

1 23

I If

4 56

I- - - - - - - - - H

7 8 9

H—I

1 23 4 56 7 8 9

Mdm2 null —|—1|---1--- 1|---1-|---1---1 Neo' j--- allele

B TUNEL assay - E3,5

Figure 5: Characterization of Mdm2 inactivation in vivo in mice. (A) The structure of the wild- type allele and Mdm2 null allele are depicted. Exons are represented by black vertical bars.

The targeted allele contained the neomycine résistance gene (Neo'^). (B) Mdm2 wild type and

mutant embryos at E3.5 stained by TUNEL (right panel) and viable p53-/- Mdm2-/- mice (left

panel). (Adapted from Montes de Oca Luna et ai., 1995; Chavez-Reyes et ai., 2003)

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intron of Mdm2, and p53 stimulâtes only transcription of the downstream P2 promoter (Barak ef a/., 1993; Wu étal., 1993).

Most of the data presented above results from in vitro or overexpression experiments. Therefore, in vivo experiments were needed to study if the low/physiological levels of Mdm2 are necessary and sufficient to induce proteasome-dependent p53 dégradation under physiological conditions.

(ii) in vivo

The central rôle of Mdm2 in regulating p53 in vivo has been shown by the observation that Mdm2-r\uW embryos die in uteri and their embryonic lethality is completely overcome by délétion of the p53 gene (Jones et al., 1995; Montes de Oca Luna et al., 1995) (Figure 5).

In the first mouse model, exons 10-12 encoding for the Ct part of the protein, composed of the acidic région, the zinc-finger domain and the RING finger domain, are deleted (Montes de Oca Luna et al., 1995). In the second model, exons 7-12 are deleted, leading to the loss of the same domains plus the NLS (Jones et al., 1995). Mdm2-nu\\ embryos die because of increased spontaneous apoptosis initiated as early as blastocyst stage (E3.5) (Chavez-Reyes et al., 2003). This phenotype was rescued by concomitant délétion of p53, providing evidence for the genetic interaction of Mdm2 and p53 in vivo.

Another mouse model, expressing a hypomorphic allele of

Mdm2, allows the study of Mdm2 fonction in adult tissues (Mendrysa

et ai, 2003). These viable mice express reduced levels of Mdm2

without concomitant increase in p53 protein levels. Thus, this resuit

suggests that reduced levels of Mdm2 in these mice are sufficiently

high to allow for adéquate dégradation of p53. An increase of

spontaneous apoptosis is observed in crypts of small intestines and

lymphoid compartment of these mice. Therefore, Mdm2 régulâtes p53

activity in homeostatic tissues by inhibiting its growth-suppressing

effects. In addition, this study suggests that Mdm2 is a critical survival

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Mdm2 locus

1 23

ir-

PI P2 ATG

I H

78 9 10 11 12

ttH—

Mdm4 locus

1

f

P

-H

ATG

3 4 5 6 7 8

I I I- - - - - - - - - -H- - - - - -1

1kb

Figure 6: Schematic représentation of the Mdm2 and Mdm4 locus. Promoters are shown

by blue arrows. The red diamond represent two p53 response éléments and the stars are

STOP codon. The ATG start codons are indicated In green.

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factor, limiting the apoptotic function of p53, in a subset of adult homeostatic tissues. Moreover, since no phenotype was observed in differentiated adult tissues, these data suggest that Mdm2 could be dispensable in post-mitotic cells in vivo.

Despite these in vivo studies, clear understanding of the physiological contributions of Mdm2 to the régulation of p53 stability and transcriptional activity is lacking.

In most cases, induction of p53 in response to stress involves the inhibition of Mdm2 function, which is achieved through several different and indépendant pathways, depending on the stress signal (see below) (Woods and Vousden, 2001).

(b) Mdm4:

(i) in vitro

A search for novel p53-interacting proteins, identified a cDNA clone encoding Mdmx or Mdm4 (Shvarts et ai., 1996). Mdm4 was found to encode for a protein structurally homologous to Mdm2. The human ortholog, human Mdm4, was identified later (Shvarts et al., 1997), and the ability of both Mdm4 and human Mdm4 proteins to interact with p53 was confirmed in vitro by co-immunoprecipitation experiments. In addition, similar to Mdm2, Mdm4 overexpression inhibited p53-activated transcription (Shvarts et al., 1996).

Conservation between Mdm4 and Mdm2 at the genomic level is striking (de Oca Luna et al., 1996; Jones et al., 1996a; Parant et al., 2001b) (Figure 6). Exons 4-12 of Mdm2 are structurally well conserved in Mdm4 as exons 3-11, with the last exon encoding for the et 189 aa containing both zinc- and RING finger domains. A significant divergence is observed, however, at the 5’ end of the genes. One non- coding exon was found in the Mdm4 locus instead of two for Mdm2.

AIso, the intron between exon 1 and 2 in Mdm4 is about 6 kb, while in

Mdm2 the first three exons are spaced of 1 kb. In contrast to Mdm2,

no p53 response éléments was found in the Mdm4 locus and

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Mdm2

p53-binding acidic

domain NLSNES domain finger RING

18 101 178192 237 288 289 331 436 482

Percentage

identity 53 . 6 % 41 . 9 % 53 . 2 %

Figure 7: Comparison of Mdm2 and Mdm4 protein primary structure. The p53 binding

domain (red), Zinc finger domain (yellow) and RING finger domain (green) are well

conserved. Percentage identity between these domains is indicated. NLS, Nuclear

Localization Signal; NES, Nuclear Export Signal. (Adapted from Marine et al., 2004)

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accordingly, no p53-dependent induction of Mdm4 expression could be detected after DNA damage (Shvarts et al., 1996).

Mdm4/human Mdm4 and Mdm2/Human Mdm2 share a common primary structure: a p53-binding domain at the Nt part of the protein, an acidic domain, a zinc-finger domain and a RING finger domain at the et part of the protein (Figure 7).

The highest degree of similarity between the Mdm2 and Mdm4 proteins is found in the p53 interaction domain (residue 18-101) (Figure 7). Consistent with the observation that both Mdm2 and Mdm4 proteins interact with p53, the amino acids of human Mdm2 required for the interaction with p53 (Freedman et al., 1997) are strictiy conserved in human Mdm4. Even more convincing, these amino acids are strictiy conserved in ail known orthologs of human Mdm2 and human Mdm4 (Marine and Jochemsen, 2005). Another well-conserved région is the RING-finger domain, located at the Ct end of both proteins (Figure 7). Yeast two-hybrid screen and co- immunoprecipitation experiments hâve shown that Mdm2 and Mdm4 interact physically through their RING finger domain (Sharp et al., 1999). Hetero-oligomerization between Mdm4 and Mdm2 appears much more stable than homo-oligomerization of each protein (Tanimura et al., 1999). Moreover, the structural integrity of the RING- finger domains is required for Mdm2-Mdm4 heterodimerization (Sharp et al., 1999; Tanimura et al., 1999).

Mdm4, in contrast to Mdm2, does not hâve functional NLS or

NES. The absence of such signais suggests that Mdm4 is intrinsically

a cytoplasmic protein and is dépendent on other proteins for nuclear

localization. Indeed, co-expression of human Mdm2 stimulâtes the

recruitment of human Mdm4 to the nucléus (Gu et al., 2002; Migliorini

et al., 2002a). This effect is indépendant of p53, but requires intact

RING finger domains on both human Mdm4 and human Mdm2

proteins, as well as the NLS of human Mdm2. p53 is aiso capable of

targeting human Mdm4 to the nucléus independently of Mdm2, and

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I. Introduction

complex formation between human Mdm4 and p53 is required for this effect (Li et al., 2002a). Recruitment of human Mdm4/Mdm4 into the nucléus appears critical for its ability to inhibit p53-mediated transcription (Gu étal., 2002; Migliorini étal., 2002a). One should note that these observations are essentially based on transfection studies using tagged version of Mdm4/human Mdm4. Therefore, it wili be important to assess the relevance of these findings under physiologicai endogenous conditions. A further complication emerges from recent studies showing that Mdm4/human Mdm4 protein stability is regulated by the ubiquitin iigase activity of Mdm2 (de Graaf et al., 2003; Kawai et al., 2003a; Pan and Chen, 2003). Indeed, Mdm4 is a substrate for the ubiquitin iigase activity of Mdm2 and Mdm4 dégradation is Mdm2-dependent (de Graaf et al., 2003; Sabbatini and McCormick, 2002). Somehov\/ the ubiquitin ligase activity towards Mdm4 is stimuiated after DNA damage, possibly by enhancing the Mdm4/Mdm2 interaction. Ubiquitination and dégradation of Mdm4 only needs an intact Mdm2 RING domain, in contrast to the régulation of p53 by Mdm2. This offers the possibiiity of distinct régulation of p53 and Mdm4 inhibition by Mdm2.

Mdm4 was reported to act as a ubiquitin iigase In vitro (Badciong and Haas, 2002), but these data were not confirmed by overexpression (Jackson and Berberich, 2000; Migliorini et al., 2002a;

Stad et al., 2001; Stad et al., 2000). Thus, in contrast to Mdm2, Mdm4 does not seem to act as an E3 ubiquitin iigase and by itseif cannot stimulate p53 ubiquitination and proteasome-dependent dégradation.

Several studies even suggested that high Mdm4/human Mdm4 levais could stabiiize p53 by inhibiting Mdm2/human Mdm2-mediated p53 dégradation (Jackson and Berberich, 2000; Migliorini et al., 2002a; Stad et al., 2001; Stad et al., 2000). This effect was proposed to be a conséquence of reduced Mdm2-induced p53 nuclear export, an event thought to be required for efficient p53 dégradation (Boyd et al., 2000; Geyer et al., 2000). However, interprétation of these findings is

IBMM-ULB PhD thesis Sarah Francoz - 2006 13

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complicated by the tact that large excess of Mdm4/human Mdm4 protein levels over Mdm2/human Mdm2 is needed for this effect. In these conditions, Mdm4/human Mdm4 might indeed compete with Mdm2/human Mdm2 for p53 binding. It was later proposed that when the ratio of Mdm4/Mdm2 is low, these proteins cooperatively decrease p53 levels (Gu et al., 2002; Iwakuma and Lozano, 2003).

Human Mdm4 binds human Mdm2 via its RING domain and most transfection studies indicate that human Mdm4 stabilizes human Mdm2, by interfering with human Mdm2 auto-ubiquitination (Stad et al., 2001). Thus, human Mdm4 may function as a stabilizing cofactor, rendering the human Mdm2 protein sufficiently stable to function at its full potential under spécifie conditions. In striking contrast, another report suggests that Mdm4 stimulâtes not only Mdm2-mediated ubiquitination of p53, but aiso Mdm2 self-ubiquitination (Linares et al., 2003). Therefore, Mdm4 interacts with Mdm2, but whether this interaction mainly serves to stabilize Mdm2, by blocking its autoubiquitination and thus indirectiy inhibits p53 or whether the association is more important for régulation of Mdm4 activity and/or cellular localization by Mdm2 in some spécifie contexte, such as following genotoxic stress, remains to be elucidated.

It is obviousiy difficult to reconcile ail these conflicting data in a unifying model. Therefore the rôle of Mdm4 in the régulation of Mdm2 and p53 stability still needs to be firmiy established in vivo with the use of more elaborate genetic models.

The contribution of Mdm4 to the régulation of p53 transcriptional activity aiso needs to be clarified. Expression of high levels of Mdm4 inhibits p53 transcriptional activity (Shvarts et al., 1997). Since the same amino acids in p53 are required for both Mdm4/p53 and Mdm2/p53 interactions (Bottger et al., 1999) and these amino acids are located in the transactivation domain of p53, Mdm4 may inhibit p53 transcriptional activity by concealing its transactivation domain.

Indeed, several studies hâve shown that Mdm4 acts as a

(38)

I. Introduction

transcriptional repressor (Kadakia et al., 2002; Wunderlich et al., 2004; Yam et al., 1999). Moreover, Mdm4 may inhibit p53 transcriptional activity by interfering with the ability of p53 to interact with the basal transcription machinery and/or to recruit spécifie essentiel co-activator(s). The ability of Mdm4 to inhibit p53-dependent transcription could be a conséquence of inhibition of p300/CBP- mediated acétylation of p53 (Sabbatini and McCormick, 2002).

Importantly, this effect is Mdm2-independent, since Mdm4 abrogates p300/CBP-mediated acétylation of p53 even in Mdm2-r\uW cells and the same resuit is aiso observed with a mutant of Mdm4 defective in Mdm2 binding (Danovi et al., 2004). These data suggest that Mdm4 primarily inhibits p53 activity by interfering with its transcriptional ability. Even though it is now clear that direct interaction between Mdm4 and p53 appears essentiel for this activity, the molecular mechanism by which Mdm4 régulâtes p53 activity has not been fully elucidated.

In conclusion, Mdm4 may primarily inhibits p53 activity by interfering with its transcriptional activity. For this effect, a direct interaction between Mdm4 and p53 appears essentiel. Mdm4 aIso interacts with Mdm2, but whether this interaction serves mainly to stabilize Mdm2 and thus indirectiy inhibits p53 or whether the association is more important for régulation of Mdm4 activity by Mdm2 remains to be elucidated.

(il) in vivo

In order to assess the physiological relevance of Mdm4, two distinct mutant mouse lines were generated and characterized by three indépendant groupe. Mutations in the Mdm4 gene resuit in a p53-dependent early embryonic léthal phenotype, indeed p53-null State completely rescues the embryonic lethality associated with Mdm4 deficiency (Finch et al., 2002; Migliorini et al., 2002b; Parant et al., 2001a) (Figure 8B). In the first reported Mdm4 mutant mouse (Parant et al., 2001a), exons 3-5 encoding for most of the p53-binding

IBMM-ULB PhD thesis Sarah Francoz - 2006 15

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Mdm4 WT — | --- 1 --- H — I

allele

1 2

Mdm4 null —|---tlTRISAI Neo^ ISDiLTRl--- 1 allele Retroviral vector

3 4 5

1-1-1

B E11,5

Figure 8; Characterization of Mdm4 inactivation in vivo in mice. (A) The partial structure of the

wild-type allele and Mdm4 null allele are depicted. Exons are represented by black vertical

bars. The retroviral construct is composed of 2 LTR (Long Terminal Repeats), the neomycine

résistance gene (Neo^, a splicing accepter site (SA) and a splicing donor site (SD). (B) Mdm4

wild type and mutant embryos at E11.5 (left panel) and viable p53-/- Mdm4-/- mice (right

panel). (C) IHC for Caspase-3* (in the neuroepithelium) and BrdU (in the fêtai liver) of Wild

type and Mdm4 mutant embryos. (Adapted from Migliorini et ai., 2002)

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I. Introduction

domain (amino acids 27-96) were deleted by homologous recombination. This mutation caused embryonic lethality as early as E7.5 and was due to severe prolifération defects. The second Mdm4 mutant mouse line was generated by random insertion mutagenesis, with a viral insertion mapped in intron 1, upstream of the ATG start codon (Finch et al., 2002; Migliorini et al., 2002b) (Figure 8A). The resulting Mdm4-homozygous mutants died between E9.5 and E11.5 and were characterized by overall growth defects and massive apoptosis in the neuroepithelium (Figure 8B and C). The timing of embryonic death is slightiy different in these two mouse models and apoptosis is only detected in the neuroepithelium of the /Wdm4-trapped embryos. These différences could be due to the fact that in the first model, the Mdm4 allele is not a null but still produces, through alternative splicing, a truncated Mdm4 protein with a deleted p53 binding domain, but retaining the RING finger domain (Parant et al., 2001a). Therefore the truncated Mdm4 produced could retain the ability to interact with Mdm2 and could affect its activity and/or stabillty. In the second mouse model extensive protein analyses failed to detect Mdm4 protein expression in the Mdm4lp53 double-null mice, rendering uniikely, even if not excluding, the possibility of a hypomorphic allele. This model supports a direct rôle for Mdm4 in regulating both apoptotic and cell cycle arrest fonctions of p53. As evidence for increased p53 transcriptional activity in the absence of Mdm4 expression, gene expression profiling experiments revealed a significant increase in the expression of more than 20 known p53 target genes in homozygous Mdm4-deficient mice, including p21 and several pro-apoptotic genes such as Bax, Apaf-1, and Noxa (Doumont et al., 2005a; Martoriati et al., 2005). It was concluded from these experiments that impaired Mdm4 expression causes embryonic lethality as a resuit of deregulated p53 activity. Both described phenotypes are quite distinct from the phenotype associated with Mdm2 loss and, importantly, the physiological levels of Mdm2 cannot

IBMM-ULB PhD thesis Sarah Francoz - 2006 16

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compensate for Mdm4 loss. Taken together, Mdm4 is critical and non- redundant to keep p53 activity in check during embryogenesis. A comparable rôle for Mdm4 in adult tissues remains, however, to be demonstrated.

Earlier lethality in Mdm2-nu\\ mice as compared to Mdm4-nu\\

mice allows at least two different scénarios to be pictured. One possibility is that Mdm2 is the master p53 regulator and can, at least to some extent, function in the absence of Mdm4. Mdm4 would then be a cofactor of Mdm2, only required in spécifie tissues or, in a given tissue, at a particular phase of the différentiation program. A particularly attractive model proposes that Mdm4 would be required during the massive expansion phase of the progenitor cells in a given cell type. These cells indeed undergo rapidiy multiple DNA réplication cycles with little time for DNA repair mechanisms to operate, conditions that would normally favor p53 activation. In this context, Mdm4 would assist Mdm2 in keeping p53 activity down. In agreement with this model, Mdm4 is overexpressed in hematopoietic stem cell (Phillips et al., 2000), suggestive of prédominant Mdm4 expression in the hematopoietic progenitor cells. Alternatively, even if less likely, Mdm4 and Mdm2 may function independently in different cell types.

For instance, there is so far no evidence for a rôle of Mdm2 in the survival of the erythroid lineage cells. Mice expressing low endogenous levels of the Mdm2 protein from a hypomorphic allele are viable and hâve fairly normal red blood counts, whereas the lymphoid compartment is strongly affected in these mice (Mendrysa et al., 2003). In contrast, impaired Mdm4 activity dramatically affects primitive as well as definitive erythropoiesis (Migliorini et al., 2002b).

These questions should be answered by tissue-specific functional inactivation of Mdm4 and/or Mdm2 in vivo.

Clear understanding of the physiological contributions of Mdm2

and Mdm4 to the régulation of p53 stability and transcriptional activity

is lacking.

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I. Introduction

(3) Additional négative regulators

Several other p53-negative regulators implicated in the control of its stability and/or activity hâve been identified. These proteins, in combination with Mdm2 and Mdm4 may keep p53 at low levels or inactive in unstressed cells.

Most of these regulators, Pirh2 (Leng et al., 2003), COP1 (Dornan et al., 2004), Yin Yang1 (Sui et al., 2004) and ARF-BP1/Mule (Chen et al., 2005) are ubiquitin E3 ligase proteins and like Mdm2, are implicated in the dégradation of p53 in a Mdm2-independent manner.

Moreover, Pirh2 and COP1 are p53-target genes and therefore are implicated, as Mdm2, in an autoregulatory feedback loop that Controls p53. Together, Mdm2, Pirh2, COP1 Yin Yang1 and ARF-BP1/Mule represent an array of E3 ligases that the cells can call upon to regulate and maintain p53 levels (Brooks and Gu, 2006). They suggest that both Mdm2-dependent and -indépendant mechanisms are used cooperatively by the celi to tight p53 régulation. It is yet uncertain exactiy how these proteins are specifically regulated and under what situations they may be differentially activated.

Another level of control of p53 fonction has been discovered with the identification of Parc (p53-associated, parkin-like cytoplasmic protein) (Nikolaev et al., 2003), a cytoplasmic anchor for p53. Nuclear localization of p53 is essential for its transcription activity. Therefore cytoplasmic séquestration of p53 by Parc is inactivating p53 fonction and inhibiting its tumor suppressor fonction (Kastan and Zambetti, 2003). Moreover, neuroblastome cells, where p53 is cytoplasmic, are expressing high levels of Parc.

The discovery of new négative regulators of p53 makes the understanding of the control of p53 more complicated. However, p53 régulation by these proteins has just been shown in vitro and in vivo experiments hâve still to be performed to assess the physiological relevance of these new putative regulators.

IBMM-ULB PhD thesis Sarah Francoz - 2006 18

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CK1 DNAPK Chk1/2 CAK p38 JNK cdk2 PKC CK2 PKR

Figure 9: p53 posttnanslational modifications. The partial p53 protein primary structure

is depicted. Residues modified by phosphorylation (black star), acétylation (red star)

and sumoylation (purple star) are indicated. The kinases are represented in black, the

acetylases in red and the SUMO E3 ligases in purple. TA, the transactivation domain

(red), Pro, Proline-rich domain (orange), tet, tetramerisation domain (yellow), reg,

regulatory domain (green) and NLS, Nuclear Localization Signal (gray). (Adapted from

Leblanc et al., 2002; Xu, 2003)

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I. Introduction

b) Activation followino DNA damage / stress Many types of stress activate p53 including DNA damage, telomere érosion, oncogenes activation, hypoxia and loss of normal growth and survival factors (Vousden and Lu, 2002). Several types of DNA damage can activate p53, including double-strand breaks produced by Y-irradiation and the presence of DNA repair intermediates after ultraviolet irradiation or Chemical damage to DNA.

This résulta in a rapid increase in the levels of p53 in the cell and activation of p53 as a transcription factor. p53 levels increase because the half-iife of the protein is augmented. This stabilization of p53 and subséquent activation occur by posttranslationai mechanisms.

Moreover, posttranslationai modifications enhance p53’s sequence- specific binding and transcriptional activities in response to stress.

Mdm2 and Mdm4 are aiso modified upon stress conditions leading to p53 activation.

Various p53 (Figure 9), Mdm2 and Mdm4 posttranslationai modifications are summarized in this section

(1) Phosphorylation on Ser and Thr residues

p53 is phosphorylated at multiple sites at the Nt and Ct part of the protein in vitro by a number of kinases, and most of these phosphorylation sites are conserved between human and mouse p53 (Xu, 2003) (Figure 9).

ATM (Ataxia Telangiectasia Mutated) and ATR (Ataxia Telangiectasia and Rad3 related protein), t\«o Ser/Thr kinases upregulate p53 by targeting Sert5 (Ser18 in mouse) after ionizing radiation and UV irradiation, respectively (Banin étal., 1998; Canman et al., 1998; Siliciano et al., 1997; Tibbetts étal., 1999). This phosphorylation contributes to p53 transactivation activity by increasing p53 binding to the transcription coactivator p300/CBP and thereby competing with Mdm2 binding (Dumaz and Meek, 1999). ATM is aIso able to induce the

IBMM-ULB PhD thesis Sarah Francoz - 2006 19

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