Arthur C Clarke

Harvey Ras

Janus Kinase

Kras Kirsten

High mobility group HRAS

Keywords:

New ideas pass through three periods: -It can't be done.

-It probably can be done, but it's not worth doing.

-I knew it was a good idea all along! »

AKNOWLEDGMENTS

As I write the last lines of this manuscript and take a review of these intense years I would like to thank many different people without the support of which I would not have made it so far. I would like to thank Dr. Robert Ballotti, Dr. Eirikur Steingrimsson and Dr. Ali Hamiche for their critical evaluation of my manuscript. I feel honoured that such high quality researchers have accepted to be members of my thesis committee. The pioneering work in your respective fields have definitely inspired my work and imprinted my current and future research interests. This work would not have been possible without the guidance of my supervisor Dr. Irwin Davidson. Irwin, joining your laboratory is one of the best and luckiest decisions I made in my life. I want to thank you for your support and trust as well as our pleasant and constructive discussions that kept me on track time after time and brought this fascinating project to what it is now. Thank you also for giving me the opportunity to take part to excellent conferences in incredible locations around the world. Sharing my work and meet worldwide leading investigators has been truly inspirational.

Many thanks go to all current and past members of the lab.

In first line I would like to thank Thomas for his mentoring, that turned into a valued friendship. I'm glad I had the chance to work with you. Thank you so much for not having been a numpty -private joke ;) -and for shaping the golden years of 4011 with jazz, Lab-Knock 'Em Over, and Apero !!! « Bref, j'ai fait une thèse !! » I want to thank Sylvia for having been an invaluable friend during these long years. I'm grateful you were present both in the best and most difficult moments of this thesis. I know I don't need to tell you to what extent this whole part wouldn't have been the same without your friendship.

Thanks to Dana, for the numerous discussions (sometimes very long indeed !) and help improving my Croatian (Govorim malo hrvatski). I feel fortunate about the chance I had to have you as lab-mate. Hvala lijepa ! Shilpy, working on your side and sharing fresh ideas and insights has definitely changed the face of this thesis but also helped me in becoming a better scientist. Beyond this contagious hard work, our cultural and personal exchange will probably be the most precious remain from my time in 4039. Thank you for all that.

Gabrielle and Isabelle, I thank you so much for your advices and constant technical support. Your help with mouse line management and the uncountable number of genotyping PCRs has been crucial. I also thank you for tolerating my (reasonable) mess, the jukebox music, and most importantly for your constant (permanent!) good mood, open to my numerous jokes.

I'm indebt to the former postdocs Anas, Diana and Daniel for their helpful comments on many parts of my project. You are great both as persons and scientists and have been very inspiring to me. I consider you as real examples in many aspects of what an accomplished researcher should be. I also thank Igor, Sébastien, Marie, Thomas, Guillaume, Mariam and Dominique for their past or present help. I'm thankful to the many other colleagues and friends in and out of IGBMC (Benoît, Carole, Diane, Marie, Gaetan, Vanessa, Audrey, Nataliia, Emmanuelle, Alexandre, Cyrille, Emmanuelle, members of the SHU Haguenau and many others) for their constant company, support, and help keeping a good balance between work and personal life. You have made this period a once in a lifetime experience! I would like to address many thanks to the common facility members, which represent a key stone of the IGBMC institute and have made my days significantly easier. I thank the cell culture facility members, especially Betty Heller; the histopathology service (special thanks to Bruno and Patrice), mice caretakers William and Vincent, and all the sequencing facility and bioinformatics people: Bernard, Muriel, Serge, Tao, Stephanie, Celine, Amandine. You all have been very professional and helpful and always available for discussion. It's been a real pleasure to work with you. I also address special thanks to Dr. Joern Pütz for his long lasting support. The year I have spent in Germany with your help has strongly (positively) impacted my life expectations and choices; I've learnt so much and had such an amazing experience that I'll be ever grateful. I thank you also for giving me the challenging opportunity to teach undergraduate students for these 2 years, which provided me with skills that are applicable to many other areas in life. I thank our collaborators including members from the Marine, Larue, Goding and Ballotti labs and more largely the people from the European melanoma community (Steingrimsson lab, Sommer lab, MD Galibert, AK Bosserhof , L Montoliu, H Arnheiter,) that I have met at conferences and workshops over the years for their inputs. I'm still very impressed by the strong cooperation, exchange and human atmosphere that exist within this community with the common aim of both understanding fundamental biological processes and curing melanoma disease (and if at some point that involves a bier it is even better).

INTRODUCTION

Figures and Tables

Figure

Résumé

Les mélanocytes sont des cellules spécialisées dans la production de pigments et colonisent de nombreux tissus au sein de l'organisme comme la peau et les follicules pileux. Les mélanomes cutanés sont des tumeurs provenant de la transformation de ces mélanocytes épidermiques ou folliculaires. Le mélanome est l'un des cancers les plus agressifs, connu pour sa grande résistance à la plupart des traitements actuels (chimioet radiothérapie).

L'apparition et la progression du mélanome sont le résultat de processus complexes impliquant la dérégulation de voie de signalisation et de réseaux transcriptionnels enchevêtrés et coordonnés. Les voies de signalisation MAPK et PI3K/AKT sont les plus fréquemment dérégulées lors de la mélanomagenèse : près de 15% des mélanomes possèdent une mutation activatrice de NRAS et près de 50% présentent la mutation activatrice BRAF V600E , fréquemment associé à une perte de PTEN, un acteur de la voir PI3K/AKT. Ces mutations sont responsable d'une activation constitutive de la voie MAPK associée, qui entraine une prolifération incontrôlée mais temporaire des cellules avant de subir un blocage prolifératif par l'entrée dans un état de senescence induite par les oncogènes. (OIS) Des mutations additionnelles et/ou des modifications épigénétiques permettent à quelques cellules de mélanome d'échapper à la senescence et de reprendre leur prolifération pour former des tumeurs malignes.

Compte tenu de son évolution rapide et de sa capacité importante à former des métastases, un diagnostique précoce et une ablation chirurgicale des tumeurs primaires en phase de croissance dans les couches superficielles de la peau reste le moyen le plus efficace de combattre cette pathologie. Une fois que la tumeur s'est engagée dans une phase de croissance verticale (invasion) elle peut atteindre le système sanguin ou lymphatique propice à la dispersion des métastases dans l'organisme et le mélanome devient difficile à combattre. Les thérapies existantes les plus efficaces cibles spécifiquement des médiateurs de la voie MAPK ou des couples récepteurs/ligands impliqués dans la stimulation des réponses spécifiques du système immunitaire (immunothérapie) et donnent des résultats exceptionnels mais ne permettent de prolonger la durée de vie des patients que temporairement en raison du développement de cellules résistantes. Ceci explique pourquoi les mécanismes d'initiation et de progression du mélanome font l'objet d'intenses recherches biomédicales et pharmacologiques.

Pour permettre de développer de nouvelles thérapies ciblées contre cette pathologie, il est important de comprendre les mécanismes d'apparition et de développement des tumeurs et les propriétés des métastases. Pour cela, nous nous intéressons aux caractéristiques d'un acteur clé jouant un rôle à la fois dans le développement embryonnaire et le fonctionnement normal des mélanocytes: le facteur de transcription MITF (MIcrophthalmia-associated Transcription Factor).

Le laboratoire d'accueil a montré précédemment que MITF est aussi un facteur au rôle clé dans la physiopathologie du mélanome car il est le régulateur transcriptionnel de la pigmentation, de la prolifération et de l'invasion des cellules de mélanome. De nombreuses études se sont penchés sur le rôle intrinsèque du facteur MITF (comment MITF est produit et comment il agit seul sur l'expression des gènes) mais très peu sur la façon dont il est lui-même régulé en terme d'activité et de stabilité.

MITF n'agit pas seul et est associé à une variété de cofacteurs qui participent au contrôle transcriptionnel de ses gènes cibles. Ces propriétés expliquent l'intérêt particulier que nous portons à l'étude des mécanismes de régulation de MITF, de ses partenaires et de l'expression de ses gènes cibles dans la progression du mélanome.

Parmi ces cofacteurs de MITF nous avons identifié les sous-unités des complexes de remodelage de la chromatine PBAF (associé à CHD7) et NURF.

MITF interagit avec le complexe PBAF comprenant CHD7.

Nos recherches ont montré que MITF interagit avec de nombreuses sous-unités du complexe BRG1/PBAF. Il existe plusieurs variantes de ce complexe de remodelage de la chromatine ATP-dépendant qui diffèrent par la nature de la sous-unité catalytique (BRG1 ou BRM) et des 12 sous-unités associées (« BAF »). Nos expériences de spectrométrie de masse et d'immunoprécipitation de protéines ont démontré que MITF interagit avec le complexe PBAF comprenant les sous unités spécifiques BRG1, ARID2 et PBRM1 ainsi que les sous-unités communes SMARCC2 (BAF170), SMARCD2 (BAF60B), et ACTL6A (BAF53A). Par ailleurs nous avons montré que CHD7, une seconde sous-unité catalytique, est associée à PBAF et MITF dans ces cellules de mélanome, conformément à une étude décrivant l'interaction de CHD7 et PBAF dans les types cellulaires dérivés de la crête neurale. L'interaction de MITF et PBAF est particulièrement intéressante car des mutations dans les sous-unités du complexe PBAF ont été mises en évidence dans plus de 20% des cancers et des études récentes de séquençage des exomes ont identifiées plusieurs de ces mutations dans des mélanomes humains, en particulier dans la sous-unité ARID2.

C'est pourquoi nous avons décidé de réaliser une analyse intégrative et génomique complète afin de caractériser le rôle du complexe PBAF dans les mélanocytes et le mélanome. Pour évaluer le rôle de BRG1 dans les cellules de mélanome, nous avons réalisé des expériences d'interférence ARN par l'expression de siRNA ou de shRNA lentiviral dirigés contre ses ARN messagers et observé une forte diminution de l'expression de MITF. Ceci explique les effets de la perte de BRG1 sur l'expression des gènes et le phénotype des cellules très similaires à ceux observés après la perte de MITF lui-même : une diminution de l'expression des gènes cibles de MITF impliqués dans la signalisation, le cycle cellulaire et la pigmentation et l'activation de gènes du sécrétome associés à la senescence (SASP). De même la perte de BRG1 induit un arrêt de la prolifération des cellules de mélanome, une morphologie aplatie et élargie avec plusieurs projections cytoplasmiques ainsi qu'un marquage positif pour la senescence.

Nous avons ensuite réalisé une analyse transcriptomique globale et constaté que la perte de BRG1 affecte l'expression de plus de 5000 gènes suggérant le rôle central de ce facteur dans la régulation des gènes dans ces cellules. Par ailleurs, la perte de BRG1 induit une perte du facteur de transcription SOX10 et la perte de ce dernier (siSOX10) suffit à réprimer l'expression de MITF endogène. En revanche supprimer l'expression de SOX10 dans une lignée cellulaire exprimant de façon constitutive MITF (avec une étiquette 3xHA) à partir d'un promoteur CMV induit uniquement la perte de la protéine endogène et non la protéine étiquetée.

Pour déterminer si les effets observés lors de la perte de BRG1 sont dû à la perte subséquente de SOX10 et MITF ou à l'interaction fonctionnelle entre MITF et BRG1, nous avons exprimé un shARN dirigé contre BRG1 dans les cellules exprimant la protéine MITF à partir du promoteur CMV. La perte de BRG1 induit donc celle de MITF endogène mais pas celle de la protéine étiquetée. Les effets observées sont comparables malgré la Résumé 11 présence de MITF suggérant que bien que BRG1 contrôle l'expression de MITF via SOX10, l'interaction fonctionnelle entre les deux facteurs est essentielle pour activer son répertoire de gènes cibles.

De même nous avons étudié la fonction de BRG1 dans des mélanocytes humains immortalisés (Hermes 3A). Par opposition aux cellules de mélanome, la perte de BRG1 a un effet mineur sur l'expression de MITF dans les mélanocytes mais induit toujours un arrêt de la prolifération et une entrée en senescence des cellules. L'analyse globale des changements transcriptomiques montre que BRG1 est essentiel pour l'expression des gènes du cycle cellulaire dans les mélanocytes et que MITF et BRG1 régulent des répertoires de gènes chevauchants.

Compte tenu du rôle de BRG1 dans les mélanocytes et les cellules de mélanome in vitro, nous avons décidé d'étudier son rôle in vivo chez la souris. La suppression embryonnaire de BRG1 étant létale, nous avons utilisé un modèle murin permettant de recombiner des allèles de Smarca4 (Brg1) floxés spécifiquement dans la lignée mélanocytaire.

Pour cela nous avons croisés ces souris floxés avec des souris exprimant la recombinase Cre sous contrôle du promoteur de la Tyrosinase (Tyr-Cre) spécifique de ce lignage.

Alors que les souris Tyr::Cre/Smarca4 mel+/-sont noires, montrant un développement normal du lignage mélanocytaire, les souris Tyr::Cre/Smarca4 mel-/-naissent totalement blanches. Ces résultats démontrent donc qu'en plus de son rôle essential dans les mélanocytes et les cellules de mélanome, BRG1 est indispensable au développement embryonnaire des mélanoblastes in vivo.

Résumé

MITF et SOX10 recrutent BRG1 vers des éléments régulateurs critiques pour le fonctionnement des cellules de mélanome.

Compte tenu du rôle essentiel de BRG1 dans le lignage mélanocytaire in vitro et in vivo, nous avons utilisé une approche de ChIP-seq pour mieux comprendre comment MITF et BRG1 régulent ensemble l'expression de leurs gènes cibles dans les cellules de mélanome. L'immunoprécipitation de BRG1 sur la chromatine digérée à la Mnase de ces cellules a permis d'identifier plus de 42000 sites occupés par BRG1 localisés aussi bien dans des régions inter-qu'intragéniques et dans une moindre mesure sur des promoteurs. La comparaison avec des données de ChIPseq de H3K27ac de tumeurs primaires de mélanome prolifératif, (collaboration Dr. Stein Aerts, Leuven, Belgique) a montré que BRG1 est présent sur environ 15 000 sites marquées par le H3K27ac comme régions régulatrices actives au niveau des promoteurs et enhancers.

Les sites occupés par BRG1 présentent un profil d'organisation très spécifique caractérisé par deux pics séparés par 450 à 800 pb. Ceci est en accord avec le concept que BRG1 occupe deux nucléosome adjacents séparés par une distance variable. Par ailleurs au niveau des promoteurs, BRG1 est associé aux nucléosomes -1 et/ou +1 par rapport au site d'initiation de la transcription (TSS).

L'intégration des données de ChIP-seq et RNA-seq montre que les gènes liés et activés par BRG1 sont impliqués dans la pigmentation, la réplication de l'ADN, et le métabolisme du glucose et des acides gras. Les gènes liés et réprimés par BRG1 sont impliqués dans les processus d'angiogenèse et de transcription, notamment un ensemble de facteurs à doigt de zinc (ZNF). En plus de MITF, BRG1 est donc recruté par une gamme bien plus large de facteurs de transcription afin de réguler ces différents répertoires.

Résumé 13

L'analyse comparative avec les 16000 sites de liaisons génomiques de MITF montre que celui-ci colocalise avec BRG1 sur près de 6000 sites. Les sites co-occupés sont non seulement enrichis en motifs liés par MITF (E-ou M-box) mais également en motifs de fixation de SOX10, CREB ou encore ETS1, indicatif de la nature des autres facteurs qui pourrait coopérer avec MITF et BRG1 pour réguler l'expression des gènes cibles.

La représentation importante du motif pour SOX10 au niveau de ces sites et connaissant la capacité décrite de SOX10 de s'associer à BRG1 nous a conduit à déterminer son profil d'occupation génomique dans les cellules de mélanome. Nous avons identifié plus de 6000 liés par SOX10 qui sont, contrairement, à MITF, très peu enrichis eu niveau de promoteurs mais eu niveau de sites de régulation distants.

L'intégration des données à notre disposition pour SOX10, MITF, BRG1, H3K27ac a permis d'identifier un signature chromatinienne caractérisé par un ensemble de sites co-occupés au niveau desquels MITF et/ou SOX10 se lient entre deux nucléosomes liés par BRG1 et marqués par H3K27ac et qui correspondent à des éléments régulateurs actifs impliqués dans la pigmentation, la formation de lysosome, la motilité cellulaire et la signalisation.

Enfin des expériences d'iimmunoprécipitation de chromatine sur BRG1 après la perte de MITF et SOX10 induite par un siARN a montré que ces facteurs de transcriptions sont indispensables pour recruter de manière active et ciblé BRG1 au génome. Nous avons également constaté que sur les sites co-occupés par MITF et SOX10, les deux facteurs peuvent être nécessaires pour recruter BRG1. Ces résultats suggèrent donc un rôle coopératif dans le recrutement du complexe de remodelage de la chromatine pour la régulation de l'expression des gènes. The skin is the largest organ of the human body and as such the most important barrier protecting the organism from its environment. Functions of the skin include physical protection (UV radiation, microorganism invasion, mechanical and chemical stresses, fluid loss), sensation (pressure, pain and temperature receptors), thermoregulation and metabolic functions (vitamin D production). This complex organ is constituted by cells of different embryonic origins and can be divided into three primary layers (Fig. 1):

INTRODUCTION

• The hypodermis is the innermost layer that anchors the skin to the underneath tissues (skeletal muscle, organs). It is a highly vascularized connective tissue essentially composed of sweat glands and adipocytes specialized in fat storage and acting as energy reserve and heat insulators.

• The dermis is the layer of dense connective tissue underneath the epidermis and the thickest part of the skin. It is derived from the embryonic mesoderm and can be divided into two regions: the papillary dermis (superficial region, close to epidermis) and the reticular dermis (deeper region) [START_REF] Smith | Embryogenesis of the dermis in human skin[END_REF]. The fibroblasts are the main cells in the dermis. They produce important components of the extracellular matrix such as collagen and elastin fibres, which provide the skin with strength and flexibility. The dermis houses capillaries, lymphatic vessels, immune cells (Langerhans cells, macrophages, mast cells), several sensory receptors (nerves; Meissner, Pacini and Ruffini corpuscles) and epidermal appendages (nails, hair follicles, sebaceous glands and sweat glands).

• The dermo-epidermal junction or basement membrane is at the junction between dermis and epidermis and regulates the adhesion and the exchanges between the two layers. It is made of a fibrillar extracellular matrix produced jointly by fibroblasts and keratinocytes and has a crucial function in the spatial organization and architecture of the epidermis as it controls the anchoring and therefor the polarity of basal proliferating keratinocytes and other epidermal cell types.

• The epidermis is the outermost part of the skin. It's a keratinized stratified squamous epithelium that ensures most protective functions of the skin. It is associated to the different epidermal appendages but relies solely on the blood supply from the dermis for nutrient and oxygen delivery.

• • Keratinocytes make up the majority of epidermal cells (90%).

They derive from embryonic ectoderm. In a process taking 4 to 6 weeks, a single row of germinative keratinocytes hold together and to the underlying basement membrane divide in the basal layer (stratum basale) of the epidermis, and constantly move up through the layers above (so called strata), undergoing morphological change as they move from one strata to the next (Fig. 2A,). Four to five overlaying strata can be distinguished (from bottom to top): stratum basale corresponding to the proliferative compartment followed by stratum spinosum, stratum granulosum, stratum lucidum (only for palms and soles) and stratum corneum corresponding to a terminally differentiated horny layer of corneocytes, which are dead cells filled with keratin, lipids and other products. Their continuous loss (desquamation) is naturally compensated by renewal of the epidermis. This organization plays a key role in the waterproof properties and in the ability of the skin to form an efficient physical barrier exposed to the environment.

• Langerhans cells (3 to 5% of epidermal cells) are mobile dendritic cells derived from hematopoietic stem cells of the bone marrow (Fig2A and B.c). In the epidermis, they reside usually in the basal layers between the keratinocytes where they initiate and sustain the immune or allergic response. They ingest invading microorganisms and present antigens on their surface. These activated Langerhans cells then migrate through epidermis and dermis [START_REF] Stoitzner | A close-up view of migrating Langerhans cells in the skin[END_REF] to reach the lymph nodes where they initiate adaptive T-cell immune response.

• Merkel cells (2 -5% of epidermal cells) are located in the basal layer among the germinative keratinocytes and melanocytes close to sensory nerve endings (isolated or grouped together in so-called corpuscles; (Fig2A and B.d). Their embryonic origin is still under debate. They serve as mechanoreceptors and are involved in sensing touch or compression. They are particularly abundant in thick skin (palms and soles), lips, fingertips and in the back [START_REF] Moll | Human Merkel cells--aspects of cell biology, distribution and functions[END_REF].

• Melanocytes (2 -4% of epidermal cells) are dendritic cells specialized in pigment production and can be found in the epidermis, hair follicles as well as a range of other tissues as the inner ear, eyes, leptomeninges, bones and the heart where their role is less understood (reviewed in [START_REF] Delmas | Beta-catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development[END_REF][START_REF] Thomas | The making of a melanocyte: the specification of melanoblasts from the neural crest[END_REF]. In human skin, melanocytes reside in the basal layer at the dermo-epidermal junction, regularly spaced by 10-15 keratinocytes and contacting up to 40 (Fig2A and B.b). Once terminally differentiated, they synthetize the melanin pigment and transfer it to the nearby keratinocytes but divide less than twice a year (Jimbow et al., 1975). All melanocytes arise from neural crest cells (NCC) that derive from the ectoderm germ layer, at the interface between the neural plate and the adjacent non-neural ectoderm [START_REF] Dupin | The contribution of the neural crest to the vertebrate body[END_REF] with the exception of retinal pigment epithelium (RPE) melanocytes that arise from the neuroectoderme (Raymond and Jackson, 1995;Planque et al., 2004) Neural crest cells are induced during gastrulation and initially reside in the neural plate border territory (Fig. 3A). During neurulation, this border territory elevates as the neural plate closes to form the neural tube. Early induction of the neural crest is dependent upon bone-morphogenic pathway (BMP) signalling and induction of the Wnt (wingless-type), FGF (Fibroblast Growth Factor) and Notch pathways (Fig. 3B) [START_REF] Ikeya | Wnt signalling required for expansion of neural crest and CNS progenitors[END_REF][START_REF] Labonne | Induction and patterning of the neural crest, a stem cell-like precursor population[END_REF][START_REF] Steventon | Differential requirements of BMP and Wnt signalling during gastrulation and neurulation define two steps in neural crest induction[END_REF]. After neural tube closure, downstream signalling results in neural crest specification (Fig. 3B) through expression of neural crest genes such as FOXD3 [START_REF] Labosky | The winged helix transcription factor Hfh2 is expressed in neural crest and spinal cord during mouse development[END_REF], SOX10 [START_REF] Southard-Smith | Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model[END_REF] and PAX3 [START_REF] Epstein | Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3[END_REF]. NCCs undergo an epithelial-mesenchymal transition (EMT), which enables them to acquire motility and migratory properties required to detach from the dorsal neural tube and colonize their different target sites in the organism. NCCs form a transient population of pluripotent progenitor cells that progressively give rise to several different lineages (Fig. 3B and Fig. 4) [START_REF] Betters | Analysis of early human neural crest development[END_REF][START_REF] Ernfors | Cellular origin and developmental mechanisms during the formation of skin melanocytes[END_REF]. Their migratory trajectory is critically connected to the route and fate of the cells [START_REF] Bronner | Development and evolution of the neural crest: an overview[END_REF] and takes place along two major paths: dorsoventrally between neural tube and somites or dorsolaterally between epidermis and dermomyotome. Most NCCs migrate dorsoventrally and give rise to multiple cell types like Schwann cells, smooth muscle cells, peripheral neurons or adipocytes while cells taking the dorsolateral path give rise to melanoblasts, the melanocyte precursors (Fig. 4) [START_REF] Le Douarin | Neural crest cell plasticity and its limits[END_REF][START_REF] Marmigere | Specification and connectivity of neuronal subtypes in the sensory lineage[END_REF][START_REF] Marmigere | Specification and connectivity of neuronal subtypes in the sensory lineage[END_REF][START_REF] Ernfors | Cellular origin and developmental mechanisms during the formation of skin melanocytes[END_REF]. Recent studies also suggest the existence of melanocytes that derive from Schwan cell precursors from the dorsoventral route [START_REF] Adameyko | Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin[END_REF]reviewed in Ernfors, 2010).

These specification processes are all driven by expression of different transcription factors and their combinations such as slug/snail, AP-2, FOXD3, PAX3, TWIST, SOX9 and SOX10, and c-MYC (Fig. 3B; reviewed in [START_REF] Gammill | Neural crest specification: migrating into genomics[END_REF][START_REF] Meulemans | Gene-regulatory interactions in neural crest evolution and development[END_REF][START_REF] Green | Evolution of vertebrates as viewed from the crest[END_REF].

b. Signal transduction involved in melanocyte development

During embryonic development, melanoblasts proliferate, migrate and differentiate on their way to their final destination in the basal layer of the epidermis or the hair follicles, although their number and distribution varies among species. crest cells migrate out to give rise to various mesodermal and ectodermal cell populations. Their migratory trajectory is critically connected to the route and fate of the cells (and takes place along two major paths. Most NCCs migrate dorsoventrally between neural tube and somites and give rise to multiple cell types like Schwann cells, smooth muscle cells, peripheral neurons, adipocytes or osteoclasts (cranio-facial skeleton). Cells taking the dorsolateral route between epidermis and dermomyotome give rise to melanoblasts, the melanocyte precursors. (Illustration: Kaltschmidt Group, Uni-bielefeld.de).

NCC specification into melanoblasts, their survival, migration and their subsequent differentiation into melanocytes relies upon action of multiple signalling pathways such as Wnt, Notch, Endothelin3/Endothelin receptor β (EDN3/EDNRB) and KIT/KITL as well as transcription factors such as PAX3, SOX10 and MITF

• Wnt/β-catenin
The WNT/β-catenin-signalling pathway is essential for neural-crest induction and development of the melanocyte lineage. β-catenin is a member of the armadillo repeat motif (ARM) superfamily [START_REF] Hatzfeld | The armadillo family of structural proteins[END_REF]. The ARM domain of β-catenin can interact with cadherins and various coactivators such as LEF/TCF (Lymphoid Enhancer Factor/ T Cell Factor) transcription factors [START_REF] Behrens | Functional interaction of beta-catenin with the transcription factor LEF-1[END_REF][START_REF] Kemler | From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion[END_REF][START_REF] Molenaar | XTcf-3 transcription factor mediates betacatenin-induced axis formation in Xenopus embryos[END_REF] and CBP/p300 proteins [START_REF] Hecht | The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates[END_REF]. Several modes of Wnt pathway activation have been described and the canonical Wnt/β-catenin pathway is the most understood (Fig. 5): in absence of triggering signal, β-catenin is associated with GSK-3β, APC and axin in the cytoplasm. GSK-3β phosphorylates β-catenin targeting it for ubiquination and subsequent proteasomal degradation (Goding, 2000;[START_REF] Saito | Microphthalmia-associated transcription factor in the Wnt signaling pathway[END_REF]. Secreted WNT proteins (cysteine-rich glycoproteins) activate the Frizzled receptor which in turn activates the Dishevelled kinase. This protein will phosphorylate and inhibit GSK-3b resulting in accumulation and nuclear translocation of β-catenin where it co-operates with Lef-1 to activate its target genes [START_REF] Larue | Beta-catenin in the melanocyte lineage[END_REF]. Wnt signals (WNT1 or WNT3A) are essential for several neural crest derivatives including melanoblasts [START_REF] Ikeya | Wnt signalling required for expansion of neural crest and CNS progenitors[END_REF][START_REF] Dorsky | Control of neural crest cell fate by the Wnt signalling pathway[END_REF] and mice lacking wild type Wnt1 and Wnt3a have pigmentation defects [START_REF] Christiansen | Molecular control of neural crest formation, migration and differentiation[END_REF]. In addition, conditional knockout of β-catenin during neural crest differentiation prevents the generation of melanoblasts [START_REF] Hari | Lineage-specific requirements of beta-catenin in neural crest development[END_REF].

• Notch Notch signalling is important in various aspects of cellular regulation such as proliferation, migration and stem cell maintenance (Hitoshi et al., 2004;[START_REF] Duncan | Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance[END_REF][START_REF] Chiba | Notch signaling in stem cell systems[END_REF].

Notch receptors are highly conserved and induced by Delta and Jagged ligands [START_REF] Artavanis-Tsakonas | Notch signaling: cell fate control and signal integration in development[END_REF]. Notch activation results in cleavage of the receptor's intracellular domain (NICD) that acts in a transcription factor complex to promote gene expression including HES (hairy/enhancer of split) repressors.

In melanocyte development, Notch signalling plays a role in the maintenance and differentiation of melanoblast and melanocyte stem cells through cell-cell contact with epidermal keratinocytes [START_REF] Moriyama | Notch signaling via Hes1 transcription factor maintains survival of melanoblasts and melanocyte stem cells[END_REF]Schouwey et al., 2007;Kumano et al., 2008;[START_REF] Aubin-Houzelstein | Melanoblasts' proper location and timed differentiation depend on Notch/RBP-J signaling in postnatal hair follicles[END_REF][START_REF] Watanabe | Multipotency of melanoblasts isolated from murine skin depends on the Notch signal[END_REF]. NOTCH1 expression for instance is sufficient to convert pigmented mature primary human melanocytes to multipotent neural crest stem-like cells [START_REF] Zabierowski | Direct reprogramming of melanocytes to neural crest stem-like cells by one defined factor[END_REF]. • Endothelin3/Endothelin receptor β (EDN3/EDNRB) Endothelins (EDN) are short signalling peptides that act on two homologous Gprotein coupled, receptors named endothelin receptor type A (EDNRA) and type B (EDNRB). Binding of EDNs to their receptor initiates a series of intracellular signal transduction events with various outputs (reviewed in [START_REF] Bouallegue | Endothelin-1-induced signaling pathways in vascular smooth muscle cells[END_REF][START_REF] Sugden | Endothelin signalling in the cardiac myocyte and its pathophysiological relevance[END_REF].

In melanocytes, binding of EDN1 or EDN3 to EDNRB causes the activation of the associated G protein and downstream components (Fig. 5) including protein kinase C (PKC) MAPK (Mitogen-associated protein kinase)/p90rsk and cAMP/PKA/CREB pathways [START_REF] Bohm | Identification of p90RSK as the probable CREB-Ser133 kinase in human melanocytes[END_REF]Imokawa et al., 1996[START_REF] Imokawa | The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanisms of mitogenesis and melanogenesis[END_REF][START_REF] Sato-Jin | Epistatic connections between microphthalmia-associated transcription factor and endothelin signaling in Waardenburg syndrome and other pigmentary disorders[END_REF]. This results both in increased Mitf expression, and direct phosphorylation-dependant stimulation of MITF activity. Endothelin signalling appears to play an important role at the early stage of melanocyte development, when melanoblasts start migrating away from the neural tube [START_REF] Shin | The temporal requirement for endothelin receptor-B signalling during neural crest development[END_REF][START_REF] Harris | Directing pathfinding along the dorsolateral paththe role of EDNRB2 and EphB2 in overcoming inhibition[END_REF]reviewed in Saldana-Caboverde and Kos, 2010). EDN3 induces active proliferation and differentiation in pigment cells during early embryogenesis [START_REF] Lahav | Endothelin 3 promotes neural crest cell proliferation and mediates a vast increase in melanocyte number in culture[END_REF] How EDNRB itself is regulated in melanocyte development is unknown. The SOX10 transcription factor was shown to directly activate EDNRB transcription in NC stem cellderived enteric neurons [START_REF] Zhu | Spatiotemporal regulation of endothelin receptor-B by SOX10 in neural crest-derived enteric neuron precursors[END_REF] and RNA interference (RNAi) as well as ChIP analyses (Chromatin immunoprécipitation) performed by [START_REF] Yokoyama | SOX10, in combination with Sp1, regulates the endothelin receptor type B gene in human melanocyte lineage cells[END_REF] confirmed this regulation in human melanocytes in vitro. However a conflicting report from [START_REF] Hakami | Genetic evidence does not support direct regulation of EDNRB by SOX10 in migratory neural crest and the melanocyte lineage[END_REF] based on genetic evidence does not support direct regulation of EDNRB by SOX10 in the melanocyte lineage.

Consistent with these roles, hypomorphic mutations in the human genes encoding EDN3 or EDNRB result in Waardenburg syndrome [START_REF] Attie | Mutation of the endothelin-receptor B gene in Waardenburg-Hirschsprung disease[END_REF][START_REF] Edery | Mutation of the endothelin-3 gene in the Waardenburg-Hirschsprung disease (Shah-Waardenburg syndrome)[END_REF]. In the mouse, mutations or deletions of the Edn3 or Ednrb genes result in lethal spotting and piebald phenotypes respectively causing severe defects such as white spotting, agangliotic megacolon [START_REF] Baynash | Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons[END_REF][START_REF] Hosoda | Targeted and natural (piebald-lethal) mutations of endothelin-B receptor gene produce megacolon associated with spotted coat color in mice[END_REF] • KIT/KITL KIT signalling is required for the development of three distinct migratory cell types: blood cells, melanocytes, and primordial germ cells [START_REF] Chabot | The protooncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus[END_REF]. c-Kit (KIT or SCFR or CD117) belongs to the receptor tyrosine kinase (RTK) family of membrane receptors. A number of important downstream signalling cascades are activated by the KIT pathway including MAPK (Fig. 5), phosphatidylinositol 3′-kinase (PI3K), JAK/STAT, and Src family members (Fig. 10) (reviewed in [START_REF] Mithraprabhu | Control of KIT signalling in male germ cells: what can we learn from other systems?[END_REF]. Binding of its cognate ligand KITL (a.k.a. as Steel or stem cell factor, SCF) leads to receptor dimerization and autophosphorylation to become a platform for the recruitment of adaptor proteins.

In the case of MAPK signalling, activated KIT recruits GRB2 (Growth Factor Receptor Bound Protein 2) and SOS (Son of Sevenless) towards the plasma membrane. SOS stimulates the exchange of GDP bound to Ras with GTP from the cytosol, which activates RAS (Rat Sarcoma) conformationally, and allows RAS to interact with different downstream effectors. RAS proteins are membrane-bound GTPases that include three canonical members HRAS, NRAS and KRAS [START_REF] Chin | Malignant melanoma: genetics and therapeutics in the genomic era[END_REF][START_REF] Karnoub | Ras oncogenes: split personalities[END_REF]. Activated RAS then induces a kinase cascade resulting in phosphorylationdependant activation of RAF, MEK and then ERK [START_REF] Gray-Schopfer | Melanoma biology and new targeted therapy[END_REF][START_REF] Lennartsson | C-Kit signal transduction and involvement in cancer[END_REF][START_REF] Russo | Melanoma: molecular pathogenesis and emerging target therapies (Review)[END_REF]. In mammals there are three conserved RAF genes (ARAF, BRAF and CRAF) [START_REF] Sardiello | A gene network regulating lysosomal biogenesis and function[END_REF]. Downstream, activated ERK regulates cytoplasmic targets or can translocate into the nucleus where it phosphorylates various proteins including transcription factors to regulate processes including cell proliferation and survival, protein synthesis and differentiation.

Both melanocytes and melanoblasts highly express KIT [START_REF] Yoshida | Distinct stages of melanocyte differentiation revealed by anlaysis of nonuniform pigmentation patterns[END_REF]. KIT signalling is required for the survival, migration, proliferation and differentiation of embryonic melanoblasts throughout their migration along the dorsolateral pathway [START_REF] Yoshida | Distinct stages of melanocyte differentiation revealed by anlaysis of nonuniform pigmentation patterns[END_REF][START_REF] Wehrle-Haller | Soluble and cell-bound forms of steel factor activity play distinct roles in melanocyte precursor dispersal and survival on the lateral neural crest migration pathway[END_REF][START_REF] Cable | Mutations at the W locus affect survival of neural crest-derived melanocytes in the mouse[END_REF] and during colonization of the epidermis and hair follicles (Mackenzie et al., 1997;Jordan and Jackson, 2000). Interestingly melanoblast survival and migration properties seem to be regulated independently [START_REF] Wehrle-Haller | Analysis of melanocyte precursors in Nf1 mutants reveals that MGF/KIT signaling promotes directed cell migration independent of its function in cell survival[END_REF]. KIT/MAPK signalling was shown to induce MITF phosphorylation by ERK kinase, enhancing its transcriptional activity and expression of its target genes (Hemesath et al., 1998;[START_REF] Wu | c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi[END_REF].

In the mouse, c-Kit and Kitl mutations in the related dominant white spotting (W) and Steel (Sl) loci, can result in an absence of melanocytes as well as other developmental defects [START_REF] Chabot | The protooncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus[END_REF]Huang et al., 1990;[START_REF] Miller | cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans[END_REF]. In humans such mutations have been identified in piebaldism, an autosomal dominant ventral pigmentary disorder [START_REF] Giebel | Mutation of the KIT (mast/stem cell growth factor receptor) protooncogene in human piebaldism[END_REF][START_REF] Syrris | Human piebaldism: six novel mutations of the proto-oncogene KIT[END_REF][START_REF] Miller | cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans[END_REF] caused by a defect in melanoblast proliferation and migration from neural crest during embryonic development. Piebaldism exists in various more or less severe forms but patients commonly exhibit a white forelock and characteristic depigmented areas of skin [START_REF] Agarwal | Piebaldism: A brief report and review of the literature[END_REF]. [START_REF] Ruthenburg | Recognition of a mononucleosomal histone modification pattern by BPTF via multivalent interactions[END_REF]

Transcription factors regulating the melanocyte lineage

For their development, melanoblasts depend on a collection of transcription factors that mediate the transcriptional output downstream of multiple signalling pathways and regulating melanocyte development, differentiation, survival and melanocyte stem cell maintenance.

The Sry-related HMG box (SOX) family consists of about 20 transcription factors named after the original member, SRY (sex-determining region Y), and sharing a conserved High mobility group (HMG) box DNA-binding domain [START_REF] Pusch | The SOX10/Sox10 gene from human and mouse: sequence, expression, and transactivation by the encoded HMG domain transcription factor[END_REF]. Based on HMG box homology, these factors are further classified in SOX A to H subfamilies [START_REF] Schepers | Twenty pairs of sox: extent, homology, and nomenclature of the mouse and human sox transcription factor gene families[END_REF]. SOX proteins bind DNA as monomers or dimers [START_REF] Peirano | The glial transcription factor Sox10 binds to DNA both as monomer and dimer with different functional consequences[END_REF][START_REF] Huang | SOXE transcription factors form selective dimers on non-compact DNA motifs through multifaceted interactions between dimerization and high-mobility group domains[END_REF], and regulate transcription of genes involved in multiple functions such as cell specification and differentiation [START_REF] Ludwig | Melanocyte-specific expression of dopachrome tautomerase is dependent on synergistic gene activation by the Sox10 and Mitf transcription factors[END_REF] many of them functioning together with cell type-specific transcription factors [START_REF] Bowles | Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators[END_REF].

The consensus motif recognized by the HMG box domain is 5'-(A/T)(A/T)CAA(A/T)G-3'. This domain is made by three alpha-helices in L-shape hold together through hydrophobic residues. Importantly, SOX proteins bind DNA on the minor groove, which severely bends the DNA toward the major groove along the shape of the HMG domain (up to 85°) and extends the minor groove thereby altering DNA conformation [START_REF] Wegner | From head to toes: the multiple facets of Sox proteins[END_REF]Reményi et al., 2003;Wißmüller et al., 2005). The ability to bind the minor groove of DNA is a rare feature of few proteins including SOX proteins, which suggests that they are important architectural players in the reorganization of chromatin, for the recruitment of transcriptional co-activators and the formation of active regulatory elements. While the majority of SOX proteins functions as transcriptional activators, some members of the Sox family including SOX9 and SOX10 may also act as transcriptional repressors in certain cellular context. [START_REF] Cruz-Solis | Glutamate-dependent transcriptional control in Bergmann glia: Sox10 as a repressor[END_REF][START_REF] Girard | Sumoylation of the SOX10 transcription factor regulates its transcriptional activity[END_REF][START_REF] Lee | SUMOylated SoxE factors recruit Grg4 and function as transcriptional repressors in the neural crest[END_REF] Among SOX family members, SOX10 is an important factor whose expression is restricted to neural crest cells and their non-ectomesenchymal derivatives [START_REF] Kelsh | Sorting out Sox10 functions in neural crest development[END_REF][START_REF] Hou | Transcriptional and signaling regulation in neural crest stem cellderived melanocyte development: do all roads lead to Mitf?[END_REF][START_REF] Hari | Temporal control of neural crest lineage generation by Wnt/betacatenin signaling[END_REF]. SOX10 plays a role in various regulatory aspects of neural crest biology including stem cell maintenance, cell fate specification and differentiation.

Consistent with these functions, germ line knockout of Sox10 in mouse or zebrafish is lethal at early stages of embryonic development [START_REF] Potterf | Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3[END_REF][START_REF] Dutton | Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates[END_REF]. Mutants in the Dominant megacolon (Dom) locus exhibit white spotting and megacolon [START_REF] Potterf | Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3[END_REF][START_REF] Wan | Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors[END_REF]. In humans, SOX10 haploinsufficiency causes Hirschsprung syndrome, characterized by aganglionic megacolon, pigmentary abnormalities and often deafness due to loss of sensory innervation [START_REF] Herbarth | Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease[END_REF][START_REF] Southard-Smith | The Sox10(Dom) mouse: modeling the genetic variation of Waardenburg-Shah (WS4) syndrome[END_REF].

Little information is available about its own transcriptional regulation but it is an active nucleocytoplasmic shuttle protein [START_REF] Rehberg | Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation[END_REF]. Multiple conserved enhancers containing SOX, PAX, AP-2 and TCF/LEF binding sites have been shown to regulate Sox10 [START_REF] Antonellis | Identification of neural crest and glial enhancers at the mouse Sox10 locus through transgenesis in zebrafish[END_REF][START_REF] Werner | Multiple conserved regulatory elements with overlapping functions determine Sox10 expression in mouse embryogenesis[END_REF] and their disruption in the murine Sox Hry insertion mutant result in melanocyte loss. SOX10 proteins have three sumoylation consensus sites that do not affect SOX10 subcellular localisation, but repress its transcriptional activity on its target genes, such as GJB1 and MITF [START_REF] Girard | Sumoylation of the SOX10 transcription factor regulates its transcriptional activity[END_REF]. Other identified targets of SOX10 include enhancer regions of several melanogenic genes including MITF, DCT, TYR and TYRP1 [START_REF] Murisier | A conserved transcriptional enhancer that specifies Tyrp1 expression to melanocytes[END_REF].

Thus, SOX10 represents one of the key players in the melanocyte lineage by directly regulating expression of genes required for survival, migration and differentiation (Wegner, 2005).

PAX proteins (paired box) constitute a family of transcription factor with paired box homeodomains. They play key roles in specifying and maintaining progenitor cells and driving the formation of tissues and organs during embryogenesis (Black and Ziman, 2014) PAX3 (paired box 3) is a member of the Pax family transcription factors possessing both a homeo-and a paired DNA binding domain. PAX3 is expressed very early in the development of the neural crest and coordinates the specification [START_REF] Meulemans | Gene-regulatory interactions in neural crest evolution and development[END_REF] and development of neural crest derivatives including brain, melanocyte and skeletal muscle [START_REF] Tassabehji | Mutations in the PAX3 gene causing Waardenburg syndrome type 1 and type 2[END_REF][START_REF] Watanabe | Epistatic relationship between Waardenburg syndrome genes MITF and PAX3[END_REF][START_REF] Wan | Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors[END_REF]. PAX3 is required to expand a pool of committed melanoblasts [START_REF] Hornyak | Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf[END_REF]. A large repertoire of genes regulated by PAX3 and important for neural crest differentiation has been identified including MITF (see B.2.a), TRP1 [START_REF] Galibert | Pax3 and regulation of the melanocyte-specific tyrosinase-related protein-1 promoter[END_REF] and DCT [START_REF] Lang | Pax3 functions at a nodal point in melanocyte stem cell differentiation[END_REF] pigmentation genes; anti-apoptotic (PTEN, BCL-XL) and differentiation (HES1, MyoD, c-MET) genes [START_REF] Medic | PAX3 across the spectrum: from melanoblast to melanoma[END_REF]. Consistently, transgenic mice expressing PAX3 driven by the tyrosinase promoter (Tyr::Pax3) exhibit skin hyperpigmentation resulting from both increased melanocyte numbers and melanin synthesis (Dong et al., 2012).

Homozygous loss of Pax3 is embryonic lethal du to neural tube and cardiac abnormalities while heterozygous mutations in the Pax3 gene results in the Splotch mouse phenotype [START_REF] Epstein | A mutation within intron 3 of the Pax-3 gene produces aberrantly spliced mRNA transcripts in the splotch (Sp) mouse mutant[END_REF] characterized by a white belly spot. Mutations in human PAX3 are associated with WS type 1 and type 3. Melanocytes are dendritic cells producing melanin. Melanin is the main contributor to pigmentation and plays an important role in protecting the DNA in skin cells from UV radiation, scavenging reactive oxygen species (ROS) or coupling oxidoreduction (redox) reactions [START_REF] Bush | Quantification of Ca(2+) binding to melanin supports the hypothesis that melanosomes serve a functional role in regulating calcium homeostasis[END_REF][START_REF] Costin | Human skin pigmentation: melanocytes modulate skin color in response to stress[END_REF][START_REF] Riley | Melanin[END_REF].

Melanin synthesis takes place in specialized organelles derived from lysosomes known as melanosomes [START_REF] Dell'angelica | Melanosome biogenesis: shedding light on the origin of an obscure organelle[END_REF][START_REF] Schiaffino | Signaling pathways in melanosome biogenesis and pathology[END_REF][START_REF] Wasmeier | Melanosomes at a glance[END_REF]. Melanosomes undergo a multistage maturation from immature endoplasmatic vesicles to fully structured and pigmented organelles (Fig. 6; [START_REF] Raposo | Melanosomes--dark organelles enlighten endosomal membrane transport[END_REF][START_REF] Wasmeier | Melanosomes at a glance[END_REF]. Natural differences in skin and hair pigmentation, that can be classified into cutaneous phototypes (Fig. 7) arise from variations in the number, size, shape, melanin content and distribution of melanosomes, while the number and spacing of melanocytes remain constant.

There are 2 main types of melanin synthesized during melanogenesis (Fig. 6): red/yellow pheomelanin and brown/black eumelanin [START_REF] Kondo | Update on the regulation of mammalian melanocyte function and skin pigmentation[END_REF]. The observed phototype is the result of a mixture between this two types of polymers: pheomelanin is the predominant species in people with fair skin and red hair while eumelanin is predominant in black or dark skin and hair. Only the latter one provide efficient photoprotection against UV radiation and ROS [START_REF] Smit | Melanin offers protection against induction of cyclobutane pyrimidine dimers and 6-4 photoproducts by UVB in cultured human melanocytes[END_REF] while pheomelanin is more photolabile and instead contribute to production of damaging superoxide anions and hydroxyl radicals and trigger oxidative stress to an extent that even appear to promote carcinogenesis, raising interesting questions about the biological function and evolutionary selection of this pigment (Lin and Fisher, 2007;[START_REF] Mitra | An ultravioletradiation-independent pathway to melanoma carcinogenesis in the red hair/fair skin background[END_REF].

b. Melanin biosynthesis and transfer

Several enzymes are involved in melanogenesis (Fig. 6) such as tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1 or TYRP1) and dopachrome tautomerase (TRP-2 or DCT). Both types of melanin derive from a tyrosinase-dependent biochemical pathway in which L-tyrosine is first hydroxylated to L-3,4-dihydroxyphenylalanine (DOPA) and then oxidized to DOPAquinone in a process catalyzed by the tyrosinase (TYR) enzyme. Tyrosinase is therefor the rate-limiting enzyme for the entire melanogenic process [START_REF] Hearing | Determination of melanin synthetic pathways[END_REF]Jimenez, 1987, Land et al., 2003). From dopaquinone, the eumelanin and pheomelanin pathways diverge [START_REF] Hearing | Determination of melanin synthetic pathways[END_REF]:

• Eumelanin synthesis: a redox reaction with another DOPAquinone molecule generates DOPAchrome. DOPAchrome undergoes tautomerisation resulting in 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in a process catalysed by the dopachrome tautomerase enzyme or in 5,6-dihydroxylindole (DHI) in absence of DCT. Finally both compounds are further oxidized (DHI by Tyr and DHICA by TYRP-1) and polymerized to produce brown to dark eumelanin. • Pheomelanin synthesis: cysteine that is freely available or derived from glutathione is spontaneously conjugated to DOPAquinone to form cysteinylDOPA isomers (5S-and 2S-forms) that are further oxidized and polymerized to pheomelanin (Kobayashi et al., 1994a;Kobayashi et al., 1994b;[START_REF] Kondo | Update on the regulation of mammalian melanocyte function and skin pigmentation[END_REF].

Melanosomes containing the melanin are transported to and through the melanocytic dendrites using the actin and tubulin fibres [START_REF] Wu | Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function In vivo[END_REF] and exported to the contacting keratinocytes. Thus while the melanocytes produce the pigments, the keratinocytes are the actual pigmented cells in the skin. Melanosome transfer is not completely understood and different models have been proposed including melanin released by exocytosis prior to uptake by the keratinocyte; melanosomes export over molecular bridges between the cells or cytophagocytosis of whole dendritic extremities by the keratinocytes [START_REF] Van Den Bossche | The quest for the mechanism of melanin transfer[END_REF][START_REF] Yamaguchi | Physiological factors that regulate skin pigmentation[END_REF]. Recent research provided evidence for a shedding vesicle system (Fig. 8) whereby pigment globules containing melanosomes are transferred [START_REF] Ando | Involvement of pigment globules containing multiple melanosomes in the transfer of melanosomes from melanocytes to keratinocytes[END_REF]2012). Following transfer to keratinocytes, the melanosomes are targeted over the cell nuclei to act as a cap and protect from UV exposure.

c. Mutations in the melanin biosynthesis pathway

Mutations resulting in absence or severe dysfunction of key enzymes involved in melanogenesis result in oculocutaneous albinism (OCA type 1 to 4), in which melanocytes are intact but unable to make pigment [START_REF] Kamaraj | Mutational analysis of oculocutaneous albinism: a compact review[END_REF]. Mutations in the TYR gene cause type I oculocutaneous albinism [START_REF] Oetting | Molecular basis of oculocutaneous albinism[END_REF][START_REF] Oetting | Oculocutaneous albinism type 1: the last 100 years[END_REF]. Mutations in the OCA2 gene, encoding a putative tyrosine transporter are responsible for type 2; TYRP1 mutations cause type 3; and changes in the SLC45A2 (or MATP) gene result in type 4 OCA. MATP is a membrane transporter that regulates melanosomal pH [START_REF] Bin | Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity[END_REF] Furthermore the formation, maturation and trafficking of melanosomes is crucial to pigmentation, and defects in this processes lead to other autosomal recessive disorders such as Hermansky-Pudlak Syndrome (HPS) and Chediak-Higashi Syndrome (CHS) (Lin and Fisher, 2007). Fibroblasts and keratinocytes stimulate melanocyte growth and differentiation by secreting multiple factors such as growth factors (bFGF, SCF, HGF), hormones like αMSH (melanocyte-stimulating hormone) and cytokines such as TGFβ (Transforming Growth Factor β). The secretion of these factors (SCF, αMSH) can increase in response to environmental cues such as UV radiation and trigger differentiation and melanin synthesis (tanning response).

To date several hundred mutations in over 130 genes have been associated to a pigmentary phenotype in the mouse. The gene encoding the type 1 melanocortin receptor (MC1R) is one of the most studied. It has a single highly polymorphic exon with at least 30 allelic variants, most of which result in a single amino-acid substitution [START_REF] Rana | High polymorphism at the human melanocortin 1 receptor locus[END_REF]Gerstenblith et al., 2007). Natural ligands for MC1R are αMSH and ACTH (Adrenocorticotropic hormone) (Fig. 9A). Both derive from pro-opiomelanocortin (POMC), a precursor polypeptide that also give rise to the opioid peptide endorphin [START_REF] Kippenberger | Transcription of melanogenesis enzymes in melanocytes: dependence upon culture conditions and co-cultivation with keratinocytes[END_REF] and produced both by keratinocytes and melanocytes.

UV exposure leads to activation of the p53 tumour suppressor resulting in increased POMC expression and subsequent αMSH and ACTH production [START_REF] Cui | Central role of p53 in the suntan response and pathologic hyperpigmentation[END_REF]. αMSH is secreted and binds its cognate melanocortin receptor resulting in activation of the coupled Gαs protein, which further induces an associated adenylate cyclase (Fig. 9B). This enzyme catalyses the hydrolysis of ATP to cAMP that acts a second messenger by activating protein kinase A which is further translocated to the nucleus and phosphorylates CREB (cAMP responsive element binding protein) transcription factors (Goding, 2000;[START_REF] Levy | MITF: master regulator of melanocyte development and melanoma oncogene[END_REF][START_REF] Denat | Malignant melanoma and the role of the paradoxal protein Microphthalmia transcription factor[END_REF], Lin et al., 2007). Activated CREB binds cAMP response elements (CRE) as a dimer in the promoter of target genes including Mitf (Bertolotto et al., 1998b, Bertolotto et al., 1998c;[START_REF] Bertolotto | Regulation of tyrosinase gene expression by cAMP in B16 melanoma cells involves two CATGTG motifs surrounding the TATA box: implication of the microphthalmia gene product[END_REF], which regulates the expression of numerous pigment enzymes and differentiation factors. Antagonists of MC1R such as the Agouti signalling protein (ASP) can prevent activation of this signalling pathway (Fig. 9B).

Mutations in MC1R affect recognition and binding by its ligands and therefor limited or absent subsequent signal transduction: the melanocytes are not able to respond to UV-radiation. Humans affected by such mutations have red hair and fair skin due to increased pheomelanin/eumelanin ratios and poor tanning response, which makes them prone to develop cutaneous diseases like melanoma (Lin and Fisher, 2007). Interestingly, the PI3K/AKT pathway is inhibited by cAMP activation (Fig. 8). This pathway regulates many important cellular processes, including proliferation, differentiation, motility, metabolism, survival, invasion and intracellular transport [START_REF] Engelman | The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism[END_REF]. Phosphatidyl Inositol-3 Kinases (PI3K) are a family of lipid kinases that are composed of an adaptor/regulatory subunit (i.e. p85) and a catalytic unit (i.e. p110) (Fig. 8). Their activity (PIP3 synthesis) can be counteracted by the lipid phosphatase PTEN (Phosphatase and Tensin Homolog), Induction through tyrosine-kinase receptors or Raf proteins, lead to PI3K activation, which phosphorylates phosphatidylinositols in the cell membrane (PIP2 into PIP3). The produced lipid species mediate recruitment of kinases such as AKT and PDK1 (Phosphoinositide-dependent kinase-1) to the cell membrane. PDK1 phosphorylates AKT which is then translocated to the cytosol where phosphorylates many substrate proteins such as mTOR, GSK3, or MITF affecting various aspects of cell biology. Noteworthy cAMP elevation results in inhibition of PI3K/AKT pathway, and prevents activation of small Rho GTPAses (RAC1/CDC42) involved in shaping cell morphology through the actin cytoskeleton (Fig. 10), leading to increased melanocyte dendricity. Furthermore, targeted inhibition of the p70S6K kinase, another downstream target of PI3K signalling, results in activation of tyrosinase expression [START_REF] Busca | Cyclic AMP a key messenger in the regulation of skin pigmentation[END_REF]. The role of cAMP in melanogenesis is also supported by work showing increased tyrosinase expression following treatment with cAMP elevating agents such as forskolin and cholera toxin [START_REF] Bertolotto | Regulation of tyrosinase gene expression by cAMP in B16 melanoma cells involves two CATGTG motifs surrounding the TATA box: implication of the microphthalmia gene product[END_REF].

In melanocytes, similar to αMSH/MC1R signalling, the stress-mediated p38 MAPK activation following UV radiation leads to CREB phosphorylation and MITF expression (Goding, 2000, Saha et al., 2006[START_REF] Mizutani | A single UVB exposure increases the expression of functional KIT in human melanocytes by up-regulating MITF expression through the phosphorylation of p38/CREB[END_REF][START_REF] Wan | Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors[END_REF]. Furthermore the p38 kinase phosphorylates USF1 (Upstream stimulatory factor), leading to transcription activation of key components of the pigmentation signalling pathway and pigment manufacture [START_REF] Galibert | The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression[END_REF]. A mouse model harbouring mutant USF1 exhibit a defective UV radiation-mediated tanning response and fail to activate POMC and MC1R expression in response to UV radiation [START_REF] Corre | UV-induced expression of key component of the tanning process, the POMC and MC1R genes, is dependent on the p-38-activated upstream stimulating factor-1 (USF-1)[END_REF].

Overall, these proteins and pathways provide spatial and temporal signals to create the proper physiological conditions for melanocyte development and function. Mutations in genes affecting this process result in hypopigmentation that arises from lack of melanocytes rather than lack of pigment in viable melanocytes, as occurs in OCA.

Anatomy of the hair follicle

While hair follicles don't have a vital function, they are important mediators of thermoregulation or sensation [START_REF] Ebling | The biology of hair[END_REF]. The hair is composed of several layers of keratinized cells inserted directly in the dermis. The main regions of a hair are the shaft (visible part) and the root (expanded end connected to the dermal papilla that delivers nutrients). Every hair follicle is surrounded by sensory nerve endings, associated with a bundle of smooth muscle cells called arrector pili muscle and a sebaceous gland (Fig. 1, 11 and 12) Main structures of a hair follicle are the dermal papilla, matrix, hair shaft, root sheath, hair fiber, and the bulge (Fig. 11). The hair follicle contains a permanent portion, subdivided into upper (UPP) and lower permanent portion (LPP), and a transient portion (Fig. 12). The transient portion of the hair follicle makes up to two thirds of the mature hair follicle regenerated in every hair cycle, while the permanent portion remains constant [START_REF] Stenn | Controls of hair follicle cycling[END_REF].

Hair follicles undergo cycles of regeneration that can be separated into 3 successive phases: anagen (growth), catagen (regression) and telogen (dormant) (Fig. 11).

• Anagen is the growth phase of the hair cycle. During this phase, the hair root is going through rapid cellular divisions, adding to the hair shaft. While the growth rate is rather similar between different body sites (0,25 to 0,50 mm/day), the duration of the anagen phase that determines the average length of the hair is variable from site to site. • Catagen is the involution or regressing phase. This transition phase lasts about 2 weeks and is characterized by proliferation arrest and regression of the transient portion of the hair follicle by apoptosis. • Telogen is the resting phase of the hair follicle during which the matrix is inactive. The transition back into the growth phase occurs when quiescent stem cells are activated to produce a new hair shaft [START_REF] Blanpain | Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche[END_REF][START_REF] Tumbar | Defining the epithelial stem cell niche in skin[END_REF].

There are two discrete melanocytic populations in hair follicles: melanocyte stem cells (MSC) and their differentiated progeny that produce the melanin pigmenting the hair. A third population of cells named Transit-amplifying cells (TAC) appears transiently during the regeneration of the hair follicle and correspond to differentiation intermediates that will give rise to the terminally differentiated bulb melanocytes.

The synthesis of melanin in the hair is similar to that of the epidermis and under the control of the same signalling molecules and hormones secreted by the surrounding cells (fibroblasts, keratinocytes). Melanogenesis is only active during anagen and is turned off during catagen by the time bulb melanocytes enter into apoptosis. The melanocyte stem cells reside in the bulge in the lower permanent portion, and get differentiated into melanocytes that migrate into the hair matrix to differentiate and produce pigment [START_REF] Osawa | Melanocyte stem cells[END_REF]. Melanocyte stem cells were first identified using BrdU incorporation approaches and described as slow cycling label-retaining cells [START_REF] Cotsarelis | Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis[END_REF]. Later, the Dct-LacZ transgenic mouse model has been used to study MSCs in vivo (Fig. 12), taking advantage of the fact Dct is one of the rare MSC markers identified to date (Mackenzie et al., 1997). Hair follicle melanocyte stem cells have important roles in both normal hair pigmentation and hair greying, and specific genetic defects resulting in non-lethal and visible phenotypes have shed further light on the establishment and maintenance properties of this cell population. The MSC population appears to be established during hair morphogenesis. Many of the melanogenic genes expressed by melanoblasts or bulb melanocytes exist at low/absent levels in MSCs. Although MSCs are not responsible for pigmenting the first morphogenetic hair [START_REF] Mak | Indispensable role of Bcl2 in the development of the melanocyte stem cell[END_REF], they are retained within the hair bulge while melanocytes of the bulb undergo apoptosis during the catagen phase [START_REF] Tobin | Do hair bulb melanocytes undergo apoptosis during hair follicle regression (catagen)?[END_REF][START_REF] Tobin | The fate of hair follicle melanocytes during the hair growth cycle[END_REF]. These MSCs then function to regenerate bulb melanocytes during subsequent hair cycles (Nishimura et al., 2005).

Specificities of mouse skin

While both human and mouse skin ensure the same biological functions and are similar at the histological level, some differences exist and should be considered when using the mouse as a model for pigment cell studies (Fig. 13):

Mice are almost completely covered by densely packed hair follicles, to an extent that reduce the interfollicular space to a minimum. As a consequence the melanocytes are mostly confined to the hair follicles within the dermis (with exception of ears, footpads, and tail) while in humans the vast majority lays in the basal layer of the epidermis. Furthermore some studies suggest that these differently located melanocytes have different signalling requirements and some mutations were shown to force or prevent epidermal colonization [START_REF] Aoki | Two distinct types of mouse melanocyte: differential signaling requirement for the maintenance of non-cutaneous and dermal versus epidermal melanocytes[END_REF]Schouwey et al., 2007;[START_REF] Garcia | Endothelin 3 induces skin pigmentation in a keratin-driven inducible mouse model[END_REF]. Alternatively, the similar location and microenvironment of the mouse-tail melanocytes and human melanocytes makes the tail an attractive tissue to induce and analyse somatic changes in melanocytes.

In addition, human skin has a thicker epidermis (with more cell layers) and a thicker dermis than mouse skin. Mice have a specific hair structure (which affects melanin incorporation) and possess whiskers, a specific type of hair. The hair follicle of whiskers is much larger than that of a typical hair, which makes it a model of choice for dissecting the role of the different cell compartments of the hair follicle. The murine hair cycle timing is quite different from the human one. Importantly the telogen length is quite extended, depending of the body region and the age of the animal, which explains why adult mice have a rather constant hair length. Finally, the human immune system differs significantly from that of a mouse [START_REF] Mestas | Of mice and not men: differences between mouse and human immunology[END_REF][START_REF] Shay | Conservation and divergence in the transcriptional programs of the human and mouse immune systems[END_REF][START_REF] Zschaler | Differences in innate immune response between man and mouse[END_REF] and it is known that the immune system profoundly affects physiological responses to tumor growth and metastasis in ways incompletely understood. The discovery of the microphthalmia locus and its gene was initiated by work from Paula Hertwig in 1942 when she described mice exhibiting pigmentary abnormalities and small 'microphthalmic' red eyes in the offspring of an X-ray irradiated mouse male. The mouse Mitf gene carrying this mutation was later cloned by two groups [START_REF] Hodgkinson | Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein[END_REF][START_REF] Hughes | A helix-loop-helix transcription factor-like gene is located at the mi locus[END_REF] while cloning of the human homolog was reported the following year [START_REF] Tachibana | Cloning of MITF, the human homolog of the mouse microphthalmia gene and assignment to chromosome 3p14.1-p12.3[END_REF]. Subsequently, MITF expression was revealed in a wide range of cell types including melanocytes, osteoclasts, mast cells and RPE cells [START_REF] Hodgkinson | Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein[END_REF]Steingrimsson et al., 2004). This explains how Mitf null mutations can lead to a complexe phenotype including defective pigmentation (absence of melanocytes), deafness (stria vascularis abnormalities in the cochlea of the inner ear) and small red eyes (due to absence of RPE). Since then, a variety of murine alleles with more or less subtle effects on pigmentation have been described (Steingrimsson et al., 2004;[START_REF] Bharti | The other pigment cell: specification and development of the pigmented epithelium of the vertebrate eye[END_REF][START_REF] Hou | Transcriptional and signaling regulation in neural crest stem cellderived melanocyte development: do all roads lead to Mitf?[END_REF].

b. MITF gene structure and splice variants

In humans, the MITF gene is located on chromosome 3 (chromosome 6 in mice). The gene structure is complex, with the mouse and human gene containing at least nine alternative promoters each displaying a specific 5' exons which is spliced to the common remaining exons 2 to 9 (Fig 14A ) encoding the major functional domains [START_REF] Hershey | Genomic analysis of the Microphthalmia locus and identification of the MITF-J/Mitf-J isoform[END_REF][START_REF] Steingrimsson | All for one, one for all: alternative promoters and Mitf[END_REF][START_REF] Udono | Structural organization of the human microphthalmia-associated transcription factor gene containing four alternative promoters[END_REF]. Alternative promoter activation allows specific spatiotemporal expression of MITF isoforms with different N-terminal regions (Steingrimsson et al., 2004) (Fig. 14B). Furthermore a number of rare variants missing entire or parts of exons such as exon 2 and/or 6 (Fig. 14C) have been described [START_REF] Hallsson | Genomic, transcriptional and mutational analysis of the mouse microphthalmia locus[END_REF][START_REF] Primot | ERK-regulated differential expression of the Mitf 6a/b splicing isoforms in melanoma[END_REF]. The biological function and specificity of each alternative MITF protein isoform has not been analyzed in detail. Cell type specific expression of isoforms and their relative abundance was reviewed in Steingrimsson et al., (2005).

Importantly, expression of the MITF-M isoform is restricted to the neural crest derived melanocyte lineage, where it represents the predominant form. In addition, Mitfmi -vga-9 mutant mice, which harbour an insertion in the sequence of the Mitf-M promoter are microphthalmic, deaf, and completely white [START_REF] Hodgkinson | Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein[END_REF] reflecting the key role of this specific isoform in the development of the melanocyte lineage. This explains why most research including ours focuses on the role of MITF-M that will simply be referred as 'MITF' unless otherwise specified. [START_REF] Pogenberg | Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF[END_REF]Genes Dev, 26: 2647-58).

c. MITF protein structure

The MITF protein is a basic-helix-loop-helix-leucine-zipper (bHLH-LZ) TF belonging to the MYC family of transcription factors [START_REF] Hodgkinson | Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein[END_REF][START_REF] Hughes | A helix-loop-helix transcription factor-like gene is located at the mi locus[END_REF].

The largest functional domain of MITF consists in 2 a-helices separated by a loop (Fig. 15). The N-terminal part of the first helix is a basic domain (encoded by exon 6) that provides most of the specific DNA major groove contacts [START_REF] Pogenberg | Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF[END_REF]. The following HLH domain is composed of the C-terminal part of the first helix and the N-terminal part of the second helix separated by a loop (exon 7) and is the core part of the protein dimerization interface in MITF. It is followed by a leucine-zipper (exon 8 and 9), which is also involved in protein interactions and is sufficient to mediate homodimerization [START_REF] Murre | Interactions between heterologous helix-loophelix proteins generate complexes that bind specifically to a common DNA sequence[END_REF]1994;[START_REF] Pogenberg | Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF[END_REF]. Beside these domains MITF contains conserved acidic activation domains (AD), which seem to allow protein-interactions with other factors and have been shown to affect the transcription activity of MITF (Mansky et al., 2002b;[START_REF] Sato | CBP/p300 as a co-factor for the Microphthalmia transcription factor[END_REF]Hemesath et al., 1994;Steingrimsson et al., 2004;[START_REF] Takebayashi | The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential[END_REF].

MITF can bind DNA as a homodimer but also as a heterodimer with the highly related TFEB, TFE3 and TFEC transcription factors (Transcription factor 3, B and C) which together form the MITF-TFE subfamily. In contrast, MITF does not form heterodimers with other bHLHZip proteins such as MYC, MAX, or USF (Hemesath et al., 1994). The crystallographic structure of residues 180-296 of MITF determined by Pogenberg and colleagues showed that this restricted heterodimerization is mediated through a kink in the leucine zipper induced by a three-residue insertion in the Nterminal part of the zipper (Fig. 16)

The biological consequence of MITF homo-vs. heterodimeric assembly is still very puzzling. TFE3, TFEB and TFEC are expressed more broadly than MITF. They are regulators of cell growth, survival and differentiation (Steingrimsson et al., 2004;[START_REF] Haq | Biology and clinical relevance of the micropthalmia family of transcription factors in human cancer[END_REF]. Recent studies have implicated MITF, TFE3 and especially TFEB as regulators of metabolic programming [START_REF] Salma | Transcription Factor Tfe3 Directly Regulates Pgc-1alpha in Muscle[END_REF][START_REF] Perera | Transcriptional control of autophagy-lysosome function drives pancreatic cancer metabolism[END_REF], lysosomal biogenesis and autophagy [START_REF] Sardiello | A gene network regulating lysosomal biogenesis and function[END_REF][START_REF] Settembre | TFEB links autophagy to lysosomal biogenesis[END_REF]Peña-Llopis and Brugarolas, 2011;[START_REF] Martina | MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB[END_REF][START_REF] Roczniak-Ferguson | The transcription factor TFEB links mTORC1 signaling to transcriptional control of lysosome homeostasis[END_REF][START_REF] Martina | Novel roles for the MiTF/TFE family of transcription factors in organelle biogenesis, nutrient sensing, and energy homeostasis[END_REF]. Murine germline knockout of the TfeB gene causes embryonic lethality but mutations in Tfe3 and TfeC do not have as severe effects (Steingrimsson et al., 1998). There appear to exist some functional redundancy between the factors in osteoclast development, however they do not seem to be important for pigmentation (Steingrimsson et al., 2002). (Goding, 2000Genes Dev, 14:1712-1728).

MITF is mainly localized in the nucleus [START_REF] Takebayashi | The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential[END_REF], where it primarily binds 5'-CATGTG-3' and 5'-CACGTG-3' E-box sequences (Fig. 16) [START_REF] Aksan | Targeting the microphthalmia basic helix-loop-helix-leucine zipper transcription factor to a subset of E-box elements in vitro and in vivo[END_REF] present in promoters and enhancers of its target genes. In addition an extended asymmetric E-box, called M-box 5 present in the promoters of melanogenic genes was also found to be bound by MITF with high specificity (Hemesath et al., 1994;Bentley et al., 1994;Bertolotto et al., 1998b[START_REF] Pogenberg | Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF[END_REF]. The MITF structure described by [START_REF] Pogenberg | Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF[END_REF] revealed that residue I212 is determinant for MITF's ability to bind this M-box.

The E-box is a frequently found motif in the genome that can be recognized by a variety of bHLH. In a given cell type, only a subset of these sites is specifically occupied by MITF. Therefor in addition to the presence of its binding site, the subset of genes occupied by MITF strongly depends on the local chromatin organization but also on the different partners that will define the functional output in gene activation or repression. These different aspects will be presented in the following section.

MITF expression and activity is finely tuned a. Regulation of MITF gene expression

The complex organization of the Microphthalmia locus enables a spatial and temporal regulation of the expression of each isoform. Several signalling pathways and downstream transcription factors are able to modulate the expression and transcriptional activity of MITF in vivo including SOX10, PAX3; CREB, Onecut-2, LEF1/TCF, BRN2 and GLI2 (Fig. 17).

The Wnt signalling pathway has been described earlier (A.2). Several targets of TCF/LEF/b-catenin have been identified in melanocytes and melanoma cells including MITF, TRP2 and SOX10 (Goding, 2000;Takeda et al., 2000b;Yasumoto et al., 2002;[START_REF] Saito | Microphthalmia-associated transcription factor in the Wnt signaling pathway[END_REF][START_REF] Vance | The transcription network regulating melanocyte development and melanoma[END_REF], Schepsky et al., 2006;[START_REF] Larue | The WNT/Beta-catenin pathway in melanoma[END_REF]. MITF is a target of the Wnt pathway in various species [START_REF] Widlund | Beta-catenin-induced melanoma growth requires the downstream target Microphthalmia-associated transcription factor[END_REF] but it can also interact directly with β-catenin and redirect its transcriptional activity away from canonical Wnt signalling-regulated genes to activate MITF-specific targets (Schepsky et al., 2006).

PAX3 is one of the factors in the transcriptional network (along with SOX10 and MITF) driving melanocyte lineage survival and differentiation [START_REF] Boissy | Molecular basis of congenital hypopigmentary disorders in humans: a review[END_REF]Nordlund 1997, Sommer 2005). It has been shown to regulate MITF expression by binding its promoter at two distinct sites and transactivation appears to occur synergistically with SOX10 [START_REF] Watanabe | Epistatic relationship between Waardenburg syndrome genes MITF and PAX3[END_REF][START_REF] Bondurand | Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome[END_REF][START_REF] Potterf | Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3[END_REF][START_REF] Yang | Inhibition of PAX3 by TGF-beta modulates melanocyte viability[END_REF]. However in melanocyte precursor cells, PAX3 also prevents MITF from binding some of its target genes by competing for promoter binding sites maintaining the balance between undifferentiated and differentiated state of melanoblasts until activation of the Wnt signalling pathway [START_REF] Lang | Pax3 functions at a nodal point in melanocyte stem cell differentiation[END_REF].

Regulation of PAX3 expression itself is poorly characterized beside IL6-/IL-6R/STAT3 mediated repression [START_REF] Kamaraju | Pax3 down-regulation and shut-off of melanogenesis in melanoma B16/F10.9 by interleukin-6 receptor signaling[END_REF][START_REF] Wan | Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors[END_REF]. Association of PAX3 mutation with Waardenburg syndrome type 1 and type further supports its regulatory effect on MITF. SOX10 plays a key role in the transcriptional control of the master regulator MITF. Several SOX10 binding sites with more or less affinity have been identified in the MITF-M promoter or distal enhancers (Steingrimsson et al., 2004) enabling direct transactivation by SOX10 both in mouse and humans [START_REF] Potterf | Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3[END_REF][START_REF] Wan | Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors[END_REF]. These sites were shown to flank a PAX3 binding site allowing synergistic activation of MITF expression by SOX10 and PAX3. A mutation in the transactivation domain of SOX10 abrogates PAX3-SOX10 interaction, and leads to loss of Mitf expression (Lang andEpstein 2003, Potterf et al., 2000). SOX10 can also cooperate with CREB to activate MITF transcription in a tissue restricted way in response to cAMP elevation [START_REF] Huber | A tissue-restricted cAMP transcriptional response: SOX10 modulates alpha-melanocytestimulating hormone-triggered expression of microphthalmia-associated transcription factor in melanocytes[END_REF] CREB is a transcription factor at the crossroad of different pathways such as aMSH/MC1R and p38MAPK able to activate MITF-M expression (see A.3.b). Interestingly in mammalian cells, CREB is expressed in a wide range of tissues while MITF expression is restricted to the melanocyte lineage. Thus CREB needs to cooperate with other transcription factors such as SOX10 and MITF itself to achieve tissue specific transcription activation. STAT3 (signal transducer and activator of transcription 3) is another regulator of MITF although the exact mechanism by which STAT3 regulates MITF remains to be investigated. STAT3 is phosphorylated by receptor-associated Janus kinase (JAK) in response to IL-6/IL-6R activation and translocates as a dimer into the nucleus to activate its target genes [START_REF] Darnell | Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins[END_REF]. STAT3 target genes include BCL-XL, c-MYC, and CCND1; factors that prevent apoptosis and promote cell proliferation. In mast cells and melanocytes PIAS3 (protein inhibitor of activated STAT3) was shown to directly and specifically interact with MITF and STAT3 resulting in inhibition of their transcriptional activity (Levy et al., 2003;[START_REF] Sonnenblick | Interplay between MITF, PIAS3, and STAT3 in mast cells and melanocytes[END_REF].

FOXD3 (forkhead box transcription factor D3) belongs to the FOX family that is characterized by a conserved DNA-binding domain of winged helix structure (Weigel et al., 1990). It is expressed in the dorsal neural tube during the first wave of neural crest migration and in migrating neural and glial precursors (Thomas and Erickson, 2009). However, its expression is reduced by the time melanoblasts begin to migrate. It has been shown that FOXD3 represses melanogenesis in the neural crest and that downregulation of FOXD3 results in an increase in the number of differentiating melanocytes. FOXD3 is able to repress MITF expression in cultured NCC and B16F10 melanoma cells but does not bind the MITF promoter directly but instead seems to prevent binding of PAX3 (Thomas and Erickson, 2009). BRN2 (a.k.a. N-OCT3 or POU3F2) is a member of the POU family characterized by a bipartite POU DNA-binding domain (POUs and POUh). It is expressed in melanocytes and melanoma cell lines [START_REF] Berlin | Phosphorylation of BRN2 modulates its interaction with the Pax3 promoter to control melanocyte migration and proliferation[END_REF] and BRN2 binding sites were found in the promoter of genes such as KITL and PDE5A [START_REF] Vance | The transcription network regulating melanocyte development and melanoma[END_REF][START_REF] Wan | Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors[END_REF]Kobi et al., 2011), which appear to play a role in melanoma metastasis [START_REF] Arozarena | Oncogenic BRAF induces melanoma cell invasion by downregulating the cGMP-specific phosphodiesterase PDE5A[END_REF]. Physical interaction among BRN2, SOX10, PAX3 and SOX9 have been reported by Smit et al. (2000) BRN2 is regulated both by Wnt/β-catenin (Goodall et al., 2004a) and MAPK/BRAF signalling (Goodall et al., 2004b). BRN2 can bind the MITF promoter and regulate MITF expression however; the transcriptional output of this regulation is still under debate [START_REF] Goodall | Brn-2 represses microphthalmia-associated transcription factor expression and marks a distinct subpopulation of microphthalmia-associated transcription factor-negative melanoma cells[END_REF][START_REF] Wellbrock | Oncogenic BRAF regulates melanoma proliferation through the lineage specific factor MITF[END_REF].

ONECUT-2 (OC-2) belongs to the ONECUT homeodomain class of factors [START_REF] Jacquemin | OC-2, a novel mammalian member of the ONECUT class of homeodomain transcription factors whose function in liver partially overlaps with that of hepatocyte nuclear factor-6[END_REF] and can bind at the MITF-M promoter at different sites [START_REF] Jacquemin | The transcription factor onecut-2 controls the microphthalmia-associated transcription factor gene[END_REF]Steingrimsson et al., 2004). The precise role of this factor still needs to be investigated although no mutations in the protein or its binding site have been described so far in WS2 patients.

Recent studies have further involved the transcription factor GLI2 as a negative regulator of MITF expression in melanoma [START_REF] Javelaud | Transforming growth factor-beta in cutaneous melanoma[END_REF](Javelaud et al., , 2011a(Javelaud et al., and 2011b;;[START_REF] Pierrat | Expression of microphthalmiaassociated transcription factor (MITF), which is critical for melanoma progression, is inhibited by both transcription factor GLI2 and transforming growth factor-beta[END_REF]. GLI2 is a member of the Kruppel family of transcription factor targeted by TGF-β/SMAD signalling that acts downstream of Hedgehog signalling.

GLI2 expression inversely correlates with MITF expression in melanoma cell lines, and they seem to repress each other (Javelaud et al., 2011). Noteworthy, the molecular mechanisms underlying GLI2-driven MITF gene repression appears to be independent of TGF-β-driven inhibition that occurs via repression of protein kinase activity leading to reduced CREB-dependent transcription [START_REF] Pierrat | Expression of microphthalmiaassociated transcription factor (MITF), which is critical for melanoma progression, is inhibited by both transcription factor GLI2 and transforming growth factor-beta[END_REF]. Whether GLI2 plays a role during embryonic development of the melanocyte lineage has not been determined yet.

ZEB2 (zing finger E-box binding protein 2), a transcription factor involved in EMT has been shown to activate MITF expression as ZEB2 depletion resulted in decreased level of MITF its target genes which has been associated with melanoma progression (Denecker et al., 2014). In contrast to activating potential of ZEB2 on the MITF promoter, ZEB1 has been found to directly repress MITF expression in retinal pigment epithelium [START_REF] Liu | Zeb1 represses Mitf and regulates pigment synthesis, cell proliferation, and epithelial morphology[END_REF].

The MITF transcript is also regulated by several microRNAs that promote mRNA degradation or suppress protein synthesis via binding to the MITF mRNA. Hundreds of miRNA candidates that putatively target MITF exist, although only a few of them have been validated in vitro and/or in vivo such as miR-137, miR-148, miR-155, miR-182 or miR-211 (Bemis et al., 2008;[START_REF] Segura | Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor[END_REF][START_REF] Haflidadottir | miR-148 regulates Mitf in melanoma cells[END_REF][START_REF] Philippidou | Signatures of microRNAs and selected microRNA target genes in human melanoma[END_REF]Dong et al., 2012;[START_REF] Kunz | MicroRNAs in melanoma biology[END_REF]. [START_REF] Hartman | MITF in melanoma: mechanisms behind its expression and activity[END_REF]Cell Mol Life Sci, 72: 1249-60).

b. MITF posttranslational modifications

Beside regulation at the transcriptional level, post-translational modification (PTM) of MITF plays an important role in the dynamic and precise control of MITF activity and stability. Several conserved residues of MITF have been identified as being subjected to these modifications that include phosphorylation, acetylation, ubiquitylation and sumoylation (Fig. 18)

Phosphorylation

Different signal transduction pathways can mediate MITF phosphorylation.

We previously mentioned that KITL and KIT play a key role in development and differentiation of the melanocyte lineage (see A.2.b, Fig. 5). Notably, C-KIT engages the MAP kinase signalling cascade and associated ERK2 and p90Rsk kinases which phosphorylate MITF on Ser73 and 409 respectively, enhancing its transcriptional activity to activate/stimulate expression of its target genes [START_REF] Hemesath | MAP kinase links the transcription factor Microphthalmia to c-Kit signalling in melanocytes[END_REF][START_REF] Wu | c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi[END_REF]. Ser73 phosphorylation seems to trigger CBP/p300 interaction [START_REF] Price | Lineage-specific signaling in melanocytes. C-kit stimulation recruits p300/CBP to microphthalmia[END_REF][START_REF] Sato | CBP/p300 as a co-factor for the Microphthalmia transcription factor[END_REF] however it targets MITF for Lys201 ubiquitination and subsequent proteasome-dependant degradation within a few hours [START_REF] Wu | c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi[END_REF][START_REF] Xu | Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9[END_REF]. Interestingly, [START_REF] Bauer | The role of MITF phosphorylation sites during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue[END_REF] were able to rescue very efficiently MITF mi-vga9 mice with BAC transgenes expressing MITF S73A/S409A double mutants showing that these residues are not essential for MITF function during mouse melanocyte development and physiological function.

MITF is both a transcriptional [START_REF] Khaled | Microphthalmia associated transcription factor is a target of the phosphatidylinositol-3kinase pathway[END_REF] and direct [START_REF] Busca | Cyclic AMP a key messenger in the regulation of skin pigmentation[END_REF] target of the PI3K pathway. This negative regulation of MITF levels could protect the cell from toxic effects of melanin overproduction. Inhibition of this PI3K/AKT signalling leads to GSK3β activation, which was shown to phosphorylate MITF on Ser298 (Fig. 5 and 18) resulting in increased tyrosinase promoter transactivation (Takeda et al., 2000a). The functional importance of this residue is supported by work that found mutated Ser298 is linked to WS2 [START_REF] Tassabehji | The mutational spectrum in Waardenburg syndrome[END_REF]. However a recent study from [START_REF] Grill | MITF mutations associated with pigment deficiency syndromes and melanoma have different effects on protein function[END_REF] characterizing the DNAbinding and transcription activation properties of MITF mutations linked to WS2A found mutated S298 to be functionally equivalent to the wild type MITF. Furthermore another study identified three novel GSK3 sites S397, S401 and S405 and mutation of these sites or inhibition of GSK3 enhanced MITF's stability (Ploper et al., 2015) MITF was shown to be phosphorylated on Ser307 (Fig. 18) by the p38 MAPK in osteoclasts stimulated with RANKL (Receptor Activator of NFkB Ligand) leading to increased osteoclast-specific gene expression (Mansky et al., 2002). This modification appears to be involved in interaction of MITF with FUS and BRG1 [START_REF] Bronisz | The multifunctional protein fused in sarcoma (FUS) is a coactivator of microphthalmiaassociated transcription factor (MITF)[END_REF].

Acetylation

Initial evolutionary sequence comparison of the Mitf protein revealed a number of potential acetylation sites including one in the basic domain and one in the leucine zipper domain [START_REF] Hallsson | Evolutionary sequence comparison of the Mitf gene reveals novel conserved domains[END_REF]. Follow up studies in the Steingrimsson (Schepsky et al., 15 th ESPCR Meeting 2009) and Goding labs (Siddaway et al., 22 nd IPCC 2014) identified K206 and K243 residues of MITF as acetylation targets of CBP/p300 under the control of BRAF/MAPK signalling consistent with previous work [START_REF] Price | Lineage-specific signaling in melanocytes. C-kit stimulation recruits p300/CBP to microphthalmia[END_REF]. Acetylation of K243, which contacts the phosphate backbone, reduces its DNA-binding affinity and release MITF from sites corresponding to low affinity motifs (degenerate Eor M-boxes) while a K243Q acetyl-mimic mutation results in MITF hyperactivation and melanoma cell hyperproliferation (Siddaway et al., 22 nd IPCC 2014). Overall these observations provide a direct link of MITF activity and function with the most predominantly deregulated pathway in melanoma [START_REF] Wellbrock | Microphthalmia-associated transcription factor in melanoma development and MAP-kinase pathway targeted therapy[END_REF].

Sumoylation

Sumoylation results in covalent but reversible conjugation of SUMO (small ubiquitin-like modifier) proteins to lysines present in a ψKXE sequence context [START_REF] Rodriguez | SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting[END_REF]. Analogous to ubiquitination, sumoylation involves E1 (UBA2/AOS1), E2 (UBC) and E3 (PIAS or RanBP2) enzymes engaged in processing, ligation and recycling of SUMO. Sumoylation is involved in various cellular processes including nucleocytoplasmic transport, transcriptional regulation, apoptosis and regulation of cell cycle proteins (Long et al., 2004, Schmidt and[START_REF] Schmidt | PIAS/SUMO: new partners in transcriptional regulation[END_REF].

MITF harbours two SUMOylation sites at the lysine residues 182 and 316. While they have been shown to interact, conflicting reports involve the PIAS3 sumo-ligase in MITF sumoylation [START_REF] Miller | Sumoylation of MITF and its related family members TFE3 and TFEB[END_REF][START_REF] Murakami | Sumoylation modulates transcriptional activity of MITF in a promoter-specific manner[END_REF]. Both studies show however that mutating these sites does not alter dimerization, DNA binding, stability, or nuclear localization of MITF but significantly affects its transcriptional activity on promoters containing multiple but not single binding sites such as tyrosinase.

Noteworthy a mutation affecting the acidic residue in the second SUMO recognition site (E318K) is the first recorded heritable MITF mutation that is correlated with significantly increased melanoma and renal cell carcinoma risk [START_REF] Bertolotto | A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma[END_REF]. It should be pointed out that in the kidney, the predominant and therefor affected isoform is MITF-A. From a functional perspective, this mutation was shown to increase MITF transactivation properties and induce a redistribution of MITF genome occupancy thus affecting the nature of the activated transcriptional program.

Additionally, MITF harbouring E318K enhances migration and invasiveness of melanoma cells as well as the ability of immortalized melanocytes to form colonies (clonogenicity) in vitro. Thus while the precise mechanism is still under investigation, this result suggest that K316 sumoylation controls the set of genes bound and regulated by MITF and could explain oncogenic effect of this mutation. Several following reports in other cohorts have confirmed the incidence and risks associated with the E318K variant [START_REF] Yokoyama | A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma[END_REF][START_REF] Ghiorzo | Prevalence of the E318K MITF germline mutation in Italian melanoma patients: associations with histological subtypes and family cancer history[END_REF][START_REF] Sturm | Phenotypic characterization of nevus and tumor patterns in MITF E318K mutation carrier melanoma patients[END_REF].

Ubiquitylation

Ubiquitylation is a post-translational modification resulting in conjugation of up to several ubiquitin molecules on one or several acceptor-lysines of the substrate protein. Ubiquitin plays a central role in targeting proteins for proteolytic degradation by the proteasome, although covalent attachment of ubiquitin to proteins can also regulate localization and/or activity independent of proteolysis such as transcription regulation DNA Repair [START_REF] Sun | The novel functions of ubiquitination in signaling[END_REF][START_REF] Gill | SUMO and ubiquitin in the nucleus: different functions, similar mechanisms?[END_REF].

The human ubiquitin ligase enzyme UBC9 was shown to interact with and modify MITF on lysine K201 (Fig. 18), targeting the transcription factor for proteasomal degradation [START_REF] Xu | Regulation of microphthalmia-associated transcription factor MITF protein levels by association with the ubiquitin-conjugating enzyme hUBC9[END_REF]. As we just mentioned, this process is dependant on MC1R and KIT stimulated phosphorylation of MITF [START_REF] Wu | c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi[END_REF]. Interestingly, a recent report identified the USP13 deubiquitinase as a partner of MITF involved in its stabilization and accumulation (Zhao et al., 2011), although this interaction could not be confirmed in our own work (Laurette et al., 2015).

Caspase cleavage

Apoptosis is a cell death program leading to activation a family of cysteinendoproteases called caspases. Caspase activation results in cleavage mediated activation or inactivation of substrates allowing controlled degradation of cellular components and cell death. MITF was shown to be a caspase substrate: binding of TRAIL (TNF-related apoptosis-inducing ligand) to its DR4 and DR5 receptors (death receptor 4 and 5) leads to activation of caspases (3,6 and 7) that cleave MITF at asparagine 345 into a large N-terminal fragment and a short C-terminal fragment. The N-terminal fragment is fully transcriptionally active but degraded rapidly and the C-terminal fragment has pro-apoptotic properties. Interestingly expression of a non-cleavable form of MITF renders melanoma cells resistant to apoptotic stimuli [START_REF] Larribere | The cleavage of microphthalmia-associated transcription factor, MITF, by caspases plays an essential role in melanocyte and melanoma cell apoptosis[END_REF]. 3. MITF is the master regulator of the melanocyte lineage MITF has been shown to be a master regulator of the melanocyte cell lineage and has a pivotal role in many different aspects of the melanocyte biology, including melanocyte survival, proliferation and differentiation [START_REF] Levy | MITF: master regulator of melanocyte development and melanoma oncogene[END_REF][START_REF] Delmas | Beta-catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development[END_REF][START_REF] Hou | Transcriptional and signaling regulation in neural crest stem cellderived melanocyte development: do all roads lead to Mitf?[END_REF]. The activation of genes is dependent on the level of functional MITF in the cell and the state of differentiation (Carreira et al., 2006;[START_REF] Goodall | Brn-2 represses microphthalmia-associated transcription factor expression and marks a distinct subpopulation of microphthalmia-associated transcription factor-negative melanoma cells[END_REF]Javelaud et al., 2011).

Through the extensive studies of white spotted mutant animals, a number of key genes for melanoblast development have been identified and characterized. These genes encode components of signalling pathways (WNT/b-catenin, KITL/KIT, EDN3/EDNRB, and transcription factors (SOX10, PAX3, SNAI2, and MITF). Importantly, at least four types of Waardenburg syndrome as well as Piebaldism are associated with mutations in these factors: In humans, mutations in MITF-M gene can lead to Waardenburg Syndrome type 2 (WS2) while mutations in any MITF isoform can lead to Tietz Syndrome (Grill et al., 2013;Takeda et al., 2000b). Both are characterized by sensorineural deafness and hypopigmentation of the hair, skin and iris.

In the specifying melanoblast, induction of MITF expression is dependent on SOX10 and PAX3 (Southard- [START_REF] Southard-Smith | Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model[END_REF][START_REF] Bondurand | Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome[END_REF][START_REF] Potterf | Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3[END_REF][START_REF] Elworthy | Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development[END_REF]Aoki et al., 2003). Intriguingly, SOX10 and PAX3 have more expansive expression patterns than MITF and are required for several lineages of NC cells; yet, MITF is activated in a small subset of NC cells. This suggests that additional extrinsic signals such as KIT-dependant expression and activation of MITF [START_REF] Opdecamp | Melanocyte development in vivo and in neural crest cell cultures: crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor[END_REF][START_REF] Hemesath | MAP kinase links the transcription factor Microphthalmia to c-Kit signalling in melanocytes[END_REF]Hou et al., 2000) to activate genes such as BCL2 and SNAI2, whose expressions are critical for melanoblast specification, survival and migration. [START_REF] Opdecamp | Melanocyte development in vivo and in neural crest cell cultures: crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor[END_REF][START_REF] Hornyak | Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf[END_REF]McGill et al., 2002;[START_REF] Sanchez-Martin | SLUG (SNAI2) deletions in patients with Waardenburg disease[END_REF]. Interestingly one of the earliest genes induced by MITF in melanoblasts is DCT and this induction occurs in synergy with SOX10 (Potterf et al., 2001;[START_REF] Jiao | Direct interaction of Sox10 with the promoter of murine Dopachrome Tautomerase (Dct) and synergistic activation of Dct expression with Mitf[END_REF][START_REF] Ludwig | Melanocyte-specific expression of dopachrome tautomerase is dependent on synergistic gene activation by the Sox10 and Mitf transcription factors[END_REF][START_REF] Hou | Interspecies difference in the regulation of melanocyte development by SOX10 and MITF[END_REF].

In adult melanocytes, MITF regulates a large range of genes involved in pigment synthesis, melanosome biogenesis and trafficking (Fig. 19). The first MITF target genes that have been discovered are involved in the synthesis of melanin (TYR, TYRP-1, DCT) and contain M-boxes in their promoters. Work from others and my lab have shown that MITF also regulates many other genes involved in melanin biosynthesis and transport [START_REF] Du | MLANA/MART1 and SILV/PMEL17/GP100 are transcriptionally regulated by MITF in melanocytes and melanoma[END_REF]Laurette et al., 2015). Expression of MITF in NIH3T3 fibroblasts is sufficient to convert them into melanocytelike cells. However, these cells express only a subset of these melanocyte-specific markers and lack functional characteristics of melanocytes [START_REF] Tachibana | Ectopic expression of MITF, a gene for Waardenburg syndrome type 2, converts fibroblasts to cells with melanocyte characteristics[END_REF].

Interestingly, a recent study reported that joint expression of SOX10, MITF and PAX3, is sufficient to directly reprogram human (foetal dermal or skin) and mouse (tail) fibroblasts to melanocytes which acquire stable phenotypical and functional characteristics of normal melanocytes (Yang et al., 2014).

Another cofactor of MITF has been identified recently: assays in zebrafish and mice revealed that IRF4 (Interferon Regulatory Factor 4) cooperates with MITF to activate expression of Tyrosinase [START_REF] Praetorius | A polymorphism in IRF4 affects human pigmentation through a tyrosinase-dependent MITF/TFAP2A pathway[END_REF]. A single nucleotide polymorphism (SNP) has been identified within an intronic enhancer of IRF4 and associated with sensitivity of skin to sun exposure, freckles, blue eyes, and brown hair color. Further investigations tend to show that this SNP lies within the binding site of TFAP2A, an activator of IRF4, and abolishes transcription activation.

We have seen previously that MITF is the downstream effector of several pathways important for melanocyte physiology and response to UV-radiation (see A.3.d). MITF has also been shown to regulate several components of these pathways such as MC1R [START_REF] Aoki | Involvement of microphthalmia-associated transcription factor (MITF) in expression of human melanocortin-1 receptor (MC1R)[END_REF].

MITF controls cell survival of melanoblasts, skin and hair follicles melanocytes and MSCs through the anti-apoptotic proteins BCL2 (B cell leukemia/lymphoma 2; [START_REF] Veis | Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair[END_REF]McGill et al., 2002), BIRC7 (a.k.a. ML-IAP or LIVIN; [START_REF] Dynek | Microphthalmiaassociated transcription factor is a critical transcriptional regulator of melanoma inhibitor of apoptosis in melanomas[END_REF], HIF1α (Hypoxia inducible factor 1; Busca et al., 2005) and MET [START_REF] Beuret | Up-regulation of MET expression by alpha-melanocyte-stimulating hormone and MITF allows hepatocyte growth factor to protect melanocytes and melanoma cells from apoptosis[END_REF]. MITF is also an important regulator of proliferation in these cells as it activates expression of cell cycle regulators such as CDK2 (cyclin dependent kinase 2; [START_REF] Du | Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF[END_REF] and TBX2 [START_REF] Prince | Tbx2 directly represses the expression of the p21(WAF1) cyclin-dependent kinase inhibitor[END_REF].

Altogether these studies demonstrate that the regulation of pigmentation involves many factors required for development, heterogeneity and regeneration of melanocytes and their precursors, as well as those involved in determining melanosome biogenesis and function (including their transport and distribution), and melanocytespecific transcription factors that control their expression and function (Yamaguchi and Hearing, 2014).

MITF is the master regulator of melanocyte development and differentiation. Judging from its role in several aspects of melanocyte biology, it was not surprising that MITF would also play a role in melanoma biology. Over the years increasing evidence has emerged from our lab and others showing how MITF is involved in and promotes malignant behaviour. Chapter II -Cutaneous melanoma: from disease to therapy Hyperproliferation of melanocytes can result in benign pigmentary lesions such as freckles, that can be divided into ephelides and solar lentigines. Ephelides and solar lentigines are benign pigment spots that share certain characteristics but differ significantly in etiology, morphology and environmental response. Ephelides are small pigmented red to light brown spots largely genetically determined but induced by sunlight, whereas lentigines are larger, dark brown spots induced by sun exposure and photodamage of the skin. Thus both are affected by sunlight but Ephelides tend to appear during childhood and become more pigmented during summertime while Lentigines is are more common after the age of 50 and their pigmentation not affected by the seasons [START_REF] Praetorius | Sun-induced freckling: ephelides and solar lentigines[END_REF].

The hyperproliferation of melanocytes can also result in melanocytic neoplasms that range from benign lesions, termed melanocytic naevi, to malignant ones, termed melanomas. Although melanomas can arise from pre-existing nevi (about 30%), most primary melanomas arise de novo [START_REF] Marks | Do all melanomas come from "moles"? A study of the histological association between melanocytic naevi and melanoma[END_REF]Tsao et al., 2004a;[START_REF] Gandini | Metaanalysis of risk factors for cutaneous melanoma: I. Common and atypical naevi[END_REF][START_REF] Weatherhead | Melanomas arising from naevi and de novo melanomas--does origin matter?[END_REF] from the uncontrolled growth of a malignantly transformed melanocyte [START_REF] Clark | A study of tumor progression: the precursor lesions of superficial spreading and nodular melanoma[END_REF][START_REF] Kaddu | Melanoma with benign melanocytic naevus components: reappraisal of clinicopathological features and prognosis[END_REF][START_REF] Weatherhead | Melanomas arising from naevi and de novo melanomas--does origin matter?[END_REF][START_REF] Yovino | Potential for development of malignant melanoma with congenital melanocytic nevi[END_REF]. A broad range of different types of naevi have been described and linked to different types of melanoma. Similarly there are phenotypically diverse types of melanoma that differ in their anatomical site of origin, exposure to ultraviolet (UV) radiation, host age, mutation burden and types of oncogenic alteration [START_REF] Shain | From melanocytes to melanomas[END_REF].

A. Epidemiology

While melanoma accounts for less than 5% of all skin cancers, it is the deadliest, being responsible for nearly 75% of all skin cancer deaths (American Cancer Society, Cancer Facts 2016). Almost 135.000 melanoma cases are diagnosed every year out of 3 million skin cancers (Ferlay et al., 2010) Importantly, melanoma incidence has increased significantly over the last 20 years in the Caucasian population worldwide and faster than any other cancer [START_REF] Jemal | Recent trends in cutaneous melanoma incidence and death rates in the United States, 1992-2006[END_REF][START_REF] Jemal | Cancer occurrence[END_REF][START_REF] Rigel | Malignant melanoma: prevention, early detection, and treatment in the 21st century[END_REF]. The incidence of melanoma shows a typical geographical variability : the highest incidence rates have been reported in Australia and New Zealand [START_REF] Dennis | Analysis of the melanoma epidemic, both apparent and real: data from the 1973 through 1994 surveillance, epidemiology, and end results program registry[END_REF][START_REF] Maclennan | Increasing incidence of cutaneous melanoma in Queensland, Australia[END_REF] with 35 to 40 cases per 100.000 people compared with nearly 11 cases per 100,000 men and women in France (INCa, 2014) or below 5 cases per 100.000 people in northern Scandinavia (Fig. 20A). As melanoma incidence has risen rapidly in many parts of the world, so has melanoma mortality (Fig. 20B) although recent data suggest that melanoma mortality may have peaked in the USA and parts of Europe. In all populations, melanoma is uncommon before the age of 40 years and extremely rare prior to teenagehood but incidence increases with age. For people with advanced disease, the melanoma has spread beyond the local area and regional nodes into other parts of the body or internal organs. The most common sites of melanoma metastases are under the skin (subcutaneous tissue), lymph nodes, the lung, liver, brain, and bones. However, metastasis to other sites in the body such as gastrointestinal tract can also occur. acral lentiginous melanoma (E) Ocular melanoma is a rare form of melanoma in the eye [START_REF] Tsao | Melanoma: from mutations to medicine[END_REF].

Despite increased incidence of melanoma, the prognosis has improved due to earlier stage of presentation and detection followed by surgical removal of primary lesions. The prognosis worsens the deeper the lesion extends beneath the skin and the 5 year survival rate drops drastically with later stages or with melanoma metastasis, due to limited existence of efficient therapeutic strategies (see below).

B. Melanoma classification

As melanocytes colonize many tissues throughout the body, melanoma can arise in all these sites, although cutaneous melanoma is by far the most frequent type of melanoma. Thus primary tumors can have diverse anatomic distribution, histopathological features and biological and clinical behaviour. They have a high propensity to become invasive and form distant metastasis (Fig. 21) in the lymph nodes, the liver, the lung, the bones and the central nervous system (Hombuckle et al., 2003;Belhocine et al., 2006).

Several classifications have been created to stage melanoma in order to predict disease progression and select best possible therapeutic approach.

• The Clark scale looks at how deeply the melanoma has gone into the different layers of the skin and describes the histologic changes accompanying different phases of melanoma progression [START_REF] Clark | A study of tumor progression: the precursor lesions of superficial spreading and nodular melanoma[END_REF]. • The Breslow scale measures the thickness of the melanoma in the skin.

• TNM staging (stands for Tumour, Node, and Metastases) of melanoma describes the thickness of the melanoma and whether there is any spread to lymph nodes or other parts of the body. Clark andcolleagues (1967) andReed (1976) proposed a melanoma classification based on morphologic aspects of the early (radial) growth phase and the body site of the primary tumor to distinguish four major subtypes (Fig. 22): superficial spreading melanoma (SSM), nodular melanoma (NM), lentigo malignant melanoma (LMM), and acral lentiginous melanoma (ALM). Since then other less common and rare variants of malignant melanoma have been described (e.g. Desmoplastic melanoma, Congenital nevus, Myxoid melanoma).

SSM is the most common melanoma, occurring in about 70% of patients and occasionally seen in association with pre-existing nevi (25% of SSM). SSM presents as a slowly growing flat patch of discoloured skin (brown or black) and tend to occur at sites of intense sun exposure, i.e. on neck and trunk in men and the legs in women. It is very common in individuals with very fair skin (skin phototype 1 and 2).

NM occurs in about 25% of patients. This type of lesions appears mostly de novo, grows rapidly over weeks to months and shows fast and high vertical growth. NM may arise on any site, but is most common on exposed areas of the head and neck. In about 30% of cases, melanoma derives from a pre-existing nevus through a multistep process regulated by a key set of genes. Cells must acquire successive genetic lesions prior to forming tumors and metastases. Asterisks indicate genetic risk susceptibility genes [START_REF] Bertolotto | Melanoma: from melanocyte to genetic alterations and clinical options[END_REF].

The LMM subtype accounts for 10% of cases. It is typically located on sundamaged skin of fair-skinned older individuals and grows slowly over 5-20 years. The development of an elevated nodule within the area of pigmentation indicates the change to vertically invasive LMM. This variant does not occur in children.

ALM accounts for 5% of cases but it makes up about 50% of diagnosed melanomas in Asians and individuals with dark skin (skin phototype 5 and 6). It arises on the palms, soles or beneath the nail (subungual).

TNM classification was first introduced in 1992 by The American Joint Committee on Cancer (AJCC) based on work from Dr Pierre Denoix (Institut Gustave Roussy). It is the most widely accepted staging system for cutaneous melanoma and takes into account the two other scales until recent revision (Clark level was removed in the 7th edition, 2010). It is based on the consideration of three parameters: 1) tumor depth (T), as described by Breslow's thickness (expressed in millimeters), 2) lymph node status (N), including in-transit metastasis, and 3) distant metastasis (M) Of note, a melanoma ABCDE system (Asymmetry, Border irregularity, Color variegation, large Diameter, Evolution of moles) was proposed by Stolz in 1994 as a "must to know" to help patients and dermatologists recognize potential early signs of malignant melanoma development from skin lesions without going through histology (Fig. 23)

The Bastian Group proposed a new classification of melanoma subtypes based on genetic alterations, primarily those affecting BRAF, NRAS and KIT, and histopathological features [START_REF] Curtin | Distinct sets of genetic alterations in melanoma[END_REF][START_REF] Viros | Improving melanoma classification by integrating genetic and morphologic features[END_REF], Bastian et al., 2014). Such a refined classification could improve and facilitate stratification for melanoma therapy as well as retrospective analysis of existing clinical trial data.

C. Melanoma initiation and progression

Numerous pathways have been found to contribute to melanomagenesis and accumulating evidence increasingly involves epigenetic alterations including DNA methylation, histone modifications, and non-coding RNAs in this process.

The Clark model suggests that melanoma develops and progresses in a stepwise manner from a melanocytic nevus lesion through to a dysplastic naevus, followed by radial and vertical growth phases to the formation of distant metastasis (Fig. 24). Only a subset of melanomas develop from a pre-existing nevi (see before) or progress through all these steps. In addition, there is an important variation in the time every tumor takes to progress through them. Some melanoma show partial regression and in very rare cases spontaneous cure.

According to this model, several successive events are required for melanoma progression (Fig. 24). Over the past decade, the knowledge in somatic genetic events related to melanoma initation and progression has made clear that genetic and epigenetic alterations are required for melanoma to develop and that it is a vastly heterogenous disease. Initial mutations in melanocytes, such as activating mutations in BRAF, NRAS, or KIT or amplification of CDK4 (cyclin-dependant kinase 4), result in increased but transient clonal expansion of melanocytes and formation of nevi.

The MAPK/ERK pathway is deregulated in most melanomas (Fig. 25) [START_REF] Fecher | The MAPK pathway in melanoma[END_REF]. The most common mechanism of this pathway is a somatic gain-of-function mutation of Ras or Raf proteins (Fig. 25 and26) [START_REF] Zhuang | Activation of the extracellular signal regulated kinase (ERK) pathway in human melanoma[END_REF]. NRAS is the first oncogene to be identifie in melanoma [START_REF] Padua | A novel transforming gene in a human malignant melanoma cell line[END_REF] and the most frequently mutated RAS family member (about 15% of melanomas) with activating mutations in about 56% of congenital nevi, 33% of primary-and 26% of metastatic melanomas [START_REF] Chin | Malignant melanoma: genetics and therapeutics in the genomic era[END_REF] while HRAS mutations have been associated with Spitz nevi [START_REF] Tsao | Melanoma: from mutations to medicine[END_REF]. The most common sites of mutations in NRAS are G12 (in the phosphate-binding loop) and Q61 (in the catalytic unit) [START_REF] Ellerhorst | Clinical correlates of NRAS and BRAF mutations in primary human melanoma[END_REF]Li et al., 2012). The most commonly mutated component of the pathway is BRAF (about 60% of melanomas). BRAFV600E represents the vast majority of activating mutations [START_REF] Davies | Mutations of the BRAF gene in human cancer[END_REF]. It is located in exon 15 of BRAF and mimics phosphorylation of its kinase domain, resulting in elevated kinase activity and increased phosphorylation of ERK. In melanomas, NRAS and BRAF mutations are in general mutually exclusive, indicating either of the two mutations is sufficient to constitutively activate the MAPK signalling pathway [START_REF] Goel | Examination of mutations in BRAF, NRAS, and PTEN in primary cutaneous melanoma[END_REF] while mutations in ERK1/2 are not very common. NF1 (Neurofibromin 1) is a GTPase-activating protein that inactivates RAS by facilitating hydrolysis of its bound GTP to GDP. To date, NF1 is the third most frequently mutated gene associated with melanoma (Fig. 25): inactivating mutations in NF1 are found in approx. 14 % of cutaneous melanomas and are present in 46% of melanomas expressing wild type BRAF and RAS [START_REF] Hodis | A landscape of driver mutations in melanoma[END_REF]Krauthammer et al., 2015). In addition, these studies showed that melanoma patients with the NF1 mutation appear to be older and to have a greater number of mutations in the tumors. Using a transgenic mouse model mutant, BRAF was shown to cooperate with NF1 loss to drive melanoma development through the abrogation of oncogene-induced senescence [START_REF] Maertens | Elucidating distinct roles for NF1 in melanomagenesis[END_REF] and another study identified NF1 loss as a key mediator of acquired and intrinsic BRAF inhibitor resistance [START_REF] Whittaker | A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition[END_REF]. Together these studies provide new insights into the signalling that underlies melanoma initiation and progression and suggests novel therapeutic strategies for patients developing BRAF mutant and NF-1 deficient melanomas. Overactivation of the MAPK/ERK and other pathway can be achieved without mutations in the signalling components but by alterations of the upstream receptors in particular KIT. Early studies have shown sequential loss of KIT from benign to primary and metastatic melanomas [START_REF] Montone | Proto-oncogene c-kit expression in malignant melanoma: protein loss with tumor progression[END_REF][START_REF] Shen | Analysis of protein tyrosine kinase expression in melanocytic lesions by tissue array[END_REF] that seem to be involved in an enhancement of melanoma tumorigenicity and metastasis [START_REF] Huang | Enforced c-KIT expression renders highly metastatic human melanoma cells susceptible to stem cell factor-induced apoptosis and inhibits their tumorigenic and metastatic potential[END_REF][START_REF] Phung | C-KIT signaling depends on microphthalmia-associated transcription factor for effects on cell proliferation[END_REF]. KIT is mutated in 2% of melanoma (Fig. 22) and a significant subset of melanomas, mainly mucosal (39%) and acral (36%), harbour an increase in copy number and/or activating mutations in KIT [START_REF] Antonescu | L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition[END_REF][START_REF] Beadling | KIT gene mutations and copy number in melanoma subtypes[END_REF][START_REF] Curtin | Somatic activation of KIT in distinct subtypes of melanoma[END_REF][START_REF] Torres-Cabala | Correlation between KIT expression and KIT mutation in melanoma: a study of 173 cases with emphasis on the acral-lentiginous/mucosal type[END_REF]. Furthermore the existence of a subset of melanomas overexpressing KIT and CDK4 offer and explanation of a mechanism of malignant transformation in melanomas that don't carry BRAF or NRAS driver mutations [START_REF] Smalley | Identification of a novel subgroup of melanomas with KIT/cyclin-dependent kinase-4 overexpression[END_REF].

The resulting lesions are mostly benign as overactivation of proliferative signals such as BRAFV600E [START_REF] Michaloglou | BRAF(E600) in benign and malignant human tumours[END_REF], or NRASQ61R (Zhuang et al., 2008) triggers cell cycle arrest through oncogene-induced senescence (OIS), acting as a barrier to melanoma development (Dhomen et al., 2009[START_REF] Michaloglou | BRAFE600-associated senescencelike cell cycle arrest of human naevi[END_REF]. This nevi are marked by p16INK4a (and downstream signalling, see below) induction and senescence-associated beta-galactosidase (SA-beta-gal), defined as beta-galactosidase activity detectable at pH 6.0 [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and in aging skin in vivo[END_REF][START_REF] Michaloglou | BRAFE600-associated senescencelike cell cycle arrest of human naevi[END_REF][START_REF] Gray-Schopfer | Cellular senescence in naevi and immortalisation in melanoma: a role for p16?[END_REF]. This is a very powerful process and most nevi stay in this proliferation arrested state over decades without progressing to melanoma. Of note, melanocyte entry into a senescent state can also be the result of oxidative stress [START_REF] Leikam | Oncogene activation in melanocytes links reactive oxygen to multinucleated phenotype and senescence[END_REF] or replicative senescence. The latter one is induced by activation of DNA damage pathways activated upon telomere shortening that result from fast and uncontrolled cell proliferation [START_REF] Schwahn | Dynamic regulation of the human dopachrome tautomerase promoter by MITF, ER-alpha and chromatin remodelers during proliferation and senescence of human melanocytes[END_REF][START_REF] Sviderskaya | p16(Ink4a) in melanocyte senescence and differentiation[END_REF][START_REF] Sviderskaya | p16/cyclindependent kinase inhibitor 2A deficiency in human melanocyte senescence, apoptosis, and immortalization: possible implications for melanoma progression[END_REF] b. Senescence escape and radial growth phase Additional mutations, such as inactivation of p16INK4A/retinoblastoma pathway and PTEN as well as activation of TERT (telomerase reverse transcriptase), are required to enable cells to overcome senescence, suppress apoptosis and promote proliferation, genomic instability and immortalization (Fig. 24).

CDKN2A encodes the p16INK4a and p14ARF (p19ARF in mice) cell cycle regulators (Fig. 24,25,27). The function of p16 is to inhibit CDK4 and CDK6. Both kinases phosphorylate the retinoblastoma protein (pRb) allowing the S phase entry [START_REF] Serrano | A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4[END_REF] and the inhibition of these kinases leads to cell cycle arrest. p14 blocks the degradation of the p53 protein by inhibiting HDM2, thus enhancing apoptosis. The inactivation of either p16 or p14 abrogate Rb and p53 pathways, abolishes oncogene induced senescence and lead to uncontrolled cell growth [START_REF] De Snoo | Cutaneous melanoma susceptibility and progression genes[END_REF]. This inactivation can occur through genetic lesions (Bennett, 2008a), DNA hypermethylation [START_REF] Richards | Epigenetic marks in melanoma[END_REF][START_REF] Rothhammer | Epigenetic events in malignant melanoma[END_REF], or repression via activation of Wnt⁄β-catenin signalling (see below).

Unlike many types of cancers, mutations or deletions of p53 are a rare occurrence in melanoma. [START_REF] Albino | Mutation and expression of the p53 gene in human malignant melanoma[END_REF][START_REF] Montano | Analysis of p53 in human cutaneous melanoma cell lines[END_REF][START_REF] Ragnarsson-Olding | Mutations in the TP53 gene in human malignant melanomas derived from sun-exposed skin and unexposed mucosal membranes[END_REF][START_REF] Soengas | Inactivation of the apoptosis effector Apaf-1 in malignant melanoma[END_REF]. p53 inactivation can be achieved by alteration of the CDKN2A locus [START_REF] Curtin | Distinct sets of genetic alterations in melanoma[END_REF] as mutations of p14ARF can impair its ability to prevent MDM2, an E3 ubiquitin ligase of p53, from targeting p53 for degradation (Zhang et al., 1998). Thus the p53 stress signal response is often defective in p14ARF mutant melanoma cells [START_REF] Yang | Small molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate p53 in cells[END_REF] and overexpression of MDM2 or MDMX, which bind and inactivate p53, is observed in melanomas [START_REF] Gembarska | MDM4 is a key therapeutic target in cutaneous melanoma[END_REF]Muthusamy et al., 2006;[START_REF] Polsky | HDM2 protein overexpression, but not gene amplification, is related to tumorigenesis of cutaneous melanoma[END_REF].

In melanoma, increased PI3K/AKT pathway activation manifests by functional loss of PTEN or AKT overexpression and triggers inhibition of apoptosis and melanoma cell survival [START_REF] Stahl | Loss of PTEN promotes tumor development in malignant melanoma[END_REF]. PI3K mediates activation of the AKT kinase (see A.3.d).

AKT exists as three isoforms (AKT1, 2 and 3) that phosphorylate and regulates numerous factors, such as BAD, CCND1, or FOXO transcription factors [START_REF] Dillon | Distinct biological roles for the akt family in mammary tumor progression[END_REF]Muller 2010, Soengas and[START_REF] Soengas | Apoptosis and melanoma chemoresistance[END_REF]. In melanoma, AKT3 seems to be the predominant isoform and hyperactivated in about 60% of melanomas due to its own overexpression or PTEN inactivation (Fig. 26) [START_REF] Bastian | Chromosomal gains and losses in primary cutaneous melanomas detected by comparative genomic hybridization[END_REF]Tsao et al., 2004;[START_REF] Stahl | Deregulated Akt3 activity promotes development of malignant melanoma[END_REF].

Indeed, PTEN acts as a tumor suppressor and negatively regulates AKT and the PI3K pathway. Loss of function mutations of PTEN occur in about 30% of melanomas, often together with activating mutations in BRAF [START_REF] Bennett | How to make a melanoma: what do we know of the primary clonal events?[END_REF]Tsao et al., 1998;[START_REF] Gast | Somatic alterations in the melanoma genome: a high-resolution array-based comparative genomic hybridization study[END_REF]Tsao et al., 2004[START_REF] Fecher | The MAPK pathway in melanoma[END_REF] and lead to a constitutive activation of the PI3K pathway. Recently a study from Dankort and colleagues showed that mice expressing oncogenic BRAFV600E in melanocytes develop benign melanocytic hyperplasia that failed to progress to melanoma efficiently and with high penetrance. Strikingly, the offspring from BRAFV600E mutated and PTEN-deficient mice develop melanoma with 100% penetrance and exhibit metastasis in the lymph nodes and the lung (Dankort et al., 2009). This suggests a strong cooperation between expression of oncogenic BRAF and PTEN silencing to promote the development and progression of metastatic melanoma. Multiple studies show that BRAFV600E or NRASQ61K mutations cooperate with loss of PTEN or INK4a to promote the development of more malignant stages of melanoma [START_REF] Goel | Examination of mutations in BRAF, NRAS, and PTEN in primary cutaneous melanoma[END_REF], Tsao et al., 2004b[START_REF] You | Genetic analysis of Pten and Ink4a/Arf interactions in the suppression of tumorigenesis in mice[END_REF] which is further supported by studies using transgenic mice [START_REF] Ackermann | Metastasizing melanoma formation caused by expression of activated N-RasQ61K on an INK4a-deficient background[END_REF]Dankort et al., 2009).

The Wnt/ β-catenin pathway has also been shown to be important in the development of more aggressive melanoma phenotype (Fig. 24) and constitutively active Wnt signalling has been identified in more than 30% of melanomas [START_REF] Rimm | Frequent nuclear/cytoplasmic localization of beta-catenin without exon 3 mutations in malignant melanoma[END_REF]. β-catenin mutations have been described in about 5% of primary, uncultured melanomas (Omholt et al., 2001;[START_REF] Demunter | Loss of membranous expression of beta-catenin is associated with tumor progression in cutaneous melanoma and rarely caused by exon 3 mutations[END_REF][START_REF] Reifenberger | Molecular genetic analysis of malignant melanomas for aberrations of the WNT signaling pathway genes CTNNB1, APC, ICAT and BTRC[END_REF] and almost always affect exon 3 phosphorylation sites [START_REF] Forbes | COSMIC (the Catalogue of Somatic Mutations in Cancer): a resource to investigate acquired mutations in human cancer[END_REF] resulting in stabilization of β-catenin.

In vivo in the mouse, expression of a stabilized mutant β-catenin (bcat-STA) lacking the exon3 that make it resistant to GSK3-mediated degradation-targeting can immortalize primary skin melanocytes by silencing the p16Ink4a promoter but doesn't induce melanoma formation [START_REF] Delmas | Beta-catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development[END_REF]. However, crossing bcat-STA mice with mice producing an oncogenic form of NRAS (NRASQ61K) in melanocytes result in melanoma development with high incidence (85%) and a markedly shorter average latency period [START_REF] Ackermann | Metastasizing melanoma formation caused by expression of activated N-RasQ61K on an INK4a-deficient background[END_REF]. Similarly, when β-catenin is stabilized in the context of Pten loss and Braf activation, melanomas are very metastatic, grow rapidly, and are highly differentiated (Damsky et al., 2011). Conversely, β-catenin inactivation in Braf/Pten melanomas delays tumor formation, extends survival, and prevents metastasis formation demonstrating the requirement for endogenous β-catenin in melanoma formation and progression (Damsky et al., 2011).

The role of non-canonical Wnt/Ca2+ signaling though WNT5A has also been shown to have a role in metastatic melanoma (Fig. 24): WTN5A overexpression in low metastatic melanoma enhances their aggressive features [START_REF] Weeraratna | Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma[END_REF] and WNT5A/PKC signaling is frequently associated with high grade melanomas in which it promotes EMT and inhibits metastasis repressors [START_REF] Dissanayake | The Wnt5A/protein kinase C pathway mediates motility in melanoma cells via the inhibition of metastasis suppressors and initiation of an epithelial to mesenchymal transition[END_REF].

Noteworthy, Wnt/β-catenin signaling regulates cell growth by controlling transcription of different target genes including cyclin D1 [START_REF] Shtutman | The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway[END_REF][START_REF] Tetsu | Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells[END_REF], c-myc [START_REF] He | Identification of c-MYC as a target of the APC pathway[END_REF] as well as MITF (Takeda et al., 2000b). and BRN2. (Goodall et al., 2004).

BRN2 is highly upregulated in melanoma and is involved in proliferation and survival. Regulation BRN2 expression by β-catenin has been shown in melanoma cell lines and in transgenic mice. Moreover, silencing of BRN2 in melanoma cell lines overexpressing β-catenin significantly reduces the proliferation rate of these cells (Goodall et al., 2004). CCND1 is a proto-oncogene encoding an activator subunit of CDK4 and CDK6 that plays an important role in numerous cancers, including melanoma. Amplification of the CCND1 locus as well as CCND1 protein overexpression is detected in 20% of melanoma cases (Fig. 24,25), and more commonly in acral melanoma [START_REF] Sauter | Cyclin D1 is a candidate oncogene in cutaneous melanoma[END_REF]. Furthermore, ablation of CCND1 in melanoma cells by siRNA induces G1/S arrest [START_REF] Bhatt | Adhesion control of cyclin D1 and p27Kip1 levels is deregulated in melanoma cells through BRAF-MEK-ERK signaling[END_REF] and reduces tumor growth in SCID mice xenografts [START_REF] Sauter | Cyclin D1 is a candidate oncogene in cutaneous melanoma[END_REF].

Mutations affecting members of the mammalian SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin-remodelling complex, particularly ARID2 and ARID1A (AT-rich interaction-2 and -1A), emerge at the transition to invasive melanoma. The SWI/SNF remodeller acts as a tumour suppressor in many cancers, including melanoma (Fig. 25), although the underlying mechanisms remain unclear. It has been proposed that SWI/SNF maintains genomic integrity, which is noteworthy, as the emergence of SWI/SNF mutations during progression of melanoma coincides with the appearance of widespread chromosomal aberrations that are seen in fully evolved melanomas. During this phase, described as the radial growth phase (RGP), melanoma cells can only proliferate between the layers of the epidermis and superficial dermis and are still dependant on exocrine factors produced by the keratinocytes for their survival (Bogenrieder and Herlyn, 2002) with significantly low risk for metastasis. c. EMT transition and vertical growth phase Acquisition of additional changes are required to allow melanoma cells to undergo epithelial-to-mesenchymal transition (EMT), escape keratinocyte dependence and invade deeper layers of the skin during the vertical growth phase (VGP) [START_REF] Bennett | How to make a melanoma: what do we know of the primary clonal events?[END_REF]. EMT is a morphogenetic process in which cells loose their epithelial characteristics and gain mesenchymal properties. It is important in various steps of development and adult physiology (e. g. neural crest, wound healing) and its aberrant regulation has been associated with malignant properties such as tumor invasiveness, drug resistance, or fibrosis (Fig. 28) (Craen et al., 2013).

Hallmark of EMT are expression changes in adhesion molecules such as cadherins (loss of E-cadherin, expression of N-cadherin) and integrins (overexpression of β1 et β3) [START_REF] Hsu | Shifts in cadherin profiles between human normal melanocytes and melanomas[END_REF]Hsu et al., 2000;[START_REF] Peinado | Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?[END_REF], Olmeda et al., 2007). In addition, secretion of matrix metalloproteases such as MMP2 and MMP9 affect the basement membrane integrity (collagen degradation) and allow invasion in the dermis [START_REF] Vaisanen | Matrix metalloproteinase-2 (72 kD type IV collagenase) expression occurs in the early stage of human melanocytic tumour progression and may have prognostic value[END_REF]. Dermal invasion also correlates with the development of autocrine secretions (Li et al., 2001), which include CTGF (connective tissue growth factor), bFGF and PDGF (platelet-derived growth factor) as well as immune modifying factors such as interleukins, chemokines and TGFβ.

Several pathways have been shown to trigger EMT, all of which function through three key downstream transcription factor families including the Snail (SNAIL, SLUG), Zeb (ZEB1, ZEB2) and basic helix-loop-helix (TWIST1) (Fig. 29) [START_REF] Lamouille | Molecular mechanisms of epithelial-mesenchymal transition[END_REF][START_REF] Li | Phenotype switching in melanoma: implications for progression and therapy[END_REF][START_REF] Vandamme | Melanoma cells revive an embryonic transcriptional network to dictate phenotypic heterogeneity[END_REF].

SNAIL and SLUG are downstream of TGFβ and PI3K/AKT signaling. They are repressors of epithelial markers such as adhesion molecules and activators of mesenchymal markers associated with migration and invasion properties such as Vimentin [START_REF] Poser | Loss of E-cadherin expression in melanoma cells involves up-regulation of the transcriptional repressor Snail[END_REF][START_REF] Gupta | The melanocyte differentiation program predisposes to metastasis after neoplastic transformation[END_REF][START_REF] Medici | Snail and Slug promote epithelial-mesenchymal transition through beta-catenin-T-cell factor-4-dependent expression of transforming growth factor-beta3[END_REF].

TGFβ and Sonic hedgehog pathways also play a significant role in the process of EMT, embryogenesis, and cancer pathogenesis. In contrast to melanocytes, which only express TGFβ1, melanomas and melanocytic nevi express all three isoforms of TGFβ. While melanocytes and less aggressive melanoma cells have been shown to be sensitive to the antiproliferative effects of TGFβ [START_REF] Hoek | Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature[END_REF], highly invasive melanoma cells are not. The mechanism underlying this evasion from a cytostatic response to TGFβ in highly invasive melanoma cells has not yet been fully determined but TGFβ-Smad pathway mutations are very rare [START_REF] Javelaud | Transforming growth factor-beta in cutaneous melanoma[END_REF][START_REF] Levy | MITF: master regulator of melanocyte development and melanoma oncogene[END_REF][START_REF] Rodeck | Independent regulation of growth and SMADmediated transcription by transforming growth factor beta in human melanoma cells[END_REF]. TGFβ signals through Smads: during EMT, SNAIL and SLUG associate with SMAD3 and SMAD4 in the to initiate cancer growth and metastasis by inhibiting or suppressing transcription of E-cadherin, occludin and claudin. The role of role TGFβ signaling in melanoma progression was further highlighted by the Mauviel lab by showing that over-expression of the inhibitory SMAD7 downregulates MMP2 and MMP9 expression, inhibits growth, invasion (Javelaud et al., 2005) and metastatic properties [START_REF] Javelaud | Stable overexpression of Smad7 in human melanoma cells impairs bone metastasis[END_REF] of melanoma cells. Conversely, TGFβ treatment of cultured melanoma cells was shown to induce MMP9 expression, downregulate E-cadherin and upregulates integrins [START_REF] Janji | Autocrine TGF-beta-regulated expression of adhesion receptors and integrin-linked kinase in HT-144 melanoma cells correlates with their metastatic phenotype[END_REF].

Other studies from the same laboratory identified GLI2, a mediator of the sonic hedgehog pathway (Fig. 29), as a transcriptional target of TGF-beta signaling [START_REF] Dennler | Cloning of the human GLI2 Promoter: transcriptional activation by transforming growth factor-beta via SMAD3/beta-catenin cooperation[END_REF][START_REF] Alexaki | GLI2-mediated melanoma invasion and metastasis[END_REF]. Increased GLI2 expression in melanoma tumors is associated with loss of E-cadherin expression. Furthermore, GLI2 also cooperates with ZEB1 in repression of CDH1 in melanoma cells, leading to more aggressive tumor phenotype [START_REF] Perrot | Insights into the Transforming Growth Factorbeta Signaling Pathway in Cutaneous Melanoma[END_REF]. GLI2 and MITF show reciprocal expression patterns in a wide range of melanoma cells in vitro (Javelaud et al., 2011). GLI2 binds to and represses the MITF promoter.

ZEB1 and ZEB2 belong to a small family of transcriptional factors characterized by a homeodomain flanked by two separated zinc finger clusters. They have been shown to be key player in tumor cell invasion and metastasis of many cancers [START_REF] Vandewalle | SIP1/ZEB2 induces EMT by repressing genes of different epithelial cell-cell junctions[END_REF][START_REF] Liu | Zeb1 links epithelialmesenchymal transition and cellular senescence[END_REF][START_REF] Wellner | The EMT-activator ZEB1 promotes tumorigenicity by repressing stemness-inhibiting microRNAs[END_REF]Sánchez-Tilló et al., 2011;Zhang et al., 2015) Recent work from the Chris Marine lab has shown that loss of Mitf leads to significant upregulation of ZEB1 in melanoma cell lines. Furthermore, as Mitf binds several sites in the vicinity of the ZEB1 locus, ZEB1 is likely a direct Mitf target. Loss of ZEB2 expression correlates with patient survival and therefor appears to be implicated in human melanoma development, (Denecker et al., 2014).

Overall this shows that during melanomagenesis different EMT-TFs have antagonistic functions and the EMT reprogramming is the result of a complex interplay between these factors: SNAIL2 and ZEB2 transcription factors drive a melanocytic differentiation program and act as oncosuppressive proteins, while ZEB1 and TWIST1 contribute to the malignant transformation through induction of TGFb and invasion associated gene signatures in concert with MITF downregulation (Caramel et al., 2013).

Remodelling of their actin cytoskeleton enable melanoma cell to become motile and migrate within the dermis. This is achieved by deregulation of the RAC1 and CDC42 small Rho GTPAses (see A.3.d). Interestingly, a recent exome sequencing study identified recurrent somatic mutations in RAC1 in malignant melanoma (Fig. 25) [START_REF] Krauthammer | Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma[END_REF]. Additionally, a RAC1P29S mutation was shown to induce spontaneous activation of the GTPase, leading to increased binding of its downstream effectors that trigger melanocyte proliferation and migration [START_REF] Krauthammer | Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma[END_REF].

Work from the Herlyn laboratory has shown that aberrant Notch signaling exerts an oncogenic effect on melanoma development and progression (Fig. 29). Notch seems to have a positive effect on MAPK, PI3K/AKT and b-catenin signalling. Activated Notch not only enhances primary melanoma cell proliferation in vitro, but also accelerates xenografted primary melanoma growth and promotes metastasis formation in vivo [START_REF] Balint | Activation of Notch1 signaling is required for beta-catenin-mediated human primary melanoma progression[END_REF][START_REF] Liu | Rate of growth in melanomas: characteristics and associations of rapidly growing melanomas[END_REF][START_REF] Pinnix | Active Notch1 confers a transformed phenotype to primary human melanocytes[END_REF].

SOX9 is another transcription factor that has been involved in melanoma progression [START_REF] Passeron | Upregulation of SOX9 inhibits the growth of human and mouse melanomas and restores their sensitivity to retinoic acid[END_REF][START_REF] Cheng | Methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognostic factor in advanced melanoma[END_REF]. In these studies, SOX9 expression was shown to be associated with poor survival in melanoma patients. SOX9 overexpression in melanoma cell lines results in G1/S arrest of proliferation but also in an increase of invasive properties. Conversely, knockdown of SOX9 in the invasive phenotype melanoma cells reduced their invasive properties. Both the in vitro and in vivo data suggest that the observed phenotype might be due to the activation of EMTlike genes and the downregulation of potential tumor suppressor genes.

Recent reports propose additional new regulators to play a role in melanoma invasion by regulating EMT-TFs or genes (Fig. 29). Several studies investigated the role of Hippo pathway and have shown that the downstream effectors YAP and TAZ contribute to the invasive and metastatic capacity of melanoma cells [START_REF] Kim | The role of the hippo pathway in melanocytes and melanoma[END_REF][START_REF] Nallet-Staub | Pro-invasive activity of the Hippo pathway effectors YAP and TAZ in cutaneous melanoma[END_REF]. In Verfaillie et al., (2015) analysis of invasive transcriptomes and functional experiments suggest AP-1 and TEAD family members as new key regulators in the melanoma-invasive gene network for the first time; although both AP-1 and TEAD have been implicated in EMT, either separately or together before (Zhang et al., 2009;Zhao et al., 2014). Invasive melanoma cells appear to be functionally dependent on TEADs, and blocking the activity of this family of TFs increases the sensitivity of invasive cells to MAPK-targeted drugs. It is interesting to note that TEAD have been shown to interact with YAP/TAZ [START_REF] Davies | Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGF-beta1 involves MAPK, Smad and AP-1 signalling pathways[END_REF][START_REF] Lamar | The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain[END_REF][START_REF] Bakiri | Fra-1/AP-1 induces EMT in mammary epithelial cells by modulating Zeb1/2 and TGFbeta expression[END_REF][START_REF] Gao | Epithelial-to-mesenchymal transition induced by TGF-beta1 is mediated by AP1-dependent EpCAM expression in MCF-7 cells[END_REF][START_REF] Qiao | AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFalpha-induced epithelial-mesenchymal transition in triple-negative breast cancer[END_REF] and in this respect there might be some regulatory crosstalks between the transcription factors networks.

d. Metastatic spreading

As the melanoma progresses through its growth phases and acquires invasive properties, metastatic spreading requires tumor vascularization, cell entry into blood and lymphatic vessels (intravasation) and travel to distant organs to establish a secondary tumor. Appearance of metastasis drastically worsens the prognosis.

Factors involved in EMT and in the VGF of melanoma development continue to have crucial roles in the establishment of melanoma metastasis. AKT hyperactivation for instance was shown to promote overexpression of VEGF (vascular endothelial growth factor), a key regulator of angiogenesis, resulting in proliferation and reorganization of blood vessel epithelium [START_REF] Murukesh | Biomarkers of angiogenesis and their role in the development of VEGF inhibitors[END_REF].

Another PI3K target, PDK1 (phosphoinositide-dependent kinase-1) has recently been reported to promote melanoma development and to be crucial for metastasis development. PDK1 is a master serine/threonine protein kinase able to phosphorylate more than 20 members of the AGC kinase family (e.g. PKA and C, AKT, p70S6k). PDK1 inactivation or pharmacological inhibition in the BrafV600E::Pten -/-melanoma model delays melanoma development and inhibits metastasis [START_REF] Scortegagna | Genetic inactivation or pharmacological inhibition of Pdk1 delays development and inhibits metastasis of Braf(V600E)::Pten(-/-) melanoma[END_REF].

The scaffolding proteins GAB1 and GAB2 get phosphorylated by activated RTKs and recruit PI3-kinase (activation of AKT and mTOR) and SHP2 (RAS/ERK signaling). GAB2, one of the most important partner and substrate for SHP2, was shown to be amplified (11% of cases) and overexpressed (50% of cases) in melanoma [START_REF] Horst | Gab2-mediated signaling promotes melanoma metastasis[END_REF]. Furthermore, recent unpublished evidence from the Larue lab suggest existence of a regulatory loop involving GAB2, that is regulated by MITF, and the KIT receptor which signals through GAB to regulate MITF activity and melanoma cell motility.

Upstream regulator of the Rac-specific GTPases, guanine nucleotide exchange factor (GEF) PREX1 appear to play important roles in the melanoma metastasis process. It is elevated in the great majority of human melanoma cell lines as well as tumor tissue and P-Rex1 -/-mice are resistant to metastasis when crossed to a murine model of melanoma (Tyr::Nras Q61K/°; INK4a -/-) [START_REF] Lindsay | P-Rex1 is required for efficient melanoblast migration and melanoma metastasis[END_REF]. Of note, PREX2, another GEF, has been shown to be frequently mutated in melanoma [START_REF] Berger | Melanoma genome sequencing reveals frequent PREX2 mutations[END_REF].

D. Molecular model of melanoma progression

While it fits well with clinical observations, a weakness of the linear progression model we have just seen is the consideration that cancer progresses from a proliferative /weakly metastatic state to a strongly metastatic state through the accumulation of molecular changes (mutations and epigenetic changes) which increase the potential of cancer cells [START_REF] Miller | Melanoma[END_REF]. However, this model does not explain the persistent melanoma heterogeneity and self-renewal properties nor does it offer satisfactory explanations for persistent therapy failure.

An alternative to this model is the melanoma 'stem cell' model, in which melanoma stem-like cells drive progression towards a metastatic fate. A standardized definition considers a cancer stem cell as a cell within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor [START_REF] Clarke | Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells[END_REF]. According to this model, a small population of stem-like cells is solely responsible for tumorogenic potential and propagation of metastases. They also give rise to other cells, but these don't have sufficient replicative potential to drive sustained progression.

Several studies have shown the existence of subpopulations of melanoma cells with increased tumorigenic potential and expressing stem cell-associated proteins such as ABCB5 (Schatton et al., 2008), CD20 [START_REF] Fang | A tumorigenic subpopulation with stem cell properties in melanomas[END_REF], CD133 [START_REF] Monzani | Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential[END_REF], BMP [START_REF] Rothhammer | Epigenetic events in malignant melanoma[END_REF], Notch [START_REF] Balint | Activation of Notch1 signaling is required for beta-catenin-mediated human primary melanoma progression[END_REF], or the KIT stem cell markers (reviewed in Schatton and Frank, 2008;Zimmerer et al., 2013). Another study showed that at least 25% of tumor cells isolated from melanoma patients were able to establish new tumors [START_REF] Quintana | Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized[END_REF]. This model fits observed cancer cell heterogeneity and offers an explanation as to how cancer can seed and maintain tumours, and provide a reservoir of therapeutically resistant cells but doesn't explain metastatic progression of melanoma.

Further clarification came from phenotypical analysis carried out on 86 cultured melanoma cell lines showing that they could be divided into two distinct classes: highly proliferative and rather differentiated cell lines with weak invasive and metastatic potential as compared to slowly dividing cell lines with stem-cell like properties and much higher migratory and invasive properties. Gene expression profiling revealed that these two populations are defined by specific expression signatures [START_REF] Hoek | Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature[END_REF][START_REF] Hoek | In vivo switching of human melanoma cells between proliferative and invasive states[END_REF][START_REF] Widmer | Systematic classification of melanoma cells by phenotype-specific gene expression mapping[END_REF]. The invasive signature is characterised by upregulation of EMT-related ZEB1 as well as several TGFβ target genes involved negative regulation of Wnt signalling (WNT5a, DKK1, CTGF) and extracellular environment remodelling (INHBA,COL5A1,and SERPINE1). MITF has been shown to be one of the most prominent markers of the proliferative signature, being present and highly expressed in proliferative melanomas, but only expressed at low level or undetectable in the invasive cultures. In addition many of the proliferative signature genes are targets of Wnt signaling, SOX10 or PAX3 [START_REF] Hoek | In vivo switching of human melanoma cells between proliferative and invasive states[END_REF]Verfaillie et al., 2015). Furthermore, a recent study found specific DNA methylation signatures for the proliferative and invasive melanoma phenotypes [START_REF] Cheng | Methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognostic factor in advanced melanoma[END_REF].

Several lines of evidence have emerged supporting a so called 'phenotype switching model' predicting that the two melanoma cell subpopulations are only temporarily distinct and that these cells can reversibly and dynamically switch transcriptional programs between proliferative and invasive states in response to different cues including increased sensitivity to microenvironmental signals [START_REF] Quintana | Phenotypic heterogeneity among tumorigenic melanoma cells from patients that is reversible and not hierarchically organized[END_REF][START_REF] Roesch | A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth[END_REF]Hoek and Goding, 2010 and see below). Accordingly, one of the ground studies showed that when MITF high and MITF low cells are used to make xenografts, the resulting tumours comprise cell expressing both high and low MITF expression levels [START_REF] Hoek | In vivo switching of human melanoma cells between proliferative and invasive states[END_REF]. Hence, while the original cells were rather homogenous with respect to MITF expression, the resulting tumours are heterogeneous. [START_REF] Cheli | Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny[END_REF] demonstrated the direct implication of MITF in this switch: a low abundance subpopulation of cells that have strongly reduced MITF expression arise spontaneously in in vitro cultures of high-MITF expressing cell Depletion of these cells from the population leads to a strong reduction in tumour formation, whereas the purified low-MITF expressing population has high tumour formation potential and rapidly recovers higher MITF expression after subcutaneous injection. Furthermore transient inhibition of MITF increases the number of melanoma initiating cells likely by stimulating expression of stem cell factors such as OCT4 and NANOG.

Work from

These observations confirm that cells can dynamically switch their MITF expression and that low-MITF expressing cells have much higher tumour initiating capacity. Thus the level of functional MITF determines many biological properties of melanoma cells and can be represented by a so called 'rheostat model' (Carreira et al., 2006). High MITF levels lead to terminal differentiation and cell cycle exit, intermediate levels to proliferation while lower levels result in slow cycling invasive cells with stem cell like phenotype which have for instance tumor initiating properties. In contrast, very low levels of MITF or its rapid and sustained depletion such as is achieved in siRNA experiments has been shown to trigger cellular senescence and eventually death (Giuliano et al., 2010;Bertolotto, 2015).

Of note, this heterogeneity has recently been evaluated and confirmed in our lab by single-cell gene expression analysis [START_REF] Ennen | Single-cell gene expression signatures reveal melanoma cell heterogeneity[END_REF].

In order to identify the entire set of MITF target genes and elucidate the role of MITF in melanoma regulation, Strub et al. (2011) used first ChIP-seq and identified about 12.000 MITF binding sites genome-wide which were annotated to almost 5000 genes. Integration of the ChIP-seq data with RNA-seq data from siMITF knockdown cells identified a restricted set of genes likely directly activated or repressed by MITF. Thus MITF acts both as an activator and a repressor in a promoter-specific manner: Repressed targets (up-regulated upon siMITF) were associated with function in cell adhesion, motility and cancer signaling pathways (e.g. MCAM, SHC4, RaLP) suggesting they may contribute to changes in cell morphology and increased motility and invasiveness. Activated targets (down-regulated upon siMITF) are rather associated with cell cycle (CCNB1), mitosis (PLK1), pigmentation, DNA replication and repair (e.g. LIG1, BRCA1, FANCA, TERT) which explains why MITF silencing results in mitotic defects and proliferation arrest (Giuliano et al., 2010;Strub et al., 2011) Additional data suggest that MITF can accelerate melanoma growth by modulating TBX2, CDK2 and CDK4 gene expression [START_REF] Cronin | Frequent mutations in the MITF pathway in melanoma[END_REF][START_REF] Du | Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF[END_REF]Garraway et al., 2005;[START_REF] Hoek | In vivo switching of human melanoma cells between proliferative and invasive states[END_REF][START_REF] Vance | The transcription network regulating melanocyte development and melanoma[END_REF][START_REF] Wellbrock | Oncogenic BRAF regulates melanoma proliferation through the lineage specific factor MITF[END_REF].

This work comforted MITF as a master regulator of transcriptional events in melanoma and enabled to understand how the level of functional MITF can drive phenotype switch in primary tumors between proliferative to invasive and in metastasis from invasive back to proliferative, and why loss of MITF leads to cell cycle arrest, mitotic defects and entry into senescence providing a molecular basis for the 'rheostat' model.

Altogether this favours a model in which cell state transition is dependent on reprogramming of the transcriptome rather than being dictated by the acquisition of specific DNA mutations, although specific genetic lesions may actually render melanoma cells susceptible to such reprogramming as was shown recently for Braf (Caramel et al., 2013). Understanding of the molecular mechanisms underpinning phenotype-switching in melanoma, and in particular the signalling events that regulate the expression of Mitf, points to new therapeutic approaches aimed at eradicating therapeutically resistant cells with high metastatic potential.

The role of MITF in melanoma is important, since it acts as a lineage survival oncogene (Garraway et al., 2005;McGill et al., 2002). Genomic amplifications of the MITF locus have been found in about 10-20% of the primary and metastatic tumors while mutations of MITF are relatively rare and happen in only 8 % of the melanoma samples analyzed. [START_REF] Bishop | Genotype/phenotype and penetrance studies in melanoma families with germline CDKN2A mutations[END_REF][START_REF] Gast | Somatic alterations in the melanoma genome: a high-resolution array-based comparative genomic hybridization study[END_REF]Garraway et al., 2005, Garraway and[START_REF] Garraway | Lineage dependency and lineage-survival oncogenes in human cancer[END_REF][START_REF] Levy | MITF: master regulator of melanocyte development and melanoma oncogene[END_REF][START_REF] Cronin | Frequent mutations in the MITF pathway in melanoma[END_REF][START_REF] Bertolotto | A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma[END_REF], Until recently only acquired mutations were known, such mutations affect mostly conserved regions encoding functional domains of the protein [START_REF] Grill | MITF mutations associated with pigment deficiency syndromes and melanoma have different effects on protein function[END_REF].

Interestingly, forced co-expression of BRAFV600E and MITF was shown to be able to transform melanocytes into melanoma [START_REF] Dynek | Microphthalmiaassociated transcription factor is a critical transcriptional regulator of melanoma inhibitor of apoptosis in melanomas[END_REF]Garraway et al., 2005). However BrafV600E in immortalized mouse and human melanocytes results in lower Mitf levels [START_REF] Wellbrock | Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation[END_REF] as it triggers phosphorylation dependant degradation targeting of Mitf. Tests with MEK inhibitors revealed that this process is MEK dependant [START_REF] Wellbrock | Oncogenic BRAF regulates melanoma proliferation through the lineage specific factor MITF[END_REF].

The groups of [START_REF] Bertolotto | A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma[END_REF] and [START_REF] Yokoyama | A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma[END_REF] independently identified a heterozygous germline missense substitution (E318K) in human patients. (See B.2.b). This mutation has been associated with a strong family history of cutaneous melanoma, increased nevus count and multiple primary melanomas; and carriers have a significantly increased risk of developing melanoma (5-fold) and renal cell carcinoma than non-carriers [START_REF] Bertolotto | A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma[END_REF][START_REF] Yokoyama | A novel recurrent mutation in MITF predisposes to familial and sporadic melanoma[END_REF].

Although melanomas with BRAF mutations have constitutively active growth signals, they exhibit diminished oxidative enzymes, mitochondrial number, and increased production of lactate. Oxidative phosphorylation depends on the ability of functionally intact mitochondria to metabolize oxygen, whereas glycolysis can occur independently of mitochondria. A metabolic switch from oxidative metabolism to glycolysis facilitates the uptake and incorporation of nutrients required for cellular proliferation especially in the setting of limited nutrient availability.

PGC-1α (peroxisome proliferator-activated receptor g coactivator 1α) is a wellknown regulator of mitochondrial biogenesis and oxidative phosphorylation. A recent study showed that MITF is a direct and essential mediator of BRAF in the regulation of mitochondrial respiration as the metabolic reprogramming from oxidative metabolism to glycolysis triggered by BRAF V600E is accompanied by a suppression of MITF and downstream PGC1α (Haq et al., 2013;[START_REF] Vazquez | PGC1α expression defines a subset of human melanoma tumors with increased mitochondrial capacity and resistance to oxidative stress[END_REF])

E. Risk factors

Several risk factors can make a person more likely to develop melanoma and include both intrinsic (genetic and phenotype) and extrinsic (environmental or exposure) factors (Fig. 30).

Exposure to UV-radiation is the main environmental hazard and a major risk factor for most melanomas as they induce direct damage to DNA. Sunlight is the main source of UV rays and other sources include tanning beds and sun lamps and imply a dose-dependent risk increase. Strikingly, UV "signature" mutations (pyrimidine dimers) in genes relevant to melanoma such as BRAF or CDKN2A seem to be much less frequent than in non-melanoma skin cancers although it is plausible that they could still arise from errorprone replication of UV-damaged DNA [START_REF] Gray-Schopfer | Cellular senescence in naevi and immortalisation in melanoma: a role for p16?[END_REF]. Only recent work based on next-generation sequencing identified hot spot mutations in RAC1, STK19, and PPP6C resulting from C > T transitions and provided first evidence of the direct mutagenic role of UV light in melanoma pathogenesis [START_REF] Hodis | A landscape of driver mutations in melanoma[END_REF][START_REF] Krauthammer | Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma[END_REF]. Another recent report shows that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model induces a neutrophilic inflammatory response that promotes metastatic progression, independent of its tumour-initiating effects [START_REF] Bald | Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma[END_REF].

Various drugs such as psoralens can increase UV-induced damage significantly increasing melanoma risk. A possible association of melanoma exists with ionizing radiation, as well as chemicals and pollutants such as arsenic [START_REF] Rigel | Epidemiology of melanoma[END_REF].

In addition, like other cancer types, there is evidence that melanoma development can be favoured by immune suppression [START_REF] Vajdic | Cutaneous melanoma is related to immune suppression in kidney transplant recipients[END_REF][START_REF] Kubica | Melanoma in immunosuppressed patients[END_REF].

An increased risk of melanoma has long been associated with characteristics of low skin phototype (see Fig. 7 and30). Subjects with red or blond hair, blue eyes or fair skin that freckles or burns easily have higher melanoma risk than subjects with brown hair and skin that tan easily [START_REF] Bataille | Melanoma--Part 1: epidemiology, risk factors, and prevention[END_REF][START_REF] Hearing | Determination of melanin synthetic pathways[END_REF]. The greatest risk is among patients with red hair and fair skin as they produce no or only little UVprotective eumelanin [START_REF] Rigel | Epidemiology of melanoma[END_REF].

Presence of dysplastic nevi is marker for increased melanoma risk [START_REF] Rigel | Epidemiology of melanoma[END_REF]. Subjects with large congenital nevi or bearing more than 50 nevi exhibit higher risk of developing melanoma [START_REF] Krengel | Melanoma risk in congenital melanocytic naevi: a systematic review[END_REF]. Changing skin lesions, most commonly increase in size, are also associated with melanoma risk [START_REF] Negin | Symptoms and signs of primary melanoma: important indicators of Breslow depth[END_REF]. Having a prior skin cancer, of either melanoma or non-melanoma type increases risk to develop a melanoma within five years [START_REF] Bradford | Increased risk of second primary cancers after a diagnosis of melanoma[END_REF][START_REF] Hemminki | Skin cancer and non-Hodgkin's lymphoma as second malignancies. markers of impaired immune function?[END_REF]Zhang et al., 2008).

Significant progress has been made toward understanding the genes that contribute to inherited susceptibility for melanoma in some patients. About 10% of melanoma patient present a family history of melanoma, defined by at least two melanomas within two or more members of the same family. Two major susceptibility genes have long been associated with increased risk to develop melanoma with high penetrance (i.e. high lifetime risk): CDKN2A and to a lesser extent CDK4 [START_REF] Ceha | Several noncontiguous domains of CDK4 are involved in binding to the P16 tumor suppressor protein[END_REF][START_REF] Zuo | Germline mutations in the p16INK4a binding domain of CDK4 in familial melanoma[END_REF][START_REF] Soufir | Prevalence of p16 and CDK4 germline mutations in 48 melanoma-prone families in France. The French Familial Melanoma Study Group[END_REF]. Interestingly, both proteins are potent tumor suppressors with distinct but equally crucial roles in cell cycle and apoptosis regulation. CDKN2A mutations are involved in about 30% of familial melanoma cases and 30% of sporadic cases [START_REF] Orlow | CDKN2A germline mutations in individuals with cutaneous malignant melanoma[END_REF]. CDK4 encodes the catalytic subunit of a heterodimeric Ser/Thr protein kinase, which together with its regulatory subunit is involved in controlling progression through the G1 phase of the cell cycle.

Multiple GWAS have also yielded several risk-associated single-nucleotide polymorphisms (SNPs) across the genome (Bressac- [START_REF] Bressac-De-Paillerets | Genetic and environmental factors in cutaneous malignant melanoma[END_REF][START_REF] Chatzinasiou | Comprehensive field synopsis and systematic meta-analyses of genetic association studies in cutaneous melanoma[END_REF]. The MC1R gene belongs to the most understood; as it is the determinant of susceptible phenotypes such as red hair, sun sensitivity, and freckles [START_REF] Kennedy | Melanocortin 1 receptor (MC1R) gene variants are associated with an increased risk for cutaneous melanoma which is largely independent of skin type and hair color[END_REF]. Carriers of MC1R variants such as V60L, R151C, R160W or D294H have a 2-4-fold increased risk of developing melanoma [START_REF] Udayakumar | Moderate-to low-risk variant alleles of cutaneous malignancies and nevi: lessons from genome-wide association studies[END_REF][START_REF] Fargnoli | MC1R variants increase melanoma risk in families with CDKN2A mutations: a meta-analysis[END_REF].

Other genes involved in pigmentation (TYR, TYRP1, ASIP, MATP, IRF4), cell growth, DNA repair (see below) and integrity (TERT), metabolism (MTAP, PLA2G6) or apoptosis (CASP8) have also more recently been identified as melanoma susceptibility genes [START_REF] Daniotti | BRAF alterations are associated with complex mutational profiles in malignant melanoma[END_REF][START_REF] Gudbjartsson | ASIP and TYR pigmentation variants associate with cutaneous melanoma and basal cell carcinoma[END_REF][START_REF] Kvaskoff | Polymorphisms in nevus-associated genes MTAP, PLA2G6, and IRF4 and the risk of invasive cutaneous melanoma[END_REF][START_REF] Horn | TERT promoter mutations in familial and sporadic melanoma[END_REF][START_REF] Huang | Highly recurrent TERT promoter mutations in human melanoma[END_REF][START_REF] Praetorius | A polymorphism in IRF4 affects human pigmentation through a tyrosinase-dependent MITF/TFAP2A pathway[END_REF].

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease that affects ability of skin cells to perform nucleotide excision repair and thus impairs the body's response to UV-induced damage. People with XP display a high risk of developing melanoma and other skin cancers in childhood, especially on sun-exposed areas of their skin [START_REF] Lehmann | Xeroderma pigmentosum[END_REF][START_REF] Budden | The role of altered nucleotide excision repair and UVBinduced DNA damage in melanomagenesis[END_REF]. Up to 20% of individuals with an XPC mutation are diagnosed with cutaneous melanoma in their lifetime [START_REF] Kraemer | Xeroderma Pigmentosum[END_REF].

F. Therapeutic approaches

Metastatic melanoma has very poor prognosis. Although over the last decade, substantial advances have been made in the understanding of melanoma molecular biology (development, progression, resistance to medical therapy), there are extensive cross-talk between the different signaling pathways involved in melanomagenesis, which complicates targeting them with drugs and all treatments developed to date for metastatic melanoma failed to be successful in the long run. This section will review the main therapeutic approaches available to treat melanoma.

Early Surgery that removes radial growth phase tumours that have not yet initiated vertical growth phase can be very effective and prevent further development. A wide local excision is used to remove the melanoma and some of the normal tissue around it. The prognosis worsens the deeper the lesion extends beneath the skin and average survival drops to 6 months once melanoma started spreading due to high resistance of metastasis to current therapeutic approaches.

Melanoma frequently spreads to the brain. If the spread is limited to one or a very limited number of spots within the brain, surgery may be indicated to remove the tumor. However, if the tumor is in a location in the brain that cannot be easily removed, or if there are several tumors, radiation therapy may be useful to shrink the tumors and prevent the development of additional tumors.

There are three main categories of drug treatments:

• Chemotherapy: drugs that stop or slow the growth of cancer cells by interfering with their ability to divide or reproduce themselves • Immunotherapy: drugs that stimulate or unleash the immune system to attack and kill the cancer cells • Targeted therapy: drugs that inhibit specific enzymes or molecules important to the cancer cells Cytotoxic chemotherapy has been used for the treatment of metastatic melanoma for over 3 decades. The way the chemotherapy is given depends on the type and stage of the cancer being treated. Because most of an adult's normal cells are not actively growing, they are not affected by chemotherapy, with the exception of bone marrow, the hair, and the gastrointestinal tract. Common Chemotherapeutic agents include alkylating agents (dacarbazine, temozolomide, fotemustin, nitrosoureas), platinum analogues and micro tubular toxins.

While they may have a palliative benefit in some patients, these drugs have modest antitumor efficacy in metastatic melanoma and have not been proven to have a survival benefit overall (Tsao et al., 2004).

Melanoma is considered to be highly immunogenic and responsive to immunotherapy. Some immunotherapeutic approaches have led to durable complete responses in a small subset of patients, although it has been challenging to predict which patients will respond to immunotherapy. Different types of immunotherapeutic approaches are investigated including adoptive T cell transfer, blocking antibodies against inhibitory immune molecules, stimulatory signals or antibodies for immune cells, and immunization with cancer cell antigens some of which are being used in the treatment of melanoma (IL-2, IFN-α2b, TNF)

The concept of immune checkpoint inhibitor therapy is based on following evidence: some types of immune cells, such as T cells, present checkpoint proteins on their surface that keep immune responses in check. Cancer cells hijack this system and express large amounts of these proteins, in order to prevent the proliferation, survival and effector functions of the activated T-cells, resulting in limited duration of the immune response.

Corresponding immunotherapy consists in the use of immune checkpoint inhibitors to block these proteins and restore the ability of T cells to kill cancer cells. Recent characterization of mechanisms regarding cancer cell evasion of the immune detection have been characterized and led to the development of two major immune checkpoint inhibitors (Fig. 31):

CTLA4 (cytotoxic T-lymphocyte-associated antigen-4) is an inhibitory molecule expressed on T cells that is involved in the negative regulation of the T cell interaction with antigen presenting dendritic cells (binding of CD28 on T cells to B7 proteins on APC, thus weakening the costimulation of T cells). Cancer cells express the B7 proteins (CTLA-4 ligands) to look like APCs and escape T-cell targeting. CTLA-4 inhibitors such as Ipilimumab, a monoclonal antibody, bind to CTLA-4 and allow the T cells to kill the cancer cell. PD-1 (programed cell death protein 1), like CTLA-4, is an inhibitory receptor; however, its expression is not limited to T cells and is found in B cells and some myeloid cells. PD-1 is a protein on the surface of T cells that helps keep the body's immune responses in check. PD-1 ligand PD-L1 is expressed by cancer cells and binds the cognate T-cell receptor, enabling them to escape from host immune system detection and degradation. Monoclonal antibodies targeting PD-1 and PD-L1 (e.g. pembrolizumab and nivolumab) were shown to enhance T-cell expansion, cytokine production and cytolytic functions. Identification and characterization of the different signalling pathway alterations driving melanoma initiation and progression have led to the development of targeted therapies, especially against the different mutated kinases involved (Fig. 32) BRAF is the most frequently mutated kinase in human melanomas and tumor cells appear to become addicted to the resulting MAPK pathway hyperactivation (oncogene addiction).

While large spectrum kinase inhibitors have shown poor efficiency [START_REF] Eisen | Sorafenib in advanced melanoma: a Phase II randomised discontinuation trial analysis[END_REF][START_REF] Hauschild | Results of a phase III, randomized, placebo-controlled study of sorafenib in combination with carboplatin and paclitaxel as second-line treatment in patients with unresectable stage III or stage IV melanoma[END_REF], several specific small molecule inhibitors including vemurafenib (BRAFi), dabrafenib (BRAFi), and trametinib (MEKi) have been developed to inhibit MAPK pathway components (Fig. 32). Treatment of BRAF-mutant melanomas with Vemurafenib was shown to induce detectable reduction of tumor size, and prolonge the lifetime of patients to an average of 6 months [START_REF] Chapman | Improved survival with vemurafenib in melanoma with BRAF V600E mutation[END_REF]. All patients, including the ones with initial strong and full response eventually developed fast and aggressive resistance [START_REF] Johannessen | COT drives resistance to RAF inhibition through MAP kinase pathway reactivation[END_REF][START_REF] Nazarian | Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation[END_REF][START_REF] Sullivan | Resistance to BRAF-targeted therapy in melanoma[END_REF]. Dabrafenib had similar response rates to treatment as vemurafenib, but it showed less severe adverse effects and was shown to be effective in treatment of melanoma brain metastasis [START_REF] Shah | Latest advances in chemotherapeutic, targeted, and immune approaches in the treatment of metastatic melanoma[END_REF]. Trametinib is a potent and highly sensitive MEK1/2 inhibitor and was shown to have better effect on the overall survival that chemotherapy (Flaherty et al., 2012a). Finally a combined therapy of BRAF inhibitors with MEK inhibitors has been shown to be more effective and no more toxic than either of the inhibitors alone (Flaherty et al., 2012b).

As mentioned earlier, KIT is mutated in 2% of melanoma (Fig. 25) and a significant subset of melanomas, mainly mucosal and acral melanomas harbour an increase in copy number and/or activating mutations in KIT. This led to intense search for small molecule inhibitors targeting mutated KIT and downstream effectors. Interestingly, activating KIT mutations described in melanomas overlap with those reported for gastrointestinal stromal tumors, a disease for which imatinib mesylate has been used with good results. Further studies have shown that imagine inhibits the pathways downstream of KIT, suppresses proliferation and induces apoptosis of melanoma cells with hyper activated KIT [START_REF] Jiang | Imatinib targeting of KIT-mutant oncoprotein in melanoma[END_REF]. Unfortunately, recent phase II trials produced only modest responses to imatinib treatment [START_REF] Antonescu | L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition[END_REF][START_REF] Hodi | Major response to imatinib mesylate in KIT-mutated melanoma[END_REF][START_REF] Lutzky | Dose-dependent, complete response to imatinib of a metastatic mucosal melanoma with a K642E KIT mutation[END_REF] except for specific cases [START_REF] Vidwans | A melanoma molecular disease model[END_REF]. Poor response and high toxicity was also observed in patients treated with dasatinib inhibitor that targets the Src kinase immediately downstream of KIT [START_REF] Antonescu | L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition[END_REF][START_REF] Kluger | A phase 2 trial of dasatinib in advanced melanoma[END_REF]. Other RTK inhibitors (e.g., sunitinib, nilotinib) are being tested in KIT-mutated melanomas with hopes of being more efficient [START_REF] Antonescu | L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition[END_REF]. It is also conceivable that future trials will include combinations of KIT inhibitors with other targeted drugs and immunotherapy. [START_REF] Carvajal | KIT as a therapeutic target in metastatic melanoma[END_REF] Recent melanoma research has focussed on the molecular characterization of signal processes underlying melanoma progression and heterogeneity in order to understand current therapy failures and develop alternative treatment strategies. While targeted therapies turned out to be very efficient and show exceptional results in clinics (Yang et al., 2011) average patient survival doesn't exceed few month as resistant tumors may arise under selective pressure of therapy from pre-existing resistant subclones and/or as a result of an evolutionary process during treatment selecting cells with additional driver mutations such as NRAS or CRAF in BRAF mutates tumors. The second main reason for such a high mortality rate might be the resistance of invasive phenotype cells to any of the available single or combination drug treatments [START_REF] Eggermont | New drugs in melanoma: it's a whole new world[END_REF]. These cells have stem cell like properties (see C.4.) and seem to form distant metastasis by a phenotype switch, which gives a rationale for the resistance to available therapies. Genotoxic drugs and radiotherapy are not suitable to target slow-cycling cell that will be able to generate proliferative tumors as soon as treatment stops. Chapter III. Epigenetic regulation by the SWI/SNF remodelling complex A. Chromatin structure and organization Chromatin was first defined by Flemming in 1882 as the 'substance in the cell nucleus which is readily stained' that is retractile to digestion'. Today 'chromatin' usually refers to DNA but also the associated proteins as over the past two decades, it has become clear that these associated proteins play a key role in the nuclear programm including transcription, replication and recombination of the DNA.

The basic structural component of chromatin is the nucleosome [START_REF] Kornberg | Chromatin structure: a repeating unit of histones and DNA[END_REF][START_REF] Oudet | Electron microscopic and biochemical evidence that chromatin structure is a repeating unit[END_REF] and has been shown to be dynamic and regulated at several levels.

The nucleosome (Fig. 33) consists of 146 base pairs of DNA wrapped around two molecules of the four core histone protein (H2A, H2B, H3 and H4) and a linker histone H1 which connects adjacent nucleosomes through physically binding to linker DNA located between histone beads enhancing the level of compaction [START_REF] Zhou | Position and orientation of the globular domain of linker histone H5 on the nucleosome[END_REF][START_REF] Hayes | Nucleosomes and the chromatin fiber[END_REF][START_REF] Wolffe | Transcriptional regulation in the context of chromatin structure[END_REF]. Each histone forms a structure consisting of a three-helix domain termed the histone fold. These domains form "handshake" structure connecting the histone heterodimers H2A-H2B and H3-H4 [START_REF] Arents | The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein assembly and a left-handed superhelix[END_REF][START_REF] Harp | Asymmetries in the nucleosome core particle at 2.5 A resolution[END_REF][START_REF] Khorasanizadeh | The nucleosome: from genomic organization to genomic regulation[END_REF][START_REF] Zlatanova | The nucleosome family: dynamic and growing[END_REF].

H3 has a unique role in the nucleosome, since a 2-fold symmetry in the nucleosome is set directly along the dimer interface of the two H3 histones. H3 forms heterodimers with H4, but it also forms direct contacts with H2A. The H2A-H2Bd dimers are positioned so the two H2A proteins interact with each other. In addition to structured histone fold core, each histone forms extensions protruding out from the nucleosome consisted of disordered N-terminal and/or C-terminal histone tails.

The level on chromatin compaction affects DNA accessibility and thus determines the functional output of the related biological processes. Luckily, nucleosomes can be evicted or shifted in position by ATP-dependent remodelling proteins, opening up DNA sequences. The histone tails show no secondary structures but can be subjected to post-translational modification and get covalently modified in different ways.

B. Chromatin modification and transcription regulation

Thus in order for gene transcription to take place highly compacted chromatin must be converted into a relaxed, exposed state. There are two major classes of enzymes that cooperate to remodel chromatin: ATP-dependent remodelling complexes and enzymes that post-translationally and covalently modify chromatin structure.

Histone modifications alter the DNA-histone interactions, thereby promoting changes in nucleosome structure and conformation and thus making the DNA more accessible [START_REF] Bannister | Regulation of chromatin by histone modifications[END_REF].

Many histone modifications have been described and include acetylation, methylation, phosphorylation, ubiquitylation, SUMOylation, ADP-ribosylation, deamination, glycosylation [START_REF] Martin | The diverse functions of histone lysine methylation[END_REF][START_REF] Bannister | Regulation of chromatin by histone modifications[END_REF][START_REF] Greer | Histone methylation: a dynamic mark in health, disease and inheritance[END_REF][START_REF] Kouzarides | Chromatin modifications and their function[END_REF][START_REF] Oki | Role of histone phosphorylation in chromatin dynamics and its implications in diseases[END_REF]. These modifications are catalyzed by a large group of enzymes such as histone acetyl-transferases (HATs), histone deacetylases (HDACs), kinases, methyltransferase and demethylase [START_REF] Jenuwein | Translating the histone code[END_REF][START_REF] Tamaru | Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa[END_REF][START_REF] Cuthbert | Histone deimination antagonizes arginine methylation[END_REF].

Common histone tail modifications include the methylation on lysine 4 of H3 (H3K4) or the acetylation on lysine 12 of H4 (H4K12) which serve as active or repressive marks on the chromatin. Methylation of H3K4, H3K20, and acetylation of H4 (H4K5, K8, K12, K16) serve as active marks of transcription [START_REF] Chicoine | A single histone acetyltransferase from Tetrahymena macronuclei catalyzes deposition-related acetylation of free histones and transcription-related acetylation of nucleosomal histones[END_REF][START_REF] Li | The role of chromatin during transcription[END_REF][START_REF] Parthun | Hat1: the emerging cellular roles of a type B histone acetyltransferase[END_REF][START_REF] Ruthenburg | Methylation of lysine 4 on histone H3: intricacy of writing and reading a single epigenetic mark[END_REF][START_REF] Sobel | Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4[END_REF] while methylation of H3 on lysine 9 and 27 serve as major marks of repression of transcription [START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF][START_REF] Stewart | Relationship between histone H3 lysine 9 methylation, transcription repression, and heterochromatin protein 1 recruitment[END_REF][START_REF] Hansen | A model for transmission of the H3K27me3 epigenetic mark[END_REF][START_REF] Kim | Recruitment and biological consequences of histone modification of H3K27me3 and H3K9me3[END_REF].

Interestingly, recent studies have highlighted a role for 'chromatin writers' such as the histone methyltransferases SETDB1 and EZH2, which catalyze methylation of lysine 9 and 27 in histone H3, respectively. SETDB1 is amplified in human melanoma and its expression accelerates melanoma development in a zebrafish BRAFV600E model system [START_REF] Ceol | The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset[END_REF] Similarly, EZH2 expression is elevated and associated with poor survival in melanoma, and its conditional ablation inhibits tumor growth and metastases in a melanoma mouse model [START_REF] Zingg | The epigenetic modifier EZH2 controls melanoma growth and metastasis through silencing of distinct tumour suppressors[END_REF].

DNA methylation is the single known modification of DNA and has a profound impact on genome stability, transcription and development. In prokaryotes, DNA methylation occurs on adenin and cytosine bases and serves as a regulator of DNA replication and mismatch repair [START_REF] Noyer-Weidner | Methylation of DNA in prokaryotes[END_REF]. In eukaryotes, DNA methylation only occurs on cytosines in a CpG context with exception of the enriched CpG islands [START_REF] Bird | CpG-rich islands and the function of DNA methylation[END_REF] and is catalysed by Dnmt1, Dnmt3a ET Dnmt3b [START_REF] Klose | Genomic DNA methylation: the mark and its mediators[END_REF]. Further modification to 5-hydroxymethylcytosine by TET enzymes provides a key intermediate in active demethylation pathways that can either be passively depleted through DNA replication or actively reverted to cytosine through iterative oxidation and thymine DNA glycosylase-mediated base excision repair [START_REF] Kohli | TET enzymes, TDG and the dynamics of DNA demethylation[END_REF]. Noteworthy, more than 50 genes have been shown to be silenced through epigenetic changes during melanoma development, progression, and metastasis, mainly by promoter CpG island hypermethylation [START_REF] Rothhammer | Epigenetic events in malignant melanoma[END_REF][START_REF] Howell | Epigenetics in human melanoma[END_REF]. The canonical histones ensure nucleosome integrity and regulate access to DNA, but along the canonical histone molecules, a set of specialized histone variants has evolved to regulate various cellular processes [START_REF] Weber | Histone variants: dynamic punctuation in transcription[END_REF]. Histone variants are important regulatory molecules in cancer [START_REF] Vardabasso | Histone variants: emerging players in cancer biology[END_REF] and Histones H1, H2A and H3 have been shown to have multiple variants [START_REF] Yuan | Histone variants and epigenetic inheritance[END_REF].

H2A variants are the most diverse. H2A.Z arises in early eukaryotic evolution is expressed ubiquitously, and is distinct from H2A [START_REF] Talbert | Histone variants--ancient wrap artists of the epigenome[END_REF]. Even though H2A and H2A.Z share only 60% of the amino-acid sequence, the nucleosomes comprising these two variants are very similar, but have key structural differences [START_REF] Suto | Crystal structure of a nucleosome core particle containing the variant histone H2A.Z[END_REF]. On the surface H2A.Z has an extended acidic patch, stimulating remodelling activity by ISWI class of chromatin remodelers [START_REF] Goldman | Chromatin remodeling by imitation switch (ISWI) class ATP-dependent remodelers is stimulated by histone variant H2A.Z[END_REF]. It was also reported that H2A.Z regulates nucleosome positioning around promoters and thus influence accessibility of cis-regulatory regions [START_REF] Guillemette | Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning[END_REF][START_REF] Marques | Reconciling the positive and negative roles of histone H2A.Z in gene transcription[END_REF].

Importantly, [START_REF] Kapoor | The histone variant macroH2A suppresses melanoma progression through regulation of CDK8[END_REF] recently identified a global loss of macroH2A isoforms in malignant melanoma, both in cells and cohorts of human tissue samples. They propose that this loss contributes to melanoma progression affects both gene expression programs and chromatin structure. To support this, they show that CDK8 is a macroH2AZ targets upregulated upon its loss and that CDK8 likely promotes tumor development. Another recent study confirmed these observations [START_REF] Lei | MacroH2A suppresses the proliferation of the B16 melanoma cell line[END_REF] Similarly, [START_REF] Vardabasso | Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma[END_REF] have found that histone variant H2A.Z.2 is highly expressed in metastatic melanoma and is associated with poor prognosis. They have shown that the expression of E2F1 and E2F4 target genes, which include many cell cycle regulators, is affected by H2A.Z.2. The genes appear to exhibit a specific H2A.Z.2 'occupancy signature' that is enriched at the promoter region and depleted in the gene body. Interestingly, H2A.Z.2-regulated genes also appear to be BRD2 targets and knocking down H2A.Z.2 sensitizes melanoma cells to both BET and MEK inhibitors, which are potential therapies for melanoma.

C. ATP-dependant chromatin remodelling

The second class of chromatin remodelers corresponds to ATP-dependent chromatin remodelling machines [START_REF] Becker | ATP-dependent nucleosome remodeling[END_REF]. They function by utilizing energy from ATP hydrolysis and disrupt contacts between histone proteins and DNA resulting in an altered nucleosome structure [START_REF] Kingston | ATP-dependent remodeling and acetylation as regulators of chromatin fluidity[END_REF][START_REF] Wang | The SWI/SNF family of ATP-dependent chromatin remodelers: similar mechanisms for diverse functions[END_REF].

All the remodeler contain an ATPase subunit with Dexx and HELICc domains, which are involved in DNA binding and ATP hydrolysis. Based on the nature of additional functional domains in the catalytic ATPase subunit, four subclasses of such remodelers can be distinguished (Fig. 34): SWI/SNF, ISWI (Imitation SWI), Mi-2/CHD (Chromodomain, helicase DNA binding) and INO80 (inositol requiring 80) [START_REF] Boyer | The SANT domain: a unique histone-tailbinding module?[END_REF][START_REF] Eberharter | ATP-dependent nucleosome remodelling: factors and functions[END_REF][START_REF] Marfella | The Chd family of chromatin remodelers[END_REF][START_REF] Sif | ATP-dependent nucleosome remodeling complexes: enzymes tailored to deal with chromatin[END_REF].

The INO80 (INO80, swr1, p400/Tip60) family

The INO80 (Inositol requiring 80) family remodelers include more than 10 subunits and were initially purified from S. cerevisiae. INO80 has been shown to enhance transcriptional activation and play a role in DNA repair [START_REF] Downs | Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites[END_REF]Ebbert, et al., 1998;[START_REF] Flaus | Identification of multiple distinct Snf2 subfamilies with conserved structural motifs[END_REF]. In humans, the INO80 family includes INO80, SRCAP (SNF2related CREB-activator protein), and TRRAP/Tip60, with p400 that has both ATPase and HAT activity [START_REF] Ikura | Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis[END_REF][START_REF] Bao | Chromatin remodeling in DNA double-strand break repair[END_REF]Shen 2007, Clapier and[START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF].

A characteristic feature of INO80 members is the presence of an extended insertion within the ATPase domain due to the presence of a spacer region and hence is referred to as the split ATPase domain. This characteristic domain retains ATPase activity and also acts as a scaffold protein.

Another unique aspect of this complex is that it exhibits DNA helicase activity and has an affinity for H2A variants, H2AZ and H2AX. Thus the complex plays an important role I transcription regulation but also in DNA repair where it mediates nucleosome eviction at double strand breaks and also promotes homologous recombination repair [START_REF] Peterson | Cellular machineries for chromosomal DNA repair[END_REF][START_REF] Sarkar | The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair[END_REF][START_REF] Watanabe | The INO80 family of chromatin-remodeling enzymes: regulators of histone variant dynamics[END_REF][START_REF] Kobor | A protein complex containing the conserved Swi2/Snf2related ATPase Swr1p deposits histone variant H2A.Z into euchromatin[END_REF][START_REF] Papamichos-Chronakis | Global regulation of H2A.Z localization by the INO80 chromatin-remodeling enzyme is essential for genome integrity[END_REF]Wang et al., 2014.).

The CHD-Mi2 family

The CHD (chromodomain, helicase, DNA binding) family remodelers consist in 9 CHD proteins (CHD1-9) subdivided into three subfamilies with distinct structures and domain composition (Plant Homeo Domains (PHD), BRM and Kismet domains (BRK), and SANT domains) [START_REF] Hall | CHD proteins: a diverse family with strong ties[END_REF]Sims, et al., 2005;[START_REF] Marfella | The Chd family of chromatin remodelers[END_REF]Murawska and Brehm 2011). These factors interact with methylated histone tails through their two chromodomains [START_REF] Bannister | Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain[END_REF][START_REF] Flanagan | Double chromodomains cooperate to recognize the methylated histone H3 tail[END_REF][START_REF] Lachner | Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins[END_REF]. The functions of the CHD family members seem to be very diverse : nucleosome sliding or emitting by CHD remodelers can result in enhanced transcription of genes or have repressive roles depending of type CHD protein and associated subunits.

The ISWI family

The ISWI (imitation switch) family remodelers include NURF (nucleosome remodeling factor), CHRAC, RSF and ACF complexes and were initially identified and purified from Drosophila embryo extracts. The ISWI family members are characterized by the presence of SLIDE and SANT domain which helps in the preferential interaction with nucleosomes containing linker DNA over core nucleosomes (Längst et al., 1999).

ISWI complexes have one of the two subunits Snf2h and Snf2l [START_REF] He | Diverse regulation of SNF2h chromatin remodeling by noncatalytic subunits[END_REF]Längst et al., 1999). These complexes have been implicated in regulation of several functions such as heterochromatin formation, DNA replication, transcriptional repression as well as in embryonic stem (ES) pluripotency. In human cells, ATPase SNF2L is ubiquitously expressed in different human tissues. Silencing of SNF2L in HeLa cells led to increase in proliferation and migration. Transcriptome profiling showed SNF2L was involved in modulation of the Wnt signaling pathway regulatory network. SNF2L is expressed at high levels in normal melanocytes, while its expression level is very low or undetectable in melanoma (Eckey et al., 2012).

D. The SWI/SNF chromatin remodelling complex family

The SWI/SNF family was the first chromatin remodelling complex to be identified and is conserved from yeast to humans. They use ATP to alter nucleosome structure and facilitate DNA-binding of transcriptional activators at the promoter regions of actively transcribed genes [START_REF] Vignali | ATP-dependent chromatinremodeling complexes[END_REF]de la Serna et al., 2006). Founding members were identified in yeast screens for mutants affecting mating-type switching (SWI) and sucrose fermentation ("Sucrose Non-Fermenting", SNF) [START_REF] Breeden | Cell cycle control of the yeast HO gene: cis-and trans-acting regulators[END_REF][START_REF] Neigeborn | Genes affecting the regulation of SUC2 gene expression by glucose repression in Saccharomyces cerevisiae[END_REF][START_REF] Stern | Five SWI genes are required for expression of the HO gene in yeast[END_REF]. These mutants were affecting the HO endonuclease (for mate type switching) or the SUC2 gene coding an invertase that hydrolyzes sucrose. SWI/SNF complexes in drosophila include BAP (Brahma Associated Protein) and PBAP (Polybromo-associated BAP) complexes. In human, these two complexes are called BAF (Brg1 Associated Factors) and PBAF (Polybromo-associated BAF).

From an evolutionary point of view, SWI/SNF are multi subunit complexes with an ATPase subunit consisting in Snf2 or Sth1 (RSC complex) in yeast, brahma (BRM) in drosophila, and either BRM and brahma-related 1 (BRG1) in mammals. The ATPase subunit of the SWI/SNF family of proteins has a bromodomain, which preferentially binds acetylated histones [START_REF] Muchardt | A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor[END_REF].

E. The mammalian BAF/PBAF complexes BAF complexes are 1-2 MDa in size and consist of 9-12 BRG1/BRM associated factors, (BAFs) [START_REF] Muchardt | A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor[END_REF]) (Fig. 35). The two paralogue ATPase subunits, BRG1 and BRM, share over 70% sequence identity and can display similar biochemical activities in vitro [START_REF] Khavari | BRG1 contains a conserved domain of the SWI2/SNF2 family necessary for normal mitotic growth and transcription[END_REF][START_REF] Phelan | Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits[END_REF][START_REF] Randazzo | brg1: a putative murine homologue of the Drosophila brahma gene, a homeotic gene regulator[END_REF] but don't functionally compensate for one another in various cellular processes including proliferation and differentiation (Bultman et al., 2000;[START_REF] Kadam | Transcriptional specificity of human SWI/SNF BRG1 and BRM chromatin remodeling complexes[END_REF][START_REF] Reyes | Altered control of cellular proliferation in the absence of mammalian brahma (SNF2alpha)[END_REF]. BRG1 is composed of multiple domains and the bromodomain of BRG1 has been implicated in the recognition of acetylated lysines within histone H3 and H4 tails [START_REF] Chandrasekaran | Polybromo-1-bromodomains bind histone H3 at specific acetyl-lysine positions[END_REF][START_REF] Shen | Solution structure of human Brg1 bromodomain and its specific binding to acetylated histone tails[END_REF]. Such modifications within target promoters may serve as an interaction surface for the assembly and/or recruitment of bromodomain-containing coregulator complexes, including SWI/SNF. In conjunction with the C-terminal bromodomain, BRG1 also contains an AT-hook sequence motif, which may aid in DNA binding or recruitment of SWI/SNF to acetylated lysines within histone tails [START_REF] Singh | DNA-binding properties of the recombinant highmobility-group-like AT-hook-containing region from human BRG1 protein[END_REF].

Nevertheless, these complexes are a heterogeneous mixture of additional subunits including BAF170, BAF155 and BAF47/INI1, as well as BAF60, BAF57, BAF53 and actin (Wang et al., 1996) (Fig. 35A). The presence of either BRG1 or BRM along with BAF170, BAF155 and BAF45 is sufficient for optimal chromatin remodelling activity in vitro [START_REF] Phelan | Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits[END_REF]. The functional contribution of each BAF to complexe activity has not been determined but it is clear that combinatorial assembly mechanisms result in specific interaction with various transcriptional activators and repressors and thus specific targeting [START_REF] Belandia | Targeting of SWI/SNF chromatin remodelling complexes to estrogen-responsive genes[END_REF][START_REF] Hsiao | BAF60a mediates critical interactions between nuclear receptors and the BRG1 chromatin-remodeling complex for transactivation[END_REF][START_REF] Ito | Identification of SWI.SNF complex subunit BAF60a as a determinant of the transactivation potential of Fos/Jun dimers[END_REF][START_REF] Pal | mSin3A/histone deacetylase 2-and PRMT5-containing Brg1 complex is involved in transcriptional repression of the Myc target gene cad[END_REF][START_REF] Phelan | Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits[END_REF].

Human SWI/SNF can be subdivided into to predominant other complexes: the BAF complex, which uniquely contains BAF250A or BAF250B and PBAF (Polybromoassociated BAF) complex, which specifically contains BAF200 and BAF180 [START_REF] Lemon | Selectivity of chromatin-remodelling cofactors for ligand-activated transcription[END_REF][START_REF] Nie | A specificity and targeting subunit of a human SWI/SNF family-related chromatin-remodeling complex[END_REF][START_REF] Yan | PBAF chromatin-remodeling complex requires a novel specificity subunit, BAF200, to regulate expression of selective interferon-responsive genes[END_REF]. Of note, The BRG1 protein can also be found assembled with transcription factor and histone-modifying enzyme complexes to activate or repress nuclear processes including transcription, elongation and DNA replication [START_REF] Trotter | The BRG1 transcriptional coregulator[END_REF] The exact mechanism by which ATP-dependent chromatin remodelling enzymes function to disrupt nucleosomes is not fully understood. Several models have been proposed to account for the ability of SWI/SNF to modify chromatin structure [START_REF] Hassan | Promoter targeting of chromatin-modifying complexes[END_REF][START_REF] Peterson | Promoter targeting and chromatin remodeling by the SWI/SNF complex[END_REF][START_REF] Gangaraju | Mechanisms of ATP dependent chromatin remodeling[END_REF][START_REF] Narlikar | Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes[END_REF] Models include ATP-dependent repositioning (slide, twist, or loop) movement of histone octamers in cis along the DNA: SWI/SNF complexe binding could disrupt histone-DNA contacts, create a loop of DNA that propagates around the nucleosome in a wave-like manner and then reposition the DNA with respect to the nucleosome (Fig. 35B). Sliding may also lead to the ejection of an adjacent nucleosome. Other general models propose transfer of histone octamers from one nucleosome to another, replacement or eviction of nucleosomal histones (Saha et al., 2006;[START_REF] Smith | A conserved Swi2/Snf2 ATPase motif couples ATP hydrolysis to chromatin remodeling[END_REF]. The net result is an altered structure that is hypersensitive to nuclease digestion and increased affinity for transcription factors [START_REF] Schnitzler | Human SWI/SNF interconverts a nucleosome between its base state and a stable remodeled state[END_REF].

F. Biological functions of BRG1/PBAF in mammals BRG1 and other SWI/SNF subunits have been shown to be essential for murine development: disruption or silencing of Brg1, Ini1, Baf155, Baf60c, or Baf 180 is embryonic-lethal. Strikingly, Brm inactivation in mice produces only mild effects [START_REF] Reyes | Altered control of cellular proliferation in the absence of mammalian brahma (SNF2alpha)[END_REF]. Studies indicate that SWI/SNF enzymes are required for both embryonic stem cell pluripotency as well as cellular differentiation (de la Serna et al., 2006). In the zebrafish model, Brg1-mutated embryos showed defects in the development of pigment cells and retina, the tissues expressing MITF. This work suggests that Brg1 is involved in neural crest induction, and consequently in the development of neurons, glia, and pigment cells [START_REF] Eroglu | Critical role of Brg1 member of the SWI/SNF chromatin remodeling complex during neurogenesis and neural crest induction in zebrafish[END_REF].

The SWI/SNF chromatin remodeling complex has been described as a key regulator of skeletal muscle differentiation, where the ATPase of BRG1 is required for gene expression at different stages of skeletal myogenesis [START_REF] Ohkawa | Myogenin and the SWI/SNF ATPase Brg1 maintain myogenic gene expression at different stages of skeletal myogenesis[END_REF]. SWI/SNF chromatin remodeling enzymes play an important role in the promotion of activation of melanocyte specific genes by cooperating with MITF. MITF promotes the recruitment of SWI/SNF to target genes involved in the synthesis of melanin pigment by remodeling the chromatin structure (de la Serna et al., 2006a;Keenen et al., 2010;Laurette et al., 2015).

Other than transcriptional regulation, the complex is critical for control of cellular processes, including differentiation and proliferation, and involvement in DNA repair by altering the accessibility of UV-damaged DNA-binding proteins to DNA lesions [START_REF] Dinant | Chromatin structure and DNA damage repair[END_REF][START_REF] Klochendler-Yeivin | SWI/SNF chromatin remodeling and cancer[END_REF][START_REF] Martens | Evidence that Swi/Snf directly represses transcription in S. cerevisiae[END_REF][START_REF] Roberts | The SWI/SNF complex--chromatin and cancer[END_REF]Sif et al., 2001).

Generally, the complexes function in a broad range of basic cell processes that include mainly regulation of transcription, DNA replication and repair, and homologous recombination (Neely and Workman, 2002;[START_REF] Lusser | Chromatin remodeling by ATP-dependent molecular machines[END_REF][START_REF] Hota | Diversity of operation in ATP-dependent chromatin remodelers[END_REF]. Several studies have reported the role for SWI/SNF in DNA repair in response to UV induced DNA damage. SWI/SNF has been shown to be involved in DNA repair by altering the accessibility DNA repair proteins to DNA lesions [START_REF] Dinant | Chromatin structure and DNA damage repair[END_REF].

Park et al 2006 demonstrated inactivation of the SWI/SNF complex results in inefficient DNA double-strand break (DSB) repair and increased sensitivity to DNA damage [START_REF] Gong | Role of the mammalian SWI/SNF chromatin remodeling complex in the cellular response to UV damage[END_REF][START_REF] Gong | Rad4-Rad23 interaction with SWI/SNF links ATPdependent chromatin remodeling with nucleotide excision repair[END_REF][START_REF] Park | Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting gamma-H2AX induction[END_REF]. Furthermore BRG1 was shown to be recruited to sites of UV-induced damage to promotes chromatin relaxation, and facilitates nucleotide excision repair [START_REF] Zhao | Modulation of nucleotide excision repair by mammalian SWI/SNF chromatin-remodeling complex[END_REF]Zhang et al., 2009) Several subunits including the ATPase subunits are either silenced or over expressed in various types of cancers. SWI/SNF components are concomitantly silenced in primary lung tumors. BRG1 and BRM are lost in about 30-40% of lung cancers [START_REF] Betz | Reexpression of hSNF5/INI1/BAF47 in pediatric tumor cells leads to G1 arrest associated with induction of p16ink4a and activation of RB[END_REF][START_REF] Decristofaro | Characterization of SWI/SNF protein expression in human breast cancer cell lines and other malignancies[END_REF]. BRM has been shown to play a role in intestinal differentiation in gastric cancer. Introduction of BRM expression into gastric cancer cells that are deficient in BRM expression promotes expression of villin, a known marker of intestinal differentiation [START_REF] Yamamichi | Frequent loss of Brm expression in gastric cancer correlates with histologic features and differentiation state[END_REF]. Loss of both BRG1 and BRM expression correlates with poor prognosis of non-small cell lung cancer [START_REF] Reisman | Loss of BRG1/BRM in human lung cancer cell lines and primary lung cancers: correlation with poor prognosis[END_REF]. BRG1 and BRM have both been shown to be lost in 30-40% of lung cancer cell lines and 10-20% of primary lung cancers and other solid tumors. Multiple cancer cell lines have been shown to be missing one or more SWI/SNF subunits [START_REF] Decristofaro | Characterization of SWI/SNF protein expression in human breast cancer cell lines and other malignancies[END_REF][START_REF] Decristofaro | Alteration of hSNF5/INI1/BAF47 detected in rhabdoid cell lines and primary rhabdomyosarcomas but not Wilms' tumors[END_REF]Wong, et al., 2000). Reintroduction of SWI/SNF subunits into cancer cell lines that lack their expression typically induces a change in morphology, cell cycle arrest, apoptosis or senescence.

In addition, perturbation of BAF or PBAF assembly through the alteration of key BAF subunits, as it is observed in approximately 20% cancers were associated with aberrant biological function and numerous experimental findings suggest that this complex functions as a tumor suppressor [START_REF] Klochendler-Yeivin | SWI/SNF chromatin remodeling and cancer[END_REF][START_REF] Roberts | The SWI/SNF complex--chromatin and cancer[END_REF]Reisman et al., 2009;[START_REF] Halliday | SWI/SNF: a chromatinremodelling complex with a role in carcinogenesis[END_REF][START_REF] Wilson | SWI/SNF nucleosome remodellers and cancer[END_REF]. Case examples are malignant rhabdoid tumors (MRT) and synovial sarcomas, which are characterized by bi-allelic inactivation and translocation mutations, of BAF47 respectively. Both of these mutations result in the failure to incorporate BAF47 into the BAF complex resulting in compositionally distinct BAF complexes. Thus BAF47 might have a central role in BAF assembly through adjacent interactions with other BAF subunits. Inactivating mutations of the BRG1 gene, on chromosome arm 19p, are present in several human lung cancer cell lines [START_REF] Girard | Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering[END_REF].

Furthermore, the presence of inactivating and truncation mutations in several other BAF subunits (e.g. BAF250A, BAF180, BRG1, and BRM) may also indicate a mutual dependency among other sets of BAF subunits. The full impact of these oncogenic mutations has yet to be elucidated due to the absence of a comprehensive BAF structure model and defined assembly pathway.

G. Role of BAF/PBAF in melanocyte and melanoma development PBAF remodelling was shown to play a role in melanocyte specific gene expression and melanocyte differentiation, notably through BRG1. BRG1 has been shown to interact with MITF and SOX10 and expression of their target genes (de la Serna et al., 2006;Keenen et al., 2010;Laurette et al., 2015). Further more PBAF remodelling is required for SOX10 expression (Keenen et al., 2010;[START_REF] Ondrusova | MITF-independent prosurvival role of BRG1-containing SWI/SNF complex in melanoma cells[END_REF]Laurette et al., 2015) and for expression of SOX10 target genes during Schwann cell differentiation [START_REF] Limpert | NF-kappaB forms a complex with the chromatin remodeler BRG1 to regulate Schwann cell differentiation[END_REF][START_REF] Marathe | SWI/SNF enzymes promote SOX10-mediated activation of myelin gene expression[END_REF]. Similarly, expression of MITF is absolutely dependent on the functional SWI/SNF complex (Vachtenheim et al., 2010;Laurette et al., 2015).

Overall BAF subunits are generally highly expressed components in melanoma cell lines (Vachtenheim et al., 2010;Laurette et al., 2015) but there is increasing evidence that some SWI/SNF subunits are deregulated or mutated in melanoma and that they cooperate with MITF to promote melanoma tumorigenicity (Keenen et al., 2010;[START_REF] Becker | The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a[END_REF][START_REF] Lin | BRG1 expression is increased in human cutaneous melanoma[END_REF][START_REF] Hodis | A landscape of driver mutations in melanoma[END_REF]Nikolaev et al., 2012;[START_REF] Krauthammer | Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma[END_REF].

There have been conflicting reports of the status of BRG1 in melanoma. On one hand the levels of BRG1 were shown to be elevated in the later stages of melanoma metastasis while levels of BRM decreased [START_REF] Becker | The chromatin remodelling factor BRG1 is a novel binding partner of the tumor suppressor p16INK4a[END_REF][START_REF] Saladi | Modulation of extracellular matrix/adhesion molecule expression by BRG1 is associated with increased melanoma invasiveness[END_REF]. In contrast, other studies found that BRG1 was lost in a significant number of primary and metastatic melanoma samples. In any case, it seems that the ATPases BRG1 and BRM are rarely concomitantly disrupted, ARID2 was shown to be most frequently mutated the SWI/SNF component in melanoma so far but there has not yet been any investigation into the biological function of ARID2 in melanocytes nor studies of how ARID2 might act as a tumor suppressor. INI-1 expression has been shown to be downregulated with melanoma progression. Loss of INI-1 expression seems to be related with a decreased 5-year survival in melanoma patients. Thus INI-1 has been proposed to be a marker for human melanoma progression and prognosis as well as a potential therapeutic target [START_REF] Cronin | Frequent mutations in the MITF pathway in melanoma[END_REF].

Of note, BRD2 and BRD4 are two BET (bromodomain and extraterminal domain) proteins that have been reported to be overexpressed in melanoma tissue and required for expression of cell cycle and survival genes and thus tumor proliferation and maintainance [START_REF] Segura | Aberrant miR-182 expression promotes melanoma metastasis by repressing FOXO3 and microphthalmia-associated transcription factor[END_REF][START_REF] Vardabasso | Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma[END_REF]. Importantly, BRG1 has been shown to interact and colocalize with BRD4 on chromatin and coregulate target gene expression in other contexts [START_REF] Shi | Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation[END_REF][START_REF] Liu | BRD4 regulates Nanog expression in mouse embryonic stem cells and preimplantation embryos[END_REF] suggesting SWI/SNF might be a player in these processes.

RESULTS

Context and aims of research

Melanoma is a highly aggressive cancer that arises from the transformation of cells from the melanocyte lineage, mainly from a skin melanocyte. It is generally less sensitive to most cytotoxic chemotherapeutic agents and radiation therapy than are other types of tumors, making melanoma one of the most difficult metastatic cancers to cure and consequently one of the deadliest. The mechanisms involved in melanoma initiation, progression and distant metastasis formation are the subject of intense biomedical and pharmacological research efforts.

A key factor in melanoma is MITF (MIcrophthalmia-associated Transcription Factor), a bHLH-LZ transcription factor that is the master regulator of the development of the melanocyte lineage and the physiology of adult melanocytes. The 'rheostat model' proposes that the level of functional MITF determines the biological properties melanocyte lineage cells linked to melanoma phenotype and tumor progression. High MITF levels lead to terminal differentiation and cell cycle exit, intermediate levels to proliferation, while lower levels result in slow cycling invasive cells with a stem cell like phenotype and tumor initiating properties. This has important consequences for therapeutic strategies as drugs that modulate MITF expression could be used to drive cells either to a terminally differentiated state or a non-proliferative senescent state.

Previous work done in our laboratory showed by use of high throughput genomics that MITF directly activates genes involved in DNA replication and repair, mitosis and pigmentation, but is a repressor of invasion and cell motility genes (Strub et al., 2011). This dual ability to act as an activator and a repressor underpins how changes in MITF levels can act as a switch between the proliferative and invasive programs and the resulting states.

These results were confirmed in the first study of this manuscript. Furthermore, in order to understand how MITF regulates gene expression at the molecular level, we used tandem affinity purification and mass-spectrometry to identify proteins associated with MITF both in the soluble nuclear and the chromatin-associated fractions of human 501Mel melanoma cells. This interactome analysis revealed that MITF interacts with a large set of proteins several of which may act as cofactors in transcription regulation. We were particularly interested in the interaction between MITF and the BRG1/PBAF and BPTF/NURF chromatin remodelling complexes. MITF like all transcription factors regulates gene expression in a chromatin environment. In the first study, we further investigated the role of the BRG1 complex in MITF regulated transcription in human melanoma cells. Removing BRG1 from melanocytes and melanoma cells caused the cells to stop growing and die. Consistently, BRG1 knockout in vivo from developing mouse embryos, prevented formation of the melanocytes lineage.

Further investigation revealed that MITF, together with SOX10, actively recruit BRG1 to the genome to open up the chromatin and regulate nearby genes. Interestingly, BRG1 binds to many such elements in a characteristic manner, in which BRG1 binds the two nucleosomes flanking the stretch of DNA bound by MITF, SOX10 and several other key transcription factors that together regulate many aspects of melanocyte and melanoma cell physiology.

The results of this study are presented in Article 1: [START_REF] Laurette | Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells[END_REF] Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells. Elife, 03/2015 4, e06857.

Of note, in this study we did not investigate at which stage of melanoblast development Brg1 is required nor whether Brg1 is required to generate melanocytes from the adult melanocyte stem cell (MSC) population. We recently addressed these questions and show a specific requirement for Brg1 during the proliferation of embryonic melanoblasts, whereas generation of melanocytes from the adult MSC population appears to be independent of Brg1.

These complementary results are attached to this manuscript in Annexe as Annexe 1 to

Laurette et al. (2015).

NURF is a chromatin-remodelling complex that has been shown mainly to be involved in active transcription and has previously been implicated in development of other cancers. Despite extensive characterisation of the biochemical properties of the NURF complex, much less is known about its biological functions in mammals. Furthermore, the function of the NURF complex in the melanoma and the melanocyte lineage had not been reported so far. Work presented in Article 1 revealed that MITF associates with the NURF chromatinremodelling factor in melanoma cells and that they co-regulate overlapping gene expression programs in cell lines in vitro. Somatic and specific inactivation of Bptf in developing murine melanoblasts in vivo shows that Bptf regulates their proliferation, migration and morphology. Furthermore we have shown that these melanoblasts give rise to a defective melanocyte stem cell population in the hair follicle of adult mice that fail to regenerate the terminally differentiated melanin producing melanocytes that pigment the growing hair. We have shown that melanocyte stem cells display a transcriptionally repressed chromatin state and thus Bptf appears to be essential for reactivation of the melanocyte gene expression program during transition of the transcriptionally quiescent stem cell to the differentiated state.

Results

126

The results of this study are described in Article 2 of this manuscript: Koludrovic et al. (2015) Chromatin-remodelling complex NURF is essential for differentiation of adult melanocyte stem cells. PLoS Genet 11 (10) Of note, we recently reanalysed the RNA-seq data obtained after MITF and BPTF silencing in 501Mel cells using the GSEA software and showed MITF and BPTF coregulate a set of genes involved in oxidative phosphorylation and thus can promote a metabolic switch during melanoma development.

These complementary results are attached to this manuscript in Annexe as Annexe 2 to

Koludrovic et al. (2015).

Comparison of the previous studies presented here reveals that the BRG1/PBAF and BPTF/NURF chromatin-remodelling complexes play distinct but complementary roles in human melanoma in vitro and in the mouse melanocyte lineage. To complete these observations we have used a mouse genetic model of melanoma to address the role of Brg1 and Bptf in mouse melanoma and we find that they are both essential for melanomagenesis in vivo.

The results of this study are described in Article 3 of this manuscript:

Laurette et al., Essential role for the chromatin remodellers Brg1 and Bptf in mouse melanoma. Manuscript in preparation

In a recent study from the Marine lab, Leucci et al. report a role for the long noncoding RNA SAMMSON in driving mitochondrial metabolism in melanoma through its ability to enhance the mitochondrial localization and function of p32. SAMMSON is expressed in the vast majority (>90%) of melanomas and co-amplified along with MITF in a subset of them. SAMMSON knockdown drastically decreases the viability of melanoma cells independently of common somatic mutations in genes such as BRAF, NRAS, or p53. In the frame of this collaborative work, we identified SOX10 but not MITF as the transcriptional regulator likely contributing to the melanocyte lineage restricted expression of SAMMSON. Consistently we have shown that knockdown of SOX10, but not MITF, led to a decrease in SAMMSON expression. Thus SOX10 recruits BRG1 upstream of the SAMMSON Results 127 TSS and drives its expression in melanoma cells. (see also Annexe 2). SAMMSON targeting sensitizes melanoma to MAPK-targeting therapeutics and may therefore offer a novel therapeutic vulnerability targeting mitochondrial metabolism selectively and overcome the adaptive metabolic reprogramming that limits the response to these inhibitors

The results of this study are described in Article 4 of this manuscript: Leucci, et al. (2016) Melanoma addiction to the long non-coding RNA SAMMSON. Nature 531(7595), 518-522.

Introduction

Microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper (bHLH-Zip) factor, regulates specification, survival, and proliferation of normal melanocytes, and controls proliferation, migration and invasion of melanoma cells (Goding, 2000;Widlund and Fisher, 2003;Steingrimsson et al., 2004). The level of functional MITF expression determines many of the proliferation and invasion properties of melanoma cells (Hoek and Goding, 2010) and siRNA-mediated MITF silencing induces senescence in several melanoma lines (Strub et al., 2011).

We previously reported a genome wide analysis of MITF target genes in 501Mel melanoma cells (Strub et al., 2011). Chromatin immunoprecipitation coupled to deep sequencing (ChIPseq) identified MITF binding sites and integration with RNA-seq following siRNA-mediated MITF knockdown showed that MITF directly and positively regulates genes involved in DNA replication and repair and mitosis. In contrast, MITF represses genes involved in melanoma invasion.

*For correspondence: irwin@ igbmc.fr †

These authors contributed equally to this work

To better understand the molecular function of MITF, we used tandem affinity purification to isolate MITF from 501Mel melanoma cells and mass spectrometry to identify its interacting partners. We report here the first comprehensive characterisation of the MITF interactome and we show that MITF interacts physically and functionally with a novel form of the PBAF chromatin-remodelling complex specific for neural crest derived cells comprising both BRG1 and CHD7. We also show that MITF and SOX10 recruit BRG1/PBAF to the nucleosomes flanking critical enhancer elements in the melanocyte lineage.

Results

MITF associates with multiple complexes involved in transcription, DNA replication and repair

Using 501Mel melanoma cell lines stably expressing an N-terminal FLAG-HA epitope-tagged MITF (F-H-MITF) to levels comparable to that of endogenous MITF (Figure 1A, line J), we performed tandem affinity purification of soluble nuclear-and chromatin-associated fractions [START_REF] Dran É | The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3[END_REF]. Mass spectrometry and silver nitrate staining showed numerous proteins in the tandem immunoprecipitation from F-H-MITF cells, while almost no proteins were detected in immunoprecipitations from untagged 501Mel cells (Figure 1B and Supplementary file 1). Two independent purifications and mass spectrometry analyses were performed and only proteins identified specifically in the F-H-MITF precipitations with no peptides in the control precipitations are discussed. In addition, contaminating ribo-nucleoprotein particles, spliceosome components, and chaperone proteins have been excluded from the subsequent analysis.

Previously described MITF partners such as its heterodimerisation partners TFEB, TFE3 and TFEC (Steingrimsson et al., 2002) and its cofactor β-catenin (CTNNB1) (Schepsky et al., 2006) (Figure 1D and Supplementary file 1) were detected. We also identified novel potential MITF partners. The BPTF, SMARCA1 (SNF2L), SMARCA5 (SNF2H), and RBBP4 components of the NURF chromatin-remodelling eLife digest Melanocytes are pigment-producing cells found primarily in the skin. Many of the genes that help these cells to develop are also thought to affect the development of melanomas: an aggressive form of skin cancer that originates in these cells. One such gene encodes a protein called MITF. This protein binds to DNA and regulates genes that control the development, survival, and spread of melanocytes; it is also linked to the invasive properties of melanomas.

The MITF protein works together with partner proteins to control numerous genes, activating some while inhibiting others, by binding to nearby stretches of DNA that act as regulatory elements. Its interactions are therefore widespread and complex. Now, Laurette, Strub et al. have used techniques called tandem affinity purification and mass spectrometry to identify the proteins that interact with MITF. This investigation found many new protein partners for MITF, including proteins involved in DNA damage, repair, and replication. MITF also associates with two proteins-one of which is called BRG1-that are involved in modifying how tightly DNA is packaged inside cells. DNA wrapped around proteins is known as chromatin, and if chromatin is tightly packed, the genes in that stretch of DNA cannot be easily accessed or activated.

Removing BRG1 from melanocytes and melanoma cells caused the cells to die or stop growing. When BRG1 was removed from developing mouse embryos, melanocytes failed to form. Further investigation revealed that MITF, together with another protein, localize BRG1 to sites in the melanocyte's DNA to open up the chromatin and regulate nearby genes. Furthermore, Laurette, Strub et al. report that BRG1 binds to many such elements in a characteristic manner, in which two BRG1 proteins flank the stretch of DNA bound by MITF and several other key DNA-binding proteins that together regulate many aspects of melanocyte and melanoma cell physiology.

Laurette, Strub et al. have therefore revealed many details about the molecules that activate genes in melanomas and melanocyte cells, as well as the interactions between these molecules. The results could also help researchers to understand how the BRG1 protein organises chromatin packing in other cell types. E3-ligase HERC2, previously implicated in DNA repair [START_REF] Bekker-Jensen | HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes[END_REF], and UBR5 a second HECT domain-containing E3-ligase with functions in both DNA repair and transcription [START_REF] Cojocaru | Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B[END_REF][START_REF] Gudjonsson | TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes[END_REF]. NEURL4, a known HERC2-interacting protein [START_REF] Bashkurov | Interaction proteomics identify NEURL4 and the HECT E3 ligase HERC2 as novel modulators of centrosome architecture[END_REF], was also found along with the de-ubiquitinase enzymes USP7 and USP11 that were preferentially represented in the SNE. In contrast, USP13 shown to regulate MITF stability (Zhao et al., 2011) was not detected in our experiments. Several interactions were verified by immunoblot as HERC2, BRG1, USP7, USP11, XRCC5, and XRCC6 were detected in the F-H-MITF immunoprecipitations from the soluble nuclear fraction, but not in the untagged controls (Figure 1C), with enrichment of USP7 and USP11 in the SNE compared to the CAE and the selective presence of HERC2 in the SNE. We identified several other complexes interacting with MITF. Four subunits of TFIIIC, a RNA polymerase III cofactor were detected along with the SMCA1, SMC3, STAG2, and PDS5 cohesin subunits. TRIM28 (TIF1β, KAP1), a co-repressor belonging to a sub-family of TRIM proteins comprising TRIM24 (TIF1α) and TRIM33 (TIF1γ) was found as were the HDAC1 and HDAC2 and the HP1 proteins known to associate with TRIM-co-repressor complexes [START_REF] Herquel | The TIF1alpha-related TRIM cofactors couple chromatin modifications to transcriptional regulation, signaling and tumor suppression[END_REF]. We identified TRRAP, RUVBL1, RUVBL2, and BAF53A. These proteins are already known to form a cofactor for MYC [START_REF] Park | The ATM-related domain of TRRAP is required for histone acetyltransferase recruitment and Myc-dependent oncogenesis[END_REF][START_REF] Park | BAF53 forms distinct nuclear complexes and functions as a critical c-Mycinteracting nuclear cofactor for oncogenic transformation[END_REF][START_REF] Murr | Orchestration of chromatin-based processes: mind the TRRAP[END_REF]. Interestingly, a mutation in TRRAP has recently been associated with human melanoma [START_REF] Wei | Exome sequencing identifies GRIN2A as frequently mutated in melanoma[END_REF]) that together with its interaction with MITF suggests a role in melanomagenesis.

In addition to transcription complexes, the RFC1, RFC2, RFC4, and RFC5 subunits of DNA replication factor C associate with MITF along with the MCM3, MCM5, and MCM7 subunits of the MCM complex that forms at DNA-replication origins. AKAP8 and AKAP8L were also identified and AKAP8 has been shown to interact with the MCM complex [START_REF] Eide | Protein kinase A-anchoring protein AKAP95 interacts with MCM2, a regulator of DNA replication[END_REF], although these protein kinase A anchoring proteins have additional functions [START_REF] Collas | The A-kinase-anchoring protein AKAP95 is a multivalent protein with a key role in chromatin condensation at mitosis[END_REF]. The kinase PLK1 was found in the chromatin-associated fraction suggesting that it may phosphorylate MITF on chromatin. Finally, we detected multiple subunits of the nuclear pore complex. This may reflect control of MITF subcellular localization or a coupling of MITF-driven transcription and RNA-export. Together, these data describe a comprehensive set of MITF-interacting proteins.

MITF interacts with a BRG1-CHD7-containing PBAF complex While BRG1 was reported to interact with MITF (de la Serna et al., 2006;Keenen et al., 2010), the composition of the complex had not been determined. We identified BRG1 (SMARCA4) as well as the PBRM1 (BAF180), SMARCC2 (BAF170), SMARCD2 (BAF60B), and ACTL6A (BAF53A) subunits along with CHD7 reported to associate with BRG1 in human neural crest cells [START_REF] Bajpai | CHD7 cooperates with PBAF to control multipotent neural crest formation[END_REF] (Figure 1D). The MITF interacting complex most closely resembles the PBAF variant with the presence of PBRM1 [START_REF] Trotter | The BRG1 transcriptional coregulator[END_REF]Reisman et al., 2009). To investigate BRG1 complex composition in 501Mel cells, extracts were precipitated with an anti-BRG1 antibody showing coprecipitation of BRG1 with BAF200, BAF155, BAF53A, BAF180, and BAF170, whereas BAF60B and BAF250B were not co-precipitated (Figure 2A). Peptides for BAF60B were detected in the MITF interactome, but this subunit did not associate with BRG1 in 501Mel cells, while BAF60A coprecipitated with BRG1, suggesting that MITF interacts with BAF60B independently from the BRG1 complex. The MITF-interacting complex also comprises CHD7 as seen by the reciprocal coprecipitation of BRG1 and CHD7 and their co-precipitation with MITF (Figure 2A-B). Together, these observations show that MITF interacts with a novel form of PBAF complex comprising BRG1 and CHD7 (Figure 2C).

BRG1 regulates an extensive gene expression programme essential for proliferation of melanoma cells in vitro

To address the function of BRG1 in melanoma cells, we performed si/shRNA knockdown. si/shRNA knockdown of BRG1 strongly reduced MITF protein and mRNA levels (Vachtenheim et al., 2010 and Figure 3A-B). The effects of BRG1 silencing on gene expression were therefore very similar to the loss of MITF itself with reduced expression of MITF target genes involved in cell cycle, pigmentation and signalling and activation of several genes of the senescence-associated secretory phenotype (SASP) whose expression is also induced upon MITF-knockdown (Ohanna et al., 2011) (Figure 3B). BRG1 silencing arrested 501Mel proliferation and cells showed a flattened enlarged morphology with multiple cytoplasmic projections, similar to senescent cells upon MITF knockdown (Figure 3C [Strub et al., 2011]). Up to 40% of the BRG1 knockdown cells showed senescence-associated β-galactosidase staining (Figure 3C).

RNA-seq following shBRG1 silencing revealed a dramatic effect on gene expression with >4000 genes down-regulated and >5400 genes up-regulated (p.adj <0.05 and log2fold change >1, Figure 3-figure supplement 1A and Supplementary file 2). ShMITF knockdown resulted in cell cycle arrest and morphological changes associated with senescence (Figure 3C) accompanied by the down-regulation of around 600 genes and up-regulation of 747 genes. 60% of genes down-regulated by shMITF showed similar loss of expression upon shBRG1 consistent with the fact that MITF was strongly repressed in the shBRG1 cells (Figure 3-figure supplement 1A and Supplementary file 2).

Commonly down-regulated genes were enriched in ontology terms associated with signalling, cell cycle, and mitosis (Figure 3-figure supplement 1A). Common up-regulated genes were enriched in terms associated with angiogenesis, adhesion, and migration consistent with the dramatic changes in cell morphology. MITF silencing has been shown to induce a SASP comprising CCL2, CTGF and SERPINE1 (Strub et al., 2011). RNA-seq identified a putative SASP in shMITF cells comprising around 20 secreted factors and of these 15 were also induced in the shBRG1 cells, although several key factors such as IL8 and CCL2 were not induced upon BRG1 silencing (Figure 3-figure supplement 1A). Loss of either BRG1 or MITF therefore induced senescence of 501Mel cells. SOX10, TCF/LEF/CTNNB1 and CREB have been reported to activate MITF expression (Goding, 2000). We noted that SOX10 expression is strongly repressed in BRG1 knockdown cells, but not in MITF-knockdown cells (Supplementary file 2). SiSOX10 silencing repressed endogenous MITF expression (Figure 3-figure supplement 2A-B). In 501Mel-Cl8 cells constitutively expressing 3HAtagged MITF from the CMV promoter (Strub et al., 2011), siSOX10 repressed endogenous, but not ectopic MITF. In contrast, siCREB silencing had no effect on MITF expression. SOX10 is therefore a major regulator of MITF expression in 501Mel cells and its diminished expression upon BRG1 knockdown partly explains the concomitant MITF loss. These observations are also consistent with previous reports showing that SOX10 promotes melanoma cell proliferation and that its loss leads to senescence [START_REF] Cronin | SOX10 ablation arrests cell cycle, induces senescence, and suppresses melanomagenesis[END_REF].

To determine whether the shared phenotypes of BRG1 and MITF knockdown cells resulted from the concomitant loss of MITF upon BRG1 silencing or whether BRG1 acts also as an MITF co-factor, we performed shBRG1 silencing in the 501Mel-Cl8 cells. BRG1 knockdown in these cells repressed endogenous MITF expression, but not ectopic 3HA-MITF (Figure 3-figure supplement 2C). Nevertheless, BRG1 silencing elicited a phenotype similar to 501Mel cells characterised by arrested proliferation, and morphological changes. Many MITF target genes were similarly repressed by BRG1 silencing in both 501Mel and Cl8 cells, while SASP components were induced (Figure 3-figure supplement 2D). Together, these data show that BRG1 is essential for MITF expression and that it acts as a cofactor for MITF since ectopic MITF in the Cl8 cells does not activate target genes expression in its absence.

BRG1 and MITF regulate gene expression in human melanocytes

We also investigated BRG1 function in untransformed Hermes 3A melanocytes. In contrast to 501Mel cells, shBRG1 silencing had little effect on MITF expression in Hermes 3A cells (Figure 3D), but induced changes in cell morphology with up to 80% of cells showing staining for senescenceassociated β-galactosidase (Figure 3E). Within 8 days, the BRG1 silenced cells detached from the plate. ShMITF silencing in Hermes 3A cells also led to growth arrest and a marked changes in morphology, with flattening, enlargement of the cell body and reduced neurite projections (Figure 3D). Despite these changes indicative of senescence, <50% of shMITF-silenced cells showed staining for senescence-associated β-galactosidase. As with shBRG1, MITF silencing led to cells detaching from the plate within 7 days. Thus, both BRG1 and MITF are essential for melanocyte growth, and in their absence cells undergo growth arrest, senescence, and death. RNA-seq showed that BRG1 silencing in Hermes 3A cells down-regulated 587 genes, and upregulated 971 genes, many fewer than in 501Mel cells (Figure 3-figure supplement 1B-C, and Supplementary file 2). Down-regulated genes were involved in pigmentation, cholesterol metabolism as well as intracellular signalling cascades and general cell morphology. Up-regulated genes were involved in cell-cell signalling and adhesion as well as angiogenesis. MITF silencing downregulated 757 genes and up-regulated 664 genes. Comparisons showed that 38% of genes downregulated upon BRG1 silencing were diminished by MITF silencing, while 25% of the BRG1 up-regulated genes were also increased upon MITF silencing.

Together these results show that BRG1 is critical for Hermes 3A proliferation despite the fact that it regulated a much reduced gene expression programme in these cells. As with the 501Mel cells however, MITF and BRG1 cooperate to regulate a subset of genes involved in several essential cellular processes associated with resistance to apoptosis, cell morphology, and signalling.

BRG1 is essential for generation of melanocytes in vivo

As BRG1 is essential for proliferation of both melanocytes and melanoma cells in vitro, we asked if BRG1 is also essential for melanocytes in vivo in mice. To bypass the embryonic lethal phenotype of BRG1 germ-line knockout (Bultman et al., 2000), we crossed mice with floxed alleles of the Smarca4 gene encoding BRG1 (Indra et al., 2005) with Tyr-Cre mice to allow selective inactivation of BRG1 in the melanocyte lineage (Delmas et al., 2003). We first generated Tyr-Cre::Smarca4 lox/+ mice that were crossed to generate the resulting Smarca4 mel+/-or Smarca4 mel-/-genotypes. The Tyr-Cre::Smarca4 mel-/- mice were present in the progeny at the expected ratio indicating no loss of viability (Figure 4A). While the heterozygous Tyr-Cre::Smarca4 mel+/-mice were black, homozygous Tyr-Cre::Smarca4 mel-/-mice were either completely white or had occasional black spotting presumably arising from rare melanoblasts that had escaped Cre-driven recombination during embryogenesis (Figure 4B). Examination of the hair from two genotypes showed an absence of melanin in Smarca4 mel-/-hair.

We labelled the hair follicles of the Smarca4 mel+/-or Smarca4 mel-/-animals with antibodies against Dct and Sox10. In Smarca4 mel+/-mice, Sox10 stained the nucleus of mature bulb melanocytes whose cytoplasm was stained with Dct (Figure 4C). In contrast, there was no staining with these antibodies in Smarca4 mel-/-bulbs. There were therefore no identifiable mature melanocytes in the mutant animals. Taken together with the essential role of BRG1 in melanoma and melanocyte cells in vitro, these data indicate that BRG1 is essential for generation of mature melanocytes in vivo.

BRG1 binds widely over the 501Mel cell genome

Given the critical function of BRG1 in the melanocyte lineage in vitro and in vivo, we used ChIP-seq to better understand how MITF and BRG1 regulate gene expression in 501Mel cells. We performed BRG1 ChIP-seq on native Mnase-digested chromatin along with a GFP-control ChIP-seq and identified >42,400 BRG1-occupied sites located in inter and intragenic regions, but with relative enrichment at the promoter (Figure 5A). Comparison with public ENCODE data showed BRG1-occupied sites often co-localized with or flanking enhancer regions marked with acetylated H3K27(ac) a mark of active enhancers (Smith and Shilatifard, 2014) (Figure 5-figure supplement 1A-B). As melanocyte-specific enhancers are not represented in public ENCODE data, we integrated the BRG1 profile with public H3K27ac data from primary human foreskin melanocytes and with H3K27ac data from a proliferative primary melanoma culture (Verfaillie et al., 2015). BRG1 co-localized with enhancers active in melanocytes (Figure 5-figure supplement 1A-B) including melanocyte-specific enhancers seen at the MITF and DCT loci (see below). The primary melanocyte and proliferative melanoma H3K27ac profiles are very similar and BRG1 colocalized with around 15,000 melanocyte/melanoma H3K27ac marked regions at promoters as well as distal inter and intragenic enhancers (Figure 5B). Analysis of DNA sequence motifs at BRG1 peaks identified binding sites for a large set of transcription factors (TFs), the most abundant of which are NRF1, KLF5, SP1-family factors, CTCF, EGR1, and E-boxes (Figure 5-figure supplement 1C). Thus, BRG1 is recruited by many different TFs to its sites on the 501Mel genome.

BRG1 occupied sites displayed a variety of profiles. A set of highly occupied sites (Clusters A-C, Figure 5C) comprised two peaks separated by various distances. Re-clustering of each of these sets revealed a complex profile comprising in general two more or less sharply defined peaks separated by distances of 450-800 bp (Figure 5D). At other sites however, BRG1 occupied a single sharp peak (cluster D, Figure 5C). Note that as the Tyr-Cre transgene is present on the X chromosome, we detect black mice with the Tyr-Cre::Smarca4 lox/lox genotype, but in these animals the Cre-recombinase is subjected to X-Figure 4. continued on next page BRG1-occupied sites at the TSS also displayed different profiles. BRG1 was preferentially located either upstream or downstream of the TSS (Figure 5-figure supplement 1D, clusters A-D) or showed equivalent occupancy at both sites (cluster E), or was absent (clusters F-G). Re-analysis of cluster E revealed BRG1 localized at varying distances between -370 and +440 bp with respect to the TSS (Figure 5E, clusters A-F), although it was present only on the downstream nucleosome in cluster G. BRG1 therefore appears to bind to the last (-1) nucleosome before the nucleosome-depleted region (NDR) at the TSS and the first downstream (+1) nucleosome although the distance between these nucleosomes is variable as previously described (Fenouil et al., 2012). The metaprofile of all BRG1-occupied TSS showed preferential location of BRG1 at -200 bp and +72 bp relative to the TSS (Figure 5F). Integration of RNA Polymerase II (Pol II) ChIP-seq data from 501Mel cells showed the peak of promoter paused Pol II located immediately upstream of the BRG1-bound +1 nucleosome. BRG1 therefore binds a large subset of sites both at the TSS and non-TSS regions as paired BRG1bound nucleosomes separated by variable distances.

Annotation of BRG1-occupied sites identified 7168 potential target genes in a window of ±10 kb around the TSS and >10,000 potential target genes in a window of ±30 kb (Figure 5-figure supplement 1E). Integration with RNA-seq data showed that 34% of genes down-regulated upon BRG1 silencing had at least one BRG1-occupied site at ±10 kb, while this figure attained 47% using the ±30 kb window. In contrast, only 12% and 18% of up-regulated genes were associated with BRG1occupied sites using these windows. BRG1-associated and down-regulated genes were enriched in pigmentation, DNA replication, and glucose and fatty acid metabolism (Figure 5-figure supplement 1F). BRG1-associated up-regulated genes were involved in angiogenesis and transcription regulation, notably expression of a set of ZNF TFs that appear to be co-ordinately regulated by BRG1 (Supplementary file 3). Together these data show that BRG1 is recruited by many TFs in 501Mel cells to regulate the expression of a large set of target genes.

MITF and SOX10 co-localization with BRG1

If BRG1 acts as a cofactor for MITF, they should co-localize on the genome. Integration of BRG1 and MITF ChIP-seq data showed that of >16,000 MITF occupied sites, 5867 were co-occupied by BRG1 and were located in both inter and intragenic regions, but enriched at promoters (Figure 6A). Colocalization can be observed at the MITF locus itself, where MITF occupied multiple sites, several of which co-localized with BRG1 and melanocyte H3K27ac-marked enhancers throughout this locus (Figure 6B). A similar situation was seen at the DCT locus, where MITF and BRG1 co-localized at H3K27ac marked melanocyte-specific enhancers.

At co-occupied sites, MITF binds between two BRG1 peaks as observed at the TYR locus (Figure 6C). BRG1 peaks were either positioned almost symmetrically with respect to MITF or displayed shoulders of density either upstream or downstream presumably corresponding to additional, but less well-positioned BRG1-occupied nucleosomes (Figure 6C). Genes associated with co-occupied sites are involved in previously defined critical functions of MITF such as lysosome biogenesis (Ploper et al., 2015), pigmentation, cell cycle, apoptosis, and DNA damage response (Figure 6-figure supplement 1A, and Supplementary file 4). Co-occupied sites showed enrichment not only in E and M-boxes for MITF, but also in motifs for SOX10, CREB, and ETS1 (Figure 6-figure supplement 1B), TFs that may cooperate with MITF and BRG1 to regulate associated target genes. In addition, we also identified a set of 1065 TSS, where BRG1 occupies the nucleosomes flanking the TSS and MITF binds the NDR (Figure 6D). Genes associated with these promoters were involved in lysosome formation, cell cycle, and RNA processing and splicing (data not shown).

As the SOX10 motif was so highly represented at MITF-BRG1 sites and that it physically interacts with BRG1 complexes [START_REF] Weider | Chromatin-remodeling factor Brg1 is required for Schwann cell differentiation and myelination[END_REF], we performed SOX10 ChIP-seq in 501Mel cells to profile its genomic occupancy. Almost 6000 SOX10-occupied sites were detected, but unlike MITF they were mainly located at distal regulatory elements with only around 80 sites at the proximal promoter. (Figure 6-figure supplement 2A). SOX10 recognition motifs were strongly enriched at SOX10 bound sites some of which were organised as degenerate palindromes (Figure 6-figure supplement 2B). Recognition motifs for TCF/LEF, MITF, and TFAP2A were also enriched at these sites. Genes associated with these sites were enriched in several ontology terms associated with cell motion/adhesion, cell morphogenesis and organisation (Figure 6-figure supplement 2C). We integrated the SOX10 data with that of MITF, BRG1, and H3K27ac. SOX10 and MITF co-occupied 3674 sites where MITF was located either 3′ or 5′ to SOX10 (Figure 6-figure supplement 2D). SOX10, MITF, BRG1, and H3K27ac were found at 1929 sites (Figure 7A, clusters A-D) while varying levels of SOX10, MITF and BRG1 were found at an additional ≈2000 sites (cluster E), whereas SOX10 and BRG1 co-occupied 1159 sites (cluster F) and 972 sites displayed essentially only SOX10 (cluster G). The above data define a specific organisation of MITF-associated regulatory elements (MAREs) where MITF alone or with SOX10 bind between two BRG1-bound nucleosomes. This analysis also defined 1929 MAREs at active melanocyte/ melanoma enhancer elements associated with 1511 genes involved in pigmentation, cell motility and adhesion, actin cytoskeleton organisation, and signalling (Supplementary file 4).

Co-localization of MITF and BRG1 with YY1 and TFAP2A

We also found a large overlap between BRG1 and YY1 genome occupancy (Figure 7-figure supplement 1A). Despite the fact that YY1 ChIP-seq was performed in a distinct melanoma line (Li et al., 2012), we identified 2853 sites where YY1 co-localized with MITF and 3060 SOX10-YY1 cooccupied sites (Figure 7B). All three proteins co-localize at 1070 sites together with BRG1 and 530 of these sites are marked with H3K27ac (Figure 7C). These data define a set of MAREs comprising YY1, BRG1, MITF, and SOX10 located at active melanocyte/melanoma enhancers. These 530 MARES are associated with genes involved in pigmentation, melanocyte development, cell motion as well as apoptosis (data not shown).

TFAP2A is a TF involved in melanoma [START_REF] Huang | Loss of AP-2 results in downregulation of c-KIT and enhancement of melanoma tumorigenicity and metastasis[END_REF] and normal melanocyte function [START_REF] Brewer | Wnt1-Cre-mediated deletion of AP-2alpha causes multiple neural crest-related defects[END_REF][START_REF] Van Otterloo | Differentiation of zebrafish melanophores depends on transcription factors AP2 alpha and AP2 epsilon[END_REF] and co-localizes with MITF at promoters involved in pigment cell differentiation in primary human melanocytes (H Seberg, E van Otterloo, and RA Cornell, manuscript in preparation). In addition, TFAP2A binding motifs are enriched at BRG1 bound sites (Figure 5-figure supplement 1B). In agreement with this, of the 13,693 TFAP2A-bound sites in human melanocytes, 6432 sites co-localized with BRG1 and H3K27ac defining a large set of active TFAP2A-bound regulatory elements (Figure 7-figure supplement 1B). Also, at more than 1000 sites, MITF was located at varying distances 5′ or 3′ to TFAP2A, and YY1 and TFAP2A colocalized at 4819 sites (Figure 7-figure supplement 1C and data not shown). Integrative analysis identified 762 YY1-TFAP2A sites also occupied by BRG1 and MITF and marked by H3K27ac, defining a set of TFAP2A-YY1-containing MAREs (Figure 7-figure supplement 1D). Analysis of the genes associated with these MAREs showed enrichment in those involved in lumen organisation, melanosomes, and transcriptional regulation (data not shown).

Genome-wide recruitment of BRG1 by MITF and SOX10

To determine whether MITF and/or SOX10 actively recruit BRG1 to the genome, we performed BRG1-ChIP-seq following siRNA-mediated silencing of MITF, SOX10 or siLuciferase (Luc) as control. At the TYR locus, siMITF silencing led to the specific loss of BRG1 at a promoter proximal site (* in Figure 8A), whereas at the upstream site where MITF co-localized with SOX10 little change was observed (arrowheads in Figure 8A). In contrast, siSOX10 and consequent loss of both SOX10 and MITF (see above), diminished BRG1 at all sites. Interestingly, BRG1 occupancy was observed throughout the region between these sites. siSOX10 silencing led to BRG1 loss across the entire region suggesting that it is recruited to the SOX10/MITF sites and then may spread across the locus. A similar situation was observed at the CIT locus encoding a MITF-regulated gene essential for cell cycle progression, with two downstream regulatory elements comprising MITF and SOX10 sites (* and arrowheads in Figure 8B). At both sites, BRG1 was strongly reduced upon siMITF or siSOX10 silencing. A global analysis identified sites showing a strong and preferential loss of BRG1 following, siSOX10 (clusters B and G-H, Figure 8C), siMITF (clusters E and F) or both, (cluster D), defining sites where MITF and SOX10 independently or cooperatively recruit BRG1 (Figure 8D). These data indicate that MITF and SOX10 actively recruit BRG1 either cooperatively or specifically to a large number of genomic loci.

While many genes associated with MAREs were down-regulated upon MITF silencing, MAREs may also be involved in gene silencing. Expression of SERPINE1 and IL24, two SASP components, was strongly up-regulated in senescent siMITF cells (see above). At both loci, BRG1 and MITF were present at either the promoter and/or distal regulatory regions and were lost upon siMITF coinciding with activation of their expression (Figure 8-figure supplement 1A-B). These observations suggest that BRG1 recruitment by MITF at these genes acts to silence their expression. Furthermore, BRG1 was re-localized over the genome to new sites during this process for example to CCL2 that was strongly induced upon siMITF silencing (Figure 8-figure supplement 1C).

We also noted that different sequence motifs were enriched at MITF-bound sites associated with either up or down regulated genes (Figure 6-figure supplement 1C-D). In particular, we observed that nuclear receptor half sites were enriched at loci associated with both classes. The finding of nuclear receptor half sites associated with MITF is unexpected as little is known about nuclear receptor function in melanoma although a therapeutic role of LXRb agonists has been demonstrated [START_REF] Pencheva | Broad-spectrum therapeutic suppression of metastatic melanoma through nuclear hormone receptor activation[END_REF]. Nuclear receptors can therefore be added to the list of factors that may interact functionally with MITF.

We next investigated whether on the other hand MITF occupancy is affected by BRG1 silencing using Cl8 cells where BRG1 loss did not affect expression of ectopic 3HA-tagged MITF (see above). Anti-HA ChIP-qPCR at MITF sites not associated with BRG1 co-occupancy showed only a mild reduction in MITF occupancy (Figure 9A, group A). In contrast, at many co-occupied sites, BRG1 silencing resulted in a marked increase in MITF occupancy. This was observed for example at the GPR110, UVRAG, and SOX6 loci (group B), where MITF binds to sites located between BRG1-bound nucleosomes (Figure 9B). Increased occupancy is not however seen at all co-occupied sites since at the DCT and SOX10 loci MITF occupancy was not affected (group C). Thus, BRG1 regulates the dynamics of MITF binding to a subset of sites

Discussion

A complex network of protein interactions around MITF

A comprehensive characterisation of the MITF interactome revealed its association with multiple complexes, including PBAF and NURF, TFIIIC, cohesins and multiple enzymes of the ubiquitin cycle. TFE3, TFEB, and TFEC also co-purify with MITF raising the question of which sites are bound as MITF homodimers or MITF-TFE heterodimers and whether binding as homo-or heterodimers has functional consequences for transcription regulation. Mouse genetic studies have not revealed functions for the TFE factors in development of the melanocyte lineage (Steingrimsson et al., 2002), however their role in melanoma has not been fully evaluated.

A striking finding of our study is the interaction of MITF with multiple proteins involved in DNA damage and repair, suggesting that MITF may influence these processes. Nevertheless, we so far found no recruitment of MITF to DNA damage sites (unpublished observations), the significance of these interactions therefore remains to be determined. Similarly, MITF associates with TFIIIC suggesting that it may regulate Pol III transcription. Alternatively, the presence of cohesin subunits in the interactome suggests that MITF may associate with TFIIIC at 'Extra TFIIIC' sites that organise chromatin structure in association with the cohesin complex (Noma et al., 2006;Kirkland et al., 2013).

MITF also interacts with complexes involved in DNA replication. Assembly of DNA replication origins can be influenced by the presence of transcription regulatory elements and MYC that also interacts with MCM subunits, has been shown to directly influence DNA replication (Dominguez-Sola et al., 2007;[START_REF]Eukaryotic DNA replication origins: many choices for appropriate answers[END_REF]. Perhaps MITF interaction with these replication complexes facilitates replication origin assembly at MITF-occupied sites thereby coupling replication with transcription regulation in a melanocyte-specific manner.

A novel PBAF complex containing BRG1 and CDH7 is a cofactor for MITF MITF interacts with BRG1, but the related Brahma (BRM) protein was not detected, although both proteins are expressed in 501Mel cells (Keenen et al., 2010). MITF interacts with a PBAF-like complex comprising CHD7. We have so far been unable to co-localize BRG1 and CHD7 by ChIP-seq due to the lack of ChIP-seq grade CHD7 antibodies (our unpublished data).

In 501Mel cells, BRG1 regulates an extensive gene expression programme including MITF and SOX10 whose expression is lost upon BRG1 silencing and cells undergo growth arrest and senescence characterised by a SASP similar to that seen upon MITF silencing. This requirement for BRG1 to drive a large gene expression programme required for melanoma cell proliferation in vitro contrasts with its tumour suppressor function in human cancers including melanoma (Hargreaves and Crabtree, 2011;Shain and Pollack, 2013;Bastian, 2014;Wang et al., 2014).

BRG1 is also essential in Hermes-3A cells and the overlapping gene expression changes suggest that BRG1 acts as an MITF cofactor in these cells. Nevertheless, while the impact of BRG1 silencing is less extensive than in 501Mel cells the changes in gene expression lead to senescence and eventual cell death. As BRM is expressed in Hermes-3A cells, there is a potential for redundancy between BRG1 and BRM that could account for the less extensive effect, although it is important to note that this is not the case in 501Mel cells. BRG1 knockout in developing mouse melanocytes in vivo leads to complete loss of pigmentation and immunostaining failed to reveal the presence of Sox10 or Dct expressing cells in the hair follicle of the mutant animals. The absence of expression of these important markers indicates that there are no identifiable melanocytes in the Smarca4 mel-/-hair follicles. This together with the arrested proliferation and cell death seen in melanoma cells and melanocytes in vitro, indicate that loss of pigmentation most likely results from an absence of melanocytes in these animals. Together our data identify BRG1 as an essential MITF cofactor in melanoma and melanocyte/melanoblast cells in vitro and in vivo. BRG1-YY1-SOX10-TFAP2A-MITF co-localization defines sets of MAREs with a specific chromatin organisation BRG1 binds extensively over the melanoma cell genome often at active H3K27ac-marked enhancers consistent with previous reports (Hu et al., 2011). Nevertheless, we describe here a novel profile where BRG1 often binds as two peaks separated by 250-800 base pairs. These BRG1 sites define two classes of elements. The first is a subset of TSS, where BRG1 occupies the nucleosomes flanking the NDR with the Pol II pause site located immediately 5′ of the +1 nucleosome. This specific positioning of BRG1 at the TSS was not noted in several previous ChIP-seq studies (De et al., 2011;Euskirchen et al., 2011;Ho et al., 2009;Hu et al., 2011;Yu et al., 2013;Morris et al., 2014), however, Tolstorukov et al. (2013) reported that BRG1 occupies the nucleosomes flanking the TSS. Tolstorukov et al. also reported that inactivation of BRG1 does not affect the positioning of these two nucleosomes, but rather elicits a strong reduction in their occupancy. Nevertheless, the overall meta-profiles presented by Tolstorukov et al., analogous to that reported here, overlooked two important features: (1) that many promoters show BRG1 occupancy of only the -1 or +1 nucleosome, and ( 2) that -1 or +1 nucleosome location is variable (Fenouil et al., 2012).

The second class corresponds to TF binding sites in promoter and enhancer elements. The observed variability in distances separating the BRG1-bound nucleosomes likely reflects the number of TFs bound in the intervening regions. For example, combinations of MITF, SOX10, YY1, TFAP2A as well as other TFs such as ETS1 bind between two BRG1-bound nucleosomes. At many of these sites the BRG1-bound nucleosomes are also marked by H3K27ac, thus defining MAREs active in regulating melanocyte lineage gene expression. As SOX10, YY1, TFAP2A, and ETS1 all have important roles in melanocytes and/or melanoma, the MAREs identified here define a combinatorial signature of TFs critical for gene regulation in this lineage (Figure 9). This is further underlined by the recent finding that a combination of MITF, SOX10, and PAX3 can reprogram fibroblasts into functional melanocytes (Yang et al., 2014). Ondrusova et al. reported an MITF-independent pro-survival role for BRG1 in melanoma cells (Ondru šov á et al., 2013). This observation is in agreement with our findings that BRG1 silencing affects expression of many more genes than MITF and that the binding motifs for a variety of factors other than MITF are enriched at BRG1-occupied sites. BRG1 is likely therefore to act as a cofactor for many other TFs accounting for its MITF-independent functions.

Our data are consistent with the idea that MITF and other combinations of TFs bind the DNA between two BRG1-occupied and H3K27ac-marked nucleosomes. Such an organisation has been previously proposed based on extensive ChIP-seq profiling by the Encode consortium showing that TF binding sites are often combinatorial and correspond to GC-rich, DNaseI hypersensitive NDRs flanked by two positioned nucleosomes (Wang et al., 2012). Our data support this idea through the identification of combinatorial MAREs and they extend it by showing that the nucleosomes flanking the TF binding sites are often bound by BRG1 and marked with H3K27ac (Figure 9). These regulatory elements show an analogous organisation to that of the TSS that also comprise a NDR encompassing variable numbers of TF binding sites and the PIC, flanked by BRG1-bound nucleosomes. Although examples of association between BRG1 and TFs have been previously described [START_REF] Trotter | The BRG1 transcriptional coregulator[END_REF]Reisman et al., 2009;Euskirchen et al., 2011), this specific configuration of BRG1 binding to nucleosomes flanking the TF binding sites has not been generally recognised. In yeast, it has however been reported that TF binding sites are rather flanked by nucleosomes bound by ISWI and CHD remodellers (Zentner et al., 2013).

While there have been many examples of co-localization between BRG1 and TFs, this is the first description of active genome-wide BRG1 recruitment. siRNA-mediated MITF or SOX10 silencing identified sites to which BRG1 was recruited either individually or cooperatively by these TFs. Moreover, we show that MITF associates with PBAF in the soluble nuclear fraction suggesting that they form a complex in the nucleoplasm and are recruited simultaneously to the chromatin to create the NDRs (Figure 9C).

Chromatin remodelling has been shown to facilitate TF binding, a good example being that of TAL1 where BRG1 repositions nucleosomes flanking its binding sites (Hu et al., 2011). In contrast, we observed that MITF occupancy of many co-occupied sites either shows little change or is increased following siBRG1 silencing suggesting that BRG1 regulates the dynamics of MITF binding. TF binding to chromatin is often extremely dynamic with ChIP capturing only a snapshot of their occupancy (Voss and Hager, 2014). The increased MITF occupancy seen in ChIP upon BRG1 silencing suggests an increase in its time of residence at occupied sites.

Several mechanisms may explain the increased MITF binding upon BRG1 loss. The human genome comprises many more consensus E-box elements than are occupied by MITF. The excess of consensus as well as degenerate E-boxes could potentially act as a sink thus limiting the pool of MITF for binding to functional sites. One function of BRG1-driven dynamics may therefore be to limit MITF sequestration at non-functional sites and ensure a pool of MITF for binding to functional sites. Alternatively, BRG1 may be required to establish the NDRs for MITF binding, for example after mitosis, or at specific stages of the cell cycle. As siBRG1 cells are post-mitotic and senescent, the increased MITF occupancy may reflect a new steady state where MITF remains more stably bound to the NDRs established while BRG1 was still present compared to cycling cells where the NDRs are established and erased in a more dynamic manner. Irrespective of the underlying causes, our results indicate that BRG1 regulates dynamic MITF interactions with chromatin.

Materials and methods

Generation of 501Mel cells stably expressing F-H-MITF

501Mel cells cultured in RPMI 1640 medium (Sigma, St Louis, MO, USA) supplemented with 10% fetal calf serum (FCS) were transfected with a CMV-based vector expressing Flag-HA-tagged MITF and a vector encoding puromycin resistance. Transfected cells were selected with puromycin (3 μg/ml), and the expression of MITF verified by western blot using the MITF antibody ab-1 (C5) from Neomarkers, the 12CA5 HA antibody (Roche, Basel, Switzerland), or the M2 Flag antibody (Sigma). Details of other cell culture are provided in Supplementary information. Hermes 3A cells were obtained from the University of London St Georges repository.

Tandem immunoaffinity purification and mass-spectrometry

Cell extracts were prepared essentially as previously described and subjected to tandem Flag-HA immunoprecipitation [START_REF] Dran É | The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3[END_REF]. Cells were lysed in hypotonic buffer (10 mM Tris-HCl at pH 7.65, 1.5 mM MgCl 2 , 10 mM KCl) and disrupted by Dounce homogenizer. The cytosolic fraction was separated from the pellet by centrifugation at 4˚C. The nuclear soluble fraction was obtained by incubation of the pellet in high salt buffer (final NaCl concentration of 300 mM) and then separated by centrifugation at 4˚C. To obtain the nuclear insoluble fraction (chromatin fraction), the remaining pellet was digested with micrococcal nuclease and sonicated. Tagged proteins were immunoprecipitated with anti-Flag M2-agarose (Sigma), eluted with Flag peptide (0.5 mg/ml), further affinity purified with anti-HA antibody-conjugated agarose (Sigma), and eluted with HA peptide (1 mg/ml). The HA and Flag peptides were first buffered with 50 mM Tris-HCl (pH 8.5), then diluted to 4 mg/ml in TGEN 150 buffer (20 mM Tris at pH 7.65, 150 mM NaCl, 3 mM MgCl 2 , 0.1 mM EDTA, 10% glycerol, (C) A model for regulatory elements in the melanocyte lineage. Melanocyte lineage enhancers comprise combinations of MITF, SOX10, YY1, and also TFAP2A and ETS1 (not represented for simplicity. Note also that Pol II and the PIC are present at active enhancers where enhancer RNAs are made. For simplicity these are also not represented.) bound to a nucleosome-depleted region. MITF but also these other factors recruit BRG1/PBAF to the nucleosomes flanking the combinations of transcription factors. BRG1/PBAF also occupies the nucleosomes flanking the TSS and a subset of these promoters further comprises a MITF binding site close to the TSS. DOI: 10.7554/eLife.06857.018

Laurette et al. eLife 2015;4:e06857. DOI: 10.7554/eLife.06857 0.01% NP40), and stored at -20˚C until use. Between each step, beads were washed in TGEN 150 buffer. Complexes were resolved by SDS-PAGE and stained using the Silver Quest kit (Invitrogen, La Jolla, CA, USA). Mass-spectrometry was performed at the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA).

Cell culture and lentiviral infections

Melanoma cell lines SK-Mel-28 and 501Mel were grown in RPMI 1640 medium (Sigma) supplemented with 10% FCS. 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS and penicillin/streptomycin (7.5 μg/ml). Hermes-3A cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% FCS, 200 nM TPA, 200 pM cholera toxin, 10 ng/ml human stem cell factor (Invitrogen), 10 nM endothelin-1 (Bachem, Bubendorf, Switzerland), and penicillin/streptomycin (7.5 μg/ml). All lentiviral shRNA vectors were obtained from Sigma (Mission sh-RNA series) in the PLK0 vector. The following constructs were used. shBRG1 (TRCN0000015549) and shMITF (TRCN0000019119). In each case between 5 × 10 5 and 1 × 10 6 cells were infected with the indicated shRNA lentivirus vectors and all experiments were performed at least in triplicate. siRNA knockdowns were performed with the corresponding ON-TARGET-plus SMARTpools purchased from Dharmacon Inc. (Chicago, Il., USA). Control siRNA directed against luciferase was obtained from Eurogentec (Seraing, Belgium). siRNAs were transfected using Lipofectamine RNAiMax (Invitrogen).

Transfections, extract preparation and antibodies

Transient and stable transfections were performed with 5 μg of expression vectors and using FuGENE 6 reagent (Roche) following the manufacturer's instructions. Medium was replaced 24 hr and cells were collected 48 hr after transfection. Cells lysis was performed using LSDB 500 buffer (500 mM KCl, 25 mM Tris at pH 7.9, 10% glycerol, 0.05% NP-40, 1 mM DTT, and protease inhibitor cocktail). Up to 3 mg of whole cell extracts were diluted in LDSB without KCl to obtain a final concentration of 100 mM KCl and incubated for 12 hr with 5 μg of specific antibody and 50 μl Slurry of protein-G sepharose (GE Healthcare). Beads were washed 3 times in LSDB 300, twice in LSDB 150, and boiled in Laemmli buffer before protein separation by SDS-PAGE. For flag immunoprecipitations, extracts were incubated with 50 μl Slurry of Anti-Flag M2-agarose affinity gel (Sigma) and washed similarly prior to elution with Flag peptide (0.5 mg/ml). Immunoblots were performed with the following antibodies: MITF (MS-771-P; Interchim), BRG1 (ab110641; Abcam, Cambridge, UK), HERC2 (612366; BD Transduction Laboratories, Sparks, MD), USP11 (3263-1; Epitomics, Burlingame, CA), USP7 (#4833; Cell Signaling, Danvers, MA), TRRAP (2TRR-2D5; IGBMC), NEURL4 (sc-243603; scbt, Santa Cruz, CA), actin (2D7; IGBMC), XRCC6 (sc-17789; scbt), XRCC5 (sc-5280; scbt), BAF170 (A301-038A; Bethyl Laboratories, Montgomery, TX), BAF155 (sc-10756; scbt), BAF250A (sc-373784; scbt), BAF250B (sc-32762; scbt), BAF200 (ab56082; Abcam), BAF53A (ab131272; Abcam), CHD7 (ab31824; Abcam), BAF180 (ab137661; Abcam), BAF60A (#611728; BD Transduction Laboratories), BAF60B (ab166622; Abcam), SOX10 (ab155279; Abcam), CREB (#06-863; Upstate Millipore, Molsheim, France).

Mice and genotyping

The Smarca4 lox/lox and Tyr::Cre strains have been described previously (Indra et al., 2005;Delmas et al., 2003). Genotyping of F1 offspring was carried out by PCR analysis of genomic tail DNA with primers detailed in the respective publications. All animals were handled according to institutional and national guidelines and policies.

Chromatin immunoprecipitation and sequencing

BRG1 ChIP experiments were performed on native Mnase-digested chromatin. 5 × 10 7 to 5 × 10 8 freshly harvested 501Mel cells were resuspended in 2 ml ice-cold hypotonic buffer (0.3M Sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 15 mM Tris-HCl [pH 7.5], 0.5 mM DTT, 0.1 mM PMSF, protease inhibitor cocktail) and cytoplasmic fraction was released by incubation with 2 ml of lysis-buffer (0.3M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 15 mM Tris-HCl [pH 7.5], 0.5 mM DTT, 0.1 mM PMSF, PIC, 0.5% (vol/vol) IGEPAL CA-630) for 10 min on ice. The suspension was layered onto a sucrose cushion (1.2 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl 2 , 0.1 mM EDTA, 15 mM Tris-HCl [pH 7.5], 0.5 mM DTT, 0.1 mM PMSF, PIC) and centrifuged for 25 min at 4700 rpm in a swing rotor. The nuclear pellet was resuspended in digestion buffer (0.32M sucrose, 50 mM Tris-HCl [pH 7.5], 4 mM MgCl 2 , 1 mM CaCl 2 , 0.1 mM PMSF) and subjected to Micrococcal Nuclease digestion for 5 min at 37˚C. The reaction was stopped by addition of EDTA and suspension chilled on ice for 10 min. The suspension was cleared by centrifugation at 10,000 rpm (4˚C) for 10 min and supernatant (chromatin) was used for further purposes. Chromatin was digested to around 80% of mono-nucleosomes as judged by extraction of the DNA and agarose gel electrophoresis. SOX10 and 3HA-MITF ChIP experiments were performed on 0.4% PFA-fixed chromatin isolated from 501Mel and Cl8 cells, respectively according to standard protocols as previously described (Strub et al., 2011). ChIP-seq libraries were prepared as previously described and sequenced on the Illumina Hi-seq2500 as single-end 50-base reads (Herquel et al., 2013). After sequencing, peak detection was performed using the MACS software ( [Zhang et al., 2008] http:// liulab.dfci.harvard.edu/MACS/). Peaks were then annotated with GPAT [START_REF] Krebs | GPAT: retrieval of genomic annotation from large genomic position datasets[END_REF] using a window of ±10 kb (or as indicated in the figures) relative to the transcription start site of RefSeq transcripts. Global clustering analysis and quantitative comparisons were performed using seqMINER ( [START_REF] Ye | seqMINER: an integrated ChIPseq data interpretation platform[END_REF] http://bips.u-strasbg.fr/seqminer/) and R (http://www.r-project.org/). The public human foreskin melanocyte H3K27ac data were taken from the Geo entry GSM958157.

De novo motif discovery on FASTA sequences corresponding to windowed peaks was performed using MEME-ChIP. Motif correlation matrix was calculated with in-house algorithms using JASPAR database.

mRNA preparation, quantitative PCR and RNA-seq mRNA isolation was performed according to standard procedure (Qiagen kit, Venlo, Holland). qRT-PCR was carried out with SYBR Green I (Qiagen) and Multiscribe Reverse Transcriptase (Invitrogen) and monitored by a LightCycler 480 (Roche). Detection of Actin gene was used to normalize the results. RNAseq was performed essentially as previously described (Herquel et al., 2013). Gene ontology analyses were performed using the functional annotation clustering function of DAVID (http://david.abcc.ncifcrf.gov/). Primers for RT-qPCR and ChIP-qPCR were designed using Primer 3 and are listed in Supplementary file 5.

Motif analysis

Searching of known TF motifs from the Jaspar 2014 motif database at BRG1-bound sites was made using FIMO [START_REF] Grant | FIMO: scanning for occurrences of a given motif[END_REF] within regions of 200 bp around peak summits, FIMO results were then processed by a custom Perl script which computed the frequency of occurrence of each motif. To assess the enrichment of motifs within the regions of interest, the same analysis was done 100 times on randomly selected regions of the same length as the BRG1 bound regions and the results used to compute an expected distribution of motif occurrence. The significance of the motif occurrence at the BRG1-occupied regions was estimated through the computation of a Z-score (z) with z = (xμ)/σ, where:x is the observed value (number of motif occurrence),μ is the mean of the number of occurrences (computed on randomly selected data),σ is the standard deviation of the number of occurrences of motifs (computed on randomly selected data). The source code is accessible at https:// github.com/slegras/motif-search-significance.git.

Immunostaining

Biopsies of dorsal skin were isolated and fixed overnight in 4% paraformaldehyde, washed with PBS, dehydrated, paraffin embedded, and sectioned at 5 μm. For antigen retrieval, the sections were incubated with 10 mM sodium citrate buffer, within a closed plastic container placed in a boiling waterbath, for 20 min. Sections were permeabilised with 3 × 5 min 0.1% Triton in PBS, blocked for 1 hr in 5% skim milk in PBS, and incubated overnight in 5% milk with primary antibodies. The following antibodies were used: goat anti-Dct at dilution of 1/1000 (Santa Cruz Biotechnology, sc-10451) and rabbit anti-Sox10, at 1/2000 (Abcam, ab155279). Sections were washed 3 × 5 min 0.1% Triton in PBS, and incubated with secondary antibodies, Alexa 488 donkey-anti-goat, and Alexa 555 donkey-antirabbit (Invitrogen) for 2 hr. Sections were subsequently incubated with 1/2000 Hoechst nuclear stain for 10 min, washed 3 × 5 min in PBS, dried and mounted with Vectashild.

Senescence-associated β-galactosidase assay

The senescence-associated β-galactosidase staining kit from Cell signaling technology (Beverly, MA, USA) was used according to the manufacturer's instructions to histochemically detect β-galactosidase activity at pH 6.

Funder

Grant reference Author

Ligue

Major datasets

The following dataset was generated:

Figures and figure supplements

Introduction

MIcrophthalmia-associated Transcription Factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-Zip) factor playing an essential role in the differentiation, survival, and proliferation of normal melanocytes, and in controlling the melanoma cell physiology [1][2][3][4]. Quiescent murine adult melanocyte stem cells (MSCs) residing in the bulge region of the hair follicle do not express MITF, but its expression is induced in the proliferating transit amplifying cells (TACs) that are generated at anagen [5][6][7]. MITF expression persists as TACs migrate towards the bulb to form terminally differentiated melanin producing melanocytes [8,9]. Following malignant transformation of melanocytes, cells expressing low or no MITF are slow cycling and invasive, displaying enhanced tumour initiating properties, high MITF activity is characteristic of proliferative melanoma cells, and even higher MITF activity is associated with terminal differentiation of melanocytes [10]. MITF silencing in proliferative melanoma cells leads to cell cycle arrest and entry into senescence [11,12]. These and other observations gave rise to the proposed 'rheostat' model postulating that the level of functional MITF expression determines many biological properties of melanocytes and melanoma cells [13,14]. MSCs and slow cycling melanoma cells express low or no MITF, while TACs and proliferative melanoma cells express higher levels. High MITF activity induces terminal melanocyte differentiation and can also induce cell cycle arrest of melanoma cells [15]. This property has been exploited to derive drugs that induce terminal differentiation of melanoma cells as a therapy for melanoma [16].

MITF is both an activator and a repressor of transcription and functions through a host of cofactors. A comprehensive analysis of the MITF 'interactome' in 501Mel melanoma cells revealed that the NURF (Nucleosome Remodelling Factor) complex associates with MITF [17]. NURF was first identified in Drosophila Melanogaster [18,19] and comprises NURF301 (in mammals, BPTF, Bromodomain, PHD-finger Transcription Factor), the ISWI-related SNF2L (SMARCA1) ATPase subunit, NURF55 (RbAp46, RBBP7) and NURF38 [20][21][22][23]. NURF promotes ATP-dependent nucleosome sliding and transcription from chromatin templates in vitro [24][25][26]. Mammalian NURF comprises BPTF, RBBP4 and SNF2L and may further comprise the SNF2H (SMARCA5) ATPase subunit, BAP18 and HMG2L1 [27,28]. The 450 kDa BPTF is the defining and only unique subunit of NURF and binds active promoters via the interaction of its PHD (plant homeodomain) domain with trimethylated H3K4 and of its bromodomain with acetylated H4K16 [21,[START_REF] Ruthenburg | Recognition of a mononucleosomal histone modification pattern by BPTF via multivalent interactions[END_REF][START_REF] Li | Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF[END_REF].

Despite extensive characterisation of the biochemical properties of the NURF complex and its BPTF subunit [START_REF] Badenhorst | Biological functions of the ISWI chromatin remodeling complex NURF[END_REF], much less is known about their biological functions in mammals. Bptf inactivation in mouse leads to embryonic lethality shortly after implantation [32,[START_REF] Landry | Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells[END_REF]. Bptf loss is not however cellular lethal as it is possible to isolate viable Bptf -/-ES cells, but in vitro they show defective differentiation into mesoderm, and endoderm lineages [START_REF] Landry | Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells[END_REF]. Somatic inactivation of Bptf in CD4-CD8 double negative thymocytes has shown that it is required for their subsequent maturation [START_REF] Landry | Chromatin remodeling complex NURF regulates thymocyte maturation[END_REF]. Furthermore, BPTF may also be involved in maintaining human epidermal keratinocyte stem cells in an undifferentiated state in vitro [START_REF] Mulder | Diverse epigenetic strategies interact to control epidermal differentiation[END_REF].

The interaction of MITF with NURF prompted us to investigate its role in melanoma cells and in the melanocyte lineage. BPTF is selectively required in melanoma cells and melanocytes in vitro. Inactivation of Bptf in developing melanoblasts (Bptf mel-/-) shows that it acts during their embryonic proliferation and migration, and we observed a unique phenotype in neonatal mice where the second post-natal anagen coat is devoid of pigment resulting in rapid loss of pigmentation and a lasting white pelage. Contrary to previous models where premature postnatal greying results from loss of MSCs, in Bptf mel-/-mice an MSC population persisted throughout the life of the animal, but at anagen, Bptf is required for normal TAC proliferation, for expression of melanocyte markers and hence for terminally differentiation into mature melanin producing melanocytes.

Results

MITF associates with NURF in melanoma cells

We previously described tandem affinity purification of N-terminal FLAG-HA epitope tagged MITF from the soluble nuclear and chromatin associated fractions of 501Mel cells [17]. Amongst the identified proteins were the BPTF, SMARCA5, SMARCA1 and RBBP4 subunits of the NURF complex. Multiple peptides for these proteins were detected in the chromatinassociated fraction from the cells expressing tagged MITF, whereas no peptides for these factors were found in immunoprecipitations from the control extract (S1A Fig) . Interaction of these NURF components with MITF was confirmed in western blot experiments showing that BPTF, SMARCA1 and SMARCA5 all specifically precipitated with tagged MITF in the chromatin-associated fraction (S1B Fig) . MITF therefore associates, either directly or indirectly, with the NURF complex on chromatin in 501Mel cells.

We next interrogated transcriptome data [START_REF] Rambow | New functional signatures for understanding melanoma biology from tumor cell lineage-specific analysis[END_REF] to assess expression of the BPTF, SMARCA1 and SMARCA5 subunits of NURF in a collection of human melanoma cells. The three subunits were expressed at comparable levels in all of the tested cell lines, whether they expressed high (501Mel, 888-mel) low (SK-Mel-28, LYSE) or no (1205Lu, WM852) MITF (S1C Fig) . The expression of BPTF, SMARCA1 and SMARCA5 proteins was also assessed in extracts from a subset of these lines. Again, the levels of each protein were comparable in the different lines (S1D Fig) . NURF is therefore present in all types of melanoma cells irrespective of MITF expression and their tumourigenic properties.

Selective requirement of BPTF in melanoma cells

To address the function of BPTF in 501Mel cells, we performed both siRNA and shRNA knockdown experiments (Fig 1A). SiBPTF led to prominent morphological changes in 501Mel cells similar to those observed following siRNA silencing of MITF (Fig 1B). In both cases, cells showed an enlarged, flattened and irregular morphology with extensive cytoplasmic projections. Similar results were observed following infection with lentiviral vectors expressing two different shRNAs directed against BPTF both of which led to diminished BPTF levels whereas MITF expression was unaffected (Fig 1A). ShBPTF silencing also led to strongly diminished SMARCA5 and SMARCA1 protein levels (Fig 1A), although the expression of the corresponding genes was not reduced (see below). As BPTF is so far believed to be exclusive to the NURF complex [20], the loss of SMARCA5 and SMARCA1 suggests that a large fraction of these proteins is associated with BPTF in the NURF complex that is destabilized by BPTF silencing. ShBPTF knockdown therefore leads to a loss of NURF function possibly through its chromatin-remodeling activity.

After 5 days of shBPTF silencing, cells displayed marked morphological changes analogous to those seen following siBPTF silencing and si/shMITF silencing (Fig 1C and1D). These morphological changes were characteristic of those observed when 501Mel cells enter senescence [12,[START_REF] Giuliano | Microphthalmia-associated transcription factor controls the DNA damage response and a lineage-specific senescence program in melanomas[END_REF] and up to 90% of shBPTF or shMITF silenced cells showed staining for senescenceassociated β-galactosidase (Fig 1C and1D). BPTF silencing therefore induced senescence in 501Mel cells.

Analogous results were observed in several other MITF-expressing melanoma cell lines. ShRNA-mediated BPTF silencing in SK-Mel-28 cells led to reduced cell number and marked morphological changes with many bi-nucleate and multi-nucleate cells (S2A, S2B and S2C We also investigated BPTF function in MITF-negative 1205Lu cells. In this cell line also, BPTF silencing induced an enlarged, flattened more rounded senescence-like morphology (S2D, S2E and S2F Fig) . On the other hand, shMITF silencing had no effect on these cells consistent with the fact that they do not express MITF.

In contrast to the above, shBPTF silencing in a variety of non-melanoma cells such as HeLa (cervical cancer), HEK293T (human embryonic kidney) had no effect on either proliferation or morphology (S3A,S3B and S3C Fig). These observations show that BPTF plays a selective and essential role in melanoma cells that is not seen in non-melanoma cells. Moreover, as BPTF is essential in 1205Lu cells, it may have both MITF-dependent and independent functions in melanoma.

BPTF and MITF regulate overlapping gene expression programs in 501Mel cells

As BPTF and MITF silencing in 501Mel cells generated similar phenotypes, we performed RNA-seq following shRNA-mediated silencing and compared the de-regulated gene expression programs. Following BPTF knockdown, 494 genes were down-regulated (Fig 1E ), enriched in ontology terms associated with regulation of transcription, kinase signalling, pigmentation and cell cycle (Fig 1F and S1 Dataset). 593 genes were down-regulated by MITF knockdown, enriched in cell cycle, in particular in mitosis, consistent with previous observations that MITF silencing leads to severe mitotic defects [12,17]. Comparison of the two data sets identified 191 common repressed genes associated with transcription regulation and kinase signalling (Fig 1E and1F). Thus, 39% of genes down-regulated by BPTF silencing were also down-regulated by MITF silencing indicating a large overlap between the programs regulated by each factor. To determine the statistical significance of this overlap, we used hypergeometric probability to calculate the representation factor (RF) that determines whether the number of genes in the overlap is higher than expected by chance taking into account the number of genes shBPTF-1 cells are shown, but analogous images were made for shBPTF-2. D. Staining and quantification of 501Mel cells following shBPTF and shMITF knockdown for senescence-associated β-galactosidase. E. Venn diagrams illustrate the overlap between up and down-regulated genes following shBPTF and shMITF knockdown. Several examples of commonly regulated up and down-regulated genes are indicated based on the results of RNA-seq. RF shows the representation factor and p value for the overlaps between the up and down-regulated genes sets. F Ontology of genes up and down-regulated by shBPTF and shMITF knockdown and of commonly regulated genes. The number of genes in each category is indicated along with the p value. SASP components whose expression is induced by shMITF and ShBPTF are listed on the right. doi:10.1371/journal.pgen.1005555.g001 regulated by MITF and BPTF with respect to the total number of expressed genes. For the common down-regulated genes the RF was 9.3 (p < 6.048e-141) showing the high statistical significance of the overlap. Some examples of co-regulated genes are listed in Fig 2 . Moreover, the number of co-regulated genes was highest in the top quartiles of shMITF-regulated genes (44% in quartile 1, S4A Fig) . Genes with strongest dependency on MITF were therefore more often co-regulated by BPTF than those of the bottom quartiles whose expression is least affected by MITF loss.

Following BPTF knockdown, 669 genes were up-regulated (Fig 1E and1F), enriched in cell adhesion, morphology and motion as well as a set of secreted cytokines and growth factors that constitute the senescence associated secreted phenotype (SASP). 748 genes were up-regulated by MITF knockdown, enriched in terms analogous to those of BPTF including an extensive SASP [17,[START_REF] Ohanna | Senescent cells develop a PARP-1 and nuclear factor-{kappa}B-associated secretome (PNAS)[END_REF]. Comparison of the two data sets identified 278 common induced genes, with 41% of genes up-regulated by BPTF silencing also induced by MITF silencing. For the common up-regulated genes the RF was 7.9 (p < 3.495e-191) again showing the statistical significance of this overlap. As noted above for down-regulated genes, the number of co-regulated genes was highest in the top quartiles of shMITF-regulated genes (57% in quartile 1, S4A Fig). Consistent with the similar morphological changes, the common regulated genes were involved in morphology and adhesion as well as the SASP. None of the genes repressed by MITF knockdown were activated by BPTF knockdown and only 8 genes repressed by BPTF knockdown, showed an opposite regulation, being activated by MITF knockdown.

These data show a large and significant overlap between the gene expression programs controlled by BPTF and MITF. Together these two factors positively regulate genes required for proliferation and negatively regulate genes involved in modulating cell morphology and motility.

We determined which up and down-regulated genes are associated with MITF-occupied sites. As described [17], MITF occupies >16000 sites in 501Mel cells. Using a window of +/-30kb with respect to the TSS, taking into account potential regulation by MITF from distant enhancers, identified up to 5694 potential targets (S4B Fig) . Of these 176 genes are down-regulated upon MITF silencing consistent with them being directly activated by MITF. Several cell cycle regulators such as CCND1, ANLN and CIT were associated with multiple MITF binding sites (S2 Dataset). Up to 56 genes associated with MITF binding sites were co-regulated by BPTF, including BIRC7 SHB, and BCL2A1 critical regulators of proliferation, apoptosis and survival, NPM1 an important cell cycle regulator involved in chromosome congression, spindle and kinetochore-microtubule formation required for normal centrosome function [START_REF] Vogler | BCL2A1: the underdog in the BCL2 family[END_REF][START_REF] Okuwaki | The structure and functions of NPM1/Nucleophsmin/B23, a multifunctional nucleolar acidic protein[END_REF] and PPARGC1A (PGC1α) implicated in resistance to oxidative stress and mitochondrial function in melanoma [START_REF] Haq | Oncogenic BRAF regulates oxidative metabolism via PGC1alpha and MITF[END_REF][START_REF] Vazquez | PGC1alpha expression defines a subset of human melanoma tumors with increased mitochondrial capacity and resistance to oxidative stress[END_REF] (S4C Fig) . MITF and BPTF therefore co-regulate these critical MITF target genes. Similarly, up to 187 genes were potentially directly repressed by MITF including several SASP components such as SERPINE1, IL8, IL24 and PDGFB [see also [17]]. Of these 67 were co-regulated by BPTF such as SASP components IL8 and IL24. BPTF and MITF therefore appear to co-regulate gene expression in a positive and negative manner.

BPTF and MITF regulate proliferation of Hermes 3A melanocytes

We next investigated the role of BPTF in untransformed human melanocytes by shBPTF silencing in the Hermes 3A cell line. As previously shown, MITF silencing in these cells led to proliferation arrest, morphological changes and entry into senescence [ [17] and Fig 3A and 3C]. Fewer cells were also detected following BPTF knockdown, but the cells had a more bipolar morphology compared to the expanded and flattened morphology of the shMITF cells (Fig 3A,3B and 3C). Almost 85% of shBPTF cells showed senescence-associated β-galactosidase staining (Fig 3D). RNA-seq showed that BPTF silencing repressed 1356 genes and up-regulated 1139 genes (Fig 3E and S3 Dataset). The effects of BPTF loss on gene expression were therefore more extensive in these cells than in 501Mel. Down-regulated genes were strongly enriched in cell cycle, mitosis and pigmentation functions (Fig 3F). BPTF is therefore a major regulator of genes required for proliferation of Hermes 3A cells. The up-regulated genes on the other hand were involved in transcription regulation, cell-cell signalling, including many secreted and membrane associated proteins.

The gene expression programs regulated by BPTF and MITF overlapped significantly (RF = 5.1, p < 8.086e-162) as overall 44% of genes down-regulated by shMITF knockdown were also down-regulated by shBPTF (Fig 3E). As seen with 501Mel cells, the genes most strongly regulated by MITF were most often co-regulated by BPTF (62% in the first quartile, S4A Fig) . Co-regulated genes were associated with cell cycle/mitosis, cell adhesion and apoptosis (Figs 3F and 2). For example, several critical regulators of cell cycle and mitosis such as AURKB, CDCA2 and CCND1, genes associated with MITF occupied sites in 501Mel cells, were repressed under both conditions (Fig 2). Similarly, 37% (RF = 5, p < 1.259e-112) of genes upregulated by shMITF were also induced by shBPTF (Fig 3E and 3F) including a plethora of signalling molecules and transcriptional regulators. We next compared the gene expression programs regulated by silencing of BPTF, MITF and BRG1 in Hermes 3A cells. BPTF and BRG1 silencing commonly down-regulated 277 genes. Of these 122 were also regulated upon MITF silencing (S4D Fig) . In all, 58% of MITFregulated genes were co-regulated by either BRG1 or BPTF and 16% regulated by both. BPTF and BRG1 silencing commonly up-regulated 208 genes of which 126 were also up-regulated by MITF silencing (S4D Fig) . Overall, 56% of genes up-regulated by MITF were co-regulated by either BRG1 or BPTF and 19% regulated by both. A large fraction of MITF regulated genes are therefore co-regulated by these remodellers in Hermes 3A cells. A similar comparison could not be performed in 501Mel cells were BRG1 regulated a very large number of genes.

The transcriptional programs regulated by MITF in 501Mel and Hermes3A cells were somewhat different as only 172 genes (22%) were down-regulated and only 130 genes (19%) up-regulated in both lines (S4E Fig) . Nevertheless, the common down-regulated genes comprised regulators of cell cycle and mitosis defining a critical set of MITF-regulated cell cycle genes and up-regulated genes were principally involved in signalling and cell motion/morphogenesis. An analogous comparison of BPTF regulated genes showed 46% of common downregulated genes and 34% of up-regulated genes. Common down-regulated genes comprised regulators of cell cycle and up-regulated genes comprised genes involved in signalling and cell motion/morphogenesis (S4F Fig) . A small number of genes were also co-regulated by MITF and BPTF in both cells lines (shaded area in Fig 2).

Bptf acts during melanoblast proliferation and migration

The essential role of BPTF in melanoma and melanocyte cells in vitro prompted us to investigate its role in the murine melanocyte lineage in vivo. Interrogation of transcriptome data from purified melanoblasts (GFP+ cells, see [START_REF] Colombo | Transcriptomic analysis of mouse embryonic skin cells reveals previously unreported genes expressed in melanoblasts[END_REF]) and the GFP-cells (mainly keratinocytes) from E15.5 mouse embryos indicated that Bptf and Smarca5 were expressed in both cell types to levels comparable to those of the Smarca4 (Brg1) and Pbrm1 subunits of the PBAF complex that is essential for melanoblast development [START_REF] Laurette | Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells[END_REF], (S1C Fig) . However, no significant Smarca1 expression was seen at this stage.

We crossed mice with a floxed Bptf gene (Bptf lox/lox ) with those expressing Cre recombinase under the control of the Tyrosinase enhancer that allows selective inactivation in the developing melanocyte lineage at E9.5-E10.5 [START_REF] Delmas | Cre-mediated recombination in the skin melanocyte lineage[END_REF]. The resulting Tyr-Cre/°::Bptf lox/lox mice were further crossed with animals expressing the LacZ reporter gene under the control of the Dct promoter that marks cells of the melanocyte lineage (Tyr-Cre/°::Bptf lox/lox ::Dct-LacZ/°).

Following crosses of Tyr-Cre/°::Bptf lox/+ mice, Tyr-Cre/°::Bptf lox/lox animals in which Bptf was inactivated in the melanocyte lineage (Bptf mel-/-) displayed a pigmentation phenotype characterised by a grey belly, and grey extremities of the paws, ears and tail compared to wildtype mice and heterozygous Tyr-Cre/°::Bptf lox/+ (Bptf mel+/-) littermates (Fig 4A and S5A,S5B and S5C Fig). This phenotype was confirmed upon growth of the first hair at P14 that was grey on the belly with a small and variable white belly spot (Fig 4B and S5B Fig). Otherwise, the dorsal coat was almost indistinguishable from wild-type. Melanoblasts lacking Bptf are therefore viable, but BPTF regulates their proliferation and/or migration.

To better characterise this phenotype, we used the Tyr-Cre/°::Bptf lox/lox ::Dct-LacZ/°mice to monitor melanoblast development. The number of Dct-LacZ positive melanoblasts was counted at E15.5 on 4 mice of the Bptf mel-/-and Bptf mel+/-genotypes. The migration front on the belly and the paws was similar in both genotypes (S6A and S6B Fig) . In contrast, the number of melanoblasts was reduced by around 10% in the Bptf mel-/-foetuses (S6C Fig).

By E16.5, clear differences in the ventral and limb migration fronts were observed (Fig 5A). In Bptf mel+/-, clusters of melanocytes were clearly visible on the trunk that correspond to melanocytes colonising the nascent hair follicles [START_REF] Jordan | MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles[END_REF] (Fig 5B). Although such clusters were less prominent in the Bptf lox/lox animals, the number of melanoblasts was now reduced by around 20% in the Bptf mel-/-foetuses (Fig 5C ), perhaps accounting for their diminished prominence. Futhermore, Bptf also regulated melanoblast morphology as those lacking Bptf were less dendritic and much more rounded. Bptf therefore acts during embryogenesis to regulate melanoblast proliferation, migration and morphology.

Pigmentation of the first coat is provided by the embryonic derived melanoblasts that colonise and terminally differentiate in the hair follicles [6]. As the number of melanoblasts is lower in the ventral region than in the dorsal region even in wild-type mice, the further reduction in melanoblast numbers in the Bptf mel-/-animals during the course of development could partially account for the observed greyer phenotype of the first ventral hair coat. As BPTF regulates expression of the melanin synthesis enzymes in human melanocytes in vitro (see above), it is also possible that a reduction in melanin production by the mutant melanocytes would also contribute to the greyer ventral phenotype.

Bptf is essential for differentiation of melanocytes from adult stem cells

As the Bptf mel-/-animals grew older, their pelage showed progressive greying such that by 3-6 weeks both the ventral and dorsal coat became grey and then finally white (Fig 4C and S5D Fig). The animals maintained this completely white pelage throughout their lifespan (Fig 4D). This premature greying phenotype was fully penetrant and most animals became completely white indicating complete recombination of the Bptf alleles during embryogenesis, although some animals showed spotting (for example, animal on the right of Greying was accelerated by depilation of 3-week animals, following which the newly grown hair was white (Fig 4E and S5F Fig). These observations revealed that Bptf mel-/-animals were unable to pigment the pelage from the second post-natal anagen, growth phase of the hair cycle when the new hair follicle is generated, that requires the generation of mature melanin producing melanocytes from the post-natal MSC population. Bptf is therefore required for establishment, maintenance and/or functionality of the MSC population or its derivatives.

To investigate this, we performed immunostaining of hair shafts from the Bptf mel-/-and Bptf mel+/-genotypes at different post-natal stages using antibodies against Dct, staining the MSCs, the TACs and mature melanocytes, against Sox10, labelling TACs and mature melanocytes and against the cell cycle marker Ki67 labelling all proliferating cells in the bulb [6,[START_REF] Harris | A dual role for SOX10 in the maintenance of the postnatal melanocyte lineage and the differentiation of melanocyte stem cell progenitors[END_REF][START_REF] Nishikawa | Melanocyte system for studying stem cell niche[END_REF]. Control staining of a section from an adult wild-type mouse indeed confirmed that Dct stained the mature melanocytes in the bulb along with MSCs in the bulge and TACs (Fig 6A). Staining of dorsal hair shafts from P7 and P10 animals revealed equivalent numbers of Dct-Sox10 stained melanocytes in the bulb region (Fig 6B and6C). However at 3 weeks the number of Dct stained cells in the bulb strongly decreased in the Bptf lox/lox animals and almost half the shafts were devoid of Dct-stained cells (Fig 6D). In 1-year animals, no Dct-stained cells were detected in the bulb (Fig 6E). The loss of mature bulb melanocytes is in accordance with the progressive loss of pigmentation in the mutant animals and the sustained white coat in older animals.

In previous mouse models, premature greying was associated with a loss of the MSC population [START_REF] Schouwey | Transgenic expression of Notch in melanocytes demonstrates RBP-Jkappa-dependent signaling[END_REF][START_REF] Schouwey | Notch1 and Notch2 receptors influence progressive hair graying in a dose-dependent manner[END_REF][START_REF] Mcgill | Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability[END_REF]. To determine the fate of the MSC population upon Bptf inactivation, we stained sections from the epidermis of the Tyr-Cre/°::Bptf lox/lox ::Dct-LacZ/°and Tyr-Cre/°::Bptf lox/+ :: Dct-LacZ/°animals at different ages for the presence of LacZ to visualize melanocytes. At P10, DCT-LacZ stained melanocytes were seen in the bulbs of the Bptf mel-/-and Bptf mel+/-genotypes and the presence of melanin was clearly visible (Fig 7A). Dct-LacZ stained cells were also seen in the bulge regions at this stage. By six weeks, strong staining for melanin persisted in the Bptf mel+/-animals along with the presence of Dct-LacZ-stained melanocytes in both the bulge and bulb regions (Fig 7B). In the Bptf mel-/-animals, many shafts devoid of melanin were visible and only rare Dct-LacZ-stained melanocytes were seen in the bulb. However, Dct-LacZ-positive cells were visible in the bulge region, clearly seen in short telogen/early anagen shafts (Fig 7B,right panel). Staining of one year-old animals that were white, devoid of melanin and Dct-LacZ-stained bulb melanocytes also revealed Dct-LacZ-positive cells in the bulge region. Thus, an adult MSC population is established and maintained in the absence of Bptf, but these cells are unable to give rise to differentiated pigment producing melanocytes.

To ask whether MSCs were able to proliferate and differentiate in response to anagen stimuli, we depilated 4 month-old white animals and monitored the presence of Dct-LacZ-stained cells after 1, 3, 6, 10 and 15 days. In Bptf mel+/-animals, short telogen shafts remain in wild-type animals at 1 day following depilation where Dct-LacZ stained cells were observed in the bulge region (Fig 8A). By 3 days post-depilation, many TACs could now be seen along with melanocytes that begin to colonise the future bulb (Fig 8B ). By 6 days post-depilation, the bulb was filled with mature and pigment-producing melanocytes as seen by staining for Dct-LacZ and melanin and numerous TACs could still be observed (Fig 8C). After 10 and 15 days strong Dct-LacZ-staining was maintained in the bulb and the newly grown hair was pigmented (Fig 8D ).

Immediately after depilation of Bptf mel-/-animals, Dct-LacZ stained cells were observed in the bulge region, but after 3 days fewer TACs and bulb melanocytes were visible compared to Bptf mel+/-animals (Fig 8E and8F). Similarly, at 6 days post-depilation, while Dct-LacZ stained cells were visible in the bulge, staining of the bulbs was less prominent and more variable with many bulbs showing only few melanocytes and very few residual TACs were observed (Fig 8G). By 10 and 15 days, Dct-LacZ stained cells were no longer seen in the bulb region (Fig 8H). Moreover, no melanin was made from the bulb melanocytes and the out-growing hair was white in accordance with the fact that these animals were white both before and after depilation.

We also used immunostaining to detect endogenous Dct after depilation. At day 3, Dct labelled cells could be seen in the bulge region of Bptf In contrast, no staining for any of these proteins was seen in the Bptf mel-/-bulbs. Thus, in absence of Bptf, the Dct-LacZ-labelled cell population did not express endogenous Dct and their expansion at anagen was not associated with detectable expression of Mitf, Pax3 and Sox10. In addition, the few cells that migrated to the bulb by day 6 also did not express these melanocyte markers. These observations indicate that Bptf acts at an early stage in the generation of differentiated mature melanocytes from the adult MSC population.

It was previously reported that adult MSCs display a down-regulation of RNA polymerase II (Pol II) transcription witnessed by diminished staining for phosphorylated serine 2 of the Cterminal domain (CTD) of the largest subunit and for Cdk9 [START_REF] Freter | Adult stem cells exhibit global suppression of RNA polymerase II serine-2 phosphorylation[END_REF]. During the course of this study, we tested several antibodies for their ability to detect Bptf in the hair follicle. None of the tested antibodies detected Bptf in immunostaining of hair follicles, whereas a commercial antibody gave a strong nuclear staining for Smarca5 in the keratinocyte population as well as the Dct-expressing bulb melanocytes (Fig 10A). Interestingly however, Dct-expressing MSCs in wild-type mice selectively showed strongly diminished Smarca5 staining suggesting its down-regulation in these transcriptionally silent cells (Fig 10B). A similar result was seen for the Smarca4 (Brg1) chromatin remodeller. To further investigate the transcriptionally repressed state, we stained hair follicles for marks of active chromatin (H3K4me3, H3K9ac and H3K27ac) and the repressive mark H3K27me3. All of these antibodies gave strong nuclear staining in keratinocytes and Dct-expressing bulb melanocytes (Fig 10A). In contrast, Dctexpressing MSCs showed strongly diminished staining for all of the active marks, but not for Together these observations support the idea that MSCs enter into a transcriptionally silent state. Emergence from this state to properly reactivate expression of the melanocyte differentiation genes and generate mature melanocytes requires Bptf.

Discussion

An essential and specific role for BPTF/NURF in melanoma and melanocyte cells in vitro

We show that peptides for the NURF components BPTF, SNF2H, SNF2L and RBBP4 were found in the MITF interactome. As previously described no peptides for these proteins were found in the control FLAG-HA immunoprecipitations from 501Mel cells with native MITF [17]. We did not detect peptides for the smaller BAP18 and HMG2L1 subunits. Whether this is because the small number of peptides from these proteins was missed in the mass-spectrometry or whether they are not part of the complex in 501Mel cells remains to be determined. Massspectrometry and immunoblot showed that NURF subunits were detected only in the chromatin-associated fraction indicating that MITF and NURF preferentially interact on chromatin.

Immunoprecipitation showed that endogenous NURF was co-precipitated only in the cells expressing tagged MITF demonstrating the specificity of the interaction with MITF. Lack of ChIP-grade BPTF antibodies has hampered our attempts to identify sites on the genome where MITF and BPTF co-localize. It has previously been shown that BPTF localizes almost uniquely to the active TSS by virtue of the interactions between its PHD and bromodomain with the histone modifications at the TSS [START_REF] Ruthenburg | Recognition of a mononucleosomal histone modification pattern by BPTF via multivalent interactions[END_REF]. However, these experiments were performed with a truncated epitope tagged protein containing only the PHD and bromodomains and thus it remains possible that BPTF may be recruited to other regions of the genome not by interaction with chromatin marks, but via interactions with transcriptional activators such as MITF as is seen with the PBAF complex [17].

NURF components are expressed in all tested melanoma cell lines and shRNA-mediated BPTF silencing showed its essential role in a variety of MITF-expressing melanoma cell lines, including 501Mel, MNT1, SK-Mel-28, and 888-Mel as well as the MITF-negative 1205Lu cell line where it must act independently of MITF. In contrast, BPTF silencing in a series of nonmelanoma cell lines such as HeLa, H293T had no detectable effect, in accordance with previous observations showing Bptf -/-ES cells and MEFs proliferate almost normally in vitro, and that thymocytes do not exhibit any proliferation or survival defects in vivo [START_REF] Landry | Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells[END_REF][START_REF] Landry | Chromatin remodeling complex NURF regulates thymocyte maturation[END_REF]. There is therefore no general requirement for BPTF for proliferation, but rather a specific requirement in melanoma cells that is both MITF-dependent and independent.

Upon BPTF silencing, 501Mel cells adopted a morphology similar to that seen upon MITF knockdown, developed a SASP and showed senescence-associated β-galactosidase staining. Comparative analyses showed that 39% of shBPTF down-regulated and 41% of up-regulated genes were regulated in an analogous manner by shMITF. Genes such as BIRC7, BCL2A1, and NPM1 that have roles in survival and/or in cell cycle regulation were identified as potential direct MITF targets with multiple MITF-occupied sites often close to the transcription start sites, whose expression is co-regulated by BPTF. Similarly, MITF and BPTF co-repress SASP genes like SERPINE1, IL24, PDGFB, and CYR61 as well as ZEB1 that has a crucial role in melanoma progression [START_REF] Caramel | A switch in the expression of embryonic EMT-inducers drives the development of malignant melanoma[END_REF][START_REF] Denecker | Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression[END_REF]. The above observations support the idea that MITF and BPTF positively co-regulate expression of genes involved in proliferation, but co-repress genes controlling cell motility and invasive properties. Nevertheless, BPTF likely acts as a cofactor for other transcription factors in MITF-negative melanoma cells and there are clearly genes regulated by BPTF, but not MITF, in MITF-expressing lines. Acting as a cofactor for MITF is therefore only one facet of BPTF function in melanoma cells.

While this manuscript was in preparation, Dar et al [START_REF] Dar | The Role of BPTF in Melanoma Progression and in Response to BRAF-Targeted Therapy[END_REF] reported the implication of BPTF in human melanoma. In agreement with our results they showed that BPTF silencing in MITFnegative 1205Lu cells arrested their proliferation. More importantly, they showed that BPTF expression is increased in human melanomas, where the corresponding gene is often amplified and that these increases are predictive of poor outcome. While these observations identified BPTF as a prognostic factor for melanoma progression, they did not provide a molecular basis for BPTF function in melanoma. We show here that BPTF acts as a cofactor for MITF in regulating critical cell cycle, invasion, motility and apoptosis genes thus providing a molecular mechanism by which BPTF promotes melanoma growth and progression.

Bptf is essential for production of differentiated adult melanocytes

Inactivation of Bptf in melanoblasts does not impair their viability. Instead, Bptf regulates melanoblast proliferation and migration with by E16.5, a 20% reduction in the number of melanocytes, an alteration in their morphology and a less advanced migration front. The altered melanoblast morphology with reduced dendriticity may further reflect their impaired migration. Although expression of RAC1, an important regulator of melanoblast migration [START_REF] Li | Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod-driven motility and cell-cycle progression[END_REF] is not affected in shBPTF melanocytes in vitro, expression of RAC2 is down-regulated along with PREX1 another important regulator of melanoblast migration [START_REF] Lindsay | P-Rex1 is required for efficient melanoblast migration and melanoma metastasis[END_REF]. Moreover, MITF and BPTF co-regulate PREX1 expression in melanocytes in vitro and the corresponding gene locus comprises multiple MITF-occupied sites [START_REF] Laurette | Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells[END_REF]. PREX1 is therefore a direct MITF target gene co-regulated by BPTF in melanocytes in vitro and controlling melanoblast migration in vivo. While these in vivo effects recapitulate to some extent the function of BPTF in regulating melanocyte cell cycle and morphology in vitro, Bptf is clearly not essential as a cofactor for Mitf-driven melanoblast development as could have been implied from the observation that Bptf is essential in melanoma/melanocyte cells in vitro.

The first cycle of hair growth is pigmented by embryonic melanoblasts that colonize the developing hair follicles and differentiate into mature melanocytes. The almost normal first black dorsal coat of the Bptf mel-/-animals reflects the presence of abundant melanoblasts rendering this region less sensitive to the reduction in melanoblast numbers. The ventral region on the other hand, normally comprises less melanoblasts and hence is more sensitive to the reduced number and migration of melanoblasts in the mutant animals. Nevertheless, by 3-4 weeks as the first coat is discarded and regenerated by the second anagen phase, the mutant animals show progressive greying of both the dorsal and ventral coats. Greying is accentuated by 5-6 weeks when almost all the first coat has been exchanged. Depilation of 3 week-old animals results in the outgrowth of white hair indicating that mutant animals are unable to pigment the hair of the second anagen.

In contrast to the first anagen where pigmentation comes from terminally differentiated embryonic melanoblasts [6], the melanocytes pigmenting the second anagen are derived from post-natal MSCs. The inability of the mutant mice to pigment the hair from the second anagen phase suggests either, the lack of the MSC population, MSCs that are unable to respond to signals that induce their proliferation and/or differentiation at anagen, or a defective proliferation and differentiation of the TACs. The presence of Dct-LacZ-positive cells in the bulge region of the hair follicle at P10 and at all subsequent stages including as late as one year when the mice have been completely white for more than nine months shows that Bptf is not required to establish and maintain a MSC population. However, diminished numbers of TACs and bulb melanocytes are observed following depilation-induced anagen of mutant adult animals. These TACs do not express endogenous Dct, Mitf, Sox10 or Pax3 and by 6 days after depilation reduced numbers of Dct-LacZ-positive cells are present in the bulb while by 10 days, Dct-LacZ expressing cells can no longer be detected. The lower numbers of Dct-LacZ-stained TACs and bulb melanocytes may be accounted for by their premature death or by switching off of the Dct-LacZ reporter. We cannot exclude the possibility that cells previously stained by Dct-LacZ remain in the hair follicle in an undifferentiated state with no expression of melanocyte markers. It is also interesting to note that Dct, and Sox10 staining was also strongly reduced in three week old animals. While it is possible that the negatively stained shafts represent very early second anagen phase shafts, it is more probable that these represent first anagen shafts where there is premature death of the melanocytes or a loss of their melanocyte marker expression. Bptf may therefore be required to maintain the viability and/or the terminally differentiated character of these melanocytes.

These observations show that Bptf mel-/-MSCs are stimulated to proliferate at anagen and fulfill the events necessary to maintain the MSC population throughout the multiple anagens in the life of the animal. Nevertheless, MSCs established just after birth in absence of Bptf, inactivated at an early stage of melanoblast development, are abnormal as they do not express detectable levels of endogenous Dct. This discrepancy with Dct-LacZ staining highlights a differential requirement for Bptf for expression of the endogenous Dct locus compared to the exogenous transgene reflecting the fact that these two genes are in different chromosomal localizations (chromosome 4 for Dct-LacZ and 14 for Dct) and chromatin environments and hence show a differential requirement for Bptf/NURF for their expression.

The results reported here together with previous studies show that MSCs undergo a downregulation of Pol II transcription [START_REF] Freter | Adult stem cells exhibit global suppression of RNA polymerase II serine-2 phosphorylation[END_REF] and display strongly diminished levels of chromatin remodellers and active chromatin marks. All of these observations are consistent with the entry of MSCs into a transcriptionally repressed chromatin state, between the anagen phases. Bptf, presumably via the ATP-dependent chromatin-remodelling activity of NURF, is essential for reactivation of the melanocyte gene expression program at anagen, the subsequent normal proliferation of TACs and their differentiation into mature melanocytes. This is the major defect seen in vivo, it is a unique and complex physiological situation that cannot be easily mimicked by cell lines in vitro. A specific requirement for Bptf upon reactivation of the MSCs would also explain why Bptf is not essential for differentiation of embryonic melanoblasts that have not undergone a prolonged period of stem cell associated quiescence. It is also noteworthy that the BPTF and SMARCA5 subunits of NURF were identified in an siRNA screen as factors required to maintain keratinocyte stem cells in an undifferentiated state in vitro [START_REF] Mulder | Diverse epigenetic strategies interact to control epidermal differentiation[END_REF]. BPTF is not required to maintain MSCs in an undifferentiated state in vivo, but rather is required for their differentiation. BPTF may therefore play opposing roles in keratinocyte and melanocyte stem cells.

Wnt signaling is believed to play an important role in reactivation of MSCs at anagen, [START_REF] Rabbani | Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration[END_REF]. Deletion of Ctnnb1 in melanocytes results in a loss of differentiated progeny and hair greying, but the MSC population is maintained, a phenotype similar to that observed here. Bptf may therefore act downstream of Wnt prior to Mitf induction an observation reminiscent of the situation in Drosophila where NURF is required for Wnt signaling [START_REF] Song | The ISWI-containing NURF complex regulates the output of the canonical Wingless pathway[END_REF].

The role of BPTF/NURF in melanocytes differs from that of BRG1/PBAF that also interacts with MITF [17]. While BRG1 and BPTF are essential in melanocytes and melanoma cells in vitro, they regulate overlapping but distinct gene expression programs. Furthermore, mice lacking Brg1 in melanocytes are born with a complete absence of pigmentation and no identifiable melanocytes in the hair follicles showing an essential role for Brg1 in melanoblast development, whereas Bptf is essential only for reactivation of the MSC population. Our results therefore define specific and distinct roles for the PBAF and NURF chromatin remodelling complexes in epigenetic regulation of gene expression in melanocytes and melanoma.

The phenotype observed here is unique and distinct from previous mouse mutants where premature greying was ascribed to a progressive loss of the stem cell population [START_REF] Nishimura | Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche[END_REF]. For example, targeted ablation of Bcl2 in the melanocyte lineage results in loss of the MSC population showing that it is required for their survival [START_REF] Mcgill | Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability[END_REF]. Similarly, melanocyte-specific knockout of Notch signalling components shows the requirement of this pathway to maintain the MSC population [START_REF] Schouwey | Transgenic expression of Notch in melanocytes demonstrates RBP-Jkappa-dependent signaling[END_REF][START_REF] Osawa | Notch and melanocytes: diverse outcomes from a single signal[END_REF][START_REF] Kumano | Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis[END_REF]. Bptf knockout on the other hand does not deplete the MSC population that persists throughout life, but plays an essential role in their differentiation.

Materials and Methods

Immunopurification and western blot

Cell extracts were prepared essentially as previously described and subjected to tandem Flag-HA immunoprecipitation [17,[START_REF] Drane | The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3[END_REF]. MITF was detected by antibody ab-1 (C5) from Neomarkers, BPTF by a rabbit polyclonal antibody generated and kindly donated by Dr. J. Landry as described [START_REF] Landry | Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells[END_REF], and SMARCA1 and SMARCA5 were detected using antibodies generated and kindly donated by Dr P. Becker as described [START_REF] Eckey | Nucleosome remodeler SNF2L suppresses cell proliferation and migration and attenuates Wnt signaling[END_REF].

Mice

Mice were kept in accordance with the institutional guidelines regarding the care and use of laboratory animals and in accordance with National Animal Care Guidelines (European Commission directive 86/609/CEE; French decree no.87-848). All procedures were approved by the French national ethics committee. Mice with the following genotypes have been described elsewhere: conditional Bptf lox/lox [START_REF] Landry | Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells[END_REF], Tyr-Cre [START_REF] Delmas | Cre-mediated recombination in the skin melanocyte lineage[END_REF] and Dct-LacZ [START_REF] Mackenzie | Activation of the receptor tyrosine kinase Kit is required for the proliferation of melanoblasts in the mouse embryo[END_REF] Genotyping of F1 offspring was carried out by PCR analysis of genomic tail DNA with primers detailed in the respective publications.

LacZ-staining of embryos and epidermis E15.5 and E16.5 embryos were washed in PBS and fixed in 0.25% gluteraldehyde in PBS for 45 min at +4°C, after which they were washed with PBS for 15 min at +4°C. Embryos were incubated with permeabilization solution (100 mM phosphate buffer pH 7.4, 2mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP40) for 30-45 min at room temperature (RT). Staining was performed overnight at 37°C with permeabilization buffer containing 5mM potassium ferricyanide and potassium ferrocyanide (Sigma) and 0.04% X-gal solution (Euromedex). The samples were post-fixed for 3 h in 4% paraformaldehyde at RT and washed in PBS overnight at +4°C. To count embryonic melanoblasts, photos were taken with a Nikon AZ100 Multizoom microscope (Nikon, Tokyo, Japan) and defined regions were analyzed with Photoshop grid counter.

For epidermal samples, skin biopsies at the indicated stages were isolated, cut into small pieces (4mm x 2mm) and treated with the same protocol as the embryos, with the exceptions of overnight staining at RT and an overnight post-fixation in 4% paraformaldehyde. For further immunohistochemical analysis, samples were dehydrated, embedded in paraffin and sectioned at 10 μm. Sections were subsequently stained with nuclear fast red (Abcam) and, when indicated, the Fontana Masson kit (Abcam) and pictures were taken with a brightfield microscope.

Immunostaining

Biopsies of dorsal skin were isolated, cut into small pieces, fixed overnight in 4% paraformaldehyde, washed with PBS, dehydrated, paraffin imbedded and sectioned at 5 μm. For antigen retrieval, the sections were incubated with 10mM sodium citrate buffer, within a closed plastic container placed in a boiling waterbath, for 20 min. Sections were permeabilised with 3x5 min 0.1% Triton in PBS, blocked for 1h in 5% skin milk in PBS, and incubated overnight in 5% skin milk with primary antibodies. The following antibodies were used: goat anti-Dct at dilution of 1/1000 (Santa Cruz Biotechnology, sc-10451), rabbit anti-Ki67, at 1/500 (Novocastra Laboratories, NCL-Ki67p), rabbit anti-Sox10, at 1/1000 (Abcam, ab155279), H3K4me3 (04-745 Millipore), H3K9ac (07-352 Millipore), H3K27me3 (CS200603 Millipore), H3K27ac (39133 Active motif) SMARCA5 (Abcam ab72499). Sections were washed 3x5 min 0.1% Triton in PBS, and incubated with secondary antibodies, Alexa 488 donkey-anti-goat, and Alexa 555 donkey-antirabbit (Invitrogen) for 1 h. Sections were subsequently incubated with 1/2000 Hoechst nuclear stain for 10 min. Sections were washed 3x5 min in PBS, dried, mounted with Vectashild, and coverslip immobilized with nail polish.

Purification of melanoblasts

Melanoblasts were isolated according to a protocol adapted from Van Beuren and Scambler [START_REF] Van Bueren | FACS-GAL isolation of β-galactosidase expressing cells from mid gestation mouse embryos Protcol exchange[END_REF]. Briefly, the trunk epidermis of E14.5-E15.5 Dct-LacZ::Tyr-Cre::Bptf lox/+ embryos was dissociated into a single cell suspension and LacZ-positive cells were labelled using the Detecta-Gene Green CMFDG LacZ Gene Expression kit (Molecular Probes) and isolated by FACS prior to genotyping.

Cell culture, and shRNA silencing

Melanoma cell lines SK-Mel-28, 501Mel, MNT1, 888Mel and 1205Lu were grown in RPMI 1640 medium (Sigma, St Louis, MO, USA) supplemented with 10% foetal calf serum (FCS). HEK293T, HeLa and fibroblast cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS and penicillin/streptomycin (7.5 μg/ml). Hermes-3A cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% FCS, 200nM TPA, 200pM cholera toxin, 10ng/ml human stem cell factor (Invitrogen), 10 nM endothelin-1 (Bachem) and penicillin/streptomycin (7.5 μg/ml). Hermes 3A cells were obtained from the University of London St Georges repository.

All lentiviral shRNA vectors were obtained from Sigma (Mission sh-RNA series) in the PLK0 vector and virus was produced in HEK293T cells according to the manufacturers protocol. Cells were infected with the viral stocks and after 5 days (or as indicated in the Figure legends) of puromycin selection (3 μg/ml), cells were photographed and collected for preparation of cell lysates or isolation of RNA. In each case between 5X10 5 -1X10 6 cells were infected with the indicated shRNA lentivirus vectors and all experiments were performed at least in triplicate. The following constructs were used shBPTF (TRCN0000319152, TRCN0000319153), shMITF (TRCN0000019119). For lentivirus infection, virus was produced in 293T cells according to the manufacturers protocol described. The siRNA knockdown of BPFT was performed with ON-TARGET-plus human SMARpool (L-010431-00) purchased from Dharmacon Inc. (Chicago, Il., USA). Control siRNA directed against luciferase was obtained from Eurogentec (Seraing, Belgium). siRNAs were transfected using Lipofectamine RNAiMax (Invitrogen, La Jolla, CA, USA).

Senescence-associated β-galactosidase assay

The senescence-associated β-galactosidase staining kit from Cell Signaling Technology (Beverly, MA, USA) was used according to the manufacturer's instructions to histochemically detect β-galactosidase activity at pH 6.

mRNA preparation, quantitative PCR and RNA-seq mRNA isolation was performed according to standard procedure (Qiagen kit). qRT-PCR was carried out with SYBR Green I (Qiagen) and Multiscribe Reverse Transcriptase (Invitrogen) and monitored using a LightCycler 480 (Roche). Actin gene expression was used to normalize the results. Primer sequences for each cDNA were designed using Primer3 Software and are available upon request. RNA-seq was performed essentially as previously described [START_REF] Herquel | Trim24-repressed VL30 retrotransposons regulate gene expression by producing noncoding RNA[END_REF]. Gene ontology analyses were performed using the functional annotation clustering function of DAVID (http://david.abcc.ncifcrf.gov/). mice, C. Wu and J. Landry for the gift of anti-BPTF antibody, P. Becker for gifts of anti-SMARCA1 and SMARCA5 antibodies, E. Sviderskaya and D.C. Bennett for the Hermes cells, B. Jost, and all the staff of the IGBMC high throughput sequencing facility, a member of "France Génomique" consortium (ANR10-INBS-09-08).

Melanocyte reprogramming requires chromatin and transcription remodelling

Sarah J. Welsh and Helen Rizos e-mail: helen.rizos@mq.edu.au Microphthalmia-associated transcription factor (MITF) plays a fundamental role in melanocyte development and contributes to melanomagenesis by regulating the expression of genes involved in differentiation, proliferation and survival. Loss of MITF expression is a hallmark of quiescent melanocyte stem cells, which, when stimulated, transiently reactivate MITF expression to proliferate and restore the pool of differentiated pigment-producing melanocytes. The dual activities of MITF are also evident in melanoma; low MITF melanoma cells tend to be slow cycling, invasive and display tumourinitiating properties, whereas MITF gene amplification and overexpression is oncogenic in a subset of melanomas (Garraway et al., 2005).

Recent studies have suggested that MITF may regulate gene expression by modulating the structure of chromatin, specifically by binding to the BRG1-and PBAF-SWI/SNF (De La Serna et al., 2006;Laurette et al., 2015) and NURF-ISWI families of remodelling enzymes. MITF has been shown to activate pigment-related genes by recruiting the SWI/SNF enzymes to melanocytespecific promoters, and the report of Koludrovic et al. (2015) extends these studies by investigating the influence of nucleosome remodelling factor (NURF) on MITF activity and melanocyte development.

The NURF enzyme catalyses nucleosome sliding on DNA and is necessary for the chromatin remodelling required for transcription. The core mammalian NURF complex comprises the SMARCA1 ATPase subunit, the RBBP4 histone binding polypeptide and the 450kD BPTF bromodomain PHD finger transcription factor. BPTF binds active gene promoters and shows oncogenic activity in melanoma, the gene is frequently amplified in primary melanomas, protein overexpression in melanoma is associated with poor patient prognosis, and ectopic BPTF expression diminishes melanoma cell sensitivity to the BRAF inhibitor vemurafenib (Dar et al., 2015). Koludrovic et al. (2015) also confirmed the proliferative role of BPTF, showing that silencing of BPTF expression inhibited the proliferation of melanoma cells and melanocytes in vitro and promoted senescent-like changes, including enlarged and flattened cell morphology and increased senescence-associated b-galactosidase activity. Significantly, these phenotypic changes resembled the changes associated with MITF silencing, and predictably MITF and BPTF regulate a common set of genes. In particular, 39% of genes down-regulated by BPTF silencing and 41% of genes induced by BPTF knockdown were regulated in an analogous manner by MITF suppression. Thus, BPTF and MITF positively co-regulate genes required for cell cycle progression and negatively regulate genes involved in adhesion, morphology and senescence. Importantly, BPTF suppression also induced senescence in MITFnegative melanoma cells, but not in non-melanoma cell models. These data indicate that BPTF has MITF-dependent and MITF-independent functions that are specific to the melanocytic lineage.

The melanocyte-specific inactivation of Bptf in vivo (Tyr-Cre/ o ::Bptf lox/lox ) produced animals with premature greying that involved the progressive loss of pigmented melanocytes in the hair bulb. Critically, in the absence of BPTF, the adult melanocyte stem cells, which are located in the bulge region of the hair follicle, were established and maintained as late as nine months after the mice went completely white. Thus, BPTF promotes the differentiation or survival of mature melanocytes from the stem cell population. In contrast, hair greying in mice expressing a hypomorphic mutant MITF allele appears to result from the loss of stem cells possibly as a result of their premature differentiation, which ultimately leads to the progressive loss of pigmented mature melanocytes (Nishimura et al., 2005).

Taken together, these observations indicate that transcriptionally repressed melanocyte stem cells require BPTF/ NURF-mediated chromatin remodelling for activation. A parsimonious interpretation of these observations is that BPTF/NURF chromatin remodelling activity reactivates gene transcription and promotes proliferation and differentiation of stem cells into mature pigmented melanocytes. Based on current data, it is likely that the BPTF/NURF-regulated transcriptional program involves, but is not restricted to, MITF transcription activity. MITF is also predicted to function downstream of BPTF, to regulate melanocyte stem cell renewal. Further exploration of the complex interactions between MITF and NURF will provide valuable insights into melanocyte reprogramming and, considering these two factors regulate cell proliferation and senescence, a complete understanding of their cooperative functions may provide an opportunity for novel therapeutic strategies aimed at restoring senescence, enforcing terminal differentiation or eliminating stem cells.

Coverage on: Koludrovic, D., Laurette, P., Strub, T., Keime, C., Le Coz, M., Coassolo, S., Mengus, G., Larue, L., Davidson, I. (2015). Chromatin-remodelling complex NURF is essential for differentiation of adult melanocyte stem cells. PLoS Genet. October 6; 11 (10)

News and Views

A recent study published in the Journal of Experimental Medicine reports that melanocytes may be targets of autoreactive T cells in psoriasis. Psoriasis is a common inflammatory disease of the skin that affects 1-3% of people worldwide. It presents with well-demarcated, scaly plaques that reduce the quality of life of affected patients (Lowes et al., 2014). Historically, psoriasis was thought to be a disorder of keratinocyte hyperproliferation, but most investigators now agree the driving force behind the disease is a dysregulated immune signaling cascade that results in the characteristically inflamed psoriatic lesions (Lowes et al., 2014). There is an ongoing debate whether the disease is truly autoimmune in origin, and if it is, it remains unclear which cell types, let alone what specific antigens, are the targets of this autoimmune response.

The pathogenesis of psoriasis is mediated by several immune populations in the skin, and many of the inflammatory pathways involved are now well described. These pathways lead to continued immune cell recruitment, inflammation, and the unre-strained proliferation of keratinocytes, which gives rise to the thickened, scaly plaques (Lowes et al., 2014). In the chronic inflammatory phase of the disease, T cells play a prominent role, which may produce Th1-, Th17-or Th22-specific cytokines. Of these, the IL-23/IL-17/TNF-a axis appears to be central to disease pathogenesis, as targeting this axis for treatment results in vast improvement of the disease (Lowes et al., 2014). Classically, IL-17producing cells were thought to be ab T cells, but more recent studies of psoriasis in both humans and mice have revealed an emerging role of cd T cells as additional contributors (Lowes et al., 2014). This has important implications for antigen targeting in the disease, as ab T cells recognize specific antigens in the context of HLA presentation, while cd T cells do not.

Despite advancements in our understanding of the key role T cells play in the self-amplifying inflammatory signaling loop, it is not known whether some of these T cells are responding to an autoantigen. Several potential antigens have been identified, but thus far none are reportedly cell specific. For example, patients with psoriasis have autoreactive T-cell clones against the antimicrobial peptide LL37, which is also implicated in initiation of psoriasis (Lande et al., 2014). Likewise, experimental animal models have implicated a number of other potential autoantigens (Lowes et al., 2014). Important evidence in support of an autoantigen includes preferential Va3S1/Vb13S1 chain rearrangement in the TCR among the CD8 + T-cell clones present in pso-riatic skin [START_REF] Kim | Recruitment and biological consequences of histone modification of H3K27me3 and H3K9me3[END_REF]. This suggests that the specific antigen of this prevalent TCR is present in the skin, which could lead to the continued recruitment and retention of these cells. Coupled with the fact that a prominent risk allele for psoriasis, HLA-C*06:02, is involved in antigen display (Lowes et al., 2014), it is conceivable that an autoantigen leads to activation of T cells with a Va3S1/Vb13S1 TCR, which then promotes psoriatic inflammation.

Based on this hypothesis, Arakawa and colleagues sought to identify potential psoriatic autoantigens using a T-cell hybridoma reconstituted with a Va3S1/ Vb13S1 TCR derived from a HLA-C*06:02-positive psoriatic patient. This particular T-cell hybridoma reported TCR signaling by robust GFP expression under the control of NFAT, which allowed the authors to detect low-level TCR stimulation by TCR-transfected cell lines. This became a powerful tool through which they could investigate potential skin resident cells as the source of psoriatic autoantigen. TCR signaling, indicated by GFP signal, only occurred during co-culture with melanocyte cell lines, and only in the context of HLA-C*06:02. Histologic studies of psoriatic plaques revealed a higher frequency of CD8 + T-cell-melanocyte contacts. This also revealed that CD8 + T cells directed lytic granules toward contact sites with melanocytes. Taken together, these results support the hypothesis that some of the recruited CD8 + T cells in psoriatic skin recognize and respond to a melanocyte-specific antigen.

The authors then sought to identify the antigen recognized by their T-cell Coverage on: Arakawa, A., Stewert, K., Stohr, J., Besgen, P., Kim, S.-M., Ruhl, G., Nickel, J., Vollmer, S., Thomas, P., Krebs, S., Pinkert, S., Spannagl, M., Held, K., Kammerbauer, C., Besch, R., Dornmair, K., and Prinz, J.C. (2015). Melanocyte antigen triggers autoimmunity in human psoriasis. J Exp Med. 212 (13), 2203-2212. doi: 10.1111

Abstract/ Introduction

In our two previous studies (Laurette et al. 2015;Koludrovic et al. 2015), we showed that the BRG1/PBAF and BPTF/NURF chromatin remodelling complexes interact with Microphthalmia-associated transcription factor (MITF) in human melanoma cells and we defined their functions in melanoma cells in vitro and in mouse melanocytes in vivo. We found that BRG1 is essential for the cell cycle of proliferative type melanoma cells and for regulation of a large set of genes. BRG1/PBAF interacts with MITF and SOX10 and these transcription factors actively recruit BRG1 to chromatin to establish the epigenetic landscape of proliferative melanoma cells. BPTF is also essential for cell cycle progression of proliferative melanoma cells, but regulates a more limited gene expression programme including those involved in cell cycle and oxidative metabolism. In vivo, BRG1 inactivation does not lead to an immediate loss of developing melanoblasts, but rather a progressive decrease in the melanoblast population such that they are almost completely absent by E15.5 (unpublished observation) and the resulting animals lack pigmentation. In contrast, BRG1 is not required for generation of melanocytes from the adult melanocyte stem cell population. BPTF on the other hand is not essential for melanoblast development, but is required for generation of differentiated melanocytes from the adult melanocyte stem cell population. These two chromatin-remodelling complexes therefore play distinct but complementary roles in human melanoma and in the mouse melanocyte lineage. To complete this study we have used a mouse genetic model of melanoma to address the role of Brg1 and Bptf in tumourigenesis in vivo. We find that both Brg1 and Bptf are essential for melanomagenesis in vivo.

Results and discussion.

To address the role of BRG1 and BPTF in mouse melanoma, we used the previously described model (Hooijkaas et al. 2012;Dhomen et al. 2009;Dankort et al. 2009;Damsky et al. 2011) where treatment with Tamoxifen (Tam) induces the expression of oncogenic Braf V600E and inactivates Pten selectively in the melanocyte lineage (Tyr::Cre-ER T2 ; Braf onc/+ ; Pten lox/lox ). 6-8 week old mice were depilated at the base of the back and treated with a 5 µl drop of 4-hydroxytamoxifen (4-OHT) leading to the rapid development within around 40 days of a large localised melanoma (Fig. 1A-B).

4-OHT painting of the ear or the tail also led to fast melanocyte hyper-proliferation leading to hyper-pigmentation and then melanoma (Fig. 1B and Fig. S1A). Histological examination showed that tumours on the back arose from the hair follicle. In nascent tumours soon after 4-OHT treatment, pigmented melanocytes could be seen invading the dermis (Fig. 1C). However, developing tumours rapidly lost pigmentation and only a few pigmented cells remained in the larger tumours (Fig. 1B-D). The vast majority of tumour cells stained with Sox10, but only a fraction of cells stained with Mitf at early stages, while at later stages only rare cells stained with Mitf (data not shown and see below). Of note, Intra-peritonial (IP) Tam injection led also to the appearance of multiple skin melanomas and, in contrast to topical application, to lung metastases (Fig. S1B).

To address the requirement for Brg1 and Bptf in tumourigenesis, we crossed these animals with mice in which the corresponding genes were floxed such that 4-OHT treatment would inactivate these proteins concomitant with tumour initiation (Tyr::Cre-ER T2 ;Braf onc/+ ;Pten lox/lox ;Smarca4 lox/lox or Bptf lox/lox ). Compared to mice that were wildtype or heterozygous for Smarca4 and Bptf, inactivation of these genes in the lox/lox animals strongly delayed tumour formation (Fig. 2A-B). While large tumours were seen on the backs of the wild-type or heterozygous mice by 38-44 days, only much smaller foci were observed upon Brg1 or Bptf inactivation. The effect was particularly marked for Bptf loss where only small foci were seen by day 44, whereas smaller tumours were seen by this time when Brg1 was inactivated. Comparison of the number of days required for the tumours to reach a 1cm 3 volume confirmed these observations showing a strong delay in tumour formation upon Bptf inactivation (50-60 days, compared to 38 for wild-type) and a less marked delay (45 days) for Brg1 inactivation.

The above data showed that Brg1 and Bptf inactivation retarded tumour growth, however tumours eventually developed in mice homozygous for the respective floxed alleles. To better understand the basis for this, sections were prepared from the tumours and stained with antibodies against Sox10 to identify the tumour cells and with antibody against Brg1. Immunostaining of tumours from mice wild-type for Smarca4 and Bptf indicated that almost all tumour cells expressed Sox10 and Brg1 (Fig. 3A). At early stages of tumour growth, Sox10 and Dct-stained melanoma cells were detected around the hair follicles and their expression persisted in the majority of cells at later stages of growth (Fig. 3B). Importantly however, immunostaining of tumours that developed in mice where the Smarca4 alleles were floxed also expressed Dct and Brg1. These data indicated that the cells that eventually contributed to tumour growth in these mice had escaped recombination of the Smarca4 alleles and maintained Brg1 expression. Similar experiments could not be performed for Bptf due to the lack of antibodies that detect murine Bptf by immunostaining.

To investigate whether the Smarca4 alleles had been recombined, we first stained tail sections 2-3 days after 4-OHT treatment. Dct-stained melanocytes lacking nuclear Brg1 signal were detected showing that at this stage Brg1 had been inactivated in these melanocytes. We also prepared DNA from tumours of the animals of different genotypes and performed PCR to detect deletion of the floxed alleles. Genotyping of tail DNA from mice lacking Cre-ER T2 expression allowed PCR amplification of fragments corresponding to the wild-type and floxed Smarca4 alleles (Fig. S2A). These fragments could also be amplified from DNA extracted from tumours of the Smarca4 lox/+ mice, whereas the wild-type allele was not detected in DNA from tumours from the Smarca4 lox/lox mice. As the predicted sizes of the PCR fragments from the floxed and deleted Smarca4 alleles were very similar, we could not distinguish whether the fragment detected in these tumours corresponded to the floxed allele, the deleted allele or both. We therefore re-analysed the tumour DNA using primers that detected only the deleted allele. Under these conditions, a fragment corresponding to the deleted allele was detected in the tumours from the Smarca4 lox/+ and Smarca4 lox/lox mice, but not in the tail DNA from mice lacking Cre-ER T2 (Fig. S2A). As the tumours that developed in the Smarca4 lox/lox mice could be stained for Brg1, these data indicated that cells from the tumour retained at least one coding allele and either remained Smarca4 lox/lox or had recombined one allele to become Smarca4 lox/-.

We performed a similar set of experiments using primers to detect recombination of the Bptf alleles. As above, analyses of tail DNA allowed the amplification of fragments corresponding to the wild-type and floxed, but not the deleted Bptf alleles (Fig. S2B). In tumours from the Bptf lox/+ mice, fragments corresponding to all 3 alleles could be detected. In tumours from the Bptf lox/lox mice, both the floxed and deleted alleles were detected. These data showed that cells from these tumours retained at least one coding floxed Bptf allele again suggesting that the cells forming the tumours expressed Bptf.

The above data are in agreement with a model in which the tumours in the Smarca4 lox/lox or Bptf lox/lox mice were formed from rare cells that retain at least one coding allele accounting for the delay in tumour growth and suggesting that Brg1 and Bptf are essential for tumour growth. To test this idea, later stage tumours were dissociated and the cells cultured in vitro. These cultures comprised a large majority of Sox10 expressing tumour cells (data not shown and see below). The cells were then treated with 4-OHT to recombine the residual floxed Smarca4 or Bptf alleles. Treatment of cells from tumours with wild-type Smarca4 and Bptf alleles had no effect on cell growth and Sox10 or Brg1 expression (Fig. 4A-B). In contrast, treatment of cells from Smarca4 lox/lox tumours led to loss of Brg1 expression a major change in cell morphology and eventual cell death (Fig. 4A-B). Sox10 expression was also lost suggesting that Brg1 is required for its expression in these cells. Treatment of cells from Bptf lox/lox tumours also lead to cell death, but had no effect on Brg1 or Sox10 expression. As mentioned above, current antibodies do not allow detection of murine Bptf, but as with Brg1, it is likely that cell death arises from 4-OHT-induced recombination of the residual coding allele and loss of Bptf expression. The molecular basis of death of the Brg1 and Bptf inactivated cells remains to be determined.

Altogether, the above data are consistent with a model in which Brg1 and Bptf are essential for growth of mouse melanoma cells. When animals are subjected to a single 4-OHT treatment these proteins are inactivated in the majority of melanocytes. However, rare cells that escaped recombination or had recombined only one Bptf or Smarca4 allele eventually expand to form tumours accounting for the delay in tumour growth compared to wild-type animals. However, if cells from these tumours are then treated with 4-OHT to recombine the residual coding allele cell death is observed. These data complement previous results showing that BRG1 and BPTF are essential for growth of proliferative human melanoma cells in vitro (Laurette et al. 2015;Koludrovic et al. 2015), that BRG1 is essential for the growth of human melanomas as xenografts in mice (Bossi et al. 2016) and they are in agreement with the observed amplification of the BPTF gene and its up-regulated expression in human melanomas (Dar et al. 2015).

Material and methods

Mice and genotyping

All animal experiments were performed in accordance with European and national guidelines and policies (2010/63/UE directive and French decree 2013-118) and with approval of the local Ethics Committee.

Mice bearing the Tyr::CreERT2 transgene (Yajima et al. 2006), Braf +/LSL-V600E (Dhomen et al. 2009), Pten (Suzuki et al. 2001) and Smarca4 (Indra et al. 2005) or Bptf (Landry et al. 2008) floxed alleles were intercrossed and further backcrossed on C57BL/6J background to generate the desired experimental mice. Genotyping was carried out by PCR analysis of genomic tail, skin or tumor DNA with primers detailed in the respective publications.

Tumor induction and growth analysis

4-hydroxytamoxifen (4-OHT) was prepared freshly as a 25mg/mL solution (65mM) of 4-HT (70% Z-isomer, Sigma) in dimethylsulfoxide (DMSO) and further diluted with 100% ethanol to a 1,9mg/mL working solution (5mM).

Localized melanoma induction was realized by topical application of about 5 µL 4-HT on the shaved back skin of 7 weeks old mice on three consecutive days. For distal tail or ear inductions, 2uL was applied instead.

Tumor growth was typically monitored twice weekly, from the time the first lesion appeared, by digital photography of the skin, including a size reference. Tumor size was then analysed in two dimensions using ImageJ software.

Primary cell culture and treatment

Primary melanoma cell cultures were derived from mouse tumors as described elsewhere (Gallagher et al. 2011). Early passage cells were then seeded on glass coverslips and let grow for 2 days. For Cre activation, fresh medium with 2μM 4-OHT or vehicle (ethanol) was added to the cells and refreshed everyday for 2 days.

Histochemistry and immunofluorescence

Immunofluorescence staining of formalin-fixed, paraffin-embedded skin and tumor samples was performed as described previously (Laurette et al. 2015).

The following antibodies were used: goat anti-Dct (Santa Cruz Biotechnology, sc-10451), rabbit anti-Sox10 (Abcam, ab155279) and rabbit anti-Brg1 (ab110641), Alexa 488 donkey anti-goat, and Alexa 555 donkey anti-rabbit (Invitrogen). For histological analysis, sections were stained with Haematoxylin and Eosin, following the standard procedures. Immunofluorescence of primary melanoma cells in vitro was performed by standard procedures using the same antibodies. and possibly Smarca4 lox/lox mice, but not in the tail DNA from mice lacking Cre-ER T2 . This observation is clearly confirmed using primers that detect only the deleted allele (lower panel). (B) A fragment corresponding to the deleted allele can be detected in the tumours from the Bptf lox/+ and Bptf lox/lox mice, but not in the tail DNA from mice lacking Cre-ER T2 . In silico analysis of single nucleotide polymorphism (SNP) array data from >300 human clinical samples from The Cancer Genome Atlas (TCGA) revealed that the chromosome 3p melanoma-specific focal amplifications invariably encompass a recently annotated long intergenic non-coding RNA (lincRNA) gene, SAMMSON, which is located ∼30 kilobase pair (kb) downstream of MITF (Fig. 1a and Extended Data Fig. 1a,b). Importantly, SAMMSON expression levels correlated with copy number gain (P < 0.001; Fig. 1a). Genome-wide copy number analysis (CNA) of melanoma cell lines and short-term cultures (MM lines 2 ) confirmed co-amplification of MITF and SAMMSON in a subset of these samples (Extended Data Fig. 1c).

Legends to figures

Results

Unexpectedly, although MITF-SAMMSON co-amplification only occurs in about 10% of melanomas, analysis of the TCGA RNAsequencing (RNA-seq) data set detected SAMMSON in >90% of both primary and metastatic skin cutaneous melanomas (SKCMs; Fig. 1b and Extended Data Fig. 1a). SAMMSON was also detected in 16 out of 17 randomly selected MM lines (Extended Data Fig. 1d). MM001 was the only line that did not express SAMMSON owing to a decrease in copy number (Extended Data Fig. 1c,d). SAMMSON levels were comparable in cultures that exhibited a 'proliferative' and 'invasive' transcriptional profile (Extended Data Fig. 1d) 3 . In contrast, MITF-M protein levels were high in the proliferative and low in the invasive cultures, respectively. Thus, the levels of SAMMSON and MITF do not strictly correlate. Likewise, there was no correlation between SAMMSON and MITF expression in the melanoma TCGA clinical samples (Extended Data Fig. 1e,f). Furthermore, there was no correlation between SAMMSON expression and any of the common melanoma somatic mutations such as BRAF, NRAS or TP53 (data not shown).

While barely detectable, if at all, in normal human melanocytes (NHMEs) and in non-invasive melanoma lesions in the radial growth phase (RGP), SAMMSON expression was readily detectable in invasive vertical growth phase (VGP) samples (Fig. 1c). This indicates that SAMMSON expression is specifically induced as melanoma cells transit from an immortalized to a fully transformed cell state and is therefore a putative biomarker of melanoma malignancy.

Quantitative polymerase chain reaction with reverse transcription (RT-qPCR) analysis in 60 different cancer cell lines detected SAMMSON exclusively in the melanoma samples (Extended Data Fig. 1g,h). Analysis of RNA-seq data from a total of 8,085 tumour specimens from 24 cancer types (TCGA) confirmed selective expression in melanoma (Fig. 1d). Whereas SAMMSON expression was detectable in normal human melanoblasts, it was undetectable in NHME cultures and in normal adult tissues (Fig. 1e and data not shown).

Consistent with SAMMSON being a lincRNA, the expression of which is driven by its own promoter, a peak of H3K4me3 chromatin immunoprecipitation followed by sequencing (ChIP-seq) was detected upstream of its transcription start site (TSS) in melanoma cells (Fig. 1f). Similarly, a peak of H3K27ac, a marker of active enhancers and promoters, overlaps with the H3K4me3 peak at the SAMMSON promoter. H3K27ac peaks are also found upstream of SAMMSON in all but one (MM001) MM lines (Extended Data Fig. 2a). Consistent with the melanoma-specific expression of SAMMSON, such H3K27ac peaks could not be detected in the non-melanoma cancer cell lines profiled by ENCODE.

Interestingly, we identified putative SOX-binding sites upstream of the SAMMSON TSS. SOX10 is a melanoblast/melanoma-specific transcription factor 4 and may therefore contribute to the melanoblast/ melanoma-specific expression of SAMMSON. Consistently, SOX10 and its co-factor BRG1 (ref. 5), but not MITF, were recruited upstream of the SAMMSON TSS in melanoma cells (Fig. 1f, g and Extended Data Fig. 2b,c). Knockdown of SOX10, but not MITF, led to a decrease in SAMMSON expression (Fig. 1h and Extended Data Fig. 2d,e). These

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data indicate that the lineage-specific expression profile of SAMMSON is a consequence of focal gene amplification and/or SOX10-mediated transcription.

Silencing SAMMSON using different locked nucleic acid (LNA)modified antisense oligonucleotides (GapmeRs), which trigger RNase-H-mediated degradation of the target, greatly reduced the clonogenicity of all SAMMSON-expressing melanoma cultures independently of their BRAF, NRAS or TP53 status (Fig. 2a,b and Extended Data Fig. 3a). The growth of 'invasive' melanoma cells (that is, MM165), and thereby intrinsically resistant to mitogen-activated protein kinase (MAPK) inhibitors 3 was also inhibited upon SAMMSON knockdown. Chronic treatment with inhibitors of BRAF(V600E) is invariably associated with the development of drug resistance 6 . Importantly, cells that acquired resistance to a BRAF(V600E) inhibitor (that is, SK-MEL-28-R) remained sensitive to SAMMSON targeting.

Compared with GapmeR11, GapmeR3 was more efficient in knocking down SAMMSON expression and had a greater growth inhibitory effect (Fig. 2a, b and Extended Data Fig. 3a,b). Although less efficient than GapmeR-mediated silencing, RNA interference (RNAi)-dependent knockdown of SAMMSON also inhibited melanoma growth (Extended Data Fig. 3c,d). In contrast, transfection of SAMMSON-targeting GapmeRs or short interfering RNA (siRNA) in MM001, NHME or non-melanoma cancer cell lines (that is, HCT116), all of which lack SAMMSON, did not cause growth inhibition (Fig. 2a, Extended Data Fig. 3b and data not shown). Moreover, forced expression of SAMMSON and SAMMSON mutants, carrying mismatches into the GapmeR target sequences, efficiently rescued growth inhibition induced by GapmeR3/11 (Extended Data Fig. 3e). Importantly, the latter experiment also indicated that SAMMSON exerts its pro-survival function in trans.

Flow cytometry and Caspase-Glo 3/7 assays indicated that this growth inhibition is, at least partly, due to a significant induction of the intrinsic mitochondrial apoptotic pathway (Fig. 2c and Extended Data Fig. 3b). Exposure to an inhibitor of cytochrome-c-induced procaspase-9 activation rescued SAMMSON knockdown-mediated growth inhibition (Extended Data Fig. 3f).

Notably, ectopic expression of SAMMSON in MM001 cells conferred them with a growth advantage (Fig. 2d, e and Extended Data Fig. 3g,h), confirming that SAMMSON exerts its pro-oncogenic function in trans.

Overcoming intrinsic and acquired resistance to MAPK inhibitors requires the simultaneous targeting of multiple pathways. Interestingly, GapmeR3 enhanced the cytotoxic effects of the BRAF(V600E) inhibitor vemurafenib and MEK inhibitor pimasertib (Fig. 2f and Extended Data Fig. 3i).

Knockdown of SAMMSON did not decrease MITF levels (Extended Data Fig. 4), ruling out the possibility that SAMMSON promotes melanoma survival by enhancing MITF transcription in cis. Instead, consistent with a function in trans, SAMMSON primarily localizes to the cytoplasm, with a fraction co-localizing with mitochondria (Extended Data Figs 5 and6).

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We next purified endogenous SAMMSON RNA complexes by adapting the RNA antisense purification-mass spectrometry (RAP-MS) and chromatin isolation by RNA purification (ChIRP)-like-MS methodologies 7,8 . After confirming that SAMMSON RNA, but not the housekeeping TBC and UBC messenger RNAs, was selectively retrieved (Fig. 3a), the SAMMSON-associated proteins were identified by mass spectrometry. Eighteen proteins were identified in multiple SAMMSON purifications from three independent biological samples (Extended Data Fig. 7a and Supplementary Tables 1,2). The majority of these (12/18) are RNA-binding proteins, including XRN2, a protein involved in several key aspects of RNA metabolism. XRN2 was previously retrieved in ChIRP-MS experiments performed with different lncRNAs 8 , indicating that XRN2 may be a bona fide lncRNAs interactor.

Among the enriched proteins, p32 attracted our attention because of its established role in mitochondrial metabolism. In addition, p32 expression is elevated in cancers 9-11 and p32 knockdown decreases the growth of various cancer cell lines, including melanoma 10,12 . Consistently, p32 levels were elevated in the MM cultures compared with NHME cultures (Extended Data Fig. 7b,c). RAP followed by

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western blotting analyses confirmed the association between p32 and SAMMSON. p32 was enriched in the SAMMSON pulldown relative to the samples bound to the negative control HPRT (Extended Data Fig. 7d). No enrichment was observed after purifications with either beads alone, in lysate pre-treated with RNase A, or using SAMMSONnull cells (Extended Data Fig. 7d and data not shown). To ensure that the interaction between SAMMSON and p32 is direct, we performed RAP followed by western blotting in cells in which covalent bonds between directly interacting RNA and proteins are created by ultraviolet crosslinking. p32 was still enriched in these conditions (Fig. 3b).

We also performed RNA-immunoprecipitation (RIP) assays using p32specific antibodies. Compared with the immunoglobulin G (IgG)bound sample, all antibody-bound complexes showed a significant increase in the amount of SAMMSON RNA, but not of unrelated RNAs such as HPRT, TBP or LINC00698 (Fig. 3c and Extended Data Fig. 7e). Importantly, although no effect was seen on total p32 levels, a rapid decrease in its mitochondrial fraction, accompanied by an increase in nuclear targeting, was observed upon SAMMSON silencing (Fig. 3d, e and Extended Data Fig. 8a). p32 is required for the maturation of mitochondrial 16S rRNA, and thereby for the expression of mitochondrially encoded polypeptides, the maintenance of mitochondrial membrane potential and oxidative phosphorylation (OXPHOS) 10,13-16 . Consistently, the decrease in mitochondrial p32 was accompanied by reduced levels of 16S ribosomal RNA (Fig. 3d) and mitochondrial-DNA-encoded (COX2 and ATP6), but not nuclear-DNA-encoded (SDHA or NDUFS3), respiratory chain complex components (Fig. 3e). Moreover, the enzymatic activity of respiratory complexes I and IV, which contain proteins translated on mitochondrial ribosomes, was decreased upon SAMMSON silencing (Fig. 3f and Extended Data Fig. 8b). This decrease occurred before outer membrane permeabilization, as evidenced by the oxygraph profiles, and before induction of caspase-3/7 activity (data not shown).

The mitochondrial respiratory chain generates a proton gradient that establishes the mitochondrial membrane potential used to drive ATP production. A marked decrease in mitochondrial membrane potential was observed in SAMMSON-knockdown cells (Fig. 3g and Extended Data Fig. 8c), before the onset of apoptosis, accompanied by a slight, but significant, decrease in intracellular ATP levels (Extended Data Fig. 8d).

p32 controls mitochondrial homeostasis and integrity 15,17,18 . Accordingly, p32 silencing caused aberrant mitochondrial structures with fewer and fragmented cristae and reduced mitochondria matrix density. These effects were phenocopied upon SAMMSON knockdown (Extended Data Fig. 9).

Importantly, reintroduction of p32, but not of an N-terminally tagged version that can no longer be imported into the mitochondria, significantly rescued the SAMMSON-knockdown-dependent defect in mitochondrial membrane potential and growth inhibition (Fig. 3g,h and Extended Data Fig. 8e-g). SAMMSON silencing therefore inhibits melanoma survival at least partly by disrupting vital p32-mediated mitochondrial functions.

There is increasing evidence that mitochondrial translational deficiency induces a cytosolic stress response, which impairs cell growth 19 . This retrograde signalling is induced by the collapse of the mitochondrial membrane potential, which is critical for the import of nuclear-encoded proteins into the matrix 20 . This leads to the 'toxic' over-accumulation of mitochondrial precursors in the cytosol and activation of a stress response, which in yeast has been referred to as mitochondria precursor over-accumulation stress (mPOS) 21,22 . In turn, mPOS triggers cell cycle checkpoints and/or induces cell death depending on the cellular context. Interestingly, several hours after the collapse in the membrane potential, a decrease in the mitochondrial levels of several nuclear-encoded proteins (for example, SDHA, HSP60) was observed in SAMMSON-knockdown cells (Fig. 3i). Moreover, nuclear-encoded proteins such as SDHA and ATPB accumulated in the cytosol (and even in the nucleus for ATPB) of SAMMSON-knockdown cells (Fig. 3j and Extended Data Fig. 8h), indicating that SAMMSON silencing may lead to mitochondrial import defects, which, in turn, could activate mPOS.

In yeast, mPOS-induced cell death can be attenuated by reducing cytosolic protein translation, thus decreasing stress imposed by protein over-accumulation and aggregation 21 . Strikingly, exposure to a cap-dependent translation inhibitor significantly rescued SAMMSONknockdown-induced apoptotic cell death (Fig. 3k). Notably, consistent with data indicating that mitochondrial translation stress may engage the p53 pathway 23 , SAMMSON silencing triggered a significant p53 response in several melanoma cultures (data not shown). However, p53 knockdown did not rescue caspase-3/7 induction and growth inhibition, indicating that p53 is not strictly required for SAMMSONknockdown-induced growth inhibition.

To test the therapeutic potential of SAMMSON targeting in vivo, we used two patient-derived xenograft (PDX) melanoma models (Mel006 and Mel010; Extended Data Fig. 10a). Intravenous treatment with the SAMMSON-targeting GapmeR3 significantly suppressed the growth of Mel006 tumours, decreased cell proliferation and increased apoptosis (Fig. 4a-c). Gene expression profiles of the GapmeR3-treated melanoma lesions showed enrichment for signatures associated with decreased cell proliferation, activation of p53 and decreased OXPHOS, mitochondrial ribosome biogenesis and respiratory chain complex activity (Extended Data Fig. 10b). Notably, this treatment did not cause any relevant adverse reaction or weight loss (data not shown). Tumour growth was also inhibited upon intra-tumour injections of GapmeR3 into Mel010 tumours (Extended Data Fig. 10c).

Importantly, whereas tumour growth was only inhibited after exposure to the BRAF(V600E) inhibitor dabrafenib alone, tumour regression and a significant increase in apoptosis were observed upon exposure to both dabrafenib and GapmeR3 (Fig. 4d, e and Extended Data Fig. 10d). Notably, these mice did not suffer from any relevant adverse events or weight loss, in contrast to mice treated with a combination of dabrafenib and MEK inhibitor (trametinib; Fig. 4f).

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The observation that some cancer cells are dependent on OXPHOS for survival led to the development of agents that exploit bioenergetics and metabolic alterations in mitochondria 24 . The use of such agents is, however, complicated by their dangerous side effects. We show here that SAMMSON is a lineage-specific lincRNA that promotes melanoma survival through its ability to enhance the mitochondrial localization and function of p32, a protein required for the maintenance of OXPHOS 10 . Given that SAMMSON is expressed specifically in the vast majority (>90%) of melanomas, but not in normal adult tissues, these data identify SAMMSON as an attractive therapeutic target for the disruption of mitochondrial metabolism selectively in melanoma.

MITF, which is co-amplified with SAMMSON in a subset of melanomas, also promotes mitochondrial respiration by inducing 26). The MITF-SAMMSON amplicon therefore favours oxidative metabolism via two distinct mechanisms, the MITF-PGC-1α axis and the SAMMSON-p32 axis, making these cells particularly OXPHOS dependent.

Treatment of melanoma with BRAF inhibitors induces the MITF-PGC-1α-dependent oxidative metabolic program and renders them highly dependent on OXPHOS 26 . This observation explains why co-targeting of SAMMSON and mutant BRAF promotes potent antitumour responses. SAMMSON targeting may therefore offer a novel therapeutic avenue to overcome the adaptive metabolic reprogramming that limits the efficacy of BRAF inhibitors. Moreover, as melanoma cells that acquire resistance to BRAF inhibitors remain addicted to SAMMSON expression, SAMMSON targeting might be a valid therapeutic approach to treat relapsed patients.

In addition, MITF-low invasive cells, although glycolytic, remain addicted to SAMMSON expression. These cells may still be dependent on mitochondria for functions other than ATP generation, such as fatty acid synthesis and glutaminolysis 27,28 . Interestingly, p32 has recently been shown to be required for MYC-induced addiction 11 . Through its ability to modulate mitochondrial protein synthesis, SAMMSON is also likely to affect processes outside the mitochondrion 19 . Consistently, we provide evidence that SAMMSON targeting decreases melanoma survival in an mPOS-dependent manner.

These findings warrant further investigation about the potential of SAMMSON as an informative biomarker of malignancy and as a highly selective and broad-spectrum anti-melanoma therapeutic target. Given the recent surge in optimism about antisense drugs, this therapeutic approach may be rapidly applicable to the clinic.

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METHODS

Cell culture. The melanoma cell lines Mel501, SK-MEL-28 (obtained from ATCC) were grown in 5% CO 2 at 37 °C in RPMI 1640-glutamax (Gibco, Invitrogen) or DMEM (for Mel501) supplemented with 10% FBS (Hyclone, Thermo Fisher Scientific). Sequences of the GapmeRs and siRNAs are indicated later.

Caspase 9 inhibitor was purchased from Merk Millipore (218761). 4EGI-1 was purchased from Selleckchem and used at a final concentration of 10 µM. The patient-derived low-passage MM cell lines (a gift from the Ghanem laboratory) were grown in F-10 (Gibco, Invitrogen), supplemented with 10% FBS (Hyclone, Thermo Fisher Scientific) and 12 mM glutamine.

NHMEs were grown in MGM-4 melanocyte growth medium (Lonza). Cells were transfected by Lipofectamine 2000 according to manufacturer instructions with 25 nM of GapmeR (Exiqon). For siRNA experiments the cells were transfected with 25-50 nM siRNAs or Dharmacon Pools and harvested 24 and/or 48 h after transfection. All cell lines used in this study were mycoplasma negative. Human melanoma samples. Early stages of melanoma from the UZ Leuven archive were isolated by laser capture microdissection and RNA extracted with Arcturus PicoPure Frozen RNA Isolation Kit (Life Technologies). RAP-MS. For affinity purification of SAMMSON protein targets, 100 µg of Streptavidin Sepharose High Performance (GE Healthcare) was coupled to 400 pmol of biotinylated probes against SAMMSON (Biosearch Technologies) overnight at 4 °C. Cells (60 × 10 6 cells per sample) were lysed in 2 ml of pull-out buffer pH 7.5,200 mM NaCl,2.5 mM MgCl 2 , 0.05% Igepal, 60 U Superase-In per ml (Ambion), 1 mM dithiothreitol (DTT) and a cocktail of protease inhibitors) and incubated for 3 h with the beads at 4 °C. As a negative control, an additional sample was digested with 10 µg ml -1 RNase A digestion for 10 min at room temperature before incubation with SAMMSON probes.

For the crosslinking experiments, cells were washed once in PBS, crosslinked dry at 400 mj cm -2 and lysed. During the washes the amount of Triton X-100 was doubled and SDS was added to a final concentration of 0.02%.

For mass spectrometry, samples were processed by a short separation on SDS-PAGE gels (Biorad) to remove contaminants possibly interfering with downstream analysis. After excision, washing and drying of the gel band, digestion buffer (50 mM NH 4 HCO 3 , 5 µg ml -1 trypsin) was added to fully submerge the dried gel band, and the sample was digested for 16 h at 37 °C. The generated peptide mixtures were acidified, dried and re-dissolved in a 2% CH 3 CN (acetonitrile), 0,1% formic acid solution.

The obtained peptide mixtures were introduced into a liquid chromatograpy-tandem mass spectrometry (LC-MS/MS) system through an ultimate 3000 RSLC nano LC (Thermo Scientific) inline connected to a Q Exactive mass spectro meter (Thermo Fisher Scientific). The sample mixture was first loaded on a trapping column (made in house, 100 µm internal diameter (i.d.) × 20 mm, 5 µm beads C18 Reprosil-HD, Dr. Maisch, Ammerbuch-Entringen). After flushing from the trapping column, the sample was loaded on an analytical column (made in house, 75 µm i.d. × 150 mm, 5 µm beads C18 Reprosil-HD, Dr. Maisch) packed in the nanospray needle (PicoFrit SELF/P PicoTip emitter, PF360-75-15-N-5, NewObjective). Peptides were loaded with loading solvent (0.1% TFA in water) and separated with a linear gradient from 98% solvent A′ (0.1% formic acid in water) to 40% solvent B′ (0.08% formic acid in water/acetonitrile, 20/80 (v/v)) in 30 min at a flow rate of 300 nl min -1 . This was followed by a 15 min wash reaching 99% solvent B′. The mass spectrometer was operated in data-dependent, positive ionization mode, automatically switching between MS and MS/MS acquisition for the 10 most abundant peaks in a given MS spectrum.

The source voltage was 3.4 kV, and the capillary temperature was 275 °C. One MS1 scan (m/z 400-2,000, AGC target 3 × 10 6 ions, maximum ion injection time 80 ms) acquired at a resolution of 70,000 (at 200 m/z) was followed by up to 10 tandem MS scans (resolution 17,500 at 200 m/z) of the most intense ions fulfilling the defined selection criteria (AGC target 5 × 10 4 ions, maximum ion injection time 60 ms, isolation window 2 Da, fixed first mass 140 m/z, spectrum data type: centroid, underfill ratio 2%, intensity threshold 1.7 × 10 4 , exclusion of unassigned, 1, 5-8, and >8 charged precursors, peptide match preferred, exclude isotopes on, dynamic exclusion time 20 s). The HCD collision energy was set to 25% normalized collision energy and the polydimethylcyclosiloxane background ions at 445.120025 Da were used for internal calibration (lock mass).

From the MS/MS data in each LC run, Mascot Generic Files were created using Distiller software (version 2.4.3.3, Matrix Science, http://www.matrixscience.com/ distiller.html). While generating these peak lists, grouping of spectra was allowed in Distiller with a maximal intermediate retention time of 30 s and a maximal intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor ion tolerance. A peak list was only generated when the MS/MS spectrum contains more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched using the Mascot search engine (MatrixScience) 29 with the Mascot Daemon interface (version 2.4.1, Matrix Science). Spectra were searched against the human protein entries in the Swiss-Prot database (SP2014_07; 20284 sequence entries). Variable modifications were set as methionine oxidation, pyro-glutamate formation of N-terminal glutamine, propionamide formation on cysteine and acetylation of the protein N terminus. The mass tolerance on precursor ions was set to 10 ppm (with Mascot's C13 option set to 1) and on fragment ions to 20 mmu. The instrument setting was put on ESI-QUAD. Enzyme was set to trypsin, allowing for one missed cleavage. Only peptides that were ranked first and scored above the threshold score, set at 99% confidence, were withheld. The protein candidates that were pursued in this work were consistently identified with at least two different peptides in the relevant conditions (no peptides detected in the bead controls). RIP. RIP was performed as previously described 7 . p32 and XRN2 were immunoprecipitated using 4 µg of specific antibody (Bethyl laboratories) coupled to 50 µl of protein G Dynabeads (Invitrogen) for 3 h. Cellular fractionation. Briefly, total nuclear and cytoplasmic extracts were isolated from 20-cm ø dishes using Nuclei EZ prep (Sigma-Aldrich) according to the manufacturer's instructions. RT-qPCR for MALAT1 and TBP were used to assess the purity of the fractions.

Mitochondria were purified from 20-cm ø dishes using mitochondria isolation kit for cultured cells (Thermo Fisher Scientific). Mitochondria enrichment was validated by western blot using antibodies directed against mitochondrial proteins (that is, ATPB). Mitoplasts were obtained by incubating mitochondria in hypotonic buffer (HEPES pH 7.2) for 20 min on ice. Antibodies. Western blotting experiments were performed using the following primary antibodies: vinculin (V9131, Sigma-Aldrich, 1:10,000), histone 3 (ab1791, Abcam, 1:1,000), GAPDH (ab9485, Abcam, 1:1,000), p32 (A302-863A, Bethyl Laboratories, 1:5,000), XRN2 (A301-103A, Bethyl Laboratories, 1:2,000), SOX10 (N-20, Santa Cruz, 1:500), MITF (ab12039, Abcam, 1:1,000) and SDHA (Abcam AB14715, 1:1,000), COX2 (molecular probes A6404, 1:10,000), HSP60 (BD 611562, 1:5,000), NDUFS3 (Abcam 14711, 1:1,000), ATP6 (Abcam 192423, 1:1,000). Cell growth and cell death assays. To detect cell death, cells were stained for 15 min with annexin V and PI using the FITC Annexin V Apoptosis Detection Kit II (BD Biosciences) according to the manufacturer's instructions. Cell death was detected on a MACSQuant VYB (Miltenyi Biotech BV) and data were analysed with FlowJo software (Tree Star).

Caspase 3 and 7 activity was measured using Caspase-Glo 3/7 luciferase assay (Promega) and VICTOR X4 Reader (PerkinElmer) 48 and 72 h after transfection of GapmeRs. Colony assay and cell count. Cells were plated in six-well plates at the appropriate density and cultured for 1 week. The cells were washed with PBS, fixed and stained for 15 min with a 1% crystal violet in 35% methanol solution.

For vital counts, cells were stained with Trypan Blue (Sigma-Aldrich) and counted with TC20 automated cell counter (Biorad).

BRAF and MEK inhibitors (vemurafenib and pisertimab) were used at a concentration of 5 and 1 µM, respectively. SAMMSON cloning, mutagenesis and lentiviral transduction. SAMMSON was synthesized by GenScript and cloned into pPGK (Addgene) lentiviral vector. Lentivirus produced in HEK293 cells were used to infect the MM001 cells. Successfully infected cells were selected in puromycin (0.5 µg ml -1 )-containing medium for 1 week. Pharmacological treatment of mice. The Mel0010 and Mel006 PDX models derived from two different metastatic melanoma lesions, both carrying the BRAF(V600E) mutation. Written informed consent was obtained from both patients and all procedures involving human samples were approved by the UZ Leuven/KU Leuven Medical Ethical Committee (# ML8713/S54185). All procedures involving animals (NMRI nude, 4-week-old females) were performed in accordance with the guidelines of the Catholic University of Leuven (KU Leuven) Animal Care and Use Ethical Committee (P147/2012).

Once tumours reached 150 mm 3 , 10 mg kg -1 of GapmeR3 or control GapmeR were injected i.v. or directly into the tumours every 2 days for up to 20 days. For combination therapy with BRAF inhibitor, cohorts of Mel006 were enrolled into the experiment once tumours reached 250 mm 3 in volume. Dabrafenib or vehicles were administrated daily by oral gavage. The GapmeRs, at a concentration of 10 mg kg -1 , were administrated i.v. every 2 days.

No specific randomization method was used. According to animal welfare guidelines, mice have to be killed when tumours reach a volume of 2,000 mm 3 or when their body weight decreases more than 20% from the initial weight. Mice used in this paper never reached or overcame these limits. The investigators were blinded for the evaluation of the results. FISH for SAMMSON. For detection of SAMMSON at a single-cell level, a pool of 48 FISH probes was designed using the Stellaris probe designer software (Biosearch LETTER RESEARCH Technologies). Cells were grown on slides and fixed in 3.7% formahldeyde and permeabilized in ethanol 70%. Hybridization was carried out overnight at 37 °C in 2× SSC, 10% formamide and 10% dextran. Cells were counterstained with DAPI and visualized using an Olimpus Fluoview FV1200 using a LD635 laser for Cy5, LD559 HeNe for Cy3.5 and LD405 for DAPI. Electron microscopy. Cells were fixed with 2.0% paraformaldehyde/2.5% EM-grade glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 37 °C overnight and collected. After fixation, samples were placed in 1% osmium tetroxide for 2 h and dehydrated in a graded series of ethyl alcohol. The agarembedded samples were cut with a Leica UCT ultramicrotome in 50-70 nm sections and imaged in a JEOL JEM1400 transmission electron microscope at 80 kV. Immunohistochemistry and immunofluorescence. Tumour biopsies formalin-fixed and paraffin embedded from the UZ Leuven archives, were cut in sections of about 4 µm. Samples were deparaffinized and dehydrated with xylene and graded alcohols, and subsequently rehydrated with demineralized water. Specimens were stained with haematoxylin and eosin and immunohistochemistry was performed using microwave pre-treatment of slides for antigen retrieval. Antibodies against Ki-67 (SP6, Thermo Fisher Scientific #RM-9106-S, clone SP6, 1:200) and cleaved caspase 3 (Asp175, Cell Signaling Technology, 1:300) were applied, in conjunction with goat anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies (DAKO) and visualized by DAB reaction. To evaluate the stainings, positive cells in the blue channel were counted in four different fields using ImageJ. Statistical significance was calculated by two-way ANOVA.

For immunofluorescence, cells were grown on slides and permeabilized in Triton X-100-containing buffer. Blocking was performed in 5% BSA and 10% goat serum. To detect p32 a rabbit antibody from Bethyl Laboratories was used 1:1,000 followed by staining with an anti-rabbit AlexaFluor-488 (Life Technologies, 1:500). Images where analysed on an Olympus fluoview FW1200 using a LD635 laser for Cy5, LD559 HeNe for Cy3.5 and LD405 for DAPI. To detect ATPB (AB14730) and SDHA (ab66484) antibodies from Abcam were used according to the manufacturer's instructions. RT-qPCR lncRNA expression profiling. Expression of 1,718 lncRNAs was measured on the NCI60 cell line panel using the SmartChip Human lncRNA1 panel (Wafergen Biosystems). NCI60 cell lines were obtained through the Developmental Therapeutics Program (National Cancer Institute (NCI)). Previously misclassified cell lines NCI/ADR-RES and MDA-MB-435 were correctly annotated in the analysis. RNA was isolated using the miRNeasy mini kit (Qiagen) and reverse transcribed (2 µg) using the iScript Advanced RT kit (Bio-Rad) according to the manufacturer's instructions. A qPCR reaction mixture containing 2 µg of cDNA and SsoAdvanced Universal SYBR Green mastermix was dispensed using the MultiSample NanoDispenser across the 5,184-nanowell SmartChip and analysed using the SmartChip Cycler (Wafergen Biosystems). lncRNA expression was measured in triplicate and median Cq values were normalized using the global mean normalization strategy 30 . RNA from normal adult tissues (Ambion and Biochain) and primary melanocytes was reverse transcribed using the iScript RT kit (Bio-Rad). SAMMSON expression was measured by qPCR on a LightCycler 480 (Roche) and normalized in qbase+ (Biogazelle) using HPRT1, TBP and SDHA as reference genes. Microarray gene expression profiling. Protein-coding gene expression in xenografts was measured using a custom gene expression microarray (SurePrint 8 × 60k, Agilent) or commercial microarray (SurePrint G3 Human Gene Expression v.2, Agilent) respectively. RNA (100 ng) was labelled using the Quick Amp polyA labelling kit and hybridized according to the manufacturer's instructions. Slides were scanned using a high-resolution microarray scanner (Agilent) and probe intensities were extracted using Feature Extraction software (Agilent). Signals were background corrected and Quantile normalized using the limma package in R. Only those probes expressed twofold above the mean signal of a negative control probe were retained for further analysis. Differential gene expression and pathway analysis. Probes were collapsed to gene level by retaining the probe with the highest average signal across all samples. Probes expressed in less than half of the samples were discarded for differential gene expression analysis. Genes differentially expressed across four cell lines (SK-MEL-28, MM034, MM057, MM087) upon knockdown of SAMMSON were identified using the limma package with cell line as a blocking variable. For differential expression analysis in xenograft tumours, no blocking variable was applied. Genes with an adjusted P value < 0.05 and twofold expression change were selected as differentially expressed. Gene set enrichment analysis was performed on mRNA lists, pre-ranked according to the limma t-statistic using all curated gene sets from the Molecular Signatures Database. Generalized additive models. Protein-coding gene expression data for the NCI60 cohort were obtained from the Developmental Therapeutics Program (NCI) data portal. Expression of each of the 1,718 lncRNAs (response variable) was analysed in relation to cancer type and transcription factor expression (predictor variables) using generalized additive models (GAMs). A total of 1,270 transcription factors were analysed. GAMs were constructed using the mgcv package in R using the following parameters: family = "Gaussian", link = "identity", method = "GCV.Cp". The GAM value for each model represents the percentage of lncRNA expression variance explained by the predictor variable. Quantification of RNA-FISH data. Tiff Z-stacks were imported using the StarSearch software package for automated spot counting of defined regions. Nuclear and total cell spots were counted for MM057, MM087 and SK-MEL-28 cells. Nuclear lncRNA copy number was defined as the average number of nuclear spots while cytoplasmic lncRNA copy number was defined as the difference between the number of total cell spots and the number of nuclear spots. Analysis of expression and copy number data from the TCGA cohort. Level 3 segmented DNA SNP array data for 386 melanoma tumours was obtained through the TCGA data portal. SAMMSON copy number was defined as the mean log ratio of the overlapping segment(s). Expression data for SAMMSON (uc003dog) and SOX10 (uc003aun) in melanoma and 23 additional cancer types were extracted from level 3 TCGA RNA-seq data, totalling 8,085 primary tumour samples. SAMMSON transcript boundaries. The SAMMSON TSS site was identified by means of CAGE-seq tags in two melanoma cell lines obtained from the FANTOM5 study (http://fantom.gsc.riken.jp/5/). The SAMMSON 3′ end was identified by means of 3′ RACE. Briefly, an anchored oligo-dT primer (AATACGACTCACTATAGGCGCTTTTTTTTTTTTTTTTTTTTTVN) was used for cDNA synthesis of SK-MEL-28 RNA using the iScript select reverse transcription kit (BioRad). The 3′ end was amplified (forward: AGCCAAATTTCAATGA GCCCCT; reverse: AATACGACTCACTATAGGC GC) and the resulting PCR amplicon was sized using the labchip (Calliper). The 3′ end was identified based on the amplicon length and location of the forward primer. High-resolution respirometry. SK-MEL-28 cells were resuspended in 20 mM HEPES, 110 mM sucrose, 10 mM KH 2 PO 4 , 20 mM taurine, 60 mM lactobionic acid, 3 mM MgCl 2 , 0.5 EGTA, pH 7.1, 1 mg ml -1 fatty-acid-free BSA and catalase 280 U ml -1 . Mitochondrial oxygen consumption was measured after plasma membrane permeabilization with digitonin 10 µg ml -1 in an Oroboros 2k apparatus at 37 °C. The oxygen consumption rates, expressed as pmol O 2 s -1 mg protein -1 , were measured after addition of the following substrates and specific inhibitors.

(1) 2.5 mM pyruvate, 1 mM malate, 10 mM glutamate in the absence of ADP to determine complex-I-driven non phosphorylating respiration (CI leak). (2) 2.5 mM ADP to determine complex-I-driven phosphorylating respiration (CI OXPHOS). The coupling efficiency between oxygen consumption and phosphorylation was estimated as the ratio between CI OXPHOS and CI leak. (3) 10 mM succinate to determine the phosphorylating oxygen consumption driven by simultaneous activation of complex I and II (CI+II OXPHOS). ( 4) Titrating concentrations of the mitochondrial uncoupler CCCP to reach the maximal, uncoupled respiration (CI+II electron transfer system). ( 5) 0.5 µg ml -1 antimycin A to block mitochondrial respiration at the level of complex III, and estimate residual nonmitochondrial oxygen consumption. ( 6) 2 mM ascorbate, 0.5 mM TMPD to measure cytochrome c oxidase (CIV)-driven respiration. ( 7) 10 µg ml -1 cytochrome c to evaluate mitochondrial outer membrane damage. ( 8) 250 µM potassium cyanide to measure residual chemical background. CIV-driven respiration was measured as the cyanide sensitive oxygen consumption. Measurement of ATP production, mitochondrial membrane potential and ROS production. Luciferase-based measurement of ATP production was obtained using Molecular Probes kit according to the manufacturer's instructions.

Mitochondrial membrane potential was measured by flow cytometry on a MACSQuant VYB (Miltenyi Biotech BV) using JC-1 dye (Molecular Probes). Data were analysed with FlowJo software (Tree Star) and the extent of depolarization was defined by the ratio between J-aggregates and J-monomers.

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Extended Data Figure 1 | SAMMSON is a lineage-specific lncRNA expressed in the vast majority of melanomas. a, SAMMSON is a polyadenylated and multi-exonic lncRNA that contains one additional exon (in red) downstream of the four GENECODE19-annotated exons (blue). For each melanoma (SKCM) sample in the TCGA, the mapped RNA-seq data were converted into a coverage plot. The coverage data are normalized for library size and log1p transformed. Subsequently, the coverage data for all primary (SKCM.01) and metastatic samples (SKCM.06) in the region chromosome 3:70,040,000-70,140,000 is plotted as two heat maps. b, Cap analysis of gene expression-sequencing (CAGE-seq), RNA-seq data and RT-qPCR analyses from short-term melanoma cultures (MM lines) confirmed that SAMMSON is not a read-through transcript from the upstream MITF locus. CAGE-seq counts as defined by the FANTOM5 mammalian promoter expression atlas for two melanoma cell lines at the SAMMSON locus (panels 1 and 2), location of the a 3′ rapid amplification of cloned/cDNA ends (RACE) fragment for SAMMSON (panel 3) and RNA-seq counts from a primary melanoma tumour in the SAMMSON locus. c, SAMMSON and MITF copy number as measured by qPCR in short-term melanoma cultures and melanoma cell lines. Reference human genomic DNA was used as scaling control. Error bars represent s.d. of qPCR replicates (n = 2). A significant correlation between MITF and SAMMSON copy number was observed (bottom; Spearman's rank rho = 0.933, P < 0.001). d, Expression of SAMMSON in human short-term melanoma (MM) and NHME cultures relative to the expression average of three housekeepings (left) and correlation with MITF expression by western blot (right; for gel source data, see Supplementary Fig. 1). Error bars represent s.d. of three replicates (n = 3). e, Expression correlation between MITF-M and SAMMSON in melanoma clinical samples from the TCGA database. f, Read counts were generated from RNA-seq data from TCGA melanoma samples (SKCM) and normalized to the library size. Samples were subdivided into proliferative and invasive groups as described previously 3 and box plots were generated for SAMMSON, ZEB1 and MITF. Differential expression analysis between the proliferative and invasive groups was done using edgeR50. lfc, log fold change; pval, uncorrected P value; fdr, false-discovery-rate-corrected pval. g, Relative expression in 60 cancer cell lines (NCI60 panel). h, Fraction of lncRNA expression variation (n = 1,472) across the NCI60 panel by cancer type according to a generalized additive model (GAM).

LETTER RESEARCH

Extended Data Figure 2 | SAMMSON expression in melanoma, but not other cancer, cell lines, is at least partly SOX10-dependent. a, H3K27ac ChIP-seq data generated in house using a series of short-term melanoma cultures 2 were integrated with cancer cell lines data retrieved from ENCODE. A clear H3K27ac peak is present upstream SAMMSON in all, but one (MM001), melanoma lines. No peak is detected in the vast majority (19/20) of non-melanoma cancer cell lines, of which 9 are shown. b, UCSC screenshots of ChIP-seq data for SOX10, 3HA-MITF and H3K27ac at the MITF and SAMMSON loci in Mel501. c, ChIP-qPCR of endogenous SOX10 in 501Mel cells at the indicated loci. The IgG antibody was used as a negative control. SOX10 recruitment to its well-established targets GPR110, TYR and SOX10 itself, but not to a non-SOX10 target PRM1, confirms the specificity of the SOX10 ChIP experiment. c, Western blotting analysis of total protein lysates of SK-MEL-28 confirming efficient knockdown of SOX10 and MITF. GAPDH was used as a loading control (for gel source data, see Supplementary Fig. 1). e, Fold change RNA expression levels in 501Mel cells transfected with a control siRNA pool (siCtrl) or pools targeting MITF (siMITF) or SOX10 (siSOX10).

LETTER RESEARCH

Extended Data Figure 7 | RAP-MS identifies the mitochondrial protein p32 as a SAMMSON interactor. a, The 'Metrics' table provides an overview of the MS experiment. For every MS analysis, the table contains the number of analysed spectra, the total number of identified spectra (peptide to spectra matches (PSMs)), the number of distinct peptide sequences, protein numbers and a false discovery rate (FDR) estimation based on searches against a reversed database. b, Western blot for p32 in NHMEs and in a panel of short-term melanoma cultures. Vinculin is used as a loading control and normalizer for the quantification. c, RNA-FISH for 16S rRNA (in red) and immunofluorescence for p32 (in yellow) in NHMEs and in a panel of melanoma cell lines. DAPI is in cyan. d, Pulldown of SAMMSON (and HRPT) under native conditions and upon incubation with RNase A, followed by western blotting. e, Western blot confirming enrichment of p32 following immunoprecipitation with anti-p32 antibodies. b, d, e, For gel source data, see Supplementary Fig Another possibility is that drivers end up in close proximity by random assortment on a limited number of chromosomes, which makes copy number changes more potent cancer drivers than individual mutations in certain genomic regions. In turn, deletion size may be delineated by the location of genes whose partial loss would confer a selective disadvantage during tumorigenesis (e.g., Davoli et al., 2013;Solimini et al., 2012).

Regardless of why cancers select for gene mutations in some settings and copy number changes in others, the new results should increase consideration of the prognostic potential of these lesions. Cai et al. show that 8p LOH compared to other SCNAs has the strongest association with poorer prognosis in breast cancer patients and that patients harboring 8p LOH who underwent chemotherapeutic treatment displayed decreased survival, an effect also seen in liver cancer [START_REF] Xue | Proc. Natl. Acad. Sci. USA[END_REF]. Liu et al. found that human myeloid leukemias harboring 17p deletions together with TP53 missense mu-tations are more aggressive than those harboring only TP53 mutations.

SCNAs might also be exploited therapeutically. Amplifications may offer the possibility to target more than one oncogene, potentially increasing the efficacy and specificity of therapeutic interventions. In case of segmental deletions the situation is more complex, but hemizygous lesions can lead to specific vulnerabilities when essential proteins encoded in the deleted region are less expressed, thereby creating a therapeutic window for inhibitors relative to normal cells (Nijhawan et al., 2012). Cai et al. used their system of isogenic cell lines to screen diverse compounds and found that 8p LOH confers decreased sensitivity to microtubule inhibitors or HMG-CoA reductase inhibitors but increased sensitivity to autophagy inhibitors, offering a potential therapeutic option for this specific SCNA. While there is still much work ahead to functionally explore SCNAs, the high frequency and distinct nature of these genomic events-apart from single gene mutations-warrants increased attention.

In a recent study, Leucci et al. report a role for the long non-coding RNA SAMMSON in driving mitochondrial function in melanoma. Targeting SAMMSON, the gene of which is frequently co-amplified with MITF, highlights a new cell-type-specific therapeutic vulnerability in melanoma irrespective of BRAF, NRAS, or p53 status.

Sequencing the human genome revealed far fewer protein-coding genes than were previously imagined. Although some of the intergenic space is taken up with regulatory elements that provide the crucial spatiotemporal regulation of mRNA expression necessary for development and homeostasis, the great majority of the genome appeared to lack function. et al., 2012), with non-coding RNAs making up the vast majority of transcripts. These comprise so-called small RNAs (<200 bases), including microRNAs and Piwi-interacting RNAs, and long non-coding RNAs (lncRNAs) (>200 bases) that include anti-sense RNAs, overlapping bidirectional transcripts, and those that are intronic or intergenic. The potential regulatory roles of lncRNAs are attracting increasing attention as a major class of biological regulators, with current estimates indicating almost 16,000 lncRNA genes. Broadly speaking, lncRNAs fall into two functional classes: those that function in cis, acting at or very close to the site of synthesis, for example by nucleating specific chromatin conformations that facilitate up-or downregulation of transcription; and those that act in trans and play a more widespread or global role. Importantly, lncRNAs can be inactivated by small interfering RNAs and/or antisense oligonucleotides (ASOs) and therefore represent potential therapeutic targets. However, to date few lncRNAs have a characterized pathophysiological role. The identification by Leucci et al. (2016) of SAMMSON (previously referred to as LINC01212) as a lncRNA with a crucial role in melanoma survival has provided proof-of-principle that targeting a lncRNA in vivo is a potentially viable therapeutic option.

Previews

Cutaneous melanoma, a highly aggressive skin cancer, arises from melanocytes that have their developmental origins in the neural crest. Oncogenic transformation of melanocytes to melanoma usually occurs via a combination of senescence bypass, for example by inactivation of the CDKN2A locus or loss of PTEN, combined with de-regulated MAPK signaling primarily driven by activating mutations in BRAF (50%) or NRAS (20%). Localized early-stage melanoma is effectively cured by surgical excision, but metastatic disease poses several challenges. Recently developed immune checkpoint inhibitors can be highly effective in some patients, but a significant proportion of patients either do not respond or develop resistance; treatment with BRAF inhibitors usually results in resistance developing within a few months. Therefore, an urgent need remains for complementary therapeutic strategies.

Like in most cancers, the tumor microenvironment also plays a key role in mela-noma by promoting metastatic spread and the development of therapeutic resistance. For melanoma, one of the hallmarks of intra-tumor phenotypic heterogeneity that arises though the influence of the microenvironment is the expression of the microphthalmia-associated transcription factor (MITF), which is required for melanoblast survival during development and melanocyte differentiation. Significantly, MITF also promotes proliferation and suppresses invasion. Dynamic expression of MITF in response to microenvironmental cues is thought to drive the reversible inter-conversion of cells between differentiated, proliferative, and invasive states, a process termed ''phenotype switching'' that underpins disease progression (Hoek and Goding, 2010). The pro-proliferative role of MITF, combined with the observation that the gene can be highly amplified in some melanomas, led to MITF being dubbed a lineage-addiction oncogene (Garraway et al., 2005). However, even though MITF can be amplified 100-fold, the change in protein levels is restricted to less than 2fold. One interpretation is that despite amplification of MITF, the protein levels must be restricted to prevent cell differentiation. The work from Leucci et al. (2016) provides another possible explanation: the chromosome 3p MITF focal amplicon invariably contains the SAMMSON lncRNA that lies around 30 kb downstream from MITF, and unlike MITF protein, SAMMSON RNA levels did correlate with its gene copy number. Moreover, SAMMSON levels did not vary substantially between MITF-high proliferative cells versus MITF-low invasive cell lines, and SAMMSON was barely detectable in melanocytes or non-invasive vertical growth phase melanomas. However, SAMMSON was readily detected in invasive vertical growth phase melanoma and in migratory melanoblasts; among different tumor types, it was selectively expressed in melanoma.

SAMMSON transcription appears to be independent of MITF; it has its own promoter marked by the classical promoter-associated H3K4me3 and H3K27ac epigenetic marks and is regulated by SOX10, a lineage-restricted transcription factor, but not by MITF. However, because SOX10 also regulates MITF expression and is frequently downregulated in MITF-low melanomas, it is not clear how SAMMSON expression would be maintained in MITF-low invasive melanoma cells or cells in which SOX10 is downregulated due to BRAF inhibition (Sun et al., 2014). One possibility is that in SOX10-low cells, SAMMSON is regulated by an alternative SOX factor such as SOX9, known to play a key antagonistic role to SOX10 in melanoma (Shakhova et al., 2015).

Crucially, SAMMSON confers a growth advantage on melanoma cells. Targeting SAMMSON for degradation reduced clonogenicity, irrespective of BRAF, NRAS, or p53 status, including in cell lines exhibiting BRAF inhibitor-resistance, but did not affect melanocytes, highlighting the ''addiction'' of melanomas to SAMMSON expression. It also reduced viability/ growth of invasive melanoma cells, known to exhibit increased resistance to MAPK therapeutics. Importantly, ectopic expression of SAMMSON in melanoma cells conferred a growth advantage, indicating that SAMMSON acts in trans, an observation consistent with the lack of effect on MITF expression following SAMMSON knockdown.

The authors next addressed the role of SAMMSON, with an early clue to its mode of action being that a fraction of SAMMSON co-localized with mitochondria. A mass spectrometry approach to identify SAMMSON-interacting proteins not surprisingly revealed interaction with a number of well-known RNA-binding proteins, including XRN2, a 5 0 -to-3 0 exonuclease that had previously been detected as a lncRNA-binding factor. However the authors' attention focused on p32, a protein implicated in mitochondrial metabolism and translation of mitochondrially encoded peptides that is required for proliferation of cancer cells, including melanoma (Fogal et al., 2008(Fogal et al., , 2010)). SAMMSON-p32 interaction was confirmed using RNA immunoprecipitation. Significantly, depletion of SAMMSON decreased the fraction of p32 associated with mitochondria, increased the nuclear fraction, and markedly diminished mitochondrial membrane potential. Consistent with SAMMSON-p32 interaction being important for mitochondrial function, depletion of SAMMSON phenocopied depletion of p32, yielding structurally aberrant mitochondria (Figure 1). The p53-independent apoptotic response triggered by 620 Cancer Cell 29, May 9, 2016 Cancer Cell Previews SAMMSON depletion appeared to arise as a result of accumulation of mitochondrial peptide precursors and mitochondrial import defects, collectively known as mitochondrial precursor-over-accumulation stress (mPOS).

Using patient-derived xenograft models, the authors were able to demonstrate that intravenous administration of a SAMMSON-specific antisense oligonucleotide decreased tumor growth and synergized with Dabrafenib, a BRAF inhibitor, to induce apoptosis in vivo. The synergy observed is likely to arise because BRAF inhibition activates an MITF-PGC1a axis to elevate mitochondrial oxidative phosphorylation (Haq et al., 2013), rendering cells more susceptible to inhibition of mitochondrial function by targeting the SAMMSON-p32 complex. Whether targeting SAMMSON will be effective in metastatic disease and whether it will be more effective than using other mitochondrial targeting agents, which unlike SAMMSON would not be melanoma specific, remains to be determined. Nevertheless, the work from Leucci et al. (2016) reveals a key role for the SAMMSON lncRNA that may explain how the MITF amplicon impacts mela-noma proliferation. It also nicely demonstrates how targeting a key lncRNA can have therapeutic benefit, at least in nonmetastatic xenograft models, and reinforces other studies (Zhang et al., 2016) showing how targeting mitochondrial function, and especially mitochondrial protein folding, is likely to prove beneficial in melanoma. In Laurette et al., we showed that inactivation of Brg1 in the melanocyte lineage during embryogenesis led to the generation of white mice lacking any identifiable melanocytes in the hair follicles. Nevertheless, we did not investigate at which stage of melanoblast development Brg1 is required. The Tyr::Cre transgene drives recombination of floxed alleles as early as E10.5 (Delmas et al. 2003;[START_REF] Luciani | Biological and mathematical modeling of melanocyte development[END_REF] and it was therefore possible that Brg1 inactivation at this stage led to an immediate loss of developing melanocytes.

To address this question, we crossed the Tyr::Cre; Smarca4 lox/lox animals with the Dct reporter mice in order to visualise at which stage melanoblast development was affected. Tyr::Cre; Smarca4 mel-/-; Dct::LacZ and Tyr::Cre; Smarca4 mel-/+ ; Dct::LacZ embryos were harvested and stained at E12.5, E14.5 and E15.5. At E12.5, LacZ-stained melanoblasts were visible in the Tyr-Cre; Smarca4 mel-/-embryos although their numbers were clearly diminished compared to the Tyr-Cre; Smarca4 mel-/+ embryos (Annexe 1, Fig. 1). By E14.5, the number of LacZ-stained melanoblasts was strongly diminished in absence of Brg1, while at E15.5 they were almost completely missing.

Assuming that defloxing of the Smarca4 alleles took place between E10.5 and E11.5 when the Tyr::Cre transgene gets activated, these data suggest that Brg1 is not absolutely required for the initial expansion and migration of the melanoblast population as numerous melanoblasts were visible throughout the embryo.

Nevertheless, Brg1 contributes to this expansion and is essential at later stages for continued proliferation and maintenance of this population as by E15.5 almost no lacZstained cells were observed in the mutant embryos and the resulting mice were born with an absence of pigmentation. Thus, Brg1 inactivation did not result in an immediate death of melanoblasts, but their progressive loss during embryogenesis.

ANNEXE 3 Expression profile of PBAF subunits and major transcription factors in representative human proliferative-and invasive-type melanoma lines

Whole cell extracts of proliferative type (MM011, MM074, MM117) and invasive type (MM029, MM047, MM099) primary human melanoma cultures were analysed by western blot with antibodies against the indicated proteins.

A complex network of protein interactions around MITF

A comprehensive characterisation of the MITF interactome revealed its association with multiple known and novel factors or complexes, including PBAF and NURF, TFIIIC, cohesins and multiple enzymes of the ubiquitin cycle. As expected, we found TFE3, TFEB, and TFEC associated with MITF, as they are well known dimerization partners. Strikingly, so far we don't know which sites are bound as MITF homodimers or MITF-TFE heterodimers and whether binding as homo-or heterodimers has functional consequences on transcription regulation, although given their nature there must be some overlap in the their target genes. Mouse genetic studies have not revealed functions for the TFE factors in development of the melanocyte lineage (Steingrimsson et al., 2002), however their role in melanoma has not been fully evaluated. Work from invasive-type cells in which MITF is not expressed will likely contribute to characterize their specific roles. A striking finding is the interaction of MITF with multiple proteins involved in DNA damage and repair, suggesting that MITF may influence these processes. Nevertheless, we so far found no recruitment of MITF to DNA damage sites (unpublished observations) and the significance of these interactions remains to be determined. Similarly, MITF associates with TFIIIC suggesting that it may regulate Pol III transcription. Alternatively, the presence of cohesin subunits in the interactome suggests that MITF may associate with TFIIIC at 'Extra TFIIIC' sites that organise chromatin structure in association with the cohesin complex. MITF also interacts with complexes involved in DNA replication. Assembly of DNA replication origins can be influenced by the presence of transcription regulatory elements and MYC that also interacts with MCM subunits, has been shown to directly influence DNA replication (Dominguez-Sola et al., 2007;Me ćhali, 2010). Perhaps MITF interaction with these replication complexes facilitates replication origin assembly at MITF-occupied sites thereby coupling replication with transcription regulation in a melanocyte-specific manner.

Interestingly, we detected the presence of deubiquitinating enzymes USP7 and USP11. USP7 has been previously reported to control the stability of p53 and a recent study further characterized its role in transcription coupled nucleotide excision repair [START_REF] Sarasin | UVSSA and USP7: new players regulating transcription-coupled nucleotide excision repair in human cells[END_REF]. Little is known about USP11 although there is good evidence that USP11 is a component of homologous repair and both USP7 and USP11 have been reported to regulate activity of the PRC1 Polycomb repressor complex (Maertens et al., 2011). In contrast, USP13, another de-ubiquitinase of this family that has been shown to regulate MITF stability (Zhao et al, 2011) was not detected in our experiments.

During the first year of my thesis, I performed extensive biochemical characterization of the interaction of MITF with USP7 and USP11, two factors that we found to be essential for melanoma cell proliferation. Both knockdowns and overexpressions of USP7 turned out to be toxic for melanoma cells, which eventually ended with cell death. Thus, we focused our efforts on trying to understand the role of USP11. Despite interesting observations on transcriptional regulation of SOX10 expression (and indirectly on MITF), we were unable to define a precise role for USP11 in melanoma cells and the functional relevance of its interaction with MITF. When the melanocyte lineage specific knockout of USP11 in the mouse embryo turned out to have no effect on hair pigmentation at birth or in older mice, we decided to change the scope of the project. Still, future directions might come from a careful look at the partners of USP11 that we identified by TAP-MS in melanoma cells. Furthermore, as our investigations were in agreement with a role for USP11 in DNA damage response and repair, it might be worth checking if USP11 contributes to genomic stability and melanomagenesis using DNA-damage inducing approaches and suitable mouse models of melanoma.

Nevertheless, parallel investigations we were conducting on the PBAF remodelling complexes turned out to be quite interesting leading me to refocus the work on the characterization of this complex.

MITF interacts with a BRG1-CHD7-containing PBAF complex

The numerous MITF partners that we identified include several subunits of the PBAF and NURF ATP-dependant chromatin remodelling complexes. BRG1, but not the related Brahma (BRM) protein were detected, although both proteins are expressed in 501Mel cells (Keenen et al., 2010, our own observations) and biochemical characterization confirmed that MITF interacts with a BRG1/PBAF-like complex comprising CHD7. This is consistent with previous reports (de la Serna et al., 2006;Keenen et al., 2010), although the composition of the complex had not been determined. Follow up work should determine whether the interaction of MITF and PBAF complex is direct or mediated by intermediate interactors. This can be easily addressed with available approaches and significant efforts should be put on this question, as it will give a much better understanding of the functional relationship between MITF and the complexe.

Approximately 20% of cancers including melanoma contain mutations to key subunits of the BAF complex resulting in a compositionally altered oncogenic BAF complex that promotes unchecked proliferation by mechanisms that are not completely understood [START_REF] Wilson | SWI/SNF nucleosome remodellers and cancer[END_REF][START_REF] Hodis | A landscape of driver mutations in melanoma[END_REF], Bastian et al., 2014[START_REF] Helming | Vulnerabilities of mutant SWI/SNF complexes in cancer[END_REF]. The contributions of the SWI/SNF complex to chromatin structure in vivo and the reasons why each subunit is associated with distinct cancer patterns remain poorly understood. Given the unique chromatin targeting and modification domains found in each subunit, disease development likely occur due to distinct mistargeting of residual SWI/SNF complexes causing disruption of lineage-specific gene expression programs.

Interestingly, we looked at the expression of all subunits of the PBAF complex in different melanoma cell lines established by the Marine Lab (3 proliferative-and 3 invasive-type) and noticed that some lines display loss of specific subunits (Annexe 3). It would be speculative to make a link between this loss and the melanoma phenotype but although we don't know about the mutational status of the remaining subunits yet, such cells lines will be a useful tool to evaluate their specific contribution to PBAF recruitment to chromatin and (de)regulation of gene expression. Studies we have initiated in the lab include evaluation of this contribution by systematic knockdown experiments and molecular and functional characterization of the resulting phenotypic and biochemical effects. Valuable information will also rise from genome occupancy profiling of relevant factors similar to those from the Snyder lab (Euskirchen et al., 2011), especially regarding specific PBAF complex subunits (ARID2, PBRM1, CHD7). Unfortunately we have so far been unable to co-localize BRG1 and CHD7 by ChIP-seq due to the lack of ChIP-seq grade CHD7 antibodies (our unpublished data).

Importantly, in a recent exome sequencing study [START_REF] Hodis | A landscape of driver mutations in melanoma[END_REF], ARID2 was found to be mutated in up to 14% of melanoma samples, likely resulting in a loss of function. Although it is well established that ARID2 is a member of the PBAF remodelling complex, the mechanisms by which ARID2 functions as a tumor suppressor remain unclear, and its role in melanoma is completely unknown. Thus, characterizing the role of ARID2 in melanomagenesis with similar approaches to the one presented here might pinpoint targetable vulnerabilities of ARID2-mutant cells in melanoma as well as other types of cancer.

BRG1, MITF and SOX10 coregulate gene expression in human melanocytes and melanoma

Our work shows that BRG1 and MITF are essential for melanocyte and melanoma growth in vitro. The two factors seem to cooperate to regulate an overlapping subset of genes involved in several essential cellular processes associated with pigmentation, resistance to apoptosis, cell morphology, and signalling; and in their absence cells undergo growth arrest and senescence characterised by a SASP similar to that seen upon MITF silencing. This requirement for BRG1 to drive a large gene expression programme required for melanoma cell proliferation in vitro contrasts with its tumour suppressor function in human cancers including melanoma (Hargreaves and Crabtree, 2011;Shain and Pollack, 2013;Bastian, 2014;Wang et al., 2014). Ondrusova et al. reported an MITF-independent pro-survival role for BRG1 in melanoma cells [START_REF] Ondrusova | MITF-independent prosurvival role of BRG1-containing SWI/SNF complex in melanoma cells[END_REF]. This is consistent with our findings that BRG1 silencing affects expression of many more genes than MITF and that the binding motifs for a variety of factors other than MITF are enriched at BRG1-occupied sites. BRG1 is likely therefore to act as a cofactor for many other TFs accounting for its MITF-independent functions.

Given this critical function of BRG1, we used ChIP-seq to identify MITF and BRG1 binding sites as well as histone marks in order to investigate regulation of transcription in melanoma cells.

We observed that BRG1 binds extensively over the melanoma cell genome often at active H3K27ac-marked enhancers consistent with previous reports (Hu et al., 2011).

BRG1 occupied sites displayed a variety of profiles at TSS and enhancers. The specific positioning of BRG1 at the TSS was not noted in several previous ChIP-seq studies (De et al., 2011;Euskirchen et al., 2011;Ho et al., 2009;Hu et al., 2011;Yu et al., 2013;Morris et al., 2014), however, Tolstorukov et al. (2013) reported that BRG1 occupies the nucleosomes flanking the TSS. Tolstorukov et al. also reported that inactivation of BRG1 does not affect the positioning of these two nucleosomes, but rather elicits a strong reduction in their occupancy. Nevertheless, the overall meta-profiles presented by Tolstorukov et al., analogous to that reported in our study, overlooked two important features: (1) many promoters show BRG1 occupancy of only the -1 or +1 nucleosome, and ( 2) -1 or +1 nucleosome location is variable (Fenouil et al., 2012).

In contrast, the observed variability in distances separating the BRG1-bound nucleosomes at TSS likely reflects the number of TFs bound in the intervening regions. Our analysis showed that combinations of MITF, SOX10, YY1, TFAP2A and probably other TFs such as ETS1 bind between two BRG1-bound nucleosomes. At many of these sites the BRG1-bound nucleosomes are also marked by H3K27ac, thus defining MAREs (Mitf Associated Regulatory Elements) active in regulating melanocyte lineage gene expression. Such an organisation has been previously proposed based on extensive ChIP-seq profiling by the Encode consortium showing that TF binding sites are often combinatorial and correspond to GC-rich, DNaseI hypersensitive NDRs flanked by two positioned nucleosomes (Wang et al., 2012).

As SOX10, YY1, TFAP2A, and ETS1 all have important roles in melanocytes and/or melanoma, the identified MAREs define a combinatorial signature of TFs critical for gene regulation in this lineage This is further underlined by the recent finding that a combination of MITF, SOX10, and PAX3 can reprogram fibroblasts into functional melanocytes (Yang et al., 2014). Given the fact that we have already data for MITF and SOX10, it would definitely be worth looking for PAX3 genome occupancy in the same melanoma cells. Subsequent comparative analysis would allow to determine the specific and overlapping contribution of each factor in driving this transdifferentiation. At the same time this would help characterize the transcriptional events staking place at other stages of melanocyte lineage development as well as the molecular origins of diseases such as pigmentary disorders resulting from their deregulation or mutation [START_REF] Bondurand | Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome[END_REF]Eccles et al., 2013).

Leucci et al. reported a role for the long non-coding RNA SAMMSON in driving mitochondrial metabolism in melanoma through its ability to enhance the mitochondrial localization and function of p32. Genome binding occupancy and knockdown experiments allowed us to identify SOX10 as the transcriptional regulator recruiting BRG1 to SAMMSON TSS and likely contributing its melanocyte lineage restricted expression (see Annexe 2). SAMMSON targeting sensitizes melanoma to MAPK-targeting therapeutics and may therefore offer a vulnerability targeting mitochondrial metabolism selectively and overcome the adaptive metabolic reprogramming that limits the response to these inhibitors.

Of note, our MITF binding occupancy experiments were done by immunopurification of an ectopically expressed tagged protein. Given the fact that the level of functional MITF is critical in regulating the biological properties of melanocyte and melanoma cells, future investigations should consider finding or developing antibodies against the endogenous protein to get a picture of its true functional output in the representative cell types.

Although examples of association between BRG1 and TFs have been previously described [START_REF] Trotter | The BRG1 transcriptional coregulator[END_REF]Reisman et al., 2009;Euskirchen et al., 2011), this specific configuration of BRG1 binding to nucleosomes flanking the TF binding sites has not been generally recognised. In yeast, it has however been reported that TF binding sites are rather flanked by nucleosomes bound by ISWI and CHD remodellers (Zentner et al., 2013).

Recent reports suggest the existence of epigenetic antagonism between BAF and the PRC2 complex. Thus oncogenesis might also arise from the resulting imbalance in this antagonism [START_REF] Johannessen | COT drives resistance to RAF inhibition through MAP kinase pathway reactivation[END_REF]Kim et al., 2014). Importantly, enhancer of zeste homologue 2 (EZH2), which is a component of the polycomb repressive complex 2 with H3K27 methylation activity, was shown to promote growth and metastasis of cutaneous melanoma through silencing of distinct tumour suppressors (Zingg et al., 2014).

Given this divergent role, it could be interesting to perform a comparative analysis of BRG1/PBAF and EZH2/PRC2 occupancy in proliferative vs. invasive melanoma cells. Recent evidence in the lab suggest that neither BRG1 nor BRM knockdown have a large effect on regulation of gene expression in invasive-type melanoma cells. This can account for dynamical changes in the contribution of different regulatory complexes during melanoma progression and phenotype switching. In this respect whether this change in complex contribution is the driver or the result of switching will be an important question to address.

Our work focused on regulation of transcription in a proliferative melanoma cell line. Given the absence of MITF, SOX10 (Annexe 3) and the minor effect of BRG1 knockdown in invasive melanoma it might not be interesting to focus on this network in first line. However SOX9 would be a good candidate as it belongs to the same family as SOX10, can bind the same sites and was reported to play a role in tumor invasion [START_REF] Cheng | Methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognostic factor in advanced melanoma[END_REF]Shakhova et al., 2015;[START_REF] Larsimont | Sox9 Controls Self-Renewal of Oncogene Targeted Cells and Links Tumor Initiation and Invasion[END_REF]. Similarly, TEADs could be other interesting regulators of melanoma invasion worth looking at. On a longer perspective, identifying their partners in invasive melanoma might enable to add a new layer in the understanding of the distinct transcriptional programs that account for melanoma heterogeneity and the molecular actors mediating the phenotype switching.

BRG1 regulates dynamic MITF and SOX10 interactions with chromatin

Our data show that MITF and SOX10 actively recruit BRG1 either cooperatively or distinctly to a large number of genomic loci. Furthermore, upon siMITF silencing, BRG1 is relocalized over the genome to new sites including SASP components, which were shown to be activated. Chromatin remodelling has been shown to facilitate TF binding, a good example being that of TAL1 where BRG1 repositions nucleosomes flanking its binding sites (Hu et al., 2011). In contrast, we observed that MITF occupancy of many co-occupied sites either shows little change or is increased following siBRG1 silencing suggesting that BRG1 regulates the dynamics of MITF binding. Several mechanisms may explain the increased MITF binding upon BRG1 silencing: TF binding to chromatin is often extremely dynamic with ChIP capturing only a snapshot of their occupancy thus increase thus MITF residence time at occupied sites could be increased. The human genome comprises many more consensus E-box elements than are occupied by MITF. The excess of consensus as well as degenerate E-boxes could potentially act as a sink thus limiting the pool of MITF for binding to functional sites. One function of BRG1-driven dynamics may therefore be to limit MITF sequestration at non-functional sites and ensure a pool of MITF for binding to functional sites. Alternatively, BRG1 may be required to establish the NDRs for MITF binding, for example after mitosis, or at specific stages of the cell cycle. Efforts should be put on further characterization of the MITF, SOX10 and BGR1 recruitment dynamics that would allow the construction of a model of sequential events resulting in transcription activation or repression. These processes would likely be applicable to other steps of melanocyte lineage development. Ideally such studies would also include nucleosome mapping as well as in vitro approaches to assay the true molecular output of BRG1/PBAF function.

BRG1 is essential for melanocyte and melanoma development in vivo

To complete these in vitro observations, we analysed the contribution of the catalytic subunit BRG1 in vivo at the major steps of melanocyte lineage development: melanoblasts, melanocyte stem cells (MSC) and their differentiated progeny, and melanoma.

Smarca4 knockout in developing mouse melanocytes in vivo leads to absent pigmentation in newborn mice likely due to absence of melanocytes in the hair follicle of the mutant animals (as suggested by negative staining for melanocyte markers).

Complementary observations using the DCT-lacZ mouse model which enables to track melanocyte precursor during embryonic development revealed a specific requirement for Brg1 during the proliferation of embryonic melanoblasts : Smarca4 inactivation doesn't result in an immediate loss but rather a progressive decrease in the melanoblast population such that they are almost completely absent by E15.5. Strikingly, generation of melanocytes from the adult melanocyte stem cell population appear to be independent of Brg1 (Annexe 1). This is in contrast with what we observed upon somatic inactivation of the Bptf subunit in the melanocyte lineage, which does not impair their viability and has more subtle effect on developing melanoblasts (Koludrovic et al., 2015). The resulting newborn Bptfmel-/-animals display mild greying on the back and a substantially lighter belly, with an occasional belly spot. While during the first anagen pigmentation comes from embryonic melanoblasts [START_REF] Nishimura | Melanocyte stem cells: a melanocyte reservoir in hair follicles for hair and skin pigmentation[END_REF], the melanocytes that provide pigmentation at the second anagen are derived from post-natal MSCs in the bulge. We showed that the inability of the mutant mice to pigment the hair from the second anagen phase didn't result from the loss of the MSC population but from their inability give rise to a differentiated progeny.

Overall, Brg1/PBAF remodelling is required in early events of lineage establishement and melanoblast commitment during embryonic development but doesn't appear to be required for the generation of differentiated melanocytes from the MSC in the hair follicle. In contrast, chromatin remodelling by Bptf has little function during embryonic developepment of the lineage but it is essential only for reactivation of the MSC population and the transition of the transcriptionally quiescent stem cell to the differentiated state. Thus the role of BPTF/NURF in melanocytes differs from that of BRG1/PBAF. While BRG1 and BPTF are essential in melanocytes and melanoma cells in vitro and regulate both overlapping but distinct gene expression programs; under physiological conditions their essential intervention occurs at different stages of melanocyte lineage developpement.

Finally we addressed the role of BRG1 and BPTF in mouse melanoma using a previously described model (Hooijkaas et al. 2012;Dhomen et al. 2009;Dankort et al. 2009;Damsky et al. 2011;[START_REF] Marsh Durban | Differential AKT dependency displayed by mouse models of BRAFV600E-initiated melanoma[END_REF]. BRAF is the earliest and most frequently mutated proto-oncogene in human melanoma and PTEN silencing is a common cooperating event in BRAF-mutated melanoma, which allows rapid progression of BRAF V600E -initiated melanocytes to malignant melanoma. Topical application of 4-OHT on the back skin of Tyr::CreERT2; Braf LSLV600E/+ ; Pten lox/lox mice resulted in activation of melanocyte-restricted Cre and subsequent recombination of floxed loci. This elicited development of melanoma with 100% penetrance, short latency and metastasis in local draining lymph nodes.

When we induced such tumors in mice bearing floxed Smarca4 or Bptf alleles, we were able to efficiently silence their expression at the very onset of melanocyte transformation and strongly delayed tumour formation. However tumours eventually developed, which did not fit with our expectations given the previous results in vitro and in vivo. Thus, while we think these proteins are inactivated in the majority of melanocytes, rare cells appear to escape recombination or recombine a single Bptf or Smarca4 allele and expand to form the tumors we see.

We have shown that it is possible to fully recombine the remaining alleles by treating the cell in vitro after derivation from the primary tumors. This raises the option of treating the growing melanoma tumors additional times with 4-OHT in order to recombine the remaining allele(s). If treatment can reach a significant number of cells in the tumor, we would then expect a growth arrest or even a regression.

Thus these data complement previous results showing that BRG1 and BPTF are essential for growth of proliferative human melanoma cells in vitro (Laurette et al. 2015;Koludrovic et al. 2015), that BRG1 is essential for the growth of human melanomas as xenografts in mice (Bossi et al. 2016) and they are in agreement with the observed amplification of the BPTF gene and its up-regulated expression in human melanomas (Dar et al. 2015).

Using this model turned out to be much more challenging than expected.

Only a few studies using the Braf/Pten mouse model have been published so far, 4 of which included one or two additional floxed gene leading to inactivation (Cdkn2a, Pdk1, Sgk3 or b-catenin) or overexpression of a mutant protein (b-catenin-STA). All four studies present a delay in tumor growth when these factors are inactivated concomitant to transformation although only little if any evidence is provided in these studies to show complete inactivation. Unlike expression of a proto-oncogene, inactivation of genes that would lead to a selective disadvantage will likely be counter-selected and contribute to emergence of escapee cells that will take over and expand the tumor as we have seen here Due to leaky CreERT2 activity, a significant number of mice tend to develop a melanoma spontaneously when they grow older (10-15 weeks) while mice harbouring 2 floxed Braf alleles are not viable. Furthermore in this model melanocyte transformation and tumor growth occur exceptionally fast: pigmented lesions appear about 3 weeks after treatment and mice need to be sacrificed around 40 days after application du to important tumor burden. While we observed systematic penetrance in the locally draining lymph node by that time, we didn't see any lesions in other organs such as the lung, suggesting that more time is needed to develop distant metastasis which makes this model less suitable as such for study of advanced melanoma. In contrast it might develop as a model of choice for studies on melanoma initiation events and early step of melanoma proliferation. Alternatively this model also represents a powerful choice to screen drugs and study emergence of resistant cells (Hooijkaas et al. 2012;Dhomen et al. 2009;Dankort et al. 2009;[START_REF] Marsh Durban | Differential AKT dependency displayed by mouse models of BRAFV600E-initiated melanoma[END_REF].

In addition some limitations have to be taken into account: the first are inherent to the physiological differences between human and mouse skin (see introduction) and the related questions about the precise cellular origin of these melanomas. Second, as all the targeted alleles are flanked with 'lox' sites, the genetic lesions induced upon CreERT2 activation occur within the same timeframe. However, in human melanoma, events cooperating with BRAFV600E in malignant progression are most likely temporally dissociated from the initiating event. In addition, the many additional genetic/epigenetic alterations that contribute to malignant progression in humans cannot be recapitulated in the mouse model for obvious reasons Nevertheless a couple of interesting questions remain and could be addressed with this model in the frame of this project

The Pavan lab recently published a good genome occupancy map of Sox10 binding sites and H3K27ac in melan-a melanocytes [START_REF] Fufa | Genomic analysis reveals distinct mechanisms and functional classes of SOX10regulated genes in melanocytes[END_REF]. In addition, our immunostainings have shown that Sox10, unlike Mitf is widely and homogenously expressed in the Braf LSLV600E/+ ; Pten -/-tumors. We recently obtained excellent data for histone marks and variants in these tumors and we are working on getting Sox10 occupancy data. Comparative analysis will help identify potential relevant changes both in the epigenetic landscape and Sox10's distribution over the genome, which might occur during melanocyte transformation, especially under BrafV600E signalling.

Altogether our results define specific and distinct roles for the PBAF and NURF chromatin remodelling complexes in epigenetic regulation of gene expression in melanocytes and melanoma.

BIBLIOGRAPHY

Patrick LAURETTE

Epigenetic regulation of gene expression during melanocyte and melanoma development

Le mélanome est un cancer très agressif en raison de sa capacité rapide à former des métastases et de développer une résistance aux traitements existants. MITF (Micropthalmia-associated Transcription Factor) est un facteur de transcription clé à toutes les étapes de développement du lignage mélanocytaire et dans la physiopathologie du mélanome. Afin de comprendre les mécanismes impliqués dans la régulation de l'activité et de la stabilité de MITF, nous avons identifié ses partenaires protéiques parmi lesquels figurent de nombreuses sous-unités des complexes de remodelage de la chromatine ATP-dépendant PBAF et NURF. Ce travail caractérise le rôle et l'étendue de la coopération entre BRG1/PBAF et plusieurs facteurs de transcription clés tels que MITF et SOX10 dans le fonctionnement des cellules de mélanome, qui recrutent activement de BRG1 à la chromatine et contribuent ainsi à la mise en place de la signature épigénétique caractéristique des cellules de mélanome prolifératives.

Par ailleurs, l'utilisation de différents modèles murins a permis de révéler in vivo la contribution fonctionnelle distincte mais complémentaire de ces deux complexes de remodelage associé à MITF aux cours de trois stades majeurs du lignage mélanocytaire : le développement embryonnaire des mélanocytes, leur différentiation ainsi que lors de l'initiation et la progression du mélanome. Ce travail contribue ainsi à une meilleure compréhension du fonctionnement biologique des mélanocytes, du mélanome et du remodelage de la chromatine chez les eucaryotes.

Mots clés: PBAF, NURF, remodelage de la chromatine, MITF, SOX10, signature épigénétique, mélanome, lignage mélanocytaire Malignant melanoma is the most deadly form of skin cancer due to its quick metastatic spread and the development of resistance to available treatments. MITF (Micropthalmia-associated Transcription Factor) is a transcription factor and master regulator of melanocyte lineage development and melanoma physiopathology.

In order to investigate the mechanisms involved in the regulation of MITF activity and stability, we identified its numerous partners by tandem affinity purification coupled to mass spectrometry, which include several subunits of the PBAF and NURF ATP-dependant chromatin remodelling complexes. The present work characterizes the role and extent of cooperation between BRG1/PBAF and several key transcription factors including MITF and SOX10 in melanoma cell function, that actively recruit BRG1 to chromatin to establish the epigenetic landscape of proliferative melanoma cells. Furthermore, using different mouse models we revealed the distinct but complementary functional contribution of these two MITF-associated chromatin remodelers in vivo at three majors stages of melanocyte lineage development: embryonic development of melanocytes, their differentiation and during melanomagenesis. Thus, this work contributes to a better understanding of processes regulating the biological function of melanocytes, melanoma and more widely chromatin remodelling events in eukaryotes.

Key words: PBAF, NURF, chromatin remodelling, MITF, SOX10, epigenetic landscape, melanoma, melanocyte lineage

  Figures and Tables

1 .

 1 Figure 1. The integumentary system Figure 2. Anatomy of the skin Figure 3. Representation of vertebrate neural crest development Figure 4. Neural crest organization and derived lineages Figure 5. Major signalling pathways and transcription factors regulating MITF-M expression Figure 6. Melanosome maturation and melanin biosynthesis Figure 7. Fitzpatrick skin type scale Figure 8. The melanosome transfer mechanism Figure 9. The tanning response pathway Figure 10. The PI3K/AKT signalling pathway Figure 11. The hair follicle cycle Figure 12. Melanocyte lineage-specific labelling in the hair follicle Figure 13. Comparative cross section of human and mouse skin Figure 14. The Microphthalmia locus organization Figure 15. Domain organization of the MITF protein Figure 16. Crystal structure of the MITF bHLH domain Figure 17. Transcription regulation of MITF gene expression Figure 18. Representation of MITF residues subjected to post-translational modifications Figure 19. MITF targets genes in the melanocyte lineage Figure 20. Geographical variations in melanoma incidence Figure 21. The distribution of metastasis across the human body Figure 22. Major subtypes of primary melanoma Figure 23. The ABCDE criteria for melanoma identification Figure 24. Hypothetical model of melanoma development Figure 25. Pathways Altered in Melanoma Figure 26. Schematic of commonly mutated MAPK and PI3K pathway components Figure 27. The CDKN2A locus organization Figure 28. Role of EMT during cancer progression.Figure 29. Embryonic signalling pathways inducing EMT transition Figure 30. Major melanoma risk factors Figure 31. Mechanisms of immune checkpoint inhibitors on cancer cell recognition Figure 32. Cell signalling pathways and drug targets in BRAF-mutant melanoma. Figure 33. The nucleosome structure Figure 34. ATPase subunits of the four main families of ATP-dependant chromatin remodeling complexes Figure 35. SWI/SNF complex composition and mechanism of action

Résumé 10 BRG1

 10 est essentiel pour la prolifération et la survie de mélanocytes et cellules de mélanome in vitro ainsi que pour la formation du lignage mélanocytaire in vivo.

Figure 1 :

 1 Figure 1: The integumentary system. The skin is composed of three main layers: epidermis, dermis and the hypodermis (left annotations). The different sensory structures and dermal and epidermal appendages (right and bottom annotations) are represented. The dermis include the hair follicle, nerve fibres associated to different sensory corpuscles, sebaceous gland, apocrine sweat gland and capillary blood vessels. The hypodermis contains the layer of adipose tissue, nerves and blood vessels. Cross-section of the human skin in three dimensions (Essentials of Human Anatomy and Physiology 5th Ed. 2003 © Pearson Education, Inc. publishing as Benjamin Cummings).

Figure 2 :

 2 Figure 2: Anatomy of the skin (A) Cross section of the skin with a focus on the structure of the epidermis. The different strates of this keratinized epithelium are labelled. Melanocytes reside in the basal layer at the dermo-epidermal junction, regularly spaced and contacting up to 40 through dendritic extensions used to transfer the melanin. (B) Representation of the different epidermal cell types: Keratinocytes (a), melanocytes (b), Langerhans cells (c) and Merkel cells (d) are in the layer of basal cells at the deepest part of the epidermis and are connected to nerves (Illustration: (A) K.Holoski for headandneckcancerguide.org and (B) Quizlet.com).

Figure 3 : 2 .

 32 Figure 3: Representation of vertebrate neural crest development. (A) Neural crest cells form a transient population of pluripotent progenitor cells that originate from the neural plate border. NCCs undergo epithelial to mesenchymal transition and acquire migratory properties to progressively colonize their target tissue and give rise to several different lineages (B) Specification of the neural plate border and differentiation of neural crest cells is driven by different signalling pathways and transcriptional networks. Reproduced by permission from Macmillan Publishers Ltd: Nature Reviews Neuroscience 4:10 (Gammill and Bronner-Fraser, 2003) © 2003 and Nature 520:7548 (Green et al., 2015) © 2015

Figure 4 :

 4 Figure 4: Neural crest organization and derived lineages. After neurulation, neural

Figure 5 :

 5 Figure 5: Major signalling pathways and transcription factors regulating MITF-M expression. EDN3/EDNRB, Wnt/β-catenin and KITL/KIT/MAPK pathway splay a role from the beginning of the establishment of the melanocyte lineage. α-MSH/MC1R signalling results in elevation of cAMP in response to UV. Downstream transcription factors regulating MITF expression are represented along with some functional outputs. (Illustration by Peter Jurek MUTAGENETIX (TM), B. Beutler and colleagues, Center for the Genetics of Host Defense, UT Southwestern, Dallas, TX)

Figure 6 :Figure 7 :

 67 Figure 6: Melanosome maturation and melanin biosynthesis. Melanogenesis is a multi-step process that takes place during melanosome maturation in differentiated melanocytes. They can produce melanin in two flavours (eumelanin and pheomelanin) with the help of three enzymes: tyrosinase (TYR), tyrosinase-related protein 1 (TYRP-1) and dopachrome tautomerase (DCT). The relative ratio between the two types of pigments determines the colour of skin, hairs and eyes. (Illustration: Biospectrum.com)

4 .

 4 Mature melanocyte pigmentation: function and signalling pathways a. Melanin localization and function

Figure 8 :

 8 Figure 8: The melanosome transfer mechanism. Melanosomes are packed in globules enclosed by the melanocyte plasma membrane, released into the extracellular space from various areas of the melanocyte dendrites, phagocytosed by keratinocytes, and then dispersed around the perinuclear area. Reproduced by permission from Elsevier: Journal of Investigative Dermatology 132:4 1222-1229 (Ando et al., 2012) © 2012

Figure 9 :

 9 Figure 9: The tanning response pathway is induced by UV-dependant activation of MC1R and stimulation of melanogenic genes. (A) UV exposure leads to activation of the p53 tumour suppressor resulting in increased POMC expression and subsequent αMSH production by keratinocytes. (B) Further MC1R activation and downstream signalling stimulates CREB-dependant expression of MITF and its target genes, which trigger the entire pigmentation process. (A) Reprinted with permission from AAAS: Science 346:6212 945-949 (Lo and Fisher, 2014) (B) Illustration by Peter Jurek MUTAGENETIX (TM), B. Beutler and colleagues, Center for the Genetics of Host Defense, UT Southwestern, Dallas, TX)

Figure 10 :

 10 Figure 10: The PI3K/AKT signalling pathway. Induction of tyrosine-kinase receptors lead to PI3K activation, which phosphorylates phosphatidylinositol in the cell membrane (PIP2 into PIP3). The resulting lipid species mediate recruitment of kinases such as AKT to the cell membrane and their activation. Activated AKT translocates to the cytosol where it promotes cell proliferation and survival by phosphorylating numerous substrate proteins including mTOR, GSK3, FOXO, and BAD, among others. Reproduced with permission from Macmillan Publishers Ltd: Nature Reviews Neuroscience 4:12 (Hennessy et al., 2005) © 2005

Figure 11 :

 11 Figure 11: The hair follicle cycle. Follicular morphogenesis starts with the first postnatal anagen, when the hair shaft is growing through different stages of anagen, protruding through the skin surface and reaching the size of the mature pigmentproducing follicle. At the end of anagen, the follicle goes through the catagen, during which the lower two-thirds of the cells of the hair matrix undergo apoptosis and regress. The final stage of the cycle (telogen) the follicle stays quiescent. Reprinted with permission from Macmillan Publishers Ltd: Nature Reviews Neuroscience 445:7130 (Fuchs, 2007) © 2007

Figure 12 :

 12 Figure 12: Melanocyte lineage-specific labelling in the hair follicle The hair follicle is divided into three sections, upper and lower permanent portions and a transient portion.The melanocyte stem cells reside in the bulge in the lower permanent portion, and get differentiated into melanocytes that migrate into the hair matrix to differentiate and produce pigment[START_REF] Osawa | Melanocyte stem cells[END_REF].

Figure 13 :

 13 Figure 13: Comparative cross section of human and mouse skin. Specificities of murine skin include dense population of hair follicles housing most of the melanocytes, thin dermis and epidermis and extended telogen hair stage. Reprinted with permission from Macmillan Publishers Ltd: Nature Reviews Cancer 6:4 (Khavary, 2006) © 2006

Figure 14 :

 14 Figure 14: The Microphthalmia locus organization. (A) Representation of 9 alternative promoters associated with their specific first 5' exon followed by common exon 2-9. (B) At least 9 different isoforms can be produced by alternative promoter usage and are represented here with position of the encoded bHLH-LZ domain. (C) Examples of Mitf-M isoforms produced by alternative splicing in the exon 2A and 6A (adapted from Denat et al., 2007).

Figure 15 :Figure 16 :

 1516 Figure 15: Domain organization of the MITF protein shown through the example of MITF-A isoform. Reprinted by permission from Elsevier: Trends in Molecular Medicine 12:9 406-414 (Levy et al., 2006) © 2006

Figure 17 :

 17 Figure 17: Transcription regulation of MITF gene expression. Major signalling pathways and downstream effectors involved in the regulation of MITF expression in melanocytes(Goding, 2000 Genes Dev, 14:1712-1728).

Figure 18 :

 18 Figure 18: Representation of MITF residues subjected to post-translational modifications with their corresponding effectors[START_REF] Hartman | MITF in melanoma: mechanisms behind its expression and activity[END_REF] Cell Mol Life Sci, 72: 1249-60).

Figure 19 :

 19 Figure 19: MITF regulates multiple targets in the melanocyte lineage. These genes are involved in various cellular processes such as cell-cycle control, survival, motility and pigmentation. Given the diverse biological activities of MITF and the antagonistic behaviours of some targets, it is likely that the repertoire of MITF targets depends on the level of functional MITF that correlates with the cellular context. Reproduced with permission from Elsevier: Trends in Molecular Medicine 12:9 406-414 (Levy et al., 2006) © 2006

Figure 20

 20 Figure 20 Geographical variations in melanoma incidence (A) and mortality (B). Melanoma incidence is highest in countries populated by fair-skinned persons living in high-UV environments (Erin M. Wolf Horrell, Kalin Wilson and John A. D'Orazio (2015). Melanoma -Epidemiology, Risk Factors, and the Role of Adaptive Pigmentation, Melanoma -Current Clinical Management and Future Therapeutics, Prof. Mandi Murph (Ed.), InTech, DOI: 10.5772/58994).

Figure 21 :

 21 Figure 21: The distribution of metastasis across the human body.For people with advanced disease, the melanoma has spread beyond the local area and regional nodes into other parts of the body or internal organs. The most common sites of melanoma metastases are under the skin (subcutaneous tissue), lymph nodes, the lung, liver, brain, and bones. However, metastasis to other sites in the body such as gastrointestinal tract can also occur.

Figure 22 :

 22 Figure 22: Major subtypes of primary melanoma. (A) Patient with multiple clinically atypical moles. According to the Clark model, cutaneous melanoma can be classified into several subgroups, including superficial spreading melanoma (C) nodular melanoma (D)acral lentiginous melanoma (E) Ocular melanoma is a rare form of melanoma in the eye[START_REF] Tsao | Melanoma: from mutations to medicine[END_REF].

Figure 23 :Figure 24 :

 2324 Figure 23: The ABCDE criteria for melanoma identification

Figure 25 :

 25 Figure 25: Pathways Altered in Melanoma (A) Percentage of recurrently altered pathways in the four melanoma subtypes (BRAF-, RASor NF1-mutants and triple WT) Manual curated pathway shows percentage pathway mutations across all samples. a, amplification; d, deletion, m, mutation. Reproduced with permission from Elsevier: Trends in Molecular Medicine 161:7 1681-1696 (Akbani et al., 2015) © 2015

Figure 26 :

 26 Figure 26: Schematic of commonly mutated Raf/MEK/ERK and PI3K/Akt pathway components. The most frequent type of patient mutations are indicated. Reprinted by permission from American Society for Clinical Investigation: Journal of clinical investigation (Chudnovsky et al., 2005) © 2005.

Figure 27 :

 27 Figure 27: The CDKN2A locus organization. Both RB and p53 are regulated by products of this locus i.e. p16INK4A and ARF respectively. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Cancer 15:7 (Sharpless et al., 2015) © 2015

Figure 28 :

 28 Figure 28: Role of EMT during cancer progression. EMT-TFs have been involved not only in migration and invasion but also in the suppression of senescence and apoptosis, attenuation of cell-cycle progression and resistance to radiotherapy and chemotherapy. The degree of EMT during the different steps in cancer progression probably depends on the imbalance of several associated regulatory networks with activated oncogenic pathways. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Cancer 13:2 (Craen et al., 2013) © 2013.

Figure 29 :

 29 Figure 29: Reactivation of embryonic pathway signaling leads to induction of epithelial-to-mesenchymal transition. Hedgehog (Hh), Notch, Wnt, and TGFβ signaling can activate EMT. Downstream signaling activates SOX9, YAP/TAZ, TEADs, SNAIL, SLUG, TWIST and ZEB transcription factors that drive cells reprogramming directly or indirectly to acquire more invasive phenotypes. By migrating into the circulatory system, cancer cells can spread and reach new sites where they initiate metastatic growth (modified from Takebe et al., 2011).

Figure 30 :

 30 Figure 30: Major melanoma risk factors. Risk factors for the development of melanoma can be divided into extrinsic and intrinsic factors and include exposure to UV radiation either via sunlight or indoor tanning salons, medications, chemical exposures, presence of nevi, family history of cancer, and pigment of skin (Erin M. Wolf Horrell, Kalin Wilson and John A. D'Orazio (2015). Melanoma -Epidemiology, Risk Factors, and the Role of Adaptive Pigmentation, Melanoma -Current Clinical Management and Future Therapeutics, Prof. Mandi Murph (Ed.), InTech, DOI: 10.5772/58994).

Figure 31 :

 31 Figure 31: Mechanisms of immune checkpoint inhibitors on cancer cell recognition. Blocking the binding of checkpoint proteins on tumor cells (B7-1/B7-2) to CTLA-4 on T-cells with an immune checkpoint inhibitor (anti-B7-1/B7-2 or anti-CTLA-4) prevent checkpoint blockade and allows the T cells to kill tumor cells (left panel). Blocking the binding of PD-L1 (cancer cells) to PD-1 (T-cells) with an immune checkpoint inhibitor (anti-PD-L1 or anti-PD-1) allows the T cells to kill tumor cells (right panel). (Illustrations: Terese Winslow for the National Cancer Institute (http://www.cancer.gov).)

Figure 32 :

 32 Figure 32: Cell signalling pathways and drug targets in BRAF-mutant melanoma Reprinted by permission from Elsevier: The Lancet Oncology 15:9 371-381 (Menzies and Long, 2014) © 2014

Figure 33 :

 33 Figure 33: The nucleosome structure. The nucleosome is the basic unit of chromatin consisting of 146bp of DNA wrapped around a histone octamer (modified from McGinty and Song Tan, 2015).

Figure 34 :

 34 Figure 34: ATPase subunits of the four main families of ATP-dependent chromatin remodelling complexes. The ATPase subunit of each ATP-dependent chromatinremodelling complex belongs to the SNF2 ATPase superfamily, whose ATPase domain comprises an N-terminal DExx and a C-terminal HELICc subdomain, separated by an insert region (Yong Zhong Xu, Cynthia Kanagaratham and Danuta Radzioch (2013). Chromatin Remodelling During Host-Bacterial Pathogen Interaction, Chromatin Remodelling, Dr. Danuta Radzioch (Ed.), InTech, DOI: 10.5772/55977)

Figure 35 :

 35 Figure 35: SWI/SNF complex composition and mechanism of action (a) The predominant SWI/SNF complexes in mammalian cells are composed of evolutionarily conserved core subunits (green) and variant subunits (yellow) (b) Mechanism of chromatin remodelling by SWI/SNF: the steps of remodelling include binding, disruption of histone-DNA contacts, the creation of a loop of DNA that propagates around the nucleosome in a wave-like manner and the repositioning of DNA with respect to the nucleosome (sliding). Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Cancer 11:7 (Wilson and Roberts, 2011) © 2011

DOI: 10 .

 10 7554/eLife.06857.002 Laurette et al. eLife 2015;4:e06857. DOI: 10.7554/eLife.06857 complex associated with MITF specifically in the chromatin fraction. Components of the DNA damage response machinery including XRCC5 and XRCC6 (Ku80 and Ku70), DNA-dependent protein kinase (PRKDC), BRCA2 as well as MSH2 and MSH6 were identified along with the HECT domain-containing

Figure 1 .

 1 Figure 1. Purification of MITF-associated complexes. (A) Western blot of 501Mel cell lines stably expressing Flag-HA-tagged-MITF (F-H-MITF). (B) The immunoprecipitated material from the soluble nuclear extract (SNE) was separated by SDS PAGE and stained with silver nitrate. F-H-MITF is indicated along with * that designates a contaminating protein seen in the control immunoprecipitations. Lane M corresponds to a molecular mass marker indicated in kDa. (C) Immunoblot detection of HERC2, BRG1, USP7, USP11, XRCC5, and XRCC6 in the MITF-associated complexes. (D) Summary of proteins and complexes interacting with MITF. Shown are the proteins found specifically in the immunopurifications of F-H-MITF classified according to their function and organisation into known complexes. DOI: 10.7554/eLife.06857.003

Figure 2 .Figure 3 .

 23 Figure 2. Composition of BRG1 complexes in 501Mel cells. (A) BRG1 associates with CHD7 in 501Mel cells. Following immunoprecipitation of 501Mel cell extracts with anti-BRG1 antibody or HA beads as control the eluted fractions were probed with antibodies for the indicated proteins. (B) Following immunoprecipitation of 501Mel cell extracts with anti-CHD7 antibody or HA beads as control the eluted fractions were probed with antibodies for the indicated proteins. (C) Tablesummarisingthe known subunits of BRG1-containing complexes highlighting catalytic subunits with ATPase activity, common subunits and specific subunits. The composition of the complex interacting with MITF based on mass-spectrometry and immunoblots is schematised. DOI: 10.7554/eLife.06857.004

Figure 3 .Figure supplement 1 .Figure supplement 2 .

 312 Figure 3. Continued stained for senescence-associated β-galactosidase. Arrowheads indicate representative stained cells. Inserts show enlargements of representative cells. (D) Immunoblots of Hermes 3A extracts following infection with lentiviral vectors expressing control, anti-BRG1, or anti-MITF shRNAs. (E) Phase contrast microscopy of Hermes 3A cells infected with the indicated shRNA vectors after 5 days of puromycin selection and stained for senescence-associated β-galactosidase. Arrowheads indicate representative stained cells. Inserts show enlargements of representative cells. DOI: 10.7554/eLife.06857.005 The following figure supplements are available for figure 3: Figure supplement 1. Gene expression changes in 501Mel and Hermes 3A cells. DOI: 10.7554/eLife.06857.006 Figure supplement 2. SOX10 regulates MITF expression in 501Mel cells. DOI: 10.7554/eLife.06857.007

Figure 4 .

 4 Figure 4. BRG1 is essential in mouse melanocytes in vivo. (A) Statistics relevant to the phenotype of the mice lacking Brg1 in the melanocyte lineage. Note that as the Tyr-Cre transgene is present on the X chromosome, we detect black mice with the Tyr-Cre::Smarca4 lox/lox genotype, but in these animals the Cre-recombinase is subjected to X-Figure 4. continued on next page

Figure 4 .Figure 5 .

 45 Figure 4. Continued inactivation such that recombination does not occur in all melanoblasts. (B) Photographs of representative mice of the indicated genotypes. Bright field images of hair from the backs of 8-week-old animals of the two genotypes are also shown. Magnification X40. (C) Labelling of dorsal hair follicle bulbs with antibodies against Sox10 in red and Dct in green along with Hoechst-stained nuclei in blue. Arrows indicate labelled melanocytes in the bulb of Brg1 expressing mice. Scale bars are 50 μm. DOI: 10.7554/eLife.06857.008

Figure 5 .Figure supplement 1 .Figure 6 .

 516 Figure 5. Continued re-clustered and representative meta-profiles are shown to illustrate the distances separating the BRG1 peaks with the large and small arrowheads indicating the stronger and weaker peaks, respectively. (E) Re-analysis of RefSeq TSS where BRG1 is present reveals different localizations of BRG1-bound nucleosomes. Several representative meta-profiles of different subclasses are shown. The peak coordinates in each class relative to the TSS are indicated. (F) The overall meta-profile of BRG1 TSS occupancy is superimposed on the RNA Pol II meta-profile. The binding profiles of BRG1 and Pol II around the TSS are schematised below the graph. DOI: 10.7554/eLife.06857.009 The following figure supplement is available for figure 5: Figure supplement 1. Profiling of BRG1 genome occupancy. DOI: 10.7554/eLife.06857.010

Figure 6 .Figure supplement 1 .Figure supplement 2 .Figure 7 .

 6127 Figure 6. Continued control. (C) Meta-profiles of BRG1 occupancy in clusters A-D of panel A. The lower panel shows a UCSC screenshot of MITF and BRG1 occupancy over the TYR locus highlighting the binding of MITF between two BRG1-occupied nucleosomes. (D) Meta-profile showing occupancy by BRG1, MITF, and Pol II at the TSS. The binding profiles of each factor BRG1 and Pol II around the TSS are schematised below the graph. DOI: 10.7554/eLife.06857.011 The following figure supplements are available for figure 6: Figure supplement 1. TF binding motifs at MITF and BRG1-bound sites. DOI: 10.7554/eLife.06857.012 Figure supplement 2. MITF co-localizes with SOX10. DOI: 10.7554/eLife.06857.013

Figure 7 .Figure supplement 1 .Figure 8 .

 718 Figure 7. Continued on YY1-occupied sites. (C) Similar to panel A, read density clustering illustrates MAREs with co-localization of H3K27ac, BRG1, MITF, and SOX10 at YY1bound regions. A meta-profile is shown to the right along with a schematic representation of these sites. DOI: 10.7554/eLife.06857.014 The following figure supplement is available for figure 7: Figure supplement 1. Identification of YY1-TFAP2A associated MAREs. DOI: 10.7554/eLife.06857.015

Figure 8 .Figure supplement 1 .Figure 9 .

 819 Figure 8. Continued indicates a region where BRG1 is diminished upon both siMITF and siSOX10 silencing. (C) Read density clustering illustrating set of sites whose BRG1 occupancy is diminished following siMITF or siSOX10 silencing compared to siLuc. The meta-profiles of several representative clusters are shown to the right of the figure. (D) UCSC screenshots showing examples of BRG1 recruitment by SOX10 or MITF. DOI: 10.7554/eLife.06857.016 The following figure supplement is available for figure 8: Figure supplement 1. BRG1 and MITF repress gene expression. DOI: 10.7554/eLife.06857.017

Figure 9 .

 9 Figure 9. BRG1 controls dynamics of MITF binding. (A) ChIP-qPCR of 3HA-MITF in 501Mel-CL8 cells at the indicated loci following transfection with siLuc or siBRG1. The protamine 1 locus (PRM1) was used as a negative control. (B) UCSC screenshots illustrating binding of MITF between two BRG1-occupied nucleosomes at selected loci assayed by ChIP-qPCR in panel A. sThe GPR110-1 and GPR110-2 sites assayed in Panel A are indicated in panel B.Figure 9. continued on next page

Figure 9

 9 Figure 9. Continued

  Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells Patrick Laurette, et al. Laurette et al. eLife 2015;4:e06857. DOI: 10.7554/eLife.06857 1 of 29

Figure 3 -Figure 3 -Figure 5 -Figure 5 -Figure 6 -Figure 6 -Figure 6 -Figure 6 -Figure 7 -Figure 7 -Figure 8 -

 33556666778 Figure 3-figure supplement 1. Gene expression changes in 501Mel and Hermes 3A cells. (A) A Comparative analysis of RNA-seq data from shBRG1 and shMITF 501Mel cells. Venn diagrams indicate the overlap between genes that are up and down-regulated in the shBRG1 and shMITF cells. The results of ontology analysis of the commonly up-and down-regulated genes are shown graphically and classed by p value and the number of genes in each category is indicated. Lists of secreted growth factors and cytokines forming the SASP in the shBRG1 and shMITF cells are indicated. (B-C) Comparative analysis of RNA-seq data from shBRG1 and shMITF Hermes 3A cells. Venn diagrams indicate the overlap between genes that are up and down-regulated in the shBRG1 and shMITF cells. The results of ontology analysis of the specifically and commonly up-and down-regulated genes are shown graphically and classed by p value and the number of genes in each category is indicated. DOI: 10.7554/eLife.06857.006

Fig 1 .

 1 Fig 1. BPTF is essential in 501Mel cells. A. Western blots showing si/shRNA knockdowns of BPTF and MITF in 501Mel cells. B. Phase contrast microscopy of 501Mel cells following siBPTF and siMITF knockdown. Magnification X20. Inlays show enlargements (X40) of representative cells. C. Phase contrast microscopy of 501Mel cells following shBPTF and shMITF knockdown. Inlays show enlargements of representative cells. For simplification only

  Fig). In melanoma MNT1 cells, shBPTF silencing led to a spindle-like bipolar morphology (S2B and S2C Fig), while in 888-Mel cells, BPTF knockdown led to strongly reduced cell numbers with the remaining cells again showing a spindle-like bipolar morphology (S2C Fig).

Fig 2 .

 2 Fig 2. Co-regulated genes in 501Mel and Hermes 3A cells. Examples of genes that are co-regulated by MITF and BPTF in each cell type. The Log2 fold change under each condition is indicated. The shaded area highlights genes that are co-regulated in both cell types. The lower portion shows examples of genes that are regulated by MITF only. All of the genes listed are direct MITF targets with associated MITF-occupied sites in 501Mel cells. The asterisk in the Hermes column indicates that these genes are direct MITF targets in 501Mel cells, but that we have no corresponding ChIP-seq data in Hermes 3A cells. doi:10.1371/journal.pgen.1005555.g002

Fig 3 .

 3 Fig 3. BPTF and MITF are required for the proliferation of Hermes-3A cells. A. Western blot showing knockdown of BPTF and MITF in Hermes-3A cells. B. Numbers of Hermes-3A cells measured using a MTT cell proliferation kit following shBPTF knockdown. C. Phase contrast microscopy of Hermes-3A cells following shBPTF and shMITF knockdown. Magnification X20. Inlays show enlargements. D. Staining and quantification of Hermes-3A cells following shBPTF and shMITF knockdown for senescence-associated βgalactosidase. E. Venn diagrams illustrate the overlap between up and down-regulated genes following shBPTF and shMITF knockdown. RF shows the representation factor and p value for the overlaps between the up and down-regulated genes sets. Several examples of commonly regulated up and down-regulated genes are indicated based on the results of RNA-seq. F. Ontology of genes up and down-regulated by shBPTF and shMITF knockdown and of commonly regulated genes in Hermes 3A cells. doi:10.1371/journal.pgen.1005555.g003

Fig 4 .Fig 5 .

 45 Fig 4. Premature greying of mice lacking Bptf in the melanocyte lineage. A Photographs of P10 mice of the indicated genotypes before onset of hair growth. B-C. Photographs of 14 day-old and 6 week-old mice of the indicated genotypes to illustrate the characteristics of the first coat and the premature greying phenotype. D. Photographs of 1 year-old mice of the indicated genotypes. E. Photographs of 6 week-old mice that had undergone depilation at 3 weeks of age. The depilated areas are outlined. doi:10.1371/journal.pgen.1005555.g004

  Fig 4D) by rare melanocytes derived from melanoblasts that escaped recombination. Recombination of the Bptf allele was confirmed by PCR-based genotyping on both neonatal mouse-tail DNA and purified E15.5 melanoblasts (S5E Fig).

Fig 6 .

 6 Fig 6. Loss of differentiated melanocytes in the bulb region of post-natal Bptf mel-/-mice. A. Staining of a dorsal wild-type hair follicle with antibody against Dct showing the presence of Dct-labelled cells in the bulge corresponding to MSCs, in the shaft corresponding to TACs and in the bulb corresponding to differentiated melanocytes. B-C. Staining of dorsal hair follicles from mice with the indicated genotypes with antibodies against Dct and Sox10 to detect differentiated melanocytes in the bulb. Quantification is shown on the right. N = 30. D. Staining of dorsal hair follicles from mice with the indicated genotypes with antibodies against Dct and Ki67 to detect melanocytes in the bulb of 21 day-old animals. Quantification is shown on the right. N = 50. E. Staining of dorsal hair follicles from one year-old mice with antibodies against Dct and Ki67 illustrating the absence of melanocytes in the bulb of Bptf-mutant animals. Scale bars represent 50μm. NS = Non significant. ***P < 0.001 doi:10.1371/journal.pgen.1005555.g006

Fig 7 .

 7 Fig 7. Persistence of a MSC population in adult Bptf-mutant animals. A. Staining for Dct-LacZ-labelled melanocytes in dorsal hair follicles of P10 mice of the indicated genotypes. Arrows indicate representative examples of MSCs in the bulge region and differentiated melanocytes in the bulb. B-C Staining for Dct-LacZ-labelled melanocytes in dorsal hair follicles of 6 week-old and one year-old mice. Arrows are as in panel A. Scales bars represent: 20μm in right of panel B and panel C, 25μm in inlays of panels A and B and 50μm in panel A and left and center images of panel B. doi:10.1371/journal.pgen.1005555.g007

  mel+/-animals (Fig 9A) as well as in migrating TACs and cells colonising the bulb (Fig 9B). Moreover, Dct-labelled cells expressing Pax3, Mitf and Sox10 were also observed at day 3 (Fig 9B and 9C). In contrast, no expression of Dct or of any of the other markers was detected in the Bptf mel-/-animals (Fig 9A). At days 6 and 10, antibody staining showed endogenous Dct in the Bptf mel+/-bulb melanocytes that were also labelled for Sox10 (Fig 9C and S7 Fig). Melanocytes in bulbs from these animals also expressed Pax3, Mitf and the HMB45 melanosome marker (S7C Fig).

Fig 8 .Fig 9 .

 89 Fig 8. Bptf is required for proliferation and terminal differentiation of adult melanocytes. A-D. Dct-LacZ staining shows generation of TACs and terminal differentiation of Bptf mel+/-melanocytes 1, 3, 6, 10 and 15 days after depilation. Staining for melanin reveals pigmentation of the newly formed hair. E-H. Dct-LacZ staining shows reduced number of TACs and lack of differentiated melanin producing Bptf mel-/-melanocytes. The lower panels of C and G show representative examples of bulbs of the two different genotypes. Note the absence of pigmentation of the newly formed hair. All sections were stained for melanin by Fontana Masson and for Dct-LacZ-expressing cells. Scales bars represent: 20μm in panels A and E, 25μm in panels B and F and in inlays of panels C and G and 50μm in panels C, D, G, and H. doi:10.1371/journal.pgen.1005555.g008

Fig 10 .

 10 Fig 10. A transcriptionally repressed chromatin state in MSCs. A. Staining of hair follicle bulbs with antibodies against Dct and the indicated chromatin remodellers and chromatin modifications. Representative Dct-expressing melanocytes are indicated by arrows. B. Staining of the bulge region with antibodies against Dct and the indicated chromatin-remodellers and chromatin modifications. Dct-stained cells with diminished expression of the corresponding marks are indicated by arrows. Scale bars represent 50μm in panel A and 20μm in panel B. doi:10.1371/journal.pgen.1005555.g010

Figure 1 .

 1 Figure 1. Histolopathological features of melanoma developpement on Tyr::Cre-

Figure 2 .

 2 Figure 2. Recombination of Brg1 or Bptf loci during melanocyte transformation

Figure 3 .

 3 Figure 3. BRG1 expression persists in transformed BRG1 lox/lox melanocytes. BRG1

Figure 4 .Supplemental figure 1 .Supplemental figure 2 .

 412 Figure 4. Brg1 and Bptf are essential for growth of mouse melanoma cells (A)

Figure 1 |

 1 Figure 1 | Gene amplification and SOX10-mediated transcription drives SAMMSON expression in melanoma. a, DNA copy number of SAMMSON and MITF in melanoma lesions (n = 386). Samples are ranked according to SAMMSON copy number, expressed as the mean log ratio of the segment encompassing SAMMSON. Samples with a SAMMSON log ratio > 0.5 are shown (Mann-Whitney P < 0.001). Chr, chromosome. b, Read counts were generated based on the GENECODE annotation (version 19) from the RNA-seq alignments of 345 SKCM samples (TCGA). 7,014 lncRNAs were selected for which the raw counts were normalized to reads per kb per million reads (RPKM) values. The rank expression of SAMMSON was determined, sorted and plotted for the primary (left) and metastatic (right) samples. c, Relative expression in NHME, RGP, VGP and metastatic melanoma. Significance was calculated by analysis of variance (ANOVA). d, Relative expression across a cohort of 8,085 primary tumour samples from different cancer types. e, Relative expression across 38 adult tissues, 5 melanoma cell lines and NHME cultures. Error bars represent standard deviations (s.d.) of qPCR replicates (n = 2). f, University of California, Santa Cruz (UCSC) Genome Browser screenshots of ChIP-seq data illustrating the selective recruitment of SOX10 to the SAMMSON loci and the co-localization of H3K4me3 and H3K27ac at the SAMMSON TSS in Mel501. g, ChIP-qPCR of SOX10 in SK-MEL-28 at the indicated loci. The IgG antibody was used as a negative control. h, Gene expression levels in untreated (Mock) SK-MEL-28 cells or cells transfected with a control siRNA pool (siCtrl) or pools targeting MITF (siMITF) or SOX10 (siSOX10). Data are an average of three biological replicates ± standard error of the mean (s.e.m.). r, Pearson correlation coefficient.

Figure 2 |Figure 3 |

 23 Figure 2 | SAMMSON is required for melanoma growth and survival. a, Quantification of colony formation assays 7 days after seeding of melanoma cells presented as the mean density (percentage of area occupancy) of three different biological replicates ± s.e.m. P values were calculated by ANOVA. mut, mutation b, Relative SAMMSON expression in cells transfected with a GapmeR control (Ctrl), GapmeR3 and GapmeR11. The results are the average of three different biological replicates ± s.e.m. P values were calculated by ANOVA. c, Caspase-Glo 3/7 assays in untreated cells (Mock) and cells transfected with GapmeR control (Ctrl), GapmeR3 and GapmeR11 (48 h post-transfection). The data are presented as the means of three different biological replicates ± s.e.m. P values were calculated by ANOVA. NS, not significant. d, Cell proliferation assays in MM001 upon ectopic SAMMSON expression. e, Quantification of colony formation assays 7 days after seeding 1,000, 5,000 or 10,000 cells described in d. The data are presented as the mean number of colonies of three different biological replicates ± s.e.m. at each density. P values were calculated by ANOVA. f, Quantification of colony formation assays 7 days after seeding of SK-MEL-28 cells transfected with a control GapmeR (Ctrl) or GapmeR3 and exposed to either vehicle or a halfmaximum effective concentration (EC 50 ) dose of vemurafenib (PLX4032) or pimasertib. The data are presented as the mean density of three different biological replicates ± s.e.m. P values were calculated by ANOVA.

Figure 4 |

 4 Figure 4 | Therapeutic potential of SAMMSON targeting in vivo. a, Tumour volume of cohorts of Mel006 PDX mice treated (intravenous injections) with a control GapmeR (Ctrl) or GapmeR3. Data are the means ± s.d. of different biological replicates (P value was calculated by two-ways ANOVA). b, c, Quantification of Ki-67-positive cells (b) and cleaved caspase 3-positive (Casp3* + ; c) of melanoma lesions treated as described in a. P value was calculated by t-test. NS, not significant. d, Tumour volume of cohorts of PDX mice (Mel006) treated with combinations of control GapmeR (Ctrl) and GapmeR3 with either vehicle or dabrafenib by daily oral gavage (vehicle or dabrafenib) and intravenous injection of the GapmeRs every 2 days. Data are means ± s.d. of different biological replicates (P value was calculated by t-test). e, Quantification of cleaved caspase 3 of melanoma lesions treated as described in d. f, Weight variation of mice treated as described in d and mice exposed daily to dabrafenib and trametinib. P values were calculated by ANOVA.

Extended Data Figure 3 |

 3 SAMMSON promotes the in vitro growth and survival of human melanomas. a, Colony formation assays 7 days after seeding of metastatic melanoma cells transfected with a GapmeR control (Ctrl), GapmeR3 and GapmeR11. b, Evaluation of cell death by co-staining for annexin V and propidium iodide (PI) followed by flow cytometric analysis. The graph is an average of three biological replicas and shows the percentage of cells alive, single positive or double positive. c, Efficiency of SAMMSON knockdown using an siRNA against SAMMSON. The expression of SAMMSON is relative to the average of three different housekeeping genes. d, Percentage of remaining living cells upon siSAMMSON, measured by flow cytometry, is indicated on y-axis ± s.e.m. e, Evaluation of the capacity of exogenus SAMMSON and SAMMSON mutants (SAMMSONgap3mut and SAMMSONgap11mut, in which mismatches were introduced into the GapmeR3 and GapmeR11 target sequences) to rescue cell death in SK-MEL-28 treated with GapmeRs. The percentage of remaining living cells is indicated on the y-axis ± s.e.m. f, Effect of a caspase-9 inhibitor on caspase-3/7 activation in SK-MEL-28 treated with GapmeRs. The graph is an average of three biological replicas; caspase-3/7 activity is relative to control sample (Ctrl) ± s.e.m. P value was calculated by ANOVA. g, Relative SAMMSON expression in MM001 cells transfected with an empty or SAMMSONencoding expression vector of three different biological replicates ± s.e.m. P values were calculated by ANOVA. h, Colony formation assays 7 days after seeding 1,000, 5,000 or 10,000 MM001 as described in g. i, Colony formation assays 7 days after seeding of SK-MEL-28 transfected with a control GapmeR (Ctrl) or GapmeR3 and exposed to either vehicle or an EC 50 dose of vemurafenib (PLX4032) or pimasertib. LETTER RESEARCH Extended Data Figure 4 | SAMMSON does not regulate MITF expression in cis. a, Relative expression of MITF as determined by microarray gene expression profiling in the indicated melanoma cell lines treated with GapmeR3 (SAMMSON knockdown) (n > 3) or expressing exogenous SAMMSON (n = 4); no significant differences in MITF expression were observed (limma, Benjamini-Hochberg adjusted P > 0.05), except in MM034, in which SAMMSON knockdown resulted in a 1.5-fold downregulation of MITF (limma, Benjamini-Hochberg adjusted P = 0.013). b, Validation of the array data in a by qPCR for SAMMSON (left) and MITF-M (right) in all the cell lines used for the arrays. Expression is relative to three different housekeeping genes. The graph shows an average of three different biological replicas ± s.e.m. The MITF-M protein levels were assessed by western blotting (bottom; for gel source data, see Supplementary Fig. 1). Extended Data Figure 5 | a, SAMMSON localizes primarily to the cytoplasm and largely co-localizes with mitochondria. Quantification of SAMMSON in nuclei and cytoplasm of SK-MEL-28. Data are expressed as nuclear/cytoplasmic ratio ± s.e.m. Data are shown for MALAT1 (exclusively nuclear) and TBP (cytoplasmic). The graph shows an average of three different fractionation experiments. b, SAMMSON RNA-FISH in a panel of melanoma cell lines and NHMEs. SAMMSON is shown in yellow and DAPI in blue. c, Quantification of SAMMSON RNA-FISH results described in b. Number of fluorescent spots in total per cell, nucleus and cytoplasm of MM057 (n = 10), MM087 (n = 10) and SK-MEL-28 (n = 7) melanoma cells are shown. d, Quantification of SAMMSON in cytoplasm, mitochondria and mitoplasts of SK-MEL-28. Data are expressed as fraction/total ratio ± s.e.m. Data are shown for mitochondrial 16S rRNA (exclusively mitochondrial) and TBP (cytoplasmic). The graph is an average of three different fractionation experiments. e, The purity of the fractions was assessed by western blotting using nuclear (UBF1), cytoplasmic (β-actin) and mitochondrial markers (HSP60 and VDAC1; for gel source data, see Supplementary Fig. 1). LETTER RESEARCH Extended Data Figure 6 | A large fraction of cytoplasmic SAMMSON co-localizes with mitochondria. SAMMSON and mitochondrial 16S rRNA RNA-FISH in four different melanoma cell lines. SAMMSON probes, labelled with Quasar570, are shown in red and 16S rRNA probes, labelled with Quasar670, are shown in yellow; DAPI is in cyan.

  .

8 |

 8 SAMMSON modulates mitochondrial metabolism in a p32-dependent manner. a, RNA-FISH for 16S rRNA (in red) and immunofluorescence for p32 (in yellow) in melanoma cells (MM034) transfected with a control GapmeR (Ctrl) or with GapmeR11. Magnification, ×200. b, Evaluation of OXPHOS complexes functionality by high-resolution respirometry in SK-MEL-28 treated with GapmeR3. The graph shows one representative experiment. c, JC-1 reveals a decrease in mitochondrial membrane potential upon GapmeR11 transfection, as assessed by the ratio between J-aggregates and J-monomers. The graph is an average of four different biological replicates ± s.e.m. P values were calculated by ANOVA. d, Evaluation of ATP production in SK-MEL-28 transfected with GapmeRs and exposed to oligomycin (used here as control). ATP production measured by luciferase is expressed as a percentage, an average of three different experiments, relative to the control sample (Ctrl) ± s.e.m. P values were calculated by ANOVA. e, Colony formation assays 5 days after seeding SK-MEL-28 transfected with a control GapmeR (Ctrl) or GapmeR3, and either with an empty or a p32-expressing vector. f, Colony formation assays 5 days after seeding, showing cell growth of SK-MEL-28 transfected with a control GapmeR (Ctrl) or GapmeR3, and either with an empty vector or a vector expressing a tagged version of p32 that cannot localize to the mitochondria. g, Quantification of the colony assay described in f. The data represent the density (occupancy area) relative to the Ctrl + pcDNA3.1 sample. The data are presented as average of three different biological replicates ± s.e.m. P values were calculated by ANOVA. h, Immunofluorescence using antibodies directed against SDHA and ATPB in MM034 melanoma cells upon GapmeR11 transfection. Magnification, ×600. Extended Data Figure 9 | SAMMSON and p32 silencing affects mitochondria integrity. a, b, Representative electron microscopy images of melanoma cells transfected with Ctrl GapmeR and GapmeR3 or with siRNA targeting p32. c, Quantification of percentage of mitochondria with intact cristae (top) and area and length (middle and bottom) in cells described in b; the total number of mitochondria evaluated per condition is indicated on the x-axis. © 2016 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH Extended Data Figure 10 | SAMMSON silencing decreases melanoma growth in vivo. a, Table describes the origin and BRAF, NRAS and TP53 mutational status of the melanoma lesions that were used to generate the Mel006 and Mel010 PDX models. b, Gene set enrichment analyses among differentially expressed genes in melanoma lesions obtained from Mel006 PDX mice treated (i.v. injections) with a control GapmeR (Ctrl) or GapmeR3. c, Tumour volume of cohorts of Mel010 PDX mice treated (intra-tumour injections) with a control GapmeR (Ctrl) or GapmeR3. Data are means ± s.d. of three different biological replicates (P value was calculated by two-ways ANOVA). d, Tumour weight of the melanoma lesions derived from PDX mice (Mel006) treated with combinations of control GapmeR (Ctrl) and GapmeR3 with either vehicle or dabrafenib by daily oral gavage (vehicle or dabrafenib) and i.v. injection of the GapmeRs every 2 days. P value was calculated by t-test. cooperating 9p tumor suppressors, in which duplication of CDKN2A and CDKN2B and epigenetic co-regulation of the encoded gene products INK4A/ARF and INK4B by polycomb complexes may coordinately regulate stem cell selfrenewal.

Figure 1 .

 1 Figure 1. Inhibition of SAMMSON Reveals Metabolic Vulnerability in MelanomaThe SAMMSON lncRNA gene is co-amplified with MITF in melanoma, and the SAMMSON-p32 complex is required for correct mitochondrial biogenesis. Depletion of SAMMSON leads to stress associated with accumulation of mitochondrial peptide precursors and mitochondrial import defects and, consequently, p53-independent apoptosis. BRAF inhibitors (BRAFi) promote dependency on mitochondrial oxidative phosphorylation and consequently synergize with SAMMSON inhibition.

Annexe 1 ,Figure 2 .Figure 1 .Annexe 2 ,Figure 2 .

 12122 Induction of Tyr::CreERT2; Smarca4 lox+-and Smarca4 lox/lox mice during adult hair cycling doesn't affect regeneration of differentiated melanocytes. (A) Tyr::CreERT2; Smarca4 lox+-and Smarca4 lox/lox mice were depilated, treated topically with 4-OHT every day for 10 days and photographed on the indicated times (0-29 days post-plucking, dpp). (B) Epifluorescence images of DAPI-(blue), DCT-(green) and BRG1-stained melanocytes (red) within the growing hair follicle (arrows) of a Tyr::CreERT2 Smarca4 lox/lox mouse harvested 10 days after depilation and daily 4-OHT application. Scale bar 50 µM. Delmas, V., S. Martinozzi, Y. Bourgeois, M. Holzenberger, and L. Larue. 2003. 'Cremediated recombination in the skin melanocyte lineage', Genesis, 36: 73-80. Luciani, F., D. Champeval, A. Herbette, L. Denat, B. Aylaj, S. Martinozzi, R. Ballotti, R. Kemler, C. R. Goding, F. De Vuyst, L. Larue, and V. Delmas. 2011. 'Biological and mathematical modeling of melanocyte development', Development, 138: 3943-54. These data reveal how the combination of transcription factors MITF and SOX10 and chromatin remodelling complexes NURF and BRG1 cooperate to drive Comparative analysis of RNA-seq data from shMITF and shBPTF in 501Mel cells. (A) Venn diagram indicating the overlap between genes commonly upregulated upon shMITF and shBPTF. GSEA analysis reveals that both datasets are enriched in genes associated with EMT and inflammatory response corresponding to the SASP. (B) Venn diagram indicating the overlap between genes commonly downregulated upon shMITF and shBPTF. GSEA analysis reveals that both datasets are enriched in E2F target genes involved in cell cycle and oxidative metabolism. MITF and SOX10 and chromatin remodelling complexes NURF and BRG1 cooperate to drive metabolic switches during melanoma growth. (A) UCSC screenshot illustrating BRG1 and MITF occupancy over the PPARGC1A locus highlighting co-localization with several H3K27ac-marked enhancers. MITF and BPTF coregulate oxidative metabolism in proliferative melanoma cells through regulation of PGC1a expression while upon MITF knockdown, BPTF activates glycolytic metabolism components (B) UCSC screenshot illustrating BRG1, MITF and SOX10 occupancy over the MITF-LINC01212 locus. SOX10 actively recruits BRG1 to activate LINC01212 expression, which drives mitochondrial function in melanoma.

  , e1005555.

	This research article has been highlighted in
	Welsh, S. J. and Rizos, H. (2016) Melanocyte reprogramming requires chromatin
	and transcription remodelling. Pigment Cell Melanoma Res. 29:260-261.

  Figure 8-figure supplement 1. ContinuedsiMITF-silenced cells where these genes are activated. (C) BRG1 re-localizes over the genome in siMITF senescent cells. UCSC screenshot illustrating that BRG1 occupancy is strongly increased over the CCL2 locus in siMITF-silenced cells where this gene is strongly activated.

	Biochemistry | Genes and chromosomes Results -Article 2
	ARTICLE 2
	Chromatin-remodelling complex NURF is essential for
	differentiation of adult melanocyte stem cells.
	Dana Koludrovic, Patrick Laurette, Thomas Strub, Céline Keime, Madeleine Le Coz,
	Sebastien Coassolo, Gabrielle Mengus, Lionel Larue, Irwin Davidson
	PLOS Genet 10/2015, 11(10), e1005555
	Highlighted in
	Welsh, S. J. and Rizos, H. (2016) Melanocyte reprogramming requires
	chromatin and transcription remodelling. Pigment Cell Melanoma Res.
	29:260-261.
	06857	27 of 29

Laurette et al. eLife 2015;4:e06857. DOI: 10.7554/eLife.06857 26 of 29 Biochemistry | Genes and chromosomes DOI: 10.7554/eLife.06857.017 Laurette et al. eLife 2015;4:e06857. DOI: 10.7554/eLife.

.1038/nature17161 Melanoma addiction to the long non-coding RNA SAMMSON Eleonora

  Leucci 1,2 , Roberto Vendramin1,2 , Marco Spinazzi 2 , Patrick Laurette 3 , Mark Fiers 2 , Jasper Wouters 4 , Enrico Radaelli 5 , Sven Eyckerman6,7 , Carina Leonelli8,9 , Katrien Vanderheyden8,9 , Aljosja Rogiers 1,2 , Els Hermans 10 , Pieter Baatsen 2 , Stein Aerts 11 , Frederic Amant 10 , Stefan Van Aelst12,13 , Joost van den Oord 4 , Bart de Strooper 2 , Irwin Davidson 3 , Denis L. J. Lafontaine 14 , Kris Gevaert6,7 , Jo Vandesompele 8,9 , Pieter Mestdagh8,9 * & Jean-Christophe Marine 1,2 *

	LETTER	doi:10	-Article 4
	ARTICLE 4
	3. Melanoma addiction to the long non-coding RNA SAMMSON
	Eleonora Leucci, Roberto Vendramin, Marco Spinazzi, Patrick Laurette, Mark Fiers, Jasper
	Wouters, Enrico Radaelli, Sven Eyckerman, Carina Leonelli, Katrien Vanderheyden,
	Aljosja Rogiers, Els Hermans, Pieter Baatsen, Stein Aerts, Frederic Amant, Stefaan Van
	Aelst, Joost van den Oord, Bart de Strooper, Irwin Davidson, Denis L.J. Lafontaine, Kris
	Gevaert, Jo Vandesompele, Pieter Mestdagh, Jean-Christophe Marine
	Nature, Vol. 531, 24 March 2016, pp. 518-522
	Highlighted in
	Goding, CR (2016) Targeting the lncRNA SAMMSON Reveals Metabolic
	Vulnerability in Melanoma. Cancer cell, 29(5), 619-621.

Focal amplifications of chromosome 3p13-3p14 occur in about 10% of melanomas and are associated with a poor prognosis. The melanoma-specific oncogene MITF resides at the epicentre of this amplicon 1 . However, whether other loci present in this amplicon also contribute to melanomagenesis is unknown. Here we show that the recently annotated long non-coding RNA (lncRNA) gene SAMMSON is consistently co-gained with MITF. In addition, SAMMSON is a target of the lineage-specific transcription factor SOX10 and its expression is detectable in more than 90% of human melanomas. Whereas exogenous SAMMSON increases the clonogenic potential in trans, SAMMSON knockdown drastically decreases the viability of melanoma cells irrespective of their transcriptional cell state and BRAF, NRAS or TP53 mutational status. Moreover, SAMMSON targeting sensitizes melanoma to MAPK-targeting therapeutics both in vitro and in patient-derived xenograft models. Mechanistically, SAMMSON interacts with p32, a master regulator of mitochondrial homeostasis and metabolism, to increase its mitochondrial targeting and pro-oncogenic function. Our results indicate that silencing of the lineage addiction oncogene SAMMSON disrupts vital mitochondrial functions in a cancer-cell-specific manner; this silencing is therefore expected to deliver highly effective and tissue-restricted anti- melanoma therapeutic responses.

  . Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551-3567 (1999). 30. Mestdagh, P. et al. A novel and universal method for microRNA RT-qPCR data normalization. Genome Biol. 10, R64 (2009).

		LETTER RESEARCH
	TGGCGGAAAAGC; reverse, CATCTTGCATGGCCCCA; TYR forward, AGCAGGCTCAGTCGATACAG; reverse, CACTGGGAATGAAGGGCAAG; AAAGTGACCGTGA; SAMMSON siRNA sense, GUCGCUAGACAUU AACC; sip32.2 sense, TCACGGTCACTTTCAACAT; antisense, ATGTTG sense, GGTTGAAGAACAGGAGCCT; antisense, AGGCTCCTGTTCTTC TTGGAGGA; non-targeting GapmeR; TCATACTATATGACAG; sip32.1 SAMMSON GapmeR3, GTGTGAACTTGGCT; GapmeR11, TTTGAGAG	UGAGGA[dA][dA]; siRNA antisense UCCUCAAAUGUCUAGCGAC[dA] [dA]; SOX10 and MITF knockdown, Dharmacon Smartpool.
		Primers and siRNAs used. HPRT forward, AGCCAGACTTTGTTGGA TTTG; reverse, TTTACTGGCGATGTCAATAAG; TBP forward, CGGC TGTTTAACTTCGCTTC; reverse, CACACGCCAAGAAACAGTGA; UBC forward, ATTTGGGTCGCGGTTCTTG; reverse, TGCCTTGACATT CTCGATGGT; MITF-M forward, CATTGTTATGCTGGAAATGCTAGAA; reverse, GGCTTGCTGTATGTGGTACTTGG; SOX10 forward, TACCCGC ACCTGCACAAC; reverse, TTCAGCAGCCTCCAGAGC; SOX9 forward, GCAAGCTCTGGAGACTTCTG; reverse, GTACTTGTAATCCGGGTGGTC; p32 forward, ACACCGACGGAGACAAAG; reverse, GGGATGCTGTTG TTAATGTTG; MALAT1 forward, GGATTCCAGGAAGGAGCGAG; re vers e, AT TGC C GAC CTCAC GGAT T T; SAMMSON for w ard , TTCCTCAACTATGCAACTCAA; reverse, TAGACTACGGGCTCATGACTT; SAMMSON forward #2, CCTCTAGATGTGTAAGGGTAGT; reverse #2, TTGAGTTGCATAGTTGAGGAA; GPR110 forward, CAGTATTG

29

Une partie du travail présenté ici a contribué à élucider le rôle de NURF dans le mélanome et les mélanocytes. Nous avons montré que la perte de BPTF, la principale sous-unité du complexe de remodelage NURF, induit un arrêt de la prolifération et une entrée en senescence des cellules de mélanome. Par ailleurs, nous avons montré que BPTF et MITF coopèrent pour réguler l'expression de gènes impliqués dans la prolifération et l'invasion. De façon inattendue, l'inactivation de Bptf spécifiquement dans les mélanocytes entraine la perte progressive et totale de la pigmentation du pelage en raison de l'incapacité des cellules souches mélanocytaire à produire une descendance fonctionnelle. C'est la première fois que l'interaction fonctionnelle entre NURF et MITF est démontrée in vitro, complétée par des observations phénotypique uniques in vivo chez la souris.Enfin de façon à compléter ces observations, nous avons inactivé les gènes exprimant BRG1 ou BPTF dans un modèle murin de mélanome récapitulant les mutations principales observées chez l'homme. Nous avons ainsi pu montrer que la perte de l'un des deux facteurs était suffisante pour ralentir significativement la progression du mélanome in vivo. Des analyses plus approfondie ont montré que les tumeurs de mélanome finissent malgré tout par croitre sous l'impulsion de cellules n'ayant par recombiné les deux allèles codant BRG1 ou BTPF.En somme, l'utilisation de différents modèles murins a permis de révéler in vivo la contribution fonctionnelle distincte mais complémentaire de ces deux complexes de remodelage associé à MITF aux cours de trois stades majeurs du lignage mélanocytaire : le développement embryonnaire des mélanocytes, leur différentiation ainsi que lors de l'initiation et la progression du mélanome.

Ensemble, ces approches ont contribué de façon significative à la caractérisation moléculaire et fonctionnelle des partenaires de MITF et ainsi à la compréhension des mécanismes d'apparition et de progression du mélanome et devraient mettre en lumière certains mécanismes généraux d'activation de la transcription chez les eucaryotes et les conséquences de leur dérégulation.

alternative promotersSpecific exonsCommon exons

Laurette et al. eLife 2015;4:e06857. DOI: 10.7554/eLife.06857

PLOS Genetics | DOI:10.1371/journal.pgen.1005555October 6, 2015

Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper.

© 2016 Macmillan Publishers Limited. All rights reserved

© 2016 Macmillan Publishers Limited. All rights reservedW W W. N A T U R E . C O M / N A T U R E | 1

Acknowledgements

We thank, S Gygi and R Tomaino for mass-spectrometry analysis, A Hamiche for advice on tandem immunopurification, L Larue for the Tyr-Cre mice, P Chambon and D Metzger for the floxed Smarca4 mice, E Sviderskaya and D Bennet for the Hermes cells, B Jost, and all the staff of the IGBMC high throughput sequencing facility, a member of 'France G énomique' consortium (ANR10-INBS-09-08). This work was supported by grants from the CNRS, the INSERM, the Fondation ARC pour la Recherche contre le Cancer, the Ligue Nationale et D épartementale R égion Alsace contre le Cancer and the Institut National du Cancer (INCa) 2011-1-PL BIO-03-INSERM 16-1, the ANR-10-LABX-0030-INRT French state fund through the Agence Nationale de la Recherche under the frame programme Investissements d'Avenir labelled ANR-10-IDEX-0002-02. ID is an ' équipe labellis ée' of the Ligue Nationale contre le Cancer. The data in this paper have been submitted to the Geo database under the reference GSE61967. Acknowledgments We thank S. Gygi and R. Tomaino for mass-spectrometry analysis, A. Hamiche for help in tandem affinity purification, Y. Davidson for help with data analysis, C. Wu for the floxed Bptf Aknowledgements We thank Lionel Larue for the Tyr::CreERT2 and floxed Braf mice, Daniel Metzger for Smarca4 and Pten floxed mice, Carl Wu for Bptf floxed mice, Isabelle Michel for help with genotyping, B. Jost, and all the staff of the IGBMC high throughput sequencing facility, a member of "France Génomique" consortium (ANR10-INBS-09-08). This work was supported by grants from the CNRS, the INSERM, the Association pour la Recherche contre le Cancer, the Ligue Nationale et Départementale Région Alsace contre le Cancer and the Institut National du Cancer (INCa) the ANR-10-LABX-0030-INRT French state fund through the Agence Nationale de la Recherche under the frame programme Investissements d'Avenir labelled ANR-10-IDEX-0002-02. PL was supported by fellowships from Ministère de l'enseignement supérieur et de la recherche and Fondation ARC pour la recherche sur le cancer. ID is an 'équipe labellisée' of the Ligue Nationale contre le Cancer.

Funding

Additional information

Competing interests ID: Reviewing editor, eLife. The other authors declare that no competing interests exist.

Additional files

Supplementary files

• Supplementary file 1. Related to Figure 1. Mass spectrometry identification of MITF partners. Excel table showing the data from the mass spectrometry analysis. Page 1 shows a summary of the number of peptides and proteins identified in the two experiments in the soluble nuclear and chromatin associated fractions. Page 2 lists the proteins identified uniquely in the F-H-MITF immunoprecipitates from the soluble nuclear fraction along with the number of peptides for each protein. Page 3 lists the proteins identified uniquely in the F-H-MITF immunoprecipitates from the chromatin-associated fraction along with the number of peptides for each protein.

DOI: 10.7554/eLife.06857.019

• Supplementary file 2. Related to Figure 2. Excel spread sheet of genes specifically and commonly regulated by BRG1 and MITF knockdown in 501Mel and Hermes 3A cells along with the appropriate gene ontology, see Figures S3B-D. DOI: 10.7554/eLife.06857.020

• Supplementary file 3. Excel spread sheet of genes with associated BRG1 occupancy (either ±10 kb, or ±30 kb with respect to TSS) and regulated in shBRG1 along with the appropriate gene ontology as described in Figure S5E. DOI: 10.7554/eLife.06857.021

• Supplementary file 4. Excel spread sheet of genes associated with BRG1 and MITF co-occupied sites or MARES along with their gene ontology.

Author Summary

The melanocytes pigmenting the coat of adult mice derive from the melanocyte stem cell population residing in the permanent bulge area of the hair follicle. At each angen phase, melanocyte stem cells are stimulated to generate proliferative transient amplifying cells that migrate to the bulb of the follicle where they differentiate into mature melanin producing melanocytes, a processes involving MIcrophthalmia-associated Transcription

Supplementary Information is available in the online version of the paper.

Acknowledgements GapmeRs were designed by J. Lai. This work was supported by the Fonds Wetenschappelijk Onderzoek (FWO; #G.0646.14N and #3G056613), Foundation Against Cancer (STK#2014-126), UGent-IOF (F2013/IOF-Advanced/676) and STK grant F/2014/376. The PDX studies were funded by GOA/14/012 (KULeuven) and KPC_29_005 (Belgian Ministries of Health). The authors wish to thank N. Samyn for help with MS, M. Van Gele for providing NHME cultures and P. Wolter for scientific discussions and his central role in the establishment of the melanoma PDX platform. E.L. is a recipient of a postdoctoral fellowship from the Marie-Curie/VIB OMICS program. M.S. is the recipient of EMBO fellowship (ALTF 648-2013). F.A. is a senior researcher from the Research Fund Flanders (FWO). P.M. and R.V. are recipients of FWO postdoctoral and PhD fellowships, respectively. D.L. is supported by the Fonds National de la Recherche (FRS/FNRS). I.D. is supported by the Institut National du Cancer PAIR melanoma (MELA13-002), the "France Génomique" consortium (ANR10-INBS-09-08), and ANR-10-LABX-0030-INRT. The I.D. laboratory is an "équipe labellisée Ligue Nationale contre le Cancer". Author Information Mass spectrometry data have been deposited in the Proteomics Identifications Database under accession numbers PXD002565. Gene expression data have been deposited in the Gene Expression Omnibus under accession number GSE70180. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to J.-C.M. (JeanChristophe.Marine@cme.vib-kuleuven.be).

Author Contributions

SUPPLEMENTARY INFORMATION

Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

We next addressed whether Brg1 is required to generate melanocytes from the adult melanocyte stem cell (MSC) population. To address this question, we generated Tyr::Cre-ER T2 ; Smarca4 lox/lox animals. At 6 weeks of age, the mice were depilated to induce and synchronise the anagen phase and treated topically with 4-OHT everyday for 10 days to inactivate Brg1 in the newly generated transient amplifying cells and the resulting mature melanocytes. Under these conditions, the newly grown coat at the depleted region was normally pigmented comparable to that of control Tyr::Cre-ER T2 ; Smarca4 lox/+ animals suggesting that Brg1 was not required for generation of pigment producing melanocytes from the adult MSC population (Annexe 1, Fig. 2A). To verify that Brg1 was indeed inactivated in these melanocytes, the epidermis was stained for Dct 10 days after depilation to identify melanocytes and for Brg1. Within the hair follicles of Tamoxifen-injected Tyr::Cre-ER T2 ; Smarca4 lox/lox mice, melanocytes with cytoplasmic Dct-staining showed loss of Brg1 labelled nuclei, whereas the surrounding keratinocytes showed strong nuclear staining (Annexe 1, Fig. 2B). These data confirm that Brg1 was inactivated in the melanocytes following Tamoxifen injection, but that it is not required for the proliferation and differentiation of adult melanocytes.

Together these data revealed a specific requirement for Brg1 during the proliferation of embryonic melanoblasts, whereas generation of melanocytes from the adult MSC population is independent of Brg1. In Koludrovic et al. (Koludrovic et al., 2015), we showed that MITF and BPTF coregulate a set of genes required for melanoma cell proliferation. We re-analysed the RNA-seq data obtained after MITF and BPTF silencing in 501Mel cells using the GSEA software. After MITF and BPTF silencing, up-regulated genes were enriched in those associated with EMT and in inflammatory response corresponding to the SASP (Annexe 2, Fig. 1A). Down-regulated genes on the other hand were enriched in E2F target genes involved in cell cycle and more interestingly in those involved in oxidative metabolism (Annexe2, Fig. 1B). One of the key genes in this pathway is PPARGC1A whose expression is activated by both MITF and BPTF. MITF binds to multiple H3K27ac-marked sites at the PPARGC1A locus showing that it is a direct target gene (Annexe 2, Fig. 2A).

Legends to figures

These data are in agreement with previous reports that MITF regulates oxidative phosphorylation in melanoma tumours and promotes resistance to BRAF inhibitors (Haq et al., 2013;Vazquez et al., 2013). Moreover, Dar and colleagues (Dar et al., 2015) showed that BPTF is required for melanoma growth and also promotes resistance to BRAF inhibitors. Together these data suggest that MITF and BPTF cooperate to regulate PPARGC1A and other genes involved in oxidative phosphorylation to promote a metabolic switch driving tumour growth and resistance to BRAF inhibitors.

A second mechanism that promotes a switch to oxidative phosphorylation is via the SAMMSON non-coding RNA (Leucci et al., 2016). In this pathway, SOX10 recruits BRG1 to the SAMMSON locus to drive its expression (Annexe 2, Fig. 2A).

Hence, we propose a model in which MITF/BPTF drive PPARGC1A expression and SOX10/BRG1 drive SAMMSON expression, the net result being an up-regulation of oxidative phosphorylation and tumour growth.