L'hématopoïèse est un processus très finement régulé qui mène à la formation de chaque cellule sanguine d'un organisme. Chez les mammifères il existe un nombre important de types cellulaires qui participent à l'établissement des mécanismes de défense du corps. Toutes ces cellules proviennent de la différenciation terminale d'une cellule unique appelée Cellule Souche Hématopoïétique (CSH) qui, par le biais de différenciations successives donnant naissance à des progéniteurs de plus en plus spécifiés, va permettre l'établissement normal de tous les types de cellules sanguines. Les CSH sont parmi les cellules les plus finement contrôlées de l'organisme, car en effet une dérégulation de leur fonctionnement normal (prolifération excessive, différentiation prématurée…) peut entrainer de graves conséquences, à savoir des maladies du sang appelées leucémies.

De nombreux facteurs moléculaires sont impliqués dans la régulation des CSH, et certains d'entre eux sont les cibles de mutations ou réarrangements chromosomiques à l'origine de leucémies, tels que le facteur de transcription RUNX1 et la Lysine Acétyl-Transférase (KAT) Monocytic Leukemia Zinc-Finger (MOZ).

Chez la Drosophile, les cellules sanguines sont apparentées au lignage myéloïde des mammifères, et les acteurs moléculaires contrôlant leur formation sont très conservés. Ainsi, les cellules à cristaux (CC), qui sont les homologues fonctionnels des mégakaryocytes, sont formées suite à l'action conjointe de l'homologue de RUNX1, Lozenge (Lz) et de GATA1, Serpent (Srp). Un crible pan génomique mené par mon équipe d'accueil visant à trouver des modulateurs de l'activité transcriptionelle de Lz et Srp, a permis de d'identifier le gène enoki mushroom (enok) comme étant un régulateur négatif de cette activité. Enok est l'homologue chez la Drosophile de MOZ, et une étude préliminaire de son rôle in vivo a pu mettre en évidence une fonction essentielle dans le développement des CC, qui disparaissent quasiment en totalité dans un contexte mutant pour enok.

L'objectif de ma thèse a été de comprendre les mécanismes par lesquels Enok régule la formation des CC chez la larve de Drosophile.

Les CC sont générées au stade larvaire à partir de la transdifférenciation de macrophages après activation par la voie Notch, et l'initiation de Lz. Au contraire de données publiées récemment par un autre groupe, j'ai démontré que Lz est requis et suffisant pour induire l'expression de l'effecteur de la voie Hippo Yorkie, et non l'inverse. De plus, grâce à des expériences de perte de fonction et de sauvetage phénotypique, j'ai montré qu'Enok est requis pour l'expression de Lz de façon autonome cellulaire dans les précurseurs de CC.

Chez les mammifères, MOZ est connu pour faire partie d'une tétrade d'acétylation, et j'ai montré qu'ici seul un de ses partenaires est requis, le facteur de type BRPF Br140. Pour approfondir l'étude de la fonction d'Enok, j'ai généré grâce au système CRISPR/Cas9 un allèle catalytiquement inactif d'enok. J'ai ainsi montré que cette fonction n'est absolument pas requise durant la différenciation des CC.

Enfin, j'ai montré qu'Enok permet la régulation de l'expression de Lz en se fixant sur un enhancer dans le troisième intron du gène. De façon intéressante, sa délétion provoque le même phénotype de perte de CC qu'une perte de fonction d'enok, montrant qu'il est requis pour l'expression de lz dans le système hématopoiétique.

Ces résultats mettent en valeur un nouveau mode d'action d'Enok dans l'hématopoïèse chez la Drosophile. De façon intéressante, une étude récente propose qu'Enok et Br140 se fixent sur la chromatine pour réguler l'expression de gènes cibles. Mes résultats sur une fonction noncatalytique d'Enok viennent enrichir ce modèle, qui montre un mode d'action peu exploré dans le domaine. Dans le futur, ce travail pourrait ouvrir des portes sur une meilleure compréhension des fonctions normales et pathologiques de MOZ chez les mammifères.

I. Preamble

Hematopoiesis is the process that leads to the normal production of every mature blood cell type in an organism; by definition, maintenance of its homeostasis is crucial throughout development, as hematopoietic deregulation has dramatic consequences on the organism.

Indeed, blood disorders are even nowadays among the deadliest pathologies. In order to maintain a normal blood content, the hematopoietic process is tightly regulated, and this regulation is extensively studied in biological research with the aim of understanding the molecular mechanisms that are deregulated in pathologies and of finding new efficient ways to cure them.

All mammalian blood cells come from the differentiation of Hematopoietic Stem Cells (HSCs). These HSCs have the essential property of being able to self-renew indefinitely; it has been shown that they can regenerate the entire hematopoietic system of irradiated mouse recipients through several successive graft experiments (Jacobson et al. 1951a;[START_REF] Spangrude | Purffication and Characterization of Mouse Hematopoietic Stem Cells[END_REF]. HSCs differentiate asymmetrically and give rise to more and more committed progenitors that ultimately generate all blood cell types.

HSC discovery led many groups to pursue the study of those cells during normal and pathologic hematopoiesis. Several models emerged over the years to study the tight regulation controlling the formation of every blood cell lineage, such as vertebrate models like mouse and zebrafish. However, the strong conservation of molecular factors controlling hematopoiesis across evolution shed light on the Drosophila model, which has been widely used for over fifteen years for the study of both normal and pathological hematopoiesis.

I will describe in a first part how mammalian blood cells are formed under normal and pathological conditions and which molecular actors are involved during those processes. In a second part, I will summarize what is known about Drosophila blood cell formation and show why it has emerged as an invaluable model to study normal and pathologic hematopoiesis.

Finally, I will describe the discovery of enoki mushroom and how it was linked to a function in hematopoiesis, which is the basis of my thesis work.

II. Introduction

A. Normal Hematopoiesis in vertebrates and mammals

Blood cell types and functions

Mammalian hematopoiesis is characterized by a great number of cell types that are formed; ten different cell types and several subtypes are produced (reviewed in [START_REF] Orkin | Hematopoiesis: An Evolving Paradigm for Stem Cell Biology[END_REF][START_REF] Weiskopf | Myeloid Cell Origins, Differentiation, and Clinical Implications[END_REF]); (Figure 1). These highly specialized cells of the myeloid and lymphoid lineages ensure a large panel of functions.

a) Myeloid lineage

Myeloid cells are responsible for innate immunity, which allows a rapid and direct defense of the organism against invading pathogens, wound healing and transport of gases. Cells involved in the innate immunity are found in many organisms, and innate immunity is the major defense system in insects and plants.

• Granulocytes are distributed in three types: neutrophils, eosinophils and basophils that are categorized based on their ability to absorb neutral, acid or basic eosin-derived dyes.

Neutrophils are phagocytes and can exocytose granules, thereby reducing inflammation.

Eosinophils are responsible for the elimination of parasites, using cytotoxic granules, and basophils produce histamine during inflammatory and allergic reactions.

• Macrophages arise from the terminal differentiation of monocytes and are responsible for phagocytosis. They eliminate apoptotic or necrotic cells, and also cancer cells that express specific membrane markers. They also have a defense function during innate immunity, as they phagocytize pathogens like bacteria. Macrophages are furthermore found as resident cells in several adult tissues where they self-maintain [START_REF] Hashimoto | Tissue-Resident Macrophages Self-Maintain Locally throughout Adult Life with Minimal Contribution from Circulating Monocytes[END_REF]; in addition, they participate in maintaining the steady state of tissues. Indeed, macrophages are able to activate BMP signaling in enteric neurons in order to control gastrointestinal motility [START_REF] Muller | Crosstalk between Muscularis Macrophages and Enteric Neurons Regulates Gastrointestinal Motility[END_REF]. It also has been shown that macrophages At the top of the tree, hematopoietic stem cells are responsible for the formation of every type of blood cell. Asymetric divisions allow self-renewal and generate progenitors of the myeloid and lymphoid lineages. These progenitors further differentiate into more committed progenitors that will eventually give rise to all mature blood cells. The blue dashed line indicates the ability of B lymphocytes to transdifferentiate into macrophages. (Adapted from [START_REF] Orkin | Hematopoiesis: An Evolving Paradigm for Stem Cell Biology[END_REF] Adapted from [START_REF] Orkin | Hematopoiesis: An Evolving Paradigm for Stem Cell Biology[END_REF] protect tissue against inflammatory response by inhibiting neutrophil recruitment [START_REF] Uderhardt | Resident Macrophages Cloak Tissue Microlesions to Prevent Neutrophil-Driven Inflammatory Damage[END_REF]).

• Megakaryocytes are responsible for the production of platelets that are released in circulation after megakaryocyte fragmentation and, upon injury, they aggregate to allow wound healing by promoting coagulation.

• Erythrocytes are the most abundant blood cell type; they are enucleated cells mostly responsible for the transport of oxygen throughout the organism during embryonic and adult life. They have other functions such as regulating blood viscosity and maintaining the shear stress necessary for vascular development and remodeling [START_REF] Baron | Concise Review: Early Embryonic Erythropoiesis: Not so Primitive After All[END_REF][START_REF] Lucitti | Vascular Remodeling of the Mouse Yolk Sac Requires Hemodynamic Force[END_REF]).

b) Lymphoid lineage

Lymphoid cells are responsible for adaptive immunity, a second, highly specific, wave of defense against invading pathogens. Adaptive immunity arose in gnathostomes, as all jawed vertebrates possess lymphocyte receptors.

• B lymphocytes are key effectors of the adaptive response. They are activated by T lymphocytes (see below) at the onset of the response to initiate the production of antibodies specific to the pathogen. A subset of B lymphocytes subsequently becomes memory B cells and allows an efficient response, if the organism has to fight the same pathogen later in life.

• T lymphocytes are classified in 3 main groups of cells: (i) T helper cells (positive for the CD4 marker) that are activated after the presentation of an antigen by Antigen Presenting Cells; the activated T cell then interacts with a B lymphocyte to trigger specific antibody production; (ii) cytotoxic T lymphocytes (positive for the CD8 marker) that directly eliminate the pathogen after their activation and (iii) memory T lymphocytes that have a long lifespan and participate in long-term immunity.

• Natural Killer cells, thanks to their cytotoxic granules, are responsible for direct destruction of cancer cells, viral-infected cells and foreign bodies.

Adult definitive blood cells all come from HSCs, localized in the bone marrow. This environment is a niche allowing HSCs to proliferate and differentiate normally (reviewed in [START_REF] Asada | Complexity of Bone Marrow Hematopoietic Stem Cell Niche[END_REF]. However, these HSCs themselves emerge after successive events taking place during embryonic development that I will describe below.

Blood cell formation

Blood cell formation is a process that initiates during early embryonic stages. Indeed, during its development, the embryo has to be supplied in oxygen and may also have to be able to fight off some pathogens that would be a threat to its survival. In order to meet those needs, three successive hematopoietic waves take place during development (Figure 2).

a) First hematopoietic wave and primitive hematopoiesis

The first hematopoietic wave consists of a transient myelopoiesis that takes place inside blood islands of the yolk sac during early embryogenesis (reviewed in [START_REF] Lacaud | Hemangioblast, Hemogenic Endothelium, and Primitive versus Definitive Hematopoiesis[END_REF]. Absence of primitive hematopoiesis in the mouse embryo, notably after loss of function of GATA-1, which is a major hematopoietic transcription factor, leads to early embryonic lethality presumably due to the absence of erythrocytes [START_REF] Fujiwara | Arrested Development of Embryonic Red Cell Precursors in Mouse Embryos Lacking Transcription Factor GATA-1[END_REF]; this phenotype underlines the importance of early hematopoiesis during development.

This first embryonic wave gives rise to primitive myeloid cells (erythrocytes, megakaryocytes and macrophages) that will migrate to the embryo proper. Some studies suggested that endothelial cells and primitive blood cells in blood islands of the yolk sac arise from a common progenitor called hemangioblast, but although it seems to be conserved from fly to mouse [START_REF] Mandal | Evidence for a Fruit Fly Hemangioblast and Similarities between Lymph-Gland Hematopoiesis in Fruit Fly and Mammal Aorta-Gonadal-Mesonephros Mesoderm[END_REF][START_REF] Vogeli | A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula[END_REF][START_REF] Huber | Haemangioblast commitment is initiated in the primitive streak of the mouse embryo[END_REF] has not yet been demonstrated in humans and is a matter of debate. In mouse, primitive erythrocytes come from erythroblasts produced in the blood islands that enter the bloodstream, continue to divide [START_REF] Bethlenfalvay | Fetal Erythropoiesis[END_REF] and finally differentiate into enucleated erythrocytes that express both embryonic and adult globins [START_REF] Kingsley | Yolk Sac-Derived Primitive Erythroblasts Enucleate during Mammalian Embryogenesis[END_REF](Kingsley , 2006; During the first wave, primitive hematopoietic cells (in red) arise from cells inside blood islands (in blue) in the yolk sac. They migrate to the embryo proper, where they differentiate into primitive erythrocytes, macrophages and megakaryocytes. B. During the second wave, the first definitive blood cells arise. These cells are Erythromyeloid Myeloid Progenitors (EMPs, in yellow); they originate from the hemogenic endothelium (in green) of the yolk sac through the Endothelial to Hematopoietic Transition process and migrate to the fetal liver, where they proliferate and differentiate. C. The third wave gives rise to the hematopoietic stem cells (HSCs). They emerge from the hemogenic endothelium of major arteries in the embryo, and in particular in the dorsal aorta. The emerging pre-HSCs will then, like EMPs, migrate to the fetal liver to expand and differentiate. At the end of embryogenesis, newly formed hematopoietic cells will migrate again to colonize the bone marrow niche. (Adapted from [START_REF] Tavian | Embryonic Development of the Human Hematopoietic System[END_REF] Endothelial cell

Hemogenic endothelium

Pre-HSC

Migration to the fetal liver

Dorsal Aorta (and other embryonic arteries)

Third hematopoietic wave First hematopoietic wave

Second hematopoietic wave

Bone marrow colonization Expansion and differentiation of HSCs

Yolk sac endoderm

Migration to the embryo proper

Endothelial cell

Yolk sac Blood Island Primitive Hematopoietic cell

Yolk sac endoderm

Endothelial cell

Hemogenic endothelium EMP

Migration to the fetal liver Expansion and differentiation of EMPs

A B C

Adapted from Tavian et al, 2005 [START_REF] Qiu | Globin Switches in Yolk Sac-like Primitive and Fetal-like Definitive Red Blood Cells Produced from Human Embryonic Stem Cells[END_REF]. It also has been shown that in the yolk sac, primitive megakaryocytes emerge from bipotent progenitors called megakaryocyte/erythroid progenitors (MEPs) that can give rise to both primitive lineages [START_REF] Tober | The Megakaryocyte Lineage Originates from Hemangioblast Precursors and Is an Integral Component Both of Primitive and of Definitive Hematopoiesis[END_REF]). These megakaryocytes participate in the formation of embryonic platelets that circulate in the embryo. Primitive macrophages in the yolk sac have two origins: a first population is composed of maternally-derived macrophages that do not express all adult macrophages markers, and a second population comes from monopotent progenitors that differentiate into macrophages exhibiting definitive adult features [START_REF] Bertrand | Three Pathways to Mature Macrophages in the Early Mouse Yolk Sac[END_REF].

Hematopoietic cells formed during the first embryonic wave are transient and thus are replaced later on during embryogenesis by definitive cells with adult features. Consequently, they are not found in post-natal stages and are not involved in definitive hematopoiesis.

However, it has recently been shown that primitive macrophages play an essential role in colonizing hematopoietic organs, notably by remodeling the extra cellular matrix [START_REF] Travnickova | Primitive Macrophages Control HSPC Mobilization and Definitive Haematopoiesis[END_REF].

b) Second hematopoietic wave and emergence of the first definitive blood cells

In addition, the second hematopoietic wave occurs in the extra embryonic yolk sac, with the emergence of a myeloid progenitor called the Erythromyeloid Progenitor (EMP; reviewed in [START_REF] Frame | Erythro-Myeloid Progenitors: "Definitive" Hematopoiesis in the Conceptus Prior to the Emergence of Hematopoietic Stem Cells[END_REF]. EMPs emerge from the hemogenic endothelium of the yolk sac [START_REF] Frame | Definitive Hematopoiesis in the Yolk Sac Emerges from Wnt-Responsive Hemogenic Endothelium Independently of Circulation and Arterial Identity: EMPs Arise from Arteriovenous Hemogenic Endothelia[END_REF]) through a process called Endothelium to Hematopoietic Transition (EHT). EMPs migrate rapidly through the new circulatory system to colonize the fetal liver [START_REF] Palis | Development of Hematopoietic Progenitors[END_REF], where they proliferate and differentiate into mature macrophages, megakaryocytes and erythrocytes. These cells are considered part of definitive hematopoiesis, since they have the same characteristics as cells arising later on from the differentiation of HSCs [START_REF] Bertrand | Three Pathways to Mature Macrophages in the Early Mouse Yolk Sac[END_REF]. The cells produced by the differentiation of EMPs are mostly transient, but some of them can be found in the adult organism, where some resident macrophages in the adult tissues derive from EMPs [START_REF] Schulz | A Lineage of Myeloid Cells Independent of Myb and Hematopoietic Stem Cells[END_REF][START_REF] Perdiguero | Tissue-Resident Macrophages Originate from Yolk-Sac-Derived Erythro-Myeloid Progenitors[END_REF].

Although EMPs lack any lymphoid potential, it has been shown that lymphocytes are present in the yolk sac during early embryogenesis, anterior to the emergence of HSCs [START_REF] Yoshimoto | Embryonic Day 9 Yolk Sac and Intra-Embryonic Hemogenic Endothelium Independently Generate a B-1 and Marginal Zone Progenitor Lacking B-2 Potential[END_REF][START_REF] Yoshimoto | Autonomous Murine T-Cell Progenitor Production in the Extra-Embryonic Yolk Sac before HSC Emergence[END_REF], and that they come from independent lymphoid progenitors.

c) Third hematopoietic wave, HSC emergence and bone marrow niche

The third and last embryonic wave gives rise to the HSCs. They were first shown to emerge in the aorta-gonad-mesonephros (AGM) region of the mouse embryo, through the process of EHT from hemogenic endothelium in the dorsal aorta [START_REF] Medvinsky | Definitive Hematopoiesis Is Autonomously Initiated by the AGM Region[END_REF][START_REF] Bruijn | Definitive Hematopoietic Stem Cells First Develop within the Major Arterial Regions of the Mouse Embryo[END_REF][START_REF] Tavian | Embryonic Development of the Human Hematopoietic System[END_REF]. After this discovery it was shown that HSCs can arise from hemogenic endothelium in several regions of the embryo, including the yolk sac, the umbilical and vitelline arteries, and the placenta ( [START_REF] Bruijn | Definitive Hematopoietic Stem Cells First Develop within the Major Arterial Regions of the Mouse Embryo[END_REF][START_REF] Gekas | The Placenta Is a Niche for Hematopoietic Stem Cells[END_REF][START_REF] Chen | Runx1 Is Required for the Endothelial to Haematopoietic Cell Transition but Not Thereafter[END_REF].

Then, newly formed hematopoietic cells called pre-HSCs delaminate from the endothelium and enter the circulation to colonize the fetal liver, probably via umbilical vessels. Once in the liver, they rapidly expand and mature to become fully competent (repopulating) HSCs [START_REF] Kieusseian | Immature Hematopoietic Stem Cells Undergo Maturation in the Fetal Liver[END_REF]. They can then yield committed progenitors that in turn give rise to the complete hematopoietic system [START_REF] Mikkola | The Journey of Developing Hematopoietic Stem Cells[END_REF]). Finally, around the time of birth, HSCs colonize the bone marrow, where their proliferation and differentiation properties are tightly controlled by the microenvironment provided by the niche during adult life.

The bone marrow compartment constitutes a very complex microenvironment (reviewed in [START_REF] Morrison | The Bone Marrow Niche for Haematopoietic Stem Cells[END_REF][START_REF] Asada | Complexity of Bone Marrow Hematopoietic Stem Cell Niche[END_REF] to protect HSCs against pathological transformation. In this niche HSCs remain mostly quiescent, with a limited rate of self-renewal activity that ensures continuous production of new hematopoietic cells in normal proportions [START_REF] Akunuru | Aging, Clonality, and Rejuvenation of Hematopoietic Stem Cells[END_REF].

d) A particular case of blood cell formation: the transdifferentiation process

Even though all cells come from the differentiation of HSCs, it has been shown that in vitro, B lymphocytes can be reprogrammed by a process called transdifferentiation (H. [START_REF] Xie | Stepwise Reprogramming of B Cells into Macrophages[END_REF]; during this process committed B cells are induced to become another type of committed cell, macrophages, without passing through a progenitor step. They simultaneously dedifferentiate from their original identity and start to express macrophages markers, meaning that at some point these cells express genes of both lineages [START_REF] Jopling | Dedifferentiation, Transdifferentiation and Reprogramming: Three Routes to Regeneration[END_REF][START_REF] Cieślar-Pobuda | Transdifferentiation and Reprogramming: Overview of the Processes, Their Similarities and Differences[END_REF]. Although rarely observed, this phenomenon does happen in mammals, yet it has been more thoroughly characterized in Drosophila (see section C-2b).

The fine equilibrium between proliferation and differentiation of HSCs can sometimes be unbalanced (notably by the mutation of key molecular actors such as transcription factors of epigenetic regulators), and this deregulation leads to the development of severe pathologies such as leukemia, which I will describe below.

B. Pathological hematopoiesis and its major actors 1. Leukemias: multifaceted malignant blood disorders

There are four major classified types of leukemia, depending on which cell lineage is affected and on the invasiveness of the disease: chronic, acute myeloid or lymphoid leukemia.

Chronic leukemia impacts 1 out of 100 000 persons per year in France, and this condition can persist for several years. In this disease, progenitor cells are not affected but do not produce mature cells able to fight off infections efficiently. With time, immature cells replace normal cells in the bone marrow niche, which is deleterious for the organism, because it completely impairs the ability of the immune system to respond to infection.

Acute leukemia has an incidence of 4 out of 100 000 cases per year in France and has a much poorer prognosis, as it is very invasive. Contrary to chronic leukemia, acute leukemia affects progenitor cells: it is characterized by the induction of immature blood cells and abnormal proliferation that rapidly inhibits production of differentiated cells in the niche.

Classically, leukemias appear as the consequence of somatic mutations accumulated during life. Chromosomal rearrangements can affect many genes, but among them some events are more represented. This is the case for rearrangements affecting the locus encoding the major hematopoietic factor RUNX1/AML1 [START_REF] Miyoshi | T(8;21) Breakpoints on Chromosome 21 in Acute Myeloid Leukemia Are Clustered within a Limited Region of a Single Gene[END_REF], or loci encoding the MOZ and MORF epigenetic enzymes of the MYST family [START_REF] Borrow | The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein[END_REF]. I will describe their functions in both normal and pathological hematopoiesis in the following sections.

The highly conserved family of RUNX transcription factors and its links to leukemia

The RUNX family is composed of Core Binding Factor (CBF) transcription factors strongly conserved across evolution of bilaterian metazoans [START_REF] Rennert | The Evolution of Runx Genes I. A Comparative Study of Sequences from Phylogenetically Diverse Model Organisms[END_REF]. Its founding member, the product of the runt gene, was described for the first time in Drosophila melanogaster in 1980 and was shown later on to be essential for embryonic segmentation [START_REF] Gergen | Dosage requirements for runt in the segmentation of Drosophila embryos[END_REF]. In Drosophila, there are four RUNX genes: runt, RUNXA, RUNXB and lozenge (lz), and in mammals only three: RUNX1, RUNX2, RUNX3 (Figure 3). These transcription factors are involved in many processes like hematopoiesis, neurogenesis and bone development [START_REF] Ito | RUNX Genes in Development and Cancer: Regulation of Viral Gene Expression and the Discovery of RUNX Family Genes[END_REF], as well as in various diseases [START_REF] Chuang | RUNX Family: Regulation and Diversification of Roles through Interacting Proteins[END_REF]C.-C. Sun et al. 2019). They all contain a highly conserved runt domain, which allows both DNA binding on a specific TGYGGTY recognition motif and heterodimerization with their partner CBFb (Adya, Castilla, et Liu 2000;[START_REF] Ito | The RUNX Family: Developmental Regulators in Cancer[END_REF]. Furthermore, they possess a C-terminal VWRPY motif responsible for the interaction with co-repressors, such as the Groucho/TLE family [START_REF] Aronson | Groucho-Dependent and -Independent Repression Activities of Runt Domain Proteins[END_REF][START_REF] Levanon | Transcriptional Repression by AML1 and LEF-1 Is Mediated by the TLE/Groucho Corepressors[END_REF].

In human, RUNX1 was first identified as the target of chromosomal translocations associated to the development of acute myeloid leukemia (AML; [START_REF] Miyoshi | T(8;21) Breakpoints on Chromosome 21 in Acute Myeloid Leukemia Are Clustered within a Limited Region of a Single Gene[END_REF][START_REF] Erickson | Identification of Breakpoints in t (8;21) Acute Myelogenous Leukemia and Isolation of a Fusion Transcript, AMLl IETO, With Similarity to Drosophila Segmentation Gene[END_REF]. It is one of the most frequently mutated loci, as RUNX1 is altered in 26% of AML cases, either by a single point mutation or by chromosomal rearrangements [START_REF] Haferlach | The <em>RUNX1</em> Gene Is Altered in 26% of AML Patients Either By Translocation, Mutation, Gain or Deletion[END_REF]. A RUNX1 mutation in AML is almost always linked with poor prognosis. This association of RUNX1 mutations with the development of severe pathologies is indicative of a prominent role of RUNX1 during normal hematopoiesis (see below). The second mammalian RUNX transcription factor, RUNX2, was identified on the basis of its sequence homology with RUNX1 [START_REF] Kamachi | Purification of a Mouse Nuclear Factor That Binds to Both the A and B Cores of the Polyomavirus Enhancer[END_REF]) and is notably involved in the ossification process [START_REF] Takarada | Genetic Analysis of Runx2 Function during Intramembranous Ossification[END_REF]. Finally, RUNX3 was identified in 1994 [START_REF] Levanon | AML1, AML2, and AML3, the Human Members of the runt domain Gene-Family: cDNA Structure, Expression, and Chromosomal Localization[END_REF]) and has been shown to be a tumor suppressor gene involved in many cancers (notably colorectal cancer; [START_REF] Bae | Tumor Suppressor Activity of RUNX3[END_REF]. Furthermore, it has been shown that in human gastric carcinoma cells, RUNX3 is responsible for the expression of miR-182, which in turn inhibits cancer growth [START_REF] Yu | RUNX3 Inhibits the Proliferation and Metastasis of Gastric Cancer through Regulating MiR-182/HOXA9[END_REF]).

Together, RUNX1 and RUNX3 are involved in breast cancer, where they have a protective effect against epithelial to mesenchyme transition [START_REF] Kulkarni | RUNX1 and RUNX3 Protect against YAP-Mediated EMT, Stem-Ness and Shorter Survival Outcomes in Breast Cancer[END_REF]. RUNX3 not only has roles in pathologies, but is also involved in normal neural development [START_REF] Appel | An Ensemble of Regulatory Elements Controls Runx3 Spatiotemporal Expression in Subsets of Dorsal Root Ganglia Proprioceptive Neurons[END_REF]) and in hematopoiesis, where it cooperates with RUNX1 for the differentiation of several subtypes of T lymphocytes (Woolf et al. 2003, 3;Li et al. 2012, 3). Furthermore, RUNX3 has a role of its own; it modifies the chromatin landscape during Cytotoxic T lymphocyte formation (D. [START_REF] Wang | The Transcription Factor Runx3 Establishes Chromatin Accessibility of Cis-Regulatory Landscapes That Drive Memory Cytotoxic T Lymphocyte Formation[END_REF].

It thus appears that RUNX genes are key regulators of normal and pathological development, and that their regulation is essential to maintain homeostasis of several tissues, including the hematopoietic tissue, where RUNX1 plays a crucial role in the development of AML.

a) RUNX1 in Acute Myeloid Leukemia

• RUNX1-ETO at the onset of AML RUNX1 is the target of several chromosomal rearrangements that lead to the induction of AML, among which the most common is the t(8;21) translocation that fuses RUNX1 with the Eight Twenty-One gene (ETO) [START_REF] Erickson | Identification of Breakpoints in t (8;21) Acute Myelogenous Leukemia and Isolation of a Fusion Transcript, AMLl IETO, With Similarity to Drosophila Segmentation Gene[END_REF][START_REF] Bae | Isolation of PEBP2 Alpha B CDNA Representing the Mouse Homolog of Human Acute Myeloid Leukemia Gene, AML1[END_REF]. Little is known about ETO function, but it is mostly involved in gene repression, since it can recruit several corepressors [START_REF] Salat | ETO, but Not Leukemogenic Fusion Protein AML1/ETO, Augments RBP-J /SHARP-Mediated Repression of Notch Target Genes[END_REF][START_REF] Kitabayashi | The AML1-MTG8 Leukemic Fusion Protein Forms a Complex with a Novel Member of the MTG8(ETO/CDR) Family, MTGR1[END_REF]) through its nervy homology (NRH) domains (Y. [START_REF] Liu | The Tetramer Structure of the Nervy Homology Two Domain, NHR2, Is Critical for AML1/ETO's Activity[END_REF]). This translocation is the most common in de novo AML [START_REF] Grimwade | Refinement of Cytogenetic Classification in Acute Myeloid Leukemia: Determination of Prognostic Significance of Rare Recurring Chromosomal Abnormalities among 5876 Younger Adult Patients Treated in the United Kingdom Medical Research Council Trials[END_REF]) and affects mostly elder people. It provokes an inhibition of wild-type RUNX1 and leads to a block in myeloid differentiation, associated with a high and abnormal self-renewal rate of progenitors [START_REF] Okuda | Expression of a Knocked-In AML1-ETO Leukemia Gene Inhibits the Establishment of Normal Definitive Hematopoiesis and Directly Generates Dysplastic Hematopoietic Progenitors[END_REF], which gives the AML phenotype. It was shown recently that the RUNX1-ETO (also called RUNX1-RUNX1T1) fusion protein interacts with several factors such as Lmo2 and the ETS factor PU.1, to inhibit the expression of differentiation genes and activate the expression of stem cell genes [START_REF] Ptasinska | Identification of a Dynamic Core Transcriptional Network in t(8;21) AML That Regulates Differentiation Block and Self-Renewal[END_REF]X.-J. Sun et al. 2013).

Furthermore, RUNX1-ETO drives changes in the chromatin landscape that modify the epigenome and facilitate leukemic functions [START_REF] Loke | RUNX1-ETO and RUNX1-EVI1 Differentially Reprogram the Chromatin Landscape in t(8;21) and t(3;21) AML[END_REF]). However, it has been proposed that the t(8;21) translocation is not able to trigger leukemogenesis on its own, and that it has to be combined with a secondary mutation [START_REF] Grisolano | An Activated Receptor Tyrosine Kinase, TEL/PDGF R, Cooperates with AML1/ETO to Induce Acute Myeloid Leukemia in Mice[END_REF][START_REF] Kelly | G ENETICS OF M YELOID L EUKEMIAS[END_REF]. In about 20-40 % of cases, a mutation in the c-KIT gene (a crucial factor in HSC development) was concomitantly identified (W. [START_REF] Xie | Acute Myeloid Leukemia with t(8;21)(Q22;Q22.1)/RUNX1-RUNX1T1 and KIT Exon 8 Mutation Is Associated with Characteristic Mastocytosis and Dismal Outcomes[END_REF][START_REF] Crowley | Detection of MOZ-CBP Fusion in Acute Myeloid Leukemia with 8;16 Translocation[END_REF].

• Other leukemogenic mutations of RUNX1

Another translocation affecting RUNX1 is the t(3;21) leading to the RUNX1-EVI1 fusion gene [START_REF] Nucifora | Consistent Intergenic Splicing and Production of Multiple Transcripts between AMLl at 21q22 and Unrelated Genes at 3q26[END_REF][START_REF] Mitani | Generation of the AML1-EVI-1 Fusion Gene in the t(3;21)(Q26;Q22) Causes Blastic Crisis in Chronic Myelocytic Leukemia[END_REF]). This translocation is mostly found as a secondary hit in already established blood pathologies, like chronic myeloid leukemia [START_REF] Nukina | Single-Cell Gene Expression Analysis Reveals Clonal Architecture of Blast-Phase Chronic Myeloid Leukaemia[END_REF]. It leads to the same phenotype as RUNX1-ETO, even if the molecular targets are not the same [START_REF] Loke | RUNX1-ETO and RUNX1-EVI1 Differentially Reprogram the Chromatin Landscape in t(8;21) and t(3;21) AML[END_REF]). In addition to chromosomal rearrangements, several point mutations in the RUNX1 gene were identified, all involved in the initiation of AML [START_REF] Gaidzik | RUNX1 Mutations in Acute Myeloid Leukemia: Results From a Comprehensive Genetic and Clinical Analysis From the AML Study Group[END_REF].

Therefore, RUNX1 appears to be a major target in pathologies affecting hematopoiesis, and its function during the malignant process is extensively studied. But the fact that its deregulation leads to severe disorders, is an indication pointing towards its important role during normal hematopoiesis.

b) RUNX1 during normal hematopoiesis

RUNX1 is expressed in the progenitors of the two definitive embryonic waves (second and third waves), as well as in some primitive macrophages (T. [START_REF] North | Cbfa2 Is Required for the Formation of Intra-Aortic Hematopoietic Clusters[END_REF]) and in adult HSCs [START_REF] Ng | A Runx1 Intronic Enhancer Marks Hemogenic Endothelial Cells and Hematopoietic Stem Cells[END_REF][START_REF] Nottingham | Runx1-Mediated Hematopoietic Stem-Cell Emergence Is Controlled by a Gata/Ets/SCL-Regulated Enhancer[END_REF]). It is a major regulator of both embryonic and adult hematopoiesis; indeed, RUNX1 is crucial for EMP and HSC emergence during EHT [START_REF] Chen | Runx1 Is Required for the Endothelial to Haematopoietic Cell Transition but Not Thereafter[END_REF], 1; T. E. [START_REF] North | Runx1 Expression Marks Long-Term Repopulating Hematopoietic Stem Cells in the Midgestation Mouse Embryo[END_REF][START_REF] Yzaguirre | Runx1 Is Sufficient for Blood Cell Formation from Non-Hemogenic Endothelial Cells in Vivo Only during Early Embryogenesis[END_REF]Liakhovitskaia et al. 2014, 41). RUNX1 mutant mouse embryos are very pale and lack any fetal liver hematopoiesis (Okuda et al. 1996, 1), phenotypes that lead to early lethality due to severe hemorrhages. This evidence underlines its importance during the establishment of hematopoietic cells, and in particular for HSC development and fetal liver colonization. Consistently, primitive hematopoiesis of the first embryonic wave does not seem to be affected in the RUNX1 mutant, showing that this wave is RUNX1 independent. It is not clear in the literature if RUNX1 is involved in HSC maintenance, but several studies show that it is expressed in the bone marrow compartment [START_REF] Ng | A Runx1 Intronic Enhancer Marks Hemogenic Endothelial Cells and Hematopoietic Stem Cells[END_REF][START_REF] Nottingham | Runx1-Mediated Hematopoietic Stem-Cell Emergence Is Controlled by a Gata/Ets/SCL-Regulated Enhancer[END_REF]). Finally, it is known that during adult hematopoiesis, RUNX1 is involved in myeloid differentiation and in particular for megakaryocytes formation [START_REF] Draper | RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis ». Édité par H. Leighton Grimes[END_REF]. RUNX1 is also responsible for B and T lymphoid development [START_REF] Chi | Loss of Runx1 Function Results in B Cell Immunodeficiency but Not T Cell in Adult Zebrafish[END_REF]Woolf et al. 2003, 3;Li et al. 2012, 3;Taniuchi et al. 2002).

RUNX1 expression and function are regulated and mediated by several factors that play a role upstream, downstream, or in collaboration with RUNX1, in order to generate every blood cell not only in the embryo but also in the adult. Here I describe RUNX1's main partners.

c) RUNX1 partners and regulators during normal hematopoiesis

• Notch is a major regulator of RUNX1 expression

The Notch pathway is crucial during development and can be involved in several processes, like proliferation, asymmetrical cell division (and thus differentiation), or even cell death. Notch is a membrane receptor composed of two parts: an intracellular domain (NICD) responsible for the transcriptional activity of the pathway and an extracellular domain (NECD) responsible for the interaction with its ligands Delta/Serrate/Jagged (DSL). Once the ligand/receptor interaction occurs, the Notch receptor is cleaved multiple times, a first time (S2 cleavage) by the ADAM metalloproteinase and a second time (S3 cleavage) by the gsecretase complex. This separates NECD from NICD, and the released intracellular part will then relocalize to the nucleus. There, it interacts with a complex composed of the Notch pathway transcription effector called CSL (CBF1/RBPJ, Supressor of Hairless(Su(H)), Lag-1), Mastermind (Mam) and other coactivators to regulate their target genes (Figure 4; reviewed in Bray 2006; [START_REF] Kovall | The Canonical Notch Signaling Pathway: Structural and Biochemical Insights into Shape, Sugar, and Force[END_REF]. In mammals, there are four Notch receptors: Notch1-4.

As described above, HSCs arise from the hemogenic endothelium of the major embryonic arteries, and in particular in the dorsal aorta. Its hemogenic potential is controlled and maintained by Notch signaling from the interaction between the ligand Jag1 and the receptor Notch1 (Kumano et al. 2003, 2;À. Robert-Moreno et al. 2008, 1;[START_REF] Clarke | The expression of Sox17 identifies and regulates haemogenic endothelium[END_REF]).

Notch1 directly regulates the expression of GATA2, a marker of hematopoietic cells within the hemogenic endothelium required for HSC development [START_REF] Robert-Moreno | RBPj -Dependent Notch Function Regulates Gata2 and Is Essential for the Formation of Intra-Embryonic Hematopoietic Cells[END_REF]de Pater et al. 2013, 2), and is at the origin of the onset of RUNX1 expression in hematopoietic cells [START_REF] Nakagawa | AML1/Runx1 Rescues Notch1-Null Mutation-Induced Deficiency of Para-Aortic Splanchnopleural Hematopoiesis[END_REF]. Then, Notch expression is downregulated in the cells undergoing EHT [START_REF] Richard | Endothelio-Mesenchymal Interaction Controls Runx1 Expression and Modulates the Notch Pathway to Initiate Aortic Hematopoiesis[END_REF]). Although Notch1 is required during the process of HSC formation, it has been shown that RUNX1 allows the emergence of EMPs from the yolk sac hemogenic endothelium independently of Notch1 [START_REF] Hadland | A Requirement for Notch1 Distinguishes 2 Phases of Definitive Hematopoiesis during Development[END_REF][START_REF] Bertrand | Notch Signaling Distinguishes 2 Waves of Definitive Hematopoiesis in the Zebrafish Embryo[END_REF]).

• GATA transcription factors are known RUNX interactors

The GATA family is composed of transcription factors conserved across evolution. They are characterized by the presence of one or two zinc finger domains that allow DNA binding

A B

or interaction with their cofactors Friend Of GATA (FOG). In mammals there are six GATA factors (GATA1-6), but only three of them (GATA1-3) are required during the hematopoietic process. GATA1 is a major actor of the hematopoietic development, as it is required for erythrocyte formation (described above, Fujiwara et al. 1996, 1) and megakaryocyte differentiation [START_REF] Chang | GATA-Factor Dependence of the Multitype Zinc-Finger Protein FOG-1 for Its Essential Role in Megakaryopoiesis[END_REF]Iwasaki et al. 2003, 1). GATA2 is absolutely required for HSC emergence and survival in the embryo ( de Pater et al. 2013, 2;Ling et al. 2004, 2;[START_REF] Tsai | An Early Haematopoietic Defect in Mice Lacking the Transcription Factor GATA-2[END_REF]. Initially, GATA3 was only shown to be required for the differentiation of T-helper-type-2 cells (G. R. [START_REF] Lee | Regulation of IL-4 Gene Expression by Distal Regulatory Elements and GATA-3 at the Chromatin Level[END_REF]Ho, Tai, et Pai 2009, 3). However, it has been shown that GATA3 is expressed in lymphoid progenitors, where it is crucial for the formation of the T cell lineage [START_REF] Rothenberg | GATA-3 Locks the Door to the B-Cell Option[END_REF], and some recent findings suggested that it also plays a role during HSC emergence in the embryo [START_REF] Zaidan | The Multi-Faceted Role of Gata3 in Developmental Haematopoiesis[END_REF].

RUNX1 is required for megakaryocyte differentiation during definitive myelopoiesis [START_REF] Draper | RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis ». Édité par H. Leighton Grimes[END_REF], and it has been shown that GATA1 cooperates with RUNX1 in this process [START_REF] Elagib | RUNX1 and GATA-1 Coexpression and Cooperation in Megakaryocytic Differentiation[END_REF][START_REF] Goldfarb | Megakaryocytic Programming by a Transcriptional Regulatory Loop: A Circle Connecting RUNX1, GATA-1, and P-TEFb[END_REF][START_REF] Pencovich | Dynamic Combinatorial Interactions of RUNX1 and Cooperating Partners Regulates Megakaryocytic Differentiation in Cell Line Models[END_REF][START_REF] Tijssen | Genome-Wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators[END_REF]. It is interesting to note that Elagib et al show that the fusion protein RUNX1-ETO inhibits GATA1 during megakaryocytic differentiation in cell culture, which they propose to be a mechanism involved in the blocking of myeloid differentiation during AML development.

Furthermore, RUNX1 and GATA2 are both present in HSCs in the bone marrow but have never been shown to interact with each other. However, they cooperate during HSC emergence in the embryo [START_REF] Wilson | Combinatorial Transcriptional Control In Blood Stem/Progenitor Cells: Genome-Wide Analysis of Ten Major Transcriptional Regulators[END_REF], and it has recently been shown that they are involved in a complex regulating the expression of the SET gene (which is a potent Inhibitor of Protein Phosphatase 2A) in two AML cell lines (HL-60 and HEL cells; [START_REF] Pippa | MYC-Dependent Recruitment of RUNX1 and GATA2 on the SET Oncogene Promoter Enhances PP2A Inactivation in Acute Myeloid Leukemia[END_REF]).

• CBFb, RUNX heterodimerization partner CBFb, a RUNX1 heterodimerization partner seems to be involved during the hematopoietic process, because a loss of function of this gene in the mouse embryo leads to the same phenotypes as a RUNX1 mutant [START_REF] Niki | Hematopoiesis in the Fetal Liver Is Impaired by Targeted Mutagenesis of a Gene Encoding a Non-DNA Binding Subunit of the Transcription Factor, Polyomavirus Enhancer Binding Protein 2/Core Binding Factor[END_REF]. It is likely that all the roles played by RUNX factors during hematopoiesis are dependent on CBFb. Indeed, its loss of function specifically in the adult, has broader phenotypes than the ones of RUNX1 alone, indicating that CBFb interacts with other partners, which may be RUNX factors during this process.

• Monocytic Leukemia Zinc-Finger protein (MOZ)

MOZ is a Lysine Acetyltransferase (KAT) involved during hematopoiesis at several levels, that I will develop later in more detail. It was shown that in cultured myeloid mouse cells, MOZ was part of the RUNX1 complex and required for its transcriptional activity, therefore participating in myeloid differentiation (I. Kitabayashi 2001, 1;Yoshida et Kitabayashi 2008, 1).

The authors propose that MOZ acts independently of its catalytic activity in this process, but this hypothesis has never been developed further. The epigenetic regulator MOZ and its paralog MOZ-Related Factor (MORF) are themselves common targets of chromosomal rearrangements leading to the induction of AML, and I will describe their role in normal and pathological hematopoiesis in the next part.

MOZ/MORF and the MYST family of Lysine Acetyl-Transferases

The MYST family is named after its founding members: MOZ, Yfb2, Sas2 and Tip60, and is a very conserved family from yeast to human (Figure 5). In mammals there are five members, MOZ/MORF, Tip60, Male absent of the First (MOF) and Human acetylase Binding to ORC1 (HBO1, reviewed in [START_REF] Yang | The Diverse Superfamily of Lysine Acetyltransferases and Their Roles in Leukemia and Other Diseases[END_REF]X.-J. Sun et al. 2015). KATs of the MYST family were first shown to acetylate histone tails, and by doing so, to participate in the remodeling of the chromatin landscape [START_REF] Grant | A Tale of Histone Modifications[END_REF][START_REF] Goll | Histone Modification and Replacement in Chromatin Activation[END_REF]. However, studies over these last years yielded increasing evidence that they have a much broader range of substrates than just histones (reviewed in [START_REF] Sapountzi | MYST-Family Histone Acetyltransferases: Beyond Chromatin[END_REF]. KATs of the MYST family are involved in a variety of pathologies, but I will focus here on the role of MOZ and its paralog MORF in hematopoietic disorders. The name MOZ comes from Monocytic leukemia Zinc-finger protein, because it was first identified as a target of chromosomal translocations responsible for the development of AML.

These rearrangements involve in particular the CREB-binding Protein (CBP) [START_REF] Borrow | The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein[END_REF][START_REF] Chaffanet | MOZ Is Fused to P300 in an Acute Monocytic[END_REF][START_REF] Crowley | Detection of MOZ-CBP Fusion in Acute Myeloid Leukemia with 8;16 Translocation[END_REF]) and TIF-2 (inv(8)(p11q13); Figure 5; [START_REF] Carapeti | Consistent Fusion of MOZ and TIF2 in AML with Inv(8)(P11q13)[END_REF]. MOZ protein contains several important domains that have been characterized and linked to specific functions. The MYST domain bears the acetylation catalytic activity and contains a C2HC zinc-finger involved in DNA binding (Holbert et al. 2007). This conserved MYST domain is also required for the interaction with MOZ partners Bromodomain-PHD finger proteins (BRPF) 1 2 and 3 [START_REF] Ullah | Molecular Architecture of Quartet MOZ/MORF Histone Acetyltransferase Complexes[END_REF]. The C-terminal domain of MOZ is called SM for Serine/Methionine-rich domain and is involved in transcription activation. This domain is also involved in the interaction with p53 (Susumu [START_REF] Rokudai | Monocytic Leukemia Zinc Finger (MOZ) Interacts with P53 to Induce P21 Expression and Cell-Cycle Arrest[END_REF][START_REF] Rokudai | MOZ Increases P53 Acetylation and Premature Senescence through Its Complex Formation with PML[END_REF][START_REF] Tham | Dominant Mutations in KAT6A Cause Intellectual Disability with Recognizable Syndromic Features[END_REF], which is a transcription factor involved in cell cycle regulation, autophagy and apoptosis [START_REF] Sabapathy | Understanding P53 Functions through P53 Antibodies ». Édité par Chandra S Verma[END_REF]. Finally the N-terminal part of MOZ, is composed of the NEMM domain (for N-terminal of Enok MOZ/MORF; these proteins are the only members of the MYST family that contain this N-terminal part) and a double PHD (Plant Homeodomain) finger, which contains a recognition domain for histones and is involved in transcription inhibition (Dreveny et al. 2014;[START_REF] Xiong | Selective Recognition of Histone Crotonylation by Double PHD Fingers of MOZ and DPF2[END_REF].

MOZ and CBP are both important factors for HSC maintenance and quiescence [START_REF] Katsumoto | MOZ Is Essential for Maintenance of Hematopoietic Stem Cells[END_REF][START_REF] Thomas | Monocytic Leukemia Zinc Finger Protein Is Essential for the Development of Long-Term Reconstituting Hematopoietic Stem Cells[END_REF][START_REF] Rebel | Distinct Roles for CREB-Binding Protein and P300 in Hematopoietic Stem Cell Self-Renewal[END_REF][START_REF] Sheikh | MOZ and BMI1 Act Synergistically to Maintain Hematopoietic Stem Cells[END_REF], and TIF2

(Transcriptional Intermediary Factor 2) is a direct partner of CBP [START_REF] Demarest | Mutual Synergistic Folding in Recruitment of CBP/P300 by P160 Nuclear Receptor Coactivators[END_REF].

The leukemogenic MOZ-CBP fusion protein contains the N-terminal part of MOZ with its intact MYST domain, and almost the entire CBP protein [START_REF] Chan | MOZ and MOZ-CBP Cooperate with NF-ΚB to Activate Transcription from NF-ΚB-Dependent Promoters[END_REF]). As a result, the recognition repertoire of CBP is aberrantly acetylated by MOZ and leads to the proliferation of leukemic progenitors and to the AML phenotype. Similarly, MOZ-TIF2 has been shown to recruit CBP via the CID interaction domain of TIF-2, which results in CBP inhibition and leads to leukemic development [START_REF] Deguchi | MOZ-TIF2-Induced Acute Myeloid Leukemia Requires the MOZ Nucleosome Binding Motif and TIF2-Mediated Recruitment of CBP[END_REF][START_REF] Kindle | MOZ-TIF2 Inhibits Transcription by Nuclear Receptors and P53 by Impairment of CBP Function[END_REF]. It also has been shown that MOZ-TIF2 represses senescence in AML stem cells and thus leads to their expansion [START_REF] Largeot | Expression of the MOZ-TIF2 Oncoprotein in Mice Represses Senescence[END_REF]. It is noteworthy that MORF, a paralog of MOZ, is also involved in a chromosomal translocation t(10;16) with CBP, associated to the development of AML [START_REF] Panagopoulos | Fusion of the MORF and CBP Genes in Acute Myeloid Leukemia with the t(10;16)(Q22;P13)[END_REF], suggesting that MORF could play a role during normal blood cell formation.

In addition to its involvement in leukemia, MOZ has also been linked to the development of other pathologies, such as intellectual disability [START_REF] Tham | Dominant Mutations in KAT6A Cause Intellectual Disability with Recognizable Syndromic Features[END_REF] and esophageal adenocarcinoma [START_REF] Dulak | Exome and Whole-Genome Sequencing of Esophageal Adenocarcinoma Identifies Recurrent Driver Events and Mutational Complexity[END_REF], and its transcriptional deregulation leads to metastasis of medulloblastoma and colorectal cancer [START_REF] Wu | Clonal Selection Drives Genetic Divergence of Metastatic Medulloblastoma[END_REF][START_REF] Mohammadi | Investigation of the Changes in the Expression Levels of MOZ Gene in Colorectal Cancer Tissues[END_REF]. It is interesting to note that in a recent study, Baell et al identified a new inhibitor of MOZ that is sufficient to stop cancer growth in a model of mouse lymphoma, by inducing senescence in cancer cells [START_REF] Baell | Inhibitors of Histone Acetyltransferases KAT6A/B Induce Senescence and Arrest Tumour Growth[END_REF].

As for RUNX1, MOZ is an important target of leukemogenic mutations and rearrangements, which prompted many research groups to study its role during normal hematopoietic development. In addition to its function in HSCs, MOZ is required for myeloid lineage and lymphoid Bcells formation: it is a coactivator of RUNX1, in particular for MIP-1a expression, and is required for normal macrophage and B-cell development [START_REF] Jiang | MOZ Forms an Autoregulatory Feedback Loop with MiR-223 in AML and Monocyte/Macrophage Development[END_REF][START_REF] Katsumoto | MOZ Is Essential for Maintenance of Hematopoietic Stem Cells[END_REF]Good-Jacobson et al. 2014 ).

As I previously mentioned, MOZ acts as a transcriptional coactivator of RUNX1, in a KATindependent manner (I. [START_REF] Kitabayashi | Activation of AML1-Mediated Transcription by MOZ and Inhibition by the MOZ-CBP Fusion Protein[END_REF]. There are consistent differences between the phenotypes of mice completely deprived of any MOZ activity [START_REF] Katsumoto | MOZ Is Essential for Maintenance of Hematopoietic Stem Cells[END_REF] proper modification of the chromatin landscape. BRPF1 has been shown to recognize in particular H3K14ac, therefore directing MOZ to its substrates [START_REF] Poplawski | Molecular Insights into the Recognition of N-Terminal Histone Modifications by the BRPF1 Bromodomain[END_REF]). In addition, BRPF1 shares common functions with MOZ; indeed, BRPF1 participates in the expression of HOX genes during development in zebrafish [START_REF] Hibiya | Brpf1, a Subunit of the MOZ Histone Acetyl Transferase Complex, Maintains Expression of Anterior and Posterior Hox Genes for Proper Patterning of Craniofacial and Caudal Skeletons[END_REF], and it is also essential for the normal establishment of fetal liver HSC [START_REF] You | BRPF1 Is Essential for Development of Fetal Hematopoietic Stem Cells[END_REF]. ING5 is a Tip60 cofactor (N. [START_REF] Liu | ING5 Is a Tip60 Cofactor That Acetylates P53 in Response to DNA Damage[END_REF] and is important for cell proliferation in human cultured cells [START_REF] Linzen | ING5 Is Phosphorylated by CDK2 and Controls Cell Proliferation Independently of P53[END_REF]. Furthermore, it has been shown to recognize histone H3 modification H3K4me3 via its PHD domains (K. S. Champagne et al. 2008, 3;Ormaza et al. 2019, 4). The function of EAF6 has not been described so far.

In summary, the ING5 complex provides an environment that enables a specific recruitment of MOZ on its target repertoire, allowing MOZ to specifically acetylate its substrates [START_REF] Ali | Tandem PHD Fingers of MORF/MOZ Acetyltransferases Display Selectivity for Acetylated Histone H3 and Are Required for the Association with Chromatin[END_REF].

Mammalian hematopoiesis is a very complex system. Indeed, the need to produce numerous cell types in physiological proportions requires a high level of regulation and therefore a lot of molecular interplay. Moreover, the classical model for hematopoiesis in mammals is the mouse model, which is complicated to manipulate. Mice possess 20 chromosomes and achieve puberty at 2 months of age, which makes it a poor genetic model.

However, the hematopoietic process is strongly conserved in vertebrates, both at the developmental and genetic levels. All the molecular factors described above (Notch, RUNX, GATA, MOZ…) are highly conserved across evolution, and in addition to vertebrate models, Drosophila melanogaster has proven these last fifteen years to be a very powerful model to study the molecular mechanisms controlling the development of innate immunity.

C. Drosophila as a model to study hematopoiesis 1. Generalities on the model

Drosophila, which means "who likes dew", also called the fruit fly, is an animal model used in many research fields, and in particular in genetic and developmental studies. In labs, the subfamily melanogaster ("black belly") is widely used.

Thomas Hunt Morgan, a zoologist, was the first to use this model in his lab (later called the Fly Room) in 1910 and received the Nobel Prize for his discovery concerning the role of chromosomes during the process of heredity. He and his group identified many genes, which are still used today as genetic markers for experiments, like the white gene that is responsible for the red color of the eye.

This animal model is particularly easy to use in genetic studies, because it only has four pairs of chromosomes, three pairs of autosomes and one pair of sexual chromosomes. In addition, meiotic recombination does not occur in males, which is of particular interest when doing genetic experiments. Moreover, Drosophila generation time is quite short, around 10 days at 25°C, which makes it easy to design in vivo experiments.

Finally, it is a highly relevant model concerning medical research, because it has been

shown that 75 % of disease-related genes have a sequence similarity in the fly, and that 50 % of fly proteins have mammalian homologs [START_REF] Reiter | A Systematic Analysis of Human Disease-Associated Gene Sequences In Drosophila Melanogaster[END_REF].

The fruit fly has a life cycle with four stages: embryo, larva, pupa and adult (Figure 8A).

The embryonic stage lasts for twenty-four hours, ending with the emergence of the larva. This larval stage is itself composed of three stages called L1, L2 and L3 and lasts for five days. Then, the larva produces an ecdysone (a steroid hormone) signal that leads to a "wandering" behavior and finally to pupariation. The pupal stage also lasts for five days, and during this stage the individual undergoes the metamorphosis process, in which tissues are remodeled and give rise to the adult morphology. Finally, the adult fly emerges from the pupa and begins to mate eight to twelve hours thereafter.

Many developmental processes are conserved throughout evolution like neurogenesis, organ formation, stem cell biology, and of course hematopoiesis. Drosophila is an ideal model to study these processes and many discoveries in Drosophila participated in our understanding of mammalian (and particularly human) development. Here, I will describe the process of hematopoiesis in flies and how it can be used as a model for this process in normal and pathological situations.

Drosophila hematopoiesis

Drosophila is an organism that possesses an open circulatory system in which circulates a liquid called the hemolymph. In larvae, the dorsal vessel (or aorta), which is positioned dorsally in the antero-posterior axis, ends with the "heart" in the posterior part. This heart beats to generate a flow of hemolymph that circulates in all the tissues and enables blood cells to migrate. In Drosophila, blood cells share functional similarities with the mammalian myeloid lineage. In 1957, Rizki characterized three cell types: the plasmatocytes, the crystal cells and the lamellocytes (Figure 7; M. T. M. Rizki 1957) which are called hemocytes.

a) Drosophila blood cell types

• Plasmatocytes

Plasmatocytes are the most abundant cells, as they represent about 95 % of all blood cells in Drosophila. They are phagocytes with functional homology to macrophages. They are crucial players of innate immunity in Drosophila, since they are responsible for the elimination of bacteria [START_REF] Charroux | Elimination of Plasmatocytes by Targeted Apoptosis Reveals Their Role in Multiple Aspects of the Drosophila Immune Response[END_REF][START_REF] Defaye | Genetic Ablation of Drosophila Phagocytes Reveals Their Contribution to Both Development and Resistance to Bacterial Infection[END_REF][START_REF] Nehme | Relative Roles of the Cellular and Humoral Responses in the Drosophila Host Defense against Three Gram-Positive Bacterial Infections ». Édité par Nick Gay[END_REF] or virally-infected cells [START_REF] Nainu | Protection of Insects against Viral Infection by Apoptosis-Dependent Phagocytosis[END_REF]. Plasmatocytes are also partly responsible for the production of antimicrobial peptides after bacterial infection [START_REF] Irving | New Insights into Drosophila Larval Haemocyte Functions through Genome-Wide Analysis[END_REF].

Moreover, they play an essential role during development, as they phagocytose apoptotic bodies both in the embryo and in the pupa, particularly after tissue remodeling during metamorphosis [START_REF] Defaye | Genetic Ablation of Drosophila Phagocytes Reveals Their Contribution to Both Development and Resistance to Bacterial Infection[END_REF][START_REF] Wood | Macrophage Functions in Tissue Patterning and Disease: New Insights from the Fly[END_REF][START_REF] Lanot | Postembryonic Hematopoiesis in Drosophila[END_REF][START_REF] Regan | Steroid Hormone Signaling Is Essential to Regulate Innate Immune Cells and Fight Bacterial Infection in Drosophila ». Édité par David S. Schneider[END_REF].

Furthermore, plasmatocytes are responsible for the secretion of extra-cellular matrix during development, in particular around the ventral nervous system and Malpighian tubules [START_REF] Olofsson | Condensation of the Central Nervous System in Embryonic Drosophila Is Inhibited by Blocking Hemocyte Migration or Neural Activity[END_REF][START_REF] Bunt | Hemocyte-Secreted Type IV Collagen Enhances BMP Signaling to Guide Renal Tubule Morphogenesis in Drosophila[END_REF]).

• Crystal cells

Crystal cells represent around 5 % of the total circulating cells. They were first identified because of the crystalline inclusions they have in their cytoplasm (M. T. M. Rizki 1957); these inclusions are in fact accumulation of prophenol-oxydases (PPOs) involved in the arthropodspecific melanization reaction [START_REF] Dudzic | Drosophila Innate Immunity: Regional and Functional Specialization of Prophenoloxidases[END_REF]. Melanization reactions are responsible for wound healing by promoting coagulation; this makes crystal cells the functional homologs of mammalian megakaryocytes. These PPOs are processed into active phenoloxydases by a proteolytic cascade [START_REF] Nam | Genetic Evidence of a Redox-Dependent Systemic Wound Response via Hayan Protease-Phenoloxidase System in Drosophila: Redox-Modulated Systemic Wound Response[END_REF], when they are released in circulation after crystal cell rupture [START_REF] Bidla | Crystal Cell Rupture after Injury in Drosophila Requires the JNK Pathway, Small GTPases and the TNF Homolog Eiger[END_REF]. This activation has also been shown to contribute to survival after bacterial infection [START_REF] Binggeli | Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila ». Édité par David S. Schneider[END_REF]). I will describe later, in more detail, how crystal cells are formed, in particular during the larval stage.

• Lamellocytes

Lamellocytes are the third and last blood cell type in Drosophila. They are large cells produced specifically when parasitoid wasps lay eggs inside the larval cuticle (M. T. M. Rizki 1957; T. M. Rizki et Rizki 1992;[START_REF] Crozatier | Cellular Immune Response to Parasitization in Drosophila Requires the EBF Orthologue Collier ». Édité par Michael Levine[END_REF]). Lamellocytes can be formed in circulation by the transdifferentiation of plasmatocytes [START_REF] Avet-Rochex | An in Vivo RNA Interference Screen Identifies Gene Networks Controlling Drosophila Melanogaster Blood Cell Homeostasis[END_REF][START_REF] Stofanko | Lineage Tracing of Lamellocytes Demonstrates Drosophila Macrophage Plasticity ». Édité par Francois Schweisguth[END_REF][START_REF] Anderl | Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila Melanogaster Larvae after Wasp Infection ». Édité par David S. Schneider[END_REF], and also directly from progenitors in a specialized organ called the lymph gland [START_REF] Oyallon | Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis ». Édité par Kenneth Söderhäll[END_REF]. Their function is to encapsulate objects too big to be phagocytized. It has been shown that in a wasp infestation condition, lamellocytes arise from the pool of intermediate progenitors in the lymph gland, and that this process occurs at the expense of crystal cell formation, suggesting that the choice between the two cell types is mutually exclusive [START_REF] Krzemien | Hematopoietic Progenitors and Hemocyte Lineages in the Drosophila Lymph Gland[END_REF]. Although lamellocytes are produced in response to an immune stress, they can also be generated both in the lymph gland and in circulation, in some genetic conditions (Avet-Rochex et al. 2010).

b) Drosophila blood cell formation

As for mammals, blood cells in Drosophila are formed during successive waves of hematopoiesis: an embryonic wave that gives rise to all circulating hemocytes in the larva, and a larval wave that generates the pool of hemocytes required during metamorphosis and later on, during adult life.

c) Embryonic wave of hematopoiesis

Blood cells in the embryo first originate from the procephalic mesoderm as bipotent progenitors (they can differentiate into both plasmatocytes and crystal cells; [START_REF] Tepass | Embryonic Origin of Hemocytes and Their Relationship to Cell Death in Drosophila[END_REF][START_REF] Holz | The Two Origins of Hemocytes in Drosophila[END_REF]). These progenitors called prohemocytes, emerge, and differentiate into the two embryonic blood cell types. Plasmatocytes then migrate throughout the embryo [START_REF] Tepass | Embryonic Origin of Hemocytes and Their Relationship to Cell Death in Drosophila[END_REF][START_REF] Wood | Distinct Mechanisms Regulate Hemocyte Chemotaxis during Development and Wound Healing in Drosophila Melanogaster[END_REF], while crystal cell lineages stay in a cluster next to the proventriculus [START_REF] Lebestky | Specification of Drosophila Hematopoietic Lineage by Conserved Transcription Factors[END_REF]Bataillé et al. 2005). At the end of embryogenesis, about 700 plasmatocytes and 30 crystal cells are generated. It is important to note that since lamellocytes are produced after an immune stress like parasitism, they only appear at the larval stage and are completely absent from the embryo (Figure 8A).

Hemocytes formed after this first hematopoietic wave during embryogenesis will persist in all stages of life. Indeed, during the larval stages, plasmatocytes will aggregate in stereotyped clusters along the cuticle that will become new hematopoietic sites called hematopoietic pockets (HPs; Figure 8B). These HPs provide a microenvironment in which plasmatocytes proliferate (K. (Leitão et Sucena 2015) or lamellocytes [START_REF] Honti | In Vivo Detection of Lamellocytes in Drosophila Melanogaster[END_REF]. It also has been shown recently that plasmatocytes located near pericardial cells of the dorsal vessel, are also able to transdifferentiate into crystal cells, seemingly after Notch induction [START_REF] Cevik | Pericardin, a Drosophila Collagen, Facilitates Accumulation of Hemocytes at the Heart[END_REF]. All the cells produced during this wave subsequently persist until adulthood.

d) Larval wave of hematopoiesis

The second wave of hematopoiesis, which takes place during the larval stage in a specialized organ called the lymph gland (Figure 8C; reviewed in Banerjee et al. 2019), has its origins in the embryo. Indeed, lymph gland progenitors emerge in the dorsal mesoderm and then migrate along the dorsal vessel. It has been shown that these precursors have a common origin with endothelial cells, both arising from hemangioblastic cells [START_REF] Mandal | Evidence for a Fruit Fly Hemangioblast and Similarities between Lymph-Gland Hematopoiesis in Fruit Fly and Mammal Aorta-Gonadal-Mesonephros Mesoderm[END_REF]; this is a common feature with mammals, where hemogenic endothelium generates all blood precursors during embryonic development.

The lymph gland is a regionalized organ, with two primary lobes apposed on the cardiac tube and multiple pairs of secondary lobes along the dorsal vessel (reviewed in Letourneau et al. 2016). Primary lobes are themselves regionalized in two regions: one medullary zone in which progenitors proliferate during the first larval stages and where they remain in a mostly quiescent state in later stages, and one cortical zone where differentiated cells (plasmatocytes and crystal cells) accumulate. One should note that a third zone is visible, the intermediary zone, where differentiating progenitors are found [START_REF] Oyallon | Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis ». Édité par Kenneth Söderhäll[END_REF]. Another compartment of interest, the Posterior Signaling Center, defines a microenvironment responsible for massive lamellocyte production after an immune stress [START_REF] Benmimoun | The EBF Transcription Factor Collier Directly Promotes Drosophila Blood Cell Progenitor Maintenance Independently of the Niche[END_REF][START_REF] Oyallon | Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis ». Édité par Kenneth Söderhäll[END_REF]. In this case, the lymph gland is prematurely disrupted and releases all its content into circulation, in order to encapsulate the wasp egg and prevent its development, which would otherwise be lethal for the fly.

In the absence of parasitism, at the end of the L3 stage, the lymph gland contains only differentiated cells, which are released in the hemolymph at the onset of pupariation. so far as playing a role during hematopoiesis [START_REF] Rehorn | A Molecular Aspect of Hematopoiesis and Endoderm Development Common to Vertebrates and Drosophila[END_REF]Lucas Waltzer et al. 2010;[START_REF] Minakhina | JAK/STAT and the GATA Factor Pannier Control Hemocyte Maturation and Differentiation in Drosophila[END_REF].

Srp is absolutely required for prohemocyte emergence, as Srp mutant embryos are completely deprived of blood cells [START_REF] Rehorn | A Molecular Aspect of Hematopoiesis and Endoderm Development Common to Vertebrates and Drosophila[END_REF]. At the cellularization stage, srp is expressed in the procephalic mesoderm, in the prohemocyte cluster [START_REF] Spahn | Multiple Regulatory Safeguards Confine the Expression of the GATA Factor Serpent to the Hemocyte Primordium within the Drosophila Mesoderm[END_REF]. Later in embryonic and larval development, srp expression is maintained in every blood cell lineage, which makes it a "panhemocyte" marker. Its interaction with its cofactor FOG U-shaped (Ush) is required for plasmatocyte differentiation, and its interaction with the RUNX factor Lz is required for crystal cell differentiation [START_REF] Fossett | Combinatorial Interactions of Serpent, Lozenge, and U-Shaped Regulate Crystal Cell Lineage Commitment during Drosophila Hematopoiesis[END_REF][START_REF] Waltzer | Two Isoforms of Serpent Containing Either One or Two GATA Zinc Fingers Have Different Roles in Drosophila Haematopoiesis[END_REF]2003a;Bataillé et al. 2005).

Srp has also been shown to be involved during lymph gland hematopoiesis. It has been proposed that the interaction with its cofactor Ush is required for the normal expression of Ecadherin in the progenitor compartment to maintain their undifferentiated state.

In summary, Srp seems to share functional similarities with GATA1 and 2 in mammals, as it is required both for the emergence of prohemocytes and later on for their proper differentiation.

b) RUNX transcription factors

In Drosophila, the RUNX ortholog Lozenge (Lz) is a crucial transcription factor during the hematopoietic process. I will, in this part, give more details concerning its history and functions during Drosophila development. Lz was first identified by the group of Thomas

Morgan in the early twentieth century [START_REF] Morgan | The genetics of Drosophila melanogaster[END_REF], and its name comes from the eye phenotype displayed by mutant flies.

Lz was initially cloned by the group of Uptal Banerjee, who showed its involvement in cell fate choice in the eye imaginal disc (Daga et al. 1996). In this study, the authors generated a series of mutant alleles, and one of them, the lz R1 allele, is the one I used in experiments I will describe in Results. Lz was linked to other processes like fertility [START_REF] Anderson | A STUDY OF THE FACTORS AFFECTING FERTILITY OF LOZENGE FEMALES OF DROSOPHILA MELANOGASTER[END_REF], as well as in the regulation of phenol-oxydase activity [START_REF] Peeples | Activity of phenol oxidases at the puparium formation stage in development of nineteen lozenge mutants of Drosophila melanogaster[END_REF][START_REF] Warner | Phenol oxidase activity and the lozenge locus of Drosophila melanogaster[END_REF]. Indeed, Lz was shown to be a suppressor of the black cells phenotype, which is caused by a mutation in the PPO1 gene (T. M. [START_REF] Rizki | Alleles of lz as suppressors of the Bc-phene in Drosophila melanogaster[END_REF]. PPOs are produced by crystal cells during embryonic and larval hematopoiesis, and it has been demonstrated that Lz is a crucial transcription factor controlling crystal cell formation (L. Waltzer 2003a).

Similarly to RUNX1, Lz can interact with several factors during the hematopoietic process.

Moreover, since RUNX1 expression is initiated in Notch activated cells in the AGM, lz expression in larval crystal cells depends on Notch signaling, and I will describe below these interactions.

• Interaction between Lz and Srp is crucial for crystal cell differentiation

In the embryo, it has been shown that Lz physically interacts with the GATA factor Srp to promote crystal cell differentiation and transcription of target genes such as PPO1 and 2 (L.

Waltzer 2003a; Ferjoux et al. 2007). This interaction is required to produce crystal cells, but it is also sufficient to induce PPO transcription ectopically in the embryo.

• CBFb factors in Drosophila, Brother (Bro) and Big brother (Bgb)

The CBFb factors are heterodimerization partners for RUNX transcription factors. Indeed, it has been shown that Bro and Bgb are involved during eye development in collaboration with Lz [START_REF] Ling-Hui | Interactions between Brother, Runt and Lozenge[END_REF][START_REF] Kaminker | Redundant Function of Big Brother and Brother[END_REF], and also in collaboration with Runt during segmentation [START_REF] Kaminker | Redundant Function of Big Brother and Brother[END_REF]. No function, however, of either Bro or Bgb in hematopoiesis has been reported yet.

• Notch signaling pathway

During larval hematopoiesis, crystal cell formation has been shown to be initiated and dependent on Notch signaling [START_REF] Duvic | Notch Signaling Controls Lineage Specification during Drosophila Larval Hematopoiesis[END_REF], both in the lymph gland and in Hematopoietic Pockets (HPs). In the lymph gland, some cells in the cortical zone have been found to express the Notch ligand Serrate (Ser), and are involved in the cell-cell contact required for Notch activation; if these cells are absent of the lymph gland, there is no crystal cell differentiation (Lebestky 2003). These Ser expressing cells are called Lineage Specifying Cells (LSCs, Ferguson et Martinez-Agosto 2014). In the HP, the Notch signaling pathway is also required, as Ser expressing cells induce plasmatocyte transdifferentiation into crystal cell precursors (Leitão et Sucena 2015). The Notch ligand Delta is not involved during this process, as its depletion does not impact crystal cell formation [START_REF] Duvic | Notch Signaling Controls Lineage Specification during Drosophila Larval Hematopoiesis[END_REF]Lebestky 2003).

Activation of the pathway specifies crystal cell lineage most likely via initiation of lz transcription. In turn, Lz switches on the specific crystal cell transcriptional program and maintains its expression by an autoregulatory loop (Ferjoux et al. 2007).

It has been shown that in addition to its requirement for the induction of lz transcription, Notch functionally interacts with Lz itself for the transcriptional activation of crystal cell specific target genes like klumpfuss and pebbled [START_REF] Terriente-Felix | Notch Cooperates with Lozenge/Runx to Lock Haemocytes into a Differentiation Programme[END_REF][START_REF] Skalska | Chromatin Signatures at Notch-Regulated Enhancers Reveal Large-Scale Changes in H3K56ac upon Activation[END_REF] thereby contributing to their differentiation. In addition to these two roles (specification and differentiation), the Notch pathway is also required for crystal cell survival. Indeed, the pathway is maintained activated in mature crystal cells, until its Lz-mediated downregulation provokes crystal cell rupture [START_REF] Mukherjee | Interaction Between Notch and Hif-a in Development and Survival of Drosophila Blood Cells[END_REF]Miller et al. 2017).

But Notch is not the only pathway involved in crystal cell differentiation; the Hippo pathway is also required for this process.

• Hippo signaling pathway

The Hippo signaling pathway is mostly known for its function during organ growth, as it is required to tightly control organ size [START_REF] Moeller | Warts Signaling Controls Organ and Body Growth through Regulation of Ecdysone[END_REF]. It is also involved in cell proliferation and is sensitive to cell contact signals [START_REF] Ota | Mammalian Tead Proteins Regulate Cell Proliferation and Contact Inhibition as Transcriptional Mediators of Hippo Signaling[END_REF].

The Hippo signaling pathway relies on a phosphorylation cascade, and most of its members are kinases. Extrinsic signals are sensed by the Tao kinase, which phosphorylates Hippo that subsequently phosphorylates Warts. Finally Warts phosphorylates Yorkie (Yki), the pathway effector, which as a consequence is sequestrated in the cytoplasm (reviewed in [START_REF] Meng | Mechanisms of Hippo Pathway Regulation[END_REF] where it remains inactive. When the pathway is inactive, Yki is present in the nucleus and interacts with molecular partners that are transcription factors; its principal partner is the TEAD factor Scalloped (Sd), and together they activate transcription of target genes like DIAP-1, which promotes cell survival (Figure 9). A recent study showed that Sd is required to regulate maintenance and proliferation of lymph gland progenitors, but it is not clear whether Yki is involved in this process or not [START_REF] Ferguson | The TEAD Family Transcription Factor Scalloped Regulates Blood Progenitor Maintenance and Proliferation in Drosophila through PDGF/VEGFR Receptor (Pvr) Signaling[END_REF].

The same group reported that Yki is required downstream of Notch activation in the lymph gland to initiate lz expression and thus allow crystal cell differentiation (Figure 10; Ferguson et Martinez-Agosto 2014). It also has been demonstrated, in cell culture with a luciferase reporter assay, that Yki is able to bind directly some enhancers in the lz gene [START_REF] Milton | The Hippo Pathway Regulates Hematopoiesis in Drosophila Melanogaster[END_REF]. This was therefore proposed to be the mechanism for the direct transcriptional activation of lz by a Yki/Sd complex during crystal cell differentiation in the lymph gland.

It is noteworthy that, depending on the context, the interactions between the Notch and Hippo signaling pathways can be very different. For instance, it has been shown in the Drosophila wing disc that Notch signaling is able to promote Yki activity in a non cellautonomous manner [START_REF] Graves | Notch Signaling Activates Yorkie Non-Cell Autonomously in Drosophila ». Édité par Madhuri Kango-Singh[END_REF] to promote cell survival. However, in the wing pouch,

Drosophila as a model for pathological hematopoiesis

Molecular actors of the hematopoietic process are highly conserved, and in particular partners of the RUNX transcription factors. This allows molecular and mechanistic studies of their functions in vivo, which is quite complicated in a mammalian model.

In addition to its relevance to studying the process of normal hematopoiesis and homeostasis of blood cell production, Drosophila is used as a model for pathological situations, as hematopoietic deregulation can be induced in vivo. This makes Drosophila an excellent tool to study the molecular mechanisms of leukemic transformation, and in the next part I will describe some of these models.

a) RUNX1-ETO as a model for human leukemic transformation

As previously mentioned, RUNX1 is a major target of single mutations or chromosomal rearrangements that lead to the development of AML. In order to gain a better understanding of the molecular mode of action by which the RUNX1-ETO fusion protein induces a leukemic transformation, two Drosophila models were developed.

In a first study aiming at the identification of suppressors of RUNX1-ETO activity [START_REF] Osman | A Drosophila Model Identifies Calpains as Modulators of the Human Leukemogenic Fusion Protein AML1-ETO[END_REF]), the authors overexpressed the RUNX1-ETO construct using the Gal4/UAS system in hemocytes. RUNX1-ETO overexpression impairs crystal cell differentiation in the embryo mostly due to the competition with Lz transcriptional activity on its target genes. When overexpressed during the larval stages with the Lz-Gal4 driver, RUNX1-ETO blocks crystal cell differentiation both in circulation and in the lymph gland, without affecting Lz expression itself.

This overexpression also induces a pupal lethality that was used by the authors to identify genes required downstream of RUNX1-ETO in a genetic screen. The suppressors of lethality were tested for their ability to suppress crystal cell differentiation defects. They identified calpainB as required for RUNX1-ETO induced blood disorders and showed that calpain inhibition affects RUNX1-ETO protein levels in the human leukemic Kasumi-1 cell line. This study in Drosophila, therefore, yields new insights into the seemingly conserved molecular mechanism at work during RUNX1-ETO dependent leukemic transformation.

The second model relied on overexpression of RUNX1-ETO in all larval hemocytes [START_REF] Sinenko | Genetic Manipulation of AML1-ETO-Induced Expansion of Hematopoietic Precursors in a Drosophila Model[END_REF]. This induces a dramatic increase of hematopoietic precursors and high levels of ROS in circulation, which necessitates an interaction between RUNX1-ETO and one CBFb factor. Indeed, overexpression of a mutated version of RUNX1-ETO that is unable to bind CBFb, is not sufficient to induce the overproliferation of progenitors. This requirement for CBFb binding during RUNX1-ETO leukemic transformation, is a conserved mechanism in mammals [START_REF] Roudaia | CBF Is Critical for AML1-ETO and TEL-AML1 Activity[END_REF]. This increased proliferation of Drosophila hemocytes is reminiscent of what is observed in AML cases, because in the pathology there is proliferation of myeloid progenitors that inhibits production of normal cells. This second model allowed the identification of RUNX1-ETO activity suppressors that are involved in the antioxidant pathway in Drosophila, and this could be relevant to human RUNX1-ETO related AML cases.

In both studies, the authors showed that expressing the human fusion protein RUNX1-ETO in Drosophila was sufficient to recapitulate the major phenotypes displayed in human AML: an increased proliferation of immature progenitors on the one hand, and an inhibition of Lz/RUNX normal activity on the other hand.

b) Myeloid Leukemia Factor

The Drosophila Myeloid leukemia factor (Mlf) belongs to a family conserved across evolution and its mammalian homologs are involved in the development of leukemia (reviewed in Gobert, Haenlin, et Waltzer 2012). It has been shown that Mlf is required for the stability of the Lz protein [START_REF] Bras | Myeloid Leukemia Factor Is a Conserved Regulator of RUNX Transcription Factor Activity Involved in Hematopoiesis[END_REF], especially by forming a chaperone complex along with DNAJ-1 that protects Lz from degradation and ensures its accumulation in crystal cells [START_REF] Dyer | Myeloid Leukemia Factor Acts in a Chaperone Complex to Regulate Transcription Factor Stability and Gene Expression[END_REF]Miller et al. 2017). By doing so, Mlf participates in the control of crystal cell differentiation, and its loss of function is accompanied by dysplastic morphology of Lz expressing cells. In addition, Dyer et al. show that Mlf binds chromatin, and their findings suggest that MLF mediates the repression of genes associated with development and differentiation, while promoting those associated with cell proliferation [START_REF] Dyer | Myeloid Leukemia Factor Acts in a Chaperone Complex to Regulate Transcription Factor Stability and Gene Expression[END_REF].

Finally it also has been shown that Mlf is required for the leukemic activity of RUNX1-ETO in Drosophila and in the human leukemia-derived Kasumi-1 cell line [START_REF] Bras | Myeloid Leukemia Factor Is a Conserved Regulator of RUNX Transcription Factor Activity Involved in Hematopoiesis[END_REF], illustrating once again the existing molecular conservation from Drosophila to human.

In conclusion, over the last 15 years, Drosophila has proven to be a valuable model system to study hematopoiesis, whether normal or pathologic. It also appears that although the cell types and functions may be very different between mammals and insects, the crystal cell lineage represents a particularly suitable context to study the conserved basis of RUNX1related blood cell formation.

Identification of enoki mushroom as a novel actor of Drosophila hematopoiesis

As described above, MOZ is a known co-activator of RUNX1 in mammalian hematopoiesis, and its deregulation leads to the development of severe blood disorders. enoki mushroom (enok) is the Drosophila homolog of MOZ, and they share a high homology, in particular in their N-terminal part, containing the NEMM domain, the tandem PHD fingers and the MYST domain. Given this high level of conservation, Drosophila seemed like an ideal model to decipher the molecular functions of this epigenetic factor.

• Identification of Enok in Drosophila

Enok was first identified in 2001 as a lucky side product of an Ethyl Methane Sulfonate (EMS) mutagenesis screen aiming at identifying mutations that fail to complement lethality of the Df(2)gek D23 deficiency [START_REF] Scott | Enok Encodes a Drosophila Putative Histone Acetyltransferase Required for Mushroom Body Neuroblast Proliferation[END_REF]. Two enok point mutation alleles were isolated: enok 1 and enok 2 . The first allele leads to the change of a Tryptophan into a Stop codon in the MYST domain, and the second allele leads to the replacement of a Cystein in the Zinc-finger motif. Both alleles are homozygous lethal, and individuals display a developmental delay and die at the beginning of the pupal stage. In this study, Scott et al showed that enok loss of function impairs proliferation of neuroblasts in the mushroom bodies. However, the authors hypothesized that the lethality observed in enok mutants is not due to the sole defect in mushroom body development. In addition, they identified a requirement for Enok in germline cells, suggesting that Enok function may affect proliferation in several tissues.

• Enok function during establishment of the female germline

In 2013, a second study led by Xin et al further demonstrated that enok is involved in female germline development [START_REF] Xin | The Drosophila Putative Histone Acetyltransferase Enok Maintains Female Germline Stem Cells through Regulating Bruno and the Niche[END_REF]. By inducing enok mutant clones specifically in germ stem cells, the authors showed that Enok function is required cell autonomously in those cells to promote their maintenance, in part by repressing bruno expression and thus inhibiting premature differentiation. In addition, the authors showed that Enok is required in the niche cells (called cap cells) for their maintenance via Notch signaling, thereby participating in the maintenance of germ stem cells in a second non-cell-autonomous manner.

• Enok is able to acetylate histone H3, in collaboration with the ING5 complex A recent study from Jerry Workman's group showed that Enok is able in vitro to acetylate histone H3 on Lysine 23 (H3K23ac). In addition, they demonstrated that after induction of enok mutant clones in eye imaginal discs, the H3K23ac staining is almost completely lost in vivo; this demonstrates for the first time that Enok is responsible for histone modification.

Here, the authors show that the H3K23ac mark is correlated with Enok-dependent expression of spir and mael genes, finally responsible for oocyte polarization.

Interestingly, a recent study reported that MOZ is able to acetylate H3 on Lysine 23 in Glioblastoma cell lines (Lv et al. 2017). In addition, BRPF1 is required for H3K23 acetylation in vivo in mouse [START_REF] You | BRPF1 Is Essential for Development of Fetal Hematopoietic Stem Cells[END_REF]). These findings suggest that H3K23 acetylation by the ING5 complex might be a conserved feature in gene regulation.

In a second study in 2016, Huang et al demonstrated that Enok catalytic activity is dependent on the entire ING5 acetylation complex (Huang et al. 2016). The complex is composed of Enok, Br140, Eaf6 and Ing5, the respective homologs of MOZ, BRPF1/2/3, EAF6 and ING5 in mammals. The authors showed in cultured S2 cells with western blot analysis that the H3K23ac levels decreased after depletion of each member of the complex. In addition, Huang et al showed that Enok interacts physically with Elg1 in S2 cells. Elg1 has been shown to be responsible for Proliferating Cell Nuclear Antigen (PCNA) unloading during DNA replication (Kubota et al. 2013, 1). The authors demonstrated that Enok is required to downregulate the PCNA unloading function of Elg1, in order to promote the G1/S transition, and thus prevent cell cycle arrest. Some in vivo experiments suggested that this interaction between Enok and Elg1 might be conserved in the female germ line, providing a new insight into Enok function in this tissue.

• Enok and Br140 are bound on Bivalent promoters

A recent study revealed that Br140, a member of the ING5 complex, binds a repertoire that largely overlaps that of the chromatin silencer Polycomb (Pc) (Kang et al. 2017). Pc is the founding member of the Polycomb group of proteins and is a part of the Polycomb Repressive Complex 1 (PRC1). PRC1 has DNA binding properties and when bound on chromatin, it can recruit Polycomb Repressor Complex 2 (PRC2), which in turn trimethylates histone H3 on lysine 27 (H3K27me3) through the activity of its catalytic subunit Enhancer of Zeste (E(z)). This histone modification is linked to gene repression and is essential for all developmental processes across evolution (reviewed in [START_REF] Papp | Histone Trimethylation and the Maintenance of Transcriptional ONand OFF States by TrxG and PcG Proteins[END_REF].

The concomitant binding of Br140 and PRC1 has been suggested as contributing to the maintenance of the bivalent state of promoters of many developmentally regulated genes in the embryo (Kang et al. 2017). Indeed, the presence of Br140 and Pc correlates with H3K4me3 and H3K27me3 chromatin marks, respectively marks of active and repressed chromatin. This chromatin landscape in mammals has been described as a specific feature of bivalent promoters (reviewed in [START_REF] Harikumar | Chromatin Remodeling and Bivalent Histone Modifications in Embryonic Stem Cells[END_REF]. Furthermore, the authors purified the PRC1 complex and after a proteomic analysis, they observed that Enok is a partner of Pc as well as Br140, suggesting that they all form a complex to maintain chromatin in this bivalent state. In this study, the authors propose that Enok/Br140 along with PRC1 bind bivalent promoters of developmental genes. As differentiation occurs, activating or repressing factors interact with them and allow the resolution of bivalency in favor of transcription activation or repression.

This novel and interesting model for Enok/Br140 function during development seems to be conserved across evolution, as BRPF2 and RING1B, respectively the Br140 homolog and the member of human PRC1 complex, are similarly co-bound on bivalent promoters in human embryonic stem cells (Kang et al. 2017).

In summary, the KAT Enok is involved in many developmental processes, and its main molecular partners are conserved from Drosophila to mammals. However, there is so far no evidence in the literature that Enok participates in Drosophila hematopoiesis, and our group is the first to establish a link between Enok and the regulation of Srp/Lz-dependent transcriptional activation (Gobert et al. 2010a).

• Enok regulates the transcriptional activity of the Srp/Lz complex As I described earlier, the GATA and RUNX factors Srp and Lz act as a complex of transcription factors, both necessary and sufficient for expression of target genes such as the PPOs, and therefore for crystal cell differentiation in the embryo (L. Waltzer 2003b;Ferjoux et al. 2007). A genome-wide screen in cell culture led to the identification of modulators of the GATA/RUNX complex transcriptional activity, including enok. [START_REF] Gobert | A Genome-Wide RNA Interference Screen Identifies a Differential Role of the Mediator CDK8 Module Subunits for GATA/ RUNX-Activated Transcription in Drosophila[END_REF].

III. Objectives of my PhD work

The genome wide screen led by our group identified the Lysine Acetyltransferase Enok as being potentially a part of the hematopoietic process. Preliminary results showed that in vivo, enok loss of function leads to a dramatic loss of crystal cells during the larval stage, establishing its involvement during blood cell formation. My project was to understand how enok participated in the production of crystal cells, by studying the molecular mechanisms involved in this process.

We can recapitulate the research project in three main questions:

• Is the KAT activity of Enok involved during the hematopoietic process in Drosophila?

• Which are Enok partners during crystal cell development?

• Does Enok regulate directly or indirectly crystal cell differentiation?

Abstract

The human KAT6 lysine acetyltransferase MOZ has been shown to be an essential player in the field of normal and malignant hematopoiesis. It belongs to a highly conserved family of epigenetic factors and remodels chromatin by acetylating histone tails in association with its partners of the ING5 complex. Here, we report that its Drosophila counterpart Enok is required during larval hematopoiesis to control the Notch-dependent process of plasmatocyte transdifferentiation into crystal cells. In particular enok is essential to allow expression of the RUNX factor Lozenge (Lz) that controls the crystal cell specific transcriptional program. We demonstrate that this function involves neither the Eaf6 and Ing5 subunits of the Drosophila ING5 complex, nor its own acetyltransferase activity. We provide evidence that Enok binds a new lz enhancer specifically required for its expression in Notch-activated crystal cell precursors. We propose that DNA-binding properties of KAT6 proteins might represent an alternative mechanism to support catalytic-independent activities in hematopoiesis.

Introduction

Hematopoiesis, the highly dynamic process of all blood cell production, is tightly regulated by key transcription and epigenetic factors [START_REF] Menegatti | Transcriptional control of blood cell emergence[END_REF][START_REF] Fujiwara | GATA Transcription Factors: Basic Principles and Related Human Disorders[END_REF][START_REF] Hu | Epigenetics of hematopoiesis and hematological malignancies[END_REF]. Many of these regulators have been identified through the pathologies induced by their deregulation/mutation. In particular, genetic aberrations such as chromosomal rearrangements have been instrumental, since they allowed immediate identification of the targeted loci. As an example, characterization of the leukemogenic translocation t(8;21) led to the discovery of the transcription factor RUNX1 that was further shown to be essential for multiple steps of mammalian hematopoiesis [START_REF] Lam | RUNX1 and RUNX1-ETO: roles in hematopoiesis and leukemogenesis[END_REF].

Similarly, the gene encoding Monocytic Leukemia Zinc finger protein (MOZ, also known as KAT6A) was originally identified as the target of myeloid leukemia associated rearrangements, which fuse it to Lysine Acetyltransferases (KAT) such as CREB-binding protein (CBP) or p300, or to the coactivator TIF2 (reviewed in [START_REF] Katsumoto | Roles of the histone acetyltransferase monocytic leukemia zinc finger protein in normal and malignant hematopoiesis[END_REF]).

MOZ/KAT6A belongs to a family of KATs conserved from yeast to human, the MYST family (named after its founding members MOZ/Ybf2/Sas2/Tip60), and is mainly known for its ability to acetylate histone H3 on lysine-9, lysine-14 and lysine-23 residues [START_REF] Voss | Moz and Retinoic Acid Coordinately Regulate H3K9 Acetylation, Hox Gene Expression, and Segment Identity[END_REF][START_REF] Qiu | Combinatorial readout of unmodified H3R2 and acetylated H3K14 by the tandem PHD finger Adya[END_REF]Dreveny et al, 2014;Lv et al, 2017). It is therefore proposed to control transcriptional events by participating in chromatin remodeling via the modification of epigenetic marks. MOZ exerts its acetylation activity in a tetrameric ING5 complex also containing the bromodomain PHD finger protein 1 (BRPF1), the human Esa1-associated factor 6 homolog (hEAF6), and the inhibitor of growth 5 (ING5) subunits [START_REF] Doyon | ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation[END_REF]Ullah et al, 2008). The analysis of a MOZ mutant murine model specifically deprived of its catalytic activity (Perez-Campo et al, 2009) reveals a drastic reduction in hematopoietic stem cells (HSCs) and committed precursor populations, directly resulting from their reduced proliferation capacities. These results highlight the essential role of MOZ-driven acetylation in HSCs. However, the phenotypic differences existing between this catalytically inactive MOZ mutant and a loss of the entire MOZ protein [START_REF] Katsumoto | MOZ is essential for maintenance of hematopoietic stem cells[END_REF] suggest that MOZ may also ensure non-acetyltransferase functions in vivo, consistent with previous observations that MOZ interaction with RUNX1 promotes transcription independently of its catalytic domain in murine cultured cells [START_REF] Kitabayashi | Activation of AML1mediated transcription by MOZ and inhibition by the MOZ-CBP fusion protein[END_REF].

Enoki mushroom (Enok), Drosophila melanogaster MOZ homolog, was first identified for its role in neuroblast proliferation [START_REF] Scott | Enok Encodes a Drosophila Putative Histone Acetyltransferase Required for Mushroom Body Neuroblast Proliferation[END_REF]. Its loss-of-function results in atrophied mushroom bodies in the fly brain, earning the gene its name, as well as a developmental delay and lethality at the early pupal stage. enok was later shown to be required for visual system wiring [START_REF] Berger | Systematic identification of genes that regulate neuronal wiring in the Drosophila visual system[END_REF] and for germline stem cell maintenance, a process during which it ensures both cell autonomous and non-autonomous functions [START_REF] Xin | The Drosophila Putative Histone Acetyltransferase Enok Maintains Female Germline Stem Cells through Regulating Bruno and the Niche[END_REF]. The first insights into the molecular mechanisms underlying its different roles came from a study that demonstrated Enok's ability to acetylate histone H3 on its lysine-23 (H3K23) [START_REF] Huang | Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire[END_REF]; in addition, it showed that enok is required for the expression of oocyte-polarizing genes and proposed that H3K23 acetylation of its targets accounts for the contribution of enok to transcriptional activation. Finally, a second study by the same group, demonstrated that Drosophila homologs of the mammalian ING5 complex Br140, Eaf6 and Ing5 interact physically and functionally with Enok for H3K23 acetylation, and identified a new role for Enok in cell cycle regulation (Huang et al, 2016). Of note, no function for Enok in hematopoiesis has ever been reported.

Drosophila is a well-established model to study both normal and pathologic hematopoiesis, since its much simpler hematopoietic system displays a high degree of conservation, whether at the functional or at the molecular level (for an exhaustive review, see (Banerjee et al, 2019)). Reminiscent of mammalian hematopoiesis, the fly blood cells (called hemocytes) are formed in two temporally distinct waves. The embryonic wave gives rises to two differentiated cell types: plasmatocytes that mainly ensure phagocytic functions, and crystal cells that are responsible for melanization reactions and therefore contribute to wound healing and immune response. During larval life, a second wave of hematopoiesis in the lymph gland yields a reservoir of plasmatocytes and crystal cells that remain separated from the circulation until they are released at the onset of metamorphosis (under normal conditions) or mobilized in response to a parasitic infestation (Letourneau et al, 2016). Cells formed during the embryonic wave persist in circulation throughout larval stages until adulthood [START_REF] Holz | The two origins of hemocytes in Drosophila[END_REF]. During larval life, a massive expansion of the plasmatocyte population occurs in segmentally dispatched clusters beneath the epidermis, called hematopoietic pockets [START_REF] Makhijani | Of blood cells and the nervous system: hematopoiesis in the Drosophila larva[END_REF]. In addition, recent live-imaging of sessile hemocytes within these hematopoietic pockets shows that upon Notch signaling pathway activation, plasmatocytes transdifferentiate into crystal cells (Leitão & Sucena, 2015) as they start to express the crystal cell lineage-specific RUNX transcription factor Lozenge (Lz), thus ensuring de novo larval crystal cell formation.

Here we provide the first evidence that like its mammalian counterpart, Enok participates in hematopoiesis, since we show that the transdifferentiation of plasmatocytes into larval circulating crystal cells is hindered in enok mutants. In particular, we demonstrate that the expression of Lz is dependent on a cell-autonomous function of Enok in Notchactivated crystal cell precursors. Unexpectedly, we establish that Enok is required independently of its KAT activity and of the ING5 acetylation complex and provide the prime example of a catalytic-independent function for a MOZ protein in vivo. Finally, we find that Enok binds a previously unidentified hematopoietic enhancer that controls expression of lz in crystal cell precursors, and we propose that the Enok non-catalytic mode of action relies on its binding to DNA.

Results

enok is essential for larval crystal cell formation.

In order to assess a possible function of enok during Drosophila hematopoiesis, we examined blood cell formation during embryonic and larval life in enok loss-of-function mutants. The three different alleles tested (enok 1 , enok 2 and enok Q253 ), placed either in hemizygote conditions (combined to the Df(2R)BSC155 deficiency that uncovers enok locus) or in allelic combination (enok 1 /enok Q253 ), exhibited a dramatic loss of circulating crystal cells at the larval stage. Functional crystal cells in hematopoietic pockets of third instar larvae can be easily visualized, as heat exposition triggers melanization reactions within the cells, which then appear as little black dots. We observed almost no melanized crystal cells in the four enok mutant contexts tested as compared to control situations (Fig. 1A), indicating a defect in lineage specification or in precursor cell differentiation. As circulating hemocytes continuously enter and exit hematopoietic pockets during larval life, we examined enok mutant hemolymph cell content. To visualize the crystal cell lineage, we used a Notch Response Element reporter construct (NRE-GFP), whose expression is detected in crystal cell precursors and persists until their terminal differentiation (Leitão & Sucena, 2015;Miller et al, 2017), and an antibody raised against Prophenoloxidase1 (PPO1), a crystal cell terminal differentiation marker. Our results show that Notch-activated hemocytes (NRE-GFP + cells) have lost PPO1 expression in enok loss-of-function contexts (Fig. 1B), confirming the absence of mature crystal cells in the sessile/circulating compartment of enok loss-of-function larvae.

enok is required for transdifferentiation of plasmatocytes into larval crystal cells.

Since the expression of PPO1 depends on the RUNX transcription factor Lz (Waltzer et al, 2003;[START_REF] Gajewski | Identification of a crystal cell-specific enhancer of the black cells prophenoloxidase gene in Drosophila[END_REF], we assessed Lz expression in circulating cells and observed that only a small fraction NRE-GFP + cells retain the ability to express Lz in enok loss-of-function mutants as compared to control larvae (Fig. 1C,D). Moreover, an increased proportion of Notch-activated hemocytes displays expression of the plasmatocyte specific marker P1 (Fig. 1E), in agreement with a previous study reporting that together with Notch, Lz controls the expression of klumpfuss, a repressor of plasmatocyte identity [START_REF] Terriente-Felix | Notch Cooperates with Lozenge/Runx to Lock Haemocytes into a Differentiation Programme[END_REF].

• controt enok'!Df-B155

While we detected a minimal increase in the number of NRE-GFP + cells in enok mutants as compared to control larvae, it was not the case with two other Notch pathway reporter constructs, rendering unlikely that the loss of Lz expression results from a generic problem in Notch signaling interpretation (Supplemental Fig. S1A-C). Altogether our results show that enok is required downstream of Notch signaling for Lz expression during the transdifferentiation of plasmatocytes into crystal cells.

enok regulates Lz expression in crystal cell precursors in a cell-autonomous fashion.

We next sought to determine in which cells enok is required to promote crystal cell production. Directing expression of a UAS-3HA-enok transgene in enok mutant larvae, either ubiquitously with the tubulin-Gal4 driver or with the Notch GMR30A01 -Gal4 driver, which allows specific expression in Notch-activated crystal cell precursors (Supplemental Fig. S2A,B Since Enok is known to cooperate with its partners in the Drosophila ING5 complex to acetylate H3K23 [START_REF] Huang | Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire[END_REF](Huang et al, , 2016)), we surmised that Enok and the whole ING5 complex might control lz expression in Notch-activated hemocytes via histone acetylation.

While a Br140 S781 loss-of-function allele already existed, no mutation for Eaf6 or Ing5 was available. We thus used the CRISPR/Cas9 genome-editing system to excise most of the coding region of these two genes and recovered the Eaf6 M26 and Ing5 ex1 alleles (methods). Likewise, we introduced in the coding sequence of enok a single amino acid substitution that specifically abrogates its lysine acetyltransferase function (enok KAT allele, see methods). As expected, the level of H3K23 acetylation in circulating cells is strongly reduced in enok 1 and Br140 S781

hemizygote contexts and more mildly in Eaf6 M26 hemizygote or Ing5 ex1 homozygote mutants (Supplemental Fig. S3); of note, the enok KAT mutation reduces the level of H3K23 acetylation to that observed with the enok null mutant context (enok 1 /Df(2R)BSC155) (SuppFig. 3), suggesting that the enok KAT allele is a genetically null allele for the acetyltransferase function.

We then observed in melanization assays that, while Br140 loss-of-function results in the same hematopoietic phenotype as enok loss-of-function, gene excision of Eaf6 or Ing5 did not affect larval crystal cell formation (Fig. 3A). More strikingly, enok KAT mutant larvae exhibited seemingly normal crystal cell differentiation in hematopoietic pockets (Fig. 3A). Additional quantification of the proportion of NRE-GFP + cells expressing Lz confirms that both Br140 S781

and enok 1 mutations prevent Notch-activated precursors from differentiating further, while enok KAT mutation has no effect on this lineage (Fig. 3B). Thus, our results demonstrate that Enok and Br140 regulate Lz through to a non-canonic mode of action that is independent of the KAT activity of Enok, and of the ING5 complex.

Lz expression precedes that of Yorkie during plasmatocyte to crystal cell transition.

During de novo hematopoiesis in the lymph gland, the onset of lozenge expression was reported to be under the control of the Hippo signaling pathway effector, Yorkie (Yki),

following Notch signaling activation [START_REF] Milton | The Hippo Pathway Regulates Hematopoiesis in Drosophila Melanogaster[END_REF]Ferguson & Martinez-Agosto, 2014b, 2014a). We postulated that the same mechanism could be at work during larval plasmatocyte transdifferentiation in the sessile/circulating compartment. We thus performed double

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- ----e:• "1••' . .. :;..~~---:----~----• :;•----: ...... :. .. . ' ~J,• :(•; _. . , . -:-..f . . : both NRE-GFP and Yki also express Lz (Fig. 4C), whereas a subset of cells that co-express NRE-GFP and Lz appear negative for Yki staining (Fig. 4D, population in the dotted ellipse). The existence of this subpopulation does not fit the model developed for de novo crystal cell differentiation in the lymph gland and raises the possibility that the transcriptional cascade controlling plasmatocyte transdifferentiation into crystal cells might be different, which prompted us to investigate it further. Quantification of Yki expression intensity in lz R1 null mutant larvae shows that crystal cell precursors deprived of lz do not express Yki, as compared to a control situation (Fig. 4E), indicating that lz is required for Yki expression in these cells.

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Furthermore, ectopically expressing lz in all larval circulating hemocytes with the hemolectin-gal4 driver is sufficient to induce a significant increase of the Yki expressing cell population (Fig. 4F). In contrast, ectopic expression of Yki or activated-Yki (Yki*) does not trigger Lz expression in circulating cells (Fig. 4F). Altogether our results suggest that following Notch signaling pathway activation in plasmatocytes, lz expression precedes that of yki, and is initiated independently of the latter. We subsequently focused on the control of Lz expression by enok.

Enok directly binds an intronic region of lozenge, which is required for its expression in crystal cell progenitors.

As it was reported that in addition to bearing the KAT-activity the MYST domain of MOZ has the ability to bind DNA (Holbert et al, 2007), we hypothesized that Enok could control lz expression by directly binding its regulatory regions. We selected a set of transgenic lines containing potential regulatory regions placed upstream of the Gal4 coding sequence and tested them for their ability to recapitulate lz expression in the NRE-GFP expressing cells (Table 1). It appears that the regulatory region covered by the Vienna Tile VT059215, either placed upstream of the Gal4 coding sequence (lz VT059215 -Gal4, Fig. 5A) or directly fused to the RedStinger reporter gene (lz VT059215 -RedStinger, Fig. 5B) is able to drive transcription in a large fraction of the NRE-GFP expressing cells, which overlaps importantly but not exclusively with endogenous Lz expression (Fig. 5A,B). This potential regulatory region is embedded in the third intron of the lz gene. In order to further establish the role played by this regulatory region, we decided to delete lz third intron using the CRISPR/Cas genome editing technique (lz int3 allele, see methods). Despite the critical roles of lz during eye development (Daga et al, 1996), the excision of lz third intron yields individuals with normal eyes, indicating that in these mutants, lz expression in the eye imaginal disc is not affected and that the Lz protein is functional. However, like enok loss-of-function, lz int3 mutation abolishes Lz expression in most Notch-activated hemocytes (Fig. 5C,D), indicating that lz third intron contributes to its expression in circulating crystal cell precursors. In addition, the lz VT059215 -Gal4 driven reexpression of lz in a lz R1 mutant background is sufficient to restore a wild-type proportion of Lz expressing cells in the NRE-GFP domain (Fig. 5E), as well as crystal cell maturation in the hematopoietic pockets (Fig. 5F). Altogether, these results show that lz third intron contains an hematopoietic enhancer both necessary and sufficient for its expression in most crystal cell precursors. We then speculated that Enok might occupy this region to regulate Lz expression in Notch-activated hemocytes. Chromatin immunoprecipitation performed in tub-Gal4/UAS-3HA-Enok embryos revealed a conspicuous enrichment in HA-Enok binding across regions of lz third intron, corresponding to the identified VT059215 regulatory element (Fig. 5G), and a

modest (yet statistically significant) binding of HA-Enok in lz first exon, which was recently reported to be bound by Br140 (Kang et al, 2017), suggesting potential co-occupancy of this region by the Enok/Br140 complex. No obvious enrichment was detected in the upstream intergenic region previously reported to contain some lz regulatory elements (Bataillé et al, 2005;Ferjoux et al, 2007). Overall, our results point to the VT059215 region as a major site of Enok binding in the lz gene, and we propose that Enok regulates lz expression by directly binding a hematopoietic enhancer located within its third intronic region.

Discussion

In the present study, we characterized the hematopoietic defects induced by the loss of enok function and demonstrated that it is required in a cell-autonomous manner in Notchactivated hemocytes during the transdifferentiation of plasmatocytes into crystal cells that occurs in the hematopoietic pockets. In particular, we presented evidence that enok acts downstream of Notch signaling pathway to ensure expression of the crystal cell fate determinant Lz in crystal cell precursors (summarized in Fig. 6). In addition, we showed that this function of Enok relies on a KAT-independent mechanism, presumably by binding the new hematopoietic enhancer we identified within the third intron of lz.

One remarkable feature of this enhancer is that it does not allow faithful recapitulation of the Lz expression pattern in Notch-activated crystal cell precursors. Indeed, only a fraction (about 65%) of the NREGFP + /Lz VT059215 -RedStinger + cells express endogenous Lz, and a large proportion (almost 30%) of the NREGFP + /Lz + cells do not express the lz VT059215 -RedStinger reporter construct. One explanation for this behavior is that the lz VT059215 enhancer might be incomplete; as a matter of fact, it corresponds to a sequence of only 2 kb in the third intron, whose size reaches 5 kb in total. However, our analysis of the lz GMR26G10 -Gal4 and lz GMR25H03 -Gal4 transgenic lines, which contain regulatory regions overlapping with the lz VT059215

sequence and allow coverage of the full intron length, did not reveal any additional transcription-promoting elements. Another possibility is that the hematopoietic enhancer located in the third intron of lz and bound by Enok is not the sole regulatory region controlling its expression in crystal cell precursors. This hypothesis is further supported by the fact that lz int3 mutation leads only to a partial defect in larval crystal cell formation, since approximately 20% of the NRE-GFP + circulating cells express Lz normally, despite the excision of the whole intron. A second regulatory region would therefore be responsible for the expression of Lz in the 20% of cells that are still able to differentiate properly following Notch signaling activation in lz int3 mutant individuals. Importantly, loss of enok function shares exactly the same partial phenotype as excision of the third intron of lz. One would therefore expect the postulated second regulatory region not to be under the influence of enok. One very obvious candidate for this additional regulatory element role is the 1.5 kb long lz-upstream region, which was shown to recapitulate Lz hematopoietic expression during embryogenesis (Bataillé et al, 2005; Ferjoux et al, 2007) and appears not to be bound by Enok in our chromatin immunoprecipitation. With the discovery of a novel enhancer contributing specifically to Lz expression in the circulating crystal cell precursors, possibly with the help of another regulatory region, the control of lozenge expression appears even more complex than previously thought.

Finally, perhaps our most striking result is that the control of Lz expression relies on a catalytic-independent mode of action of Enok likely involving its interactor Br140, but for which Eaf6 and Ing5 appear to be dispensable. Catalytic-independent functions of MOZ/KAT6A, the mammalian homolog of Enok, have been speculated based on the phenotypic differences observed between mice either totally deprived of MOZ, or in which its catalytic activity has been obliterated by a single amino acid substitution. However, there is so far no documented study of such KAT-independent functions of MOZ in vivo. Indeed, although it has been shown that MOZ potentiates RUNX1-dependent transcription independently of its catalytic domain ex vivo, this transcriptional co-activator role of MOZ has not yet been reported to occur during any developmental process. During Drosophila transdifferentiation of plasmatocytes into crystal cells, we propose that the non-canonic mode of action of Enok relies on its ability to bind DNA. Since Lz is the homolog of mammalian RUNX1 and is known to participate in an autoregulatory loop during embryogenesis (Ferjoux et al, 2007), a simple mechanistic hypothesis would be that an Enok/Lz interaction potentiates transcriptional autoregulation of lz independently of the catalytic activity of Enok.

Alternatively, since MOZ has been shown to interact with a plethora of transcription factors (RUNX1, PU.1, c-JUN, P53, TEL, NF-kB, NRF2, reviewed in [START_REF] Perez-Campo | The MYSTerious MOZ, a histone acetyltransferase with a key role in haematopoiesis[END_REF]) to control expression of various target genes, Enok could also interact with one of their Drosophila counterparts, but no such interaction has been reported yet.

More interestingly, it was shown that in Drosophila embryos Enok and Br140 interact with Polycomb (Pc), a major epigenetic silencer (Strübbe et al, 2011). This was further confirmed in a recent work highlighting the substantial overlap existing between chromatin occupancies by Br140 and by the Polycomb Repressive Complex 1, PRC1 (Kang et al, 2017) on developmentally regulated bivalent genes primed for both transcriptional activation and silencing (with activating H3K4me3 and silencing H3K27me3 methylation marks). One of the genomic regions occupied by Br140 and PRC1 is the lz locus, and even more strikingly, co-binding by the BRPF factor BRD1 and the PRC1 member RING1B on the RUNX1-encoding locus was also detected in HUES64 human ES cells. It is noteworthy that the authors did not observe any enrichment in the Enok-dependent H3K23ac mark on their set of bivalent genes, as is it compatible with a non-catalytic mode of action of Enok. The binding of the Enok/Br140 pair, which is rich in epigenetic reader domains, could allow recruitment of other chromatin modifying factors, such as the PRC1 complex, and thereby influence the choice between transcriptional activation or repression in response to a variety of developmental cues.

In conclusion, we have shown that Enok is a major actor of the plasmatocyte to crystal cell transdifferentiation during Drosophila hematopoiesis. In addition, this study provides the first example of a transcriptional regulation that relies on a non-catalytic mode of action of a KAT6 protein in an integrated model. The regulation of lz expression by an Enok/Br140 complex provides an original paradigm that can be used to address mechanistic questions on a well-defined regulatory element, in a genetically amenable and simple model. Given the large degree of evolutionary conservation of all the identified players, we believe that further elucidation of the non-catalytic mode of action of Enok during Drosophila hematopoiesis could be of high relevance to the molecular mechanisms presiding MOZ function in mammalian hematopoietic development, whether normal or malignant.

(enok KAT /Df-B155). Each dot represents the value attributed to a single cell. Statistics: control genotype was used as a reference sample; *** indicate a p-value≤0.0010.

Complements on the study of Enok function during crystal cell formation

As previously stated, during larval life, the catalytic activity of Enok is not required in circulating hemocytes for plasmatocyte to crystal cell differentiation. In order to gain further mechanistic insights about its function during this process, I initiated a structure-function study of Enok using the CRISPR/Cas9-based genome editing method.

Engineering alleles mutant for the DNA binding properties of the MYST domain

It has been shown in vitro that MOZ is able to directly bind chromatin and thus has a DNA binding function (Holbert et al. 2007). In this study the authors show that the I727E mutation drastically affects the DNA-binding ability of MOZ, without affecting its capacity to acetylate histones. A comparison of MOZ and Enok amino acid sequences reveals that the residues involved in this binding are conserved in Enok (Figure 11A), and thus I decided to mimic the I727E mutation in Drosophila. Using the CRISPR/Cas9 technique, I successfully introduced the I931E desired mutation (Figure 11A), but a melanization assay on enok I931E mutant larvae showed no obvious defect in the ability to generate larval crystal cells.

Furthermore, the mutation did not affect fly viability. These preliminary observations suggest that the mutation does not affect Enok's DNA binding ability, or that DNA-binding is not mandatory for crystal cell differentiation. I obtained this mutant fly strain late during my doctoral training and thus didn't have time to study it in depth; in order to draw a conclusion, this mutant allele has to be further characterized. In particular, it would be interesting to test its ability to bind the enhancer located in the third intron of lz, but this would require generating additional tools; one could mutate the UAS-3HA-enok construct I used previously for Chromatin Immunoprecipitation, (see section IV.A.3 ), or one could directly tag the endogenous enok I931E protein to realize the same kind of experiment. essential for cell cycle regulation and are involved in tumorigenesis (Susumu Rokudai et al. 2009, 53;S. Rokudai et al. 2013, 53). Furthermore, human mutations introducing a stop codon after the MYST domain lead to microcephaly and overall developmental delay [START_REF] Tham | Dominant Mutations in KAT6A Cause Intellectual Disability with Recognizable Syndromic Features[END_REF]. In this study the authors linked the developmental disorders to a missing interaction between MOZ and p53, as they saw differential regulation of p53 target genes in these individuals. Interestingly, it has been shown that BRPF1 mutants in human also display intellectual disability (Yan et al. 2017), which would raise the question of its interaction with MOZ and p53 during development. All this evidence led me to postulate the conservation of the interaction between Enok and p53, and that enok M31.1 mutant allele I had generated could misregulate potentially conserved Enok/p53 target genes, which would in part explain the lethality in the flies.

This hypothesis has to be investigated from the start, because it has never been shown

in Drosophila that Enok and p53 interact; furthermore, it would be interesting to study the mechanisms underlying enok M31.1 -related lethality, as the molecular mechanisms involved in this phenotype may be conserved in humans. The conservation of the MOZ/BRPF interaction in Drosophila makes it a relevant model with which to study this potential catalytic independent function of MOZ during mammalian development.

Complements on the study of the regulatory regions controlling lz expression in the crystal cell precursors

Engineering mutants carrying an excision of the lz-upstream region

As discussed in section IV.A.4, the deletion of lz third intron is not sufficient to completely abrogate its expression in circulating cells. Indeed, in lz int3 mutant larvae, around 20% of NRE-GFP positive cells still have the ability to normally initiate lz expression, and this phenotype is shared by enok loss of function. This observation suggests that there is another regulatory region responsible for lz initiation independent of enok during plasmatocyte transdifferentiation. The region located upstream of the lz gene was shown to recapitulate lz hematopoietic expression in the embryo (Bataillé et al. 2005;Ferjoux et al. 2007), and it was not bound by Enok (see section IV.A.3). I therefore generated a CRISPR-Cas9 mediated excision of this region, the lz 2AB allele, in order to assess its requirement during crystal cell formation and started to characterize its mutant phenotypes. As for the lz int3 allele, there was no eye phenotype in the adult fly, indicating that this region was not necessary for the control of lz expression in the eye imaginal disc. I observed that Lz expression is lost in circulating crystal cell precursors (Figure 13), which suggests that the lz 2AB region is responsible, at least partially, for lz expression during plasmatocyte to crystal cell transdifferentation.

To resume, we identified two enhancers that direct normal expression of lz in Notchactivated hemocytes. It would be interesting to assess the consequences of the simultaneous excision of both regulatory regions. Since the two regulatory regions seem to be partially involved in lz expression, deletion of both at the same time might completely abrogate lz expression in NRE-GFP + cells. Likewise, it would be interesting to coexpress the lz VT059215 -RedStinger (which is not expressed in all of the NRE-GFP positive cells) with the lz-LacZ reporter gene and compare their additive pattern to endogenous Lz expression, in order to see if the combination of the two regions is sufficient for the complete expression domain of lz in NRE-GFP expressing cells.

These experiments should give more insights into the mechanisms underlying lz regulation in the Hematopoietic Pockets, which is for now not characterized. enok KAT allele engineering (single amino acid substitution, Homology Directed Repair):

the K807R mutation introduced in the MYST domain of enok affects a Lysine that is highly conserved across evolution and critical for the KAT activity of yeast and human MYST proteins (Yuan et al, 2012;Yang et al, 2012). A repair DNA template called single stranded oligonucleotide donor (ssODN) was injected along with the sgRNA expression vector so as to direct the reparation (ssODN: 100 ng/uL and sgRNA: 250 ng/uL). The ssODN sequence contained the mutation of interest, as well as a sabotage mutation of the Protospacer Adjacent Motif to avoid re-cleaving of an edited chromosome. In addition, the nucleotide sequence immediately surrounding the desired site of modification was degenerated by introduction of silent mutations, in order to allow the design of a discriminant screening primer. 10-15 F1 males were crossed individually for each F0 founder; after 4-5 days in the crossing vials, each F1 male was retrieved for single fly DNA extraction. The PCR-based screen of F1 individuals relied on a triple primer PCR: two external primers amplify DNA in both positive and negative editing events, while an internal primer specifically amplifies DNA if the desired mutation is present.

lz third intron excision (Non Homologous Ends Joining): two sgRNA expression vectors targeting each extremity of lz third intron were injected at a concentration of 250 ng/uL each; 10-15 F1 males were crossed individually for each F0 founder and after 4-5 days in the crossing vials, they were retrieved and tested for their ability to generate shorter amplicons in PCR reactions.

enok I931E allele engineering (single amino acid substitution): the mutation was designed to target a conserved aminoacid crucial for MOZ binding to nucleosomes in vitro (Holbert et al. 2007). The same injection and screen protocol was used for the generation of the enok KAT allele.

-Neurofilament-like domain excision (Non Homologous Ends Joining): two rounds of injections were performed, each using a different pair of sgRNA expression vectors targeting each extremity the domain. The injection concentration was of 250 ng/uL each; F1 individuals were tested for their ability to generate shorter amplicons in PCR reactions.

lz upstream regulatory region excision (Non Homologous Ends Joining): two sgRNA expression vectors targeting each extremity of lz third intron were injected at a concentration of 250 ng/uL each; 10-15 F1 males were crossed individually for each F0 founder and after 4-5 days in the crossing vials, they were retrieved and tested for their ability to generate shorter amplicons in PCR reactions. (Leitão et Sucena 2015).

What I show in my work may therefore be a specific mechanism involved only in the transdifferentiation process that takes place in Hematopoietic Pockets.

In a recent study, Jang et al showed that when YAP, the mammalian homolog of Yki, is phosphorylated and sequestrated in the cytoplasm, a fraction of phosphorylated YAP remains in the nucleus and can interact with RUNX1 and/or RUNX3 (Jang et al. 2017). It is noteworthy that, while YAP interaction with its canonic partner TEAD promotes proliferation (Hippo pathway off), the interaction between phosphorylated YAP and RUNX3 occurs during cell cycle arrest (Hippo pathway on). Furthermore, the authors showed that lz overexpression is able to rescue the eye overgrowth phenotype induced by Yki S168A (the activated form of Yki) overexpression. This experiment showed that Lz is able to antagonize Yki activity; this antagonism might rely on a conserved interaction between RUNX factors and YAP. This study suggests that in Drosophila Lz could interact with Yki to inhibit its proliferative effects.

Interestingly, in the hematopoietic pockets plasmatocytes are able to proliferate, which is in agreement with the absence of Hippo pathway activation. In these cells, Yki could interact with Sd in the nucleus to promote proliferative genes expression. After their transdifferentiation into crystal cells, which have never been shown to have any dividing capacities, the Hippo pathway would be turned on. This model fits the observation that Yki is mostly localized in the cytoplasm of crystal cells. Considering that phosphorylated YAP is able to interact with RUNX factors, it is possible to hypothesize that Lz may form a complex with phosphorylated Yki in the crystal cell nucleus to promote the expression of their target genes.

It is further interesting to note that Milton et al showed that Yki is able to bind directly lz third intron [START_REF] Milton | The Hippo Pathway Regulates Hematopoiesis in Drosophila Melanogaster[END_REF]; among all the possible binding regions of a hypothetic Lz/Yki

Conservation of Enok/MOZ roles in cell cycle regulation and progenitor proliferation

Several studies showed that MOZ is required to regulate cell cycle progression, notably for the G1/S transition [START_REF] Sheikh | MOZ and BMI1 Act Synergistically to Maintain Hematopoietic Stem Cells[END_REF][START_REF] Perez-Campo | MOZ-Mediated Repression of P16 INK 4 a Is Critical for the Self-Renewal of Neural and Hematopoietic Stem Cells: MOZ: Key Regulator of Proliferation versus Senescence[END_REF]) in hematopoietic and neural stem cells, and in particular to maintain them in a quiescent state. Furthermore, MOZ catalytic activity has been shown to be required for the proliferation of hematopoietic progenitors (F. M. Perez-Campo et al. 2009), but not for their ability to generate all blood cell types. These molecular functions are conserved in Enok, as it is required to control the G1/S transition by controlling the PCNA unloading properties of Elg1 in S2 cells (Huang et al. 2016), and for the proliferation of neuroblasts during mushroom bodies development [START_REF] Scott | Enok Encodes a Drosophila Putative Histone Acetyltransferase Required for Mushroom Body Neuroblast Proliferation[END_REF]. However, we don't know whether Enok is also required for cell cycle control and progenitor proliferation during hematopoiesis.

I showed during my PhD training that the catalytic activity of Enok is not required for crystal cell differentiation; however, we did not assess the effect of the catalytic loss of function of Enok on the proliferative capacities of plasmatocytes, are responsible for the expansion of the circulating pool of hemocytes [START_REF] Makhijani | The Peripheral Nervous System Supports Blood Cell Homing and Survival in the Drosophila Larva[END_REF]Leitão et Sucena 2015).

Although we did not observe any reduction in circulating hemocyte number in enok mutants, it would be necessary to study properly plasmatocyte proliferative abilities in an enok KAT mutant context by using specific markers, such as phospho-Histone H3. Finally, it may be also interesting to analyze the effect of enok KAT mutation on progenitors of the lymph gland in the medullary zone and the posterior lobes.

Mechanistic hypotheses accounting for catalytic-independent regulation of lz expression by Enok in larval Hematopoietic Pockets

A recent study showed that during development, Enok and its partner Br140, along with Polycomb complexes, are detected on bivalent promoters of developmentally regulated genes (Kang et al. 2017). Bivalency is achieved on promoters with specific histone marks: the In addition to this possible scenario, some evidences in the literature led me to build another hypothesis explaining the Enok-dependent lz regulation. It has been shown in human intestinal cells that the polyadenylation factor Symplekin is required for the direct transcriptional inhibition of RUNX1 thus preventing intestinal goblet cell differentiation (Buchert et al. 2009). In addition, Symplekin is directly recruited on HOXA9 promoter in KG1

AMLO immature hematopoietic cells to prevent recruitment of MOZ and MLL, therefore regulating HOXA9 expression (Largeot et al. 2013). These mechanisms might be conserved on lz regulatory regions in Drosophila, which possesses a Symplekin homolog. It is therefore possible that the mechanism of lz regulation relies on a binding competition between Symplekin and Enok for the regulatory regions of lz .

Finally, a third mechanistic hypothesis can be elaborated based on the observation that, in hematopoietic derived M1 and Jurkat T cells, MOZ has been shown to potentiate RUNX1-mediated transcription through a direct interaction [START_REF] Kitabayashi | Activation of AML1-Mediated Transcription by MOZ and Inhibition by the MOZ-CBP Fusion Protein[END_REF][START_REF] Bristow | Transcriptional Regulation of the Human MIP-1alpha Promoter by RUNX1 and MOZ[END_REF].

This interaction is mediated by MOZ N-terminal and C-terminal parts, and has been demonstrated to be independent of MOZ catalytic activity [START_REF] Kitabayashi | Activation of AML1-Mediated Transcription by MOZ and Inhibition by the MOZ-CBP Fusion Protein[END_REF]. Thus, conservation of this catalytic-independent MOZ/RUNX1 interaction across evolution would provide a simple mechanistic explanation for the non-catalytic mode of action of Enok in the control of lz expression during plasmatocyte to crystal cell transdifferentiation. Although the enok M31.1 mutant allele revealed that the C-terminus of Enok is not required for larval crystal cell formation in the hematopoietic pockets, it is still possible that its conserved N-terminal part might be involved in an interaction with Lz itself. Interestingly, while the cis-elements required to ensure lz autoregulation during embryogenesis are located in the lz-upstream region (Ferjoux et al. 2007), I identified RUNX binding sites in the lz VT059215 region, raising the possibility that this region might also be the target of an autoregulatory loop. An interaction between Enok and Lz would therefore explain how Enok controls lz expression via the third intron enhancer. This hypothesis implies that Enok and Lz have the capacity to interact physically, which can be tested in co-immunoprecipitation experiments. Should this hypothesis be proven, subsequent ChIP-on-ChIP experiments could be used to assess if this interaction occurs on the third intron enhancer. In addition, mutagenesis of the RUNX binding site in the lz VT059215 -RedStinger reporter and/or endogenous lz intron could provide additional elements allowing us to rule out / to establish a possible role for a conserved MOZ/RUNX interaction during Enok-dependent control of lz expression in crystal cell precursors.

Mechanistic hypothesis accounting for Enok-dependent negative regulation of Srp/Lz-transactivation in cultured cells

It has been reported previously that, in Drosophila cultured Kc167 cells, Enok acts as a negative regulator of Srp/Lz-mediated transcription [START_REF] Gobert | A Genome-Wide RNA Interference Screen Identifies a Differential Role of the Mediator CDK8 Module Subunits for GATA/ RUNX-Activated Transcription in Drosophila[END_REF]. This repressor activity might also find its source in an interaction between Enok and Lz, which would antagonize Srp/Lz-dependent transcription. Indeed Srp and Enok could compete for interaction with Lz; alternatively, the formation of a ternary Srp/Lz/Enok complex might induce conformational changes that render the complex less efficient to promote transcription of the PPO1 and PPO2 target genes.

A possible conservation of KAT independent functions in the MYST family

The novel idea that there are catalytic independent functions of Enok and seemingly MOZ Hematopoiesis is a very tightly regulated process leading to the normal production of every blood cells in an organism. In mammals there is a lot of cell types that participate to the establishment of the defense mechanisms of the body. All those cells come from the terminal differentiation of a single cell called Hematopoietic Stem Cell (HSC) which will differentiate to give rise to committed progenitors that will eventually differentiate all blood cell types. HSCs are among the most controlled cells in the organism. Indeed, deregulation of their normal function (excessive proliferation, premature differentiation, …) can be at the onset of severe blood pathologies called leukemias.

Several molecular factors are involved in HSC regulation, and some of them, like RUNX1 and the Monocytic Leukemia Zinc-Finger protein (MOZ), are targets of mutations or chromosomal rearrangements that lead to a leukemic transformation.

In Drosophila, blood cells share functional homology with the mammalian myeloid lineage, and the molecular actors controlling their formation are well conserved. Indeed, differentiation of crystal cells (CC), which have similar functions than megakaryocytes, occurs following the interaction of RUNX1 homolog, Lozenge (Lz) and GATA1 Serpent (Srp). With the aim at identifying regulators of Srp/Lz transcriptional activity, a genome-wide screen led by my team allowed the identification of enoki mushroom (enok) as a strong negative regulator of this activity. Enok is the homolog of MOZ in Drosophila, and a preliminary study in vivo showed that Enok is essential for CC development during the larval stage, as CC almost completely disappear in enok loss of function context.

The objective of my PhD was to understand the mechanisms by which Enok regulates CC formation in the Drosophila larvae.

During the larval stage, CC are generated by the transdifferentiation of macrophages, after their activation by the Notch signaling pathway and onset of Lz expression. In contrast to data published by another group, I demonstrated that Lz is required and sufficient to initiate the expression of the Hippo signaling pathway effector Yorkie, and not the contrary. Furthermore, using loss of function and rescue experiments, I showed that Enok is absolutely required cell autonomously in CC precursors for Lz expression but not for the proper Notch signaling which appears normal in an enok mutant context.

In mammals, MOZ is known to be a part of an acetylation complex, and I showed that only one of its members, the BRPF factor Br140, is required alongside with Enok during CC differentiation. To go further into the characterization of Enok mode of action, I generated a catalytically inactive enok mutant using the CRISPR/Cas9 system and demonstrated that this function is completely dispensable for CC differentiation in the larvae.

Finally, I show that Enok regulates the maintenance of lz expression by binding to an enhancer located in its third intron. Hence, deletion of this enhancer provokes the same loss of CC phenotype as what is observed after enok loss of function showing that this enhancer is required for lz expression in the hematopoietic system.

These results highlight a new mode of action of Enok during Drosophila hematopoiesis. Interestingly, a recent study proposed that Enok and Br140 bind chromatin together in order to regulate gene expression. Therefore, my results on a non-catalytic activity of the drosophila Enok brings new insights into this new model, which uncovers a mode of action not much explored in the field. In the future, my work should open new avenues and might allow a better understanding of MOZ normal functions in mammals and thus give rise to more efficient ways to treat MOZ-associated pathologies.

Figure 1 :

 1 Figure 1: Representation of the mammalian hematopoietic tree

Figure 2 :

 2 Figure 2: Embryonic waves of hematopoiesis in mammalsA. During the first wave, primitive hematopoietic cells (in red) arise from cells inside blood islands (in blue) in the yolk sac. They migrate to the embryo proper, where they differentiate into primitive erythrocytes, macrophages and megakaryocytes. B. During the second wave, the first definitive blood cells arise. These cells are Erythromyeloid Myeloid Progenitors (EMPs, in yellow); they originate from the hemogenic endothelium (in green) of the yolk sac through the Endothelial to Hematopoietic Transition process and migrate to the fetal liver, where they proliferate and differentiate. C. The third wave gives rise to the hematopoietic stem cells (HSCs). They emerge from the hemogenic endothelium of major arteries in the embryo, and in particular in the dorsal aorta. The emerging pre-HSCs will then, like EMPs, migrate to the fetal liver to expand and differentiate. At the end of embryogenesis, newly formed hematopoietic cells will migrate again to colonize the bone marrow niche. (Adapted from[START_REF] Tavian | Embryonic Development of the Human Hematopoietic System[END_REF]

Figure 3 :

 3 Figure 3: RUNX transcription factors A. Schematic representation of the three human RUNX proteins, along with the Drosophila Runt and Lozenge RUNX factors. Runt is the founding member of the RUNX family; Lozenge is a RUNX gene ortholog, and the only identified RUNX protein involved Drosophila hematopoiesis. All RUNXs possess a highly conserved Runt domain responsible for DNA binding and a VWRPY motif involved in interactions with co-repressors. B. Schematic representation of the leucemogenic fusion protein RUNX1-ETO: the full-length Runt domain of RUNX1 is fused to the ETO protein, which contains four Nervy Homology Region (NRH) domains that are mostly known to allow interaction with repressors.(Adapted from[START_REF] Ito | The RUNX Family: Developmental Regulators in Cancer[END_REF]

Figure 4 :

 4 Figure 4: Overview of the Notch signaling pathway.A. When the pathway is inactive, the CSL transcription factor is bound by co-repressors and Notch target genes are repressed. B. Notch pathway is activated by the interaction between one Delta Serrate Ligand (DSL) and the Notch ExtraCellular Domain (NECD) of the receptor. Upon interaction, the receptor undergoes a series of cleavages (S2 and S3) that lead to the translocation of the Notch IntraCellular Domain (NICD) into the nucleus. There, it interacts with its CBF1/RBPJ, Supressor of Hairless (Su(H)), Lag-1 (CSL, blue) partner and with Mastermind (Mam, green) to replace corepressors and activate transcription of target genes. (Adapted from[START_REF] Bray | Notch Signalling: A Simple Pathway Becomes Complex[END_REF]

Figure 5 :

 5 Figure 5: MYST family of acetyl transferases A. Schematic representation of human and Drosophila MYST proteins. All of them possess a conserved MYST domain with catalytic activity. B. Schematic representation of MOZ and the main fusion proteins involved in leukemia. In the MOZ-CBP fusion, the N-terminal part of MOZ comprises a functional MYST domain fused to the C-terminal part of CBP. In the MOZ-TIF2 fusion, the same breakpoint in MOZ is fused to the CBP Interacting Domain (CID) domain of TIF2, which is responsible for CBP binding.(Adapted from[START_REF] Yang | The Diverse Superfamily of Lysine Acetyltransferases and Their Roles in Leukemia and Other Diseases[END_REF][START_REF] Yoshida | Chromatin Regulation by AML1 Complex[END_REF]

  b) MOZ during normal hematopoiesisMOZ was first shown to display a KAT activity on histone tails (N.[START_REF] Champagne | The Monocytic Leukemia Zinc Finger Protein MOZ Is a Histone Acetyltransferase[END_REF] and has since been studied mainly considering this property. Indeed, in a murine model carrying a point mutation in the catalytic site of MOZ, Perez-campo et al showed that MOZ-mediated acetylation is required for proliferation and expansion of hematopoietic progenitors (F. M.Perez-Campo et al. 2009). Later, the same group showed that MOZ catalytic activity is required to prevent HSCs from entering into replicative senescence, thus promoting their self-renewal capacity (Flor M.[START_REF] Perez-Campo | MOZ-Mediated Repression of P16 INK 4 a Is Critical for the Self-Renewal of Neural and Hematopoietic Stem Cells: MOZ: Key Regulator of Proliferation versus Senescence[END_REF]. MOZ is also required in for their maintenance, since its loss of function in the embryo leads to the absence of hematopoietic progenitors and adult-repopulating cells in the fetal liver[START_REF] Katsumoto | MOZ Is Essential for Maintenance of Hematopoietic Stem Cells[END_REF][START_REF] Thomas | Monocytic Leukemia Zinc Finger Protein Is Essential for the Development of Long-Term Reconstituting Hematopoietic Stem Cells[END_REF].Furthermore, specific MOZ loss of function in the adult bone marrow compartment leads to a rapid loss of HSCs (B. N.[START_REF] Sheikh | MOZ (KAT6A) Is Essential for the Maintenance of Classically Defined Adult Hematopoietic Stem Cells[END_REF], as measured by their loss of ability to reconstitute the hematopoietic system in an irradiated mouse recipient. Finally, it was shown that MOZ acts in collaboration with the polycomb group protein BMI1 to maintain HSCs in a quiescent state (Bilal N.[START_REF] Sheikh | MOZ and BMI1 Act Synergistically to Maintain Hematopoietic Stem Cells[END_REF]), ensuring their maintenance over time.

  (embryonic lethality and strong depletion of the hematopoietic system) and phenotypes of mice specifically deprived of its catalytic function (F. M.Perez-Campo et al. 2009) (viable with reduced progenitor proliferation, but no effect on differentiation). These observations strongly support the hypothesis that MOZ has other non-catalytic functions during both development and hematopoiesis.It has been shown that MOZ has broad functions during development, in addition to its roles in hematopoiesis. Indeed, it is an important player during embryonic development, as it regulates histone acetylation of HOX genes during mouse body segmentation[START_REF] Voss | Moz and Retinoic Acid Coordinately Regulate H3K9 Acetylation, Hox Gene Expression, and Segment Identity[END_REF][START_REF] Sheikh | MOZ and BMI1 Play Opposing Roles during Hox Gene Activation in ES Cells and in Body Segment Identity Specification in Vivo[END_REF].c) MOZ and the ING5 acetylation complexKATs of the MYST family generally act as members of acetylation complexes (reviewed in[START_REF] Lee | Histone Acetyltransferase Complexes: One Size Doesn't Fit All[END_REF]. It has been shown that MOZ is a part of such an acetylation complex, working within the ING5 complex, along with Esa1-Associated-Factor 6 (EAF6), Inhibitor of Growth 5 (ING5) and BRPF1 2 or 3 (Figure6;[START_REF] Ullah | Molecular Architecture of Quartet MOZ/MORF Histone Acetyltransferase Complexes[END_REF] to specifically modify histone H3. In this complex, MOZ interacts directly with BRPF factors via its MYST domain, and BRPF itself interacts directly with EAF6 and ING5[START_REF] Ullah | Molecular Architecture of Quartet MOZ/MORF Histone Acetyltransferase Complexes[END_REF]. BRPF proteins are epigenetic readers that recognize specific histone modifications in order to bind chromatin and allow

Figure 6 :

 6 Figure 6: The ING5 acetylation complexesMOZ, which is the catalytic subunit of the complex, directly interacts with the BRPF factors. The other members, EAF6 and ING5, also specifically interact with BRPF but not directly with MOZ. Together, the complex is able to recognize precise chromatin motifs that allow acetylation of a specific repertoire. The BRPF factor recognizes the histone H3 tail via its PHD fingers, ING5 recognizes trimethylation of H3K4, and MOZ recognizes acetylated H3K9 and H3K14. In turn, MOZ will acetylate another H3 tail on lysine 9 or 14. (Adapted from[START_REF] Ali | Tandem PHD Fingers of MORF/MOZ Acetyltransferases Display Selectivity for Acetylated Histone H3 and Are Required for the Association with Chromatin[END_REF]

Figure 7 :

 7 Figure 7: Drosophila blood cells Schematic representation of blood cell types in Drosophila. Progenitors, or prohemocytes, give rise to plasmatocytes, which are macrophages, and to crystal cells, which have crystalline inclusions and are functional homologs of the mammalian megakaryocytes. Upon an immune challenge like wasp infestation, prohemocytes in the larva are able to differentiate into lamellocytes; these are big cells able to encapsulate the wasp egg. It also has been shown that plasmatocytes in the larval stages are able to transdifferentiate into crystal cells or upon infestation into lamellocytes. (Adapted from Rizki1957)

Figure 8 :

 8 Figure 8: Drosophila life cycle and hematopoiesisA. During embryonic development, cells of the procephalic mesoderm and cardiogenic mesoderm (in green) develop to give rise to both circulating hemocytes (blue and purple) and to the lymh gland (green), apposed on the cardiac tube (orange). The embryo develops into a larva, then a pupa and finally an adult, where hematopoiesis occurs within Hematopoietic Hubs (HB). B. Representation of larval hematopoietic pockets during the third larval stage. In this microenvironment, plasmatocytes (brown) are in contact with neurons (green) that emit signals to promote plasmatocyte homing and proliferation. Within this microenvironment, plasmatocytes can give rise to larval crystal cells by transdifferentiating. C. Schematic representation of the lymph gland during the third larval stage. In the medullary zone of the anterior pair of lobes (MZ, blue), progenitors are mostly quiescent with a low rate of proliferation. They give rise to differentiated cells (plasmatocytes, red, and crystal cells, purple) located in the cortical zone (CZ). During wasp infestation, progenitors in the MZ are instructed by the Posterior Signaling Center (PSC) to become lamellocytes. The posterior lobes (also blue) along the cardiac tube will in the adult stage become parts of the HB. (Adapted fromLetourneau et al 2016; Gold and Brückner 2014).

  e) Hematopoiesis in the adult flyAlong with cells generated during the first hematopoietic wave, cells produced by the lymph gland will persist during the adult stage. In addition, it has been shown that there is a slight hematopoietic activity during adult life, in clusters located along the abdominal part[START_REF] Ghosh | Active Hematopoietic Hubs in Drosophila Adults Generate Hemocytes and Contribute to Immune Response[END_REF].Blood cells from lymph gland secondary lobes migrate in these clusters calledHematopoietic Hubs (HBs) to become progenitors of the new hematopoietic wave.Progenitors in HBs display proliferation abilities and give rise to both plasmatocytes and crystal cell lineages (S Ghosh,[START_REF] Ghosh | Detecting proliferation of adult hemocytes in Drosophila by BrdU incorporation and PH3 expression in response to bacterial infection[END_REF], which actively participate in protecting against infection.3. Molecular actors during Drosophila hematopoiesisIt has been shown in the last fifteen years that there is a strong conservation from Drosophila to mammals of the molecular actors regulating hematopoiesis. Indeed, key hematopoietic regulators belonging to the GATA and RUNX family of transcription factors are crucial actors during Drosophila hematopoiesis, as well as the Notch signaling pathway. I will describe below how they interact and participate in the establishment of blood cell lineages in Drosophila. a) GATA transcription factors In Drosophila there are five GATA factors: Serpent (Srp), Pannier (Pnr), GATAe, GATAd and Grain. They are major regulators of development, but only Srp and Pnr have been identified

Figure 9 :Figure 10 :

 910 Figure 9: Overview of the Hippo signaling pathway in DrosophilaWhen the Hippo pathway is inactive, its effector Yorkie competes in the nucleus with Tondu domaincontaining growth inhibitor (Tgi) for interaction with Scalloped (Sd) and activates transcription of target genes. The pathway is activated by the Tao kinase, which induces the phosphorylation cascade by phosphorylating Hippo. In turn Hippo phosphorylates Warts, which phosphorylates Yorkie. Yorkie is then sequestrated in the cytoplasm by the interaction with 14-3-3, and Tgi interacts with Sd and represses Yorkie target genes. (Adapted from[START_REF] Meng | Mechanisms of Hippo Pathway Regulation[END_REF]

Figure 1 .

 1 Figure 1. enok is required for plasmatocyte transdifferentiation into larval crystal cells. (A) Melanization assay on larvae from different enok mutant contexts. Posterior side of larvae is to the right. Melanized crystal cells appear as black dots. (B-C) Immunofluorescent staining on circulating cells from larvae of the indicated genotypes (Df-B155: abbreviation for Df(2R)BSC155); cells were stained with DAPI and either a-PPO1 (B) or a-Lz (C) antibody. (D) Quantification of the proportions of NRE-GFP + cells expressing Lz as revealed by an immunofluorescent staining. Each dot represents the proportion observed in an individual larva. Complete genotypes: NRE-GFP/+ (control) and enok 1 ,NRE-GFP/Df(2R)BSC155 (enok 1 /Df-B155). (E) Quantification of the proportions of NRE-GFP + cells expressing the plasmatocyte specific marker P1 as revealed by an immunofluorescent staining. (B-E) Hemocytes were collected on larvae carrying a NRE-GFP transgene. (D,E) Statistics: **** indicate a p-value≤0.0001.

  Figure 1

  ), restores normal crystal cell maturation in hematopoietic pockets(Fig 2A), further demonstrating that the loss of crystal cell linage is due to the loss of enok function. In addition, our result indicates that enok ensures a cell-autonomous function in the Notch-activated precursors. Furthermore, thorough quantification of the proportion of Lz-expressing cells in circulation confirms that re-expressing enok specifically in crystal cell precursors of enok mutant larvae is sufficient to restore Lz expression completely (Fig.2B). We thus conclude that enok controls the transdifferentiation of plasmatocytes into crystal cells through a cellautonomous regulation of Lz expression in Notch-activated precursors.The catalytic activity of Enok and the ING5 complex are dispensable for crystal cell formation.

Figure 2 .

 2 Figure 2. Notch GMR30A01 -Gal4-driven expression UAS-3HA-enok transgene in enok mutants restores normal crystal cell differentiation. (A) Melanization assay on larvae of the indicated genotypes. Complete genotypes: w 1118 (control), enok 1 /Df(2R)BSC155 (enok 1 /Df-B155), enok 1 /Df(2R)BSC155; tubulin-Gal4/UAS-3HA-enok (rescue tub-Gal4) and enok 1 /Df(2R)BSC155; Notch GMR30A01 -Gal4/UAS-3HA-enok (rescue Notch GMR30A01 -Gal4). (B) Quantification of the proportions of total hemocytes expressing Lz as revealed by immunofluorescent staining. Each dot represents the proportion of Lz + cells in an individual larva. Statistics: Notch GMR30A01 -Gal4/+ genotype was used as a reference sample; **** indicate a p-value≤0.0001, ns: not significantly different from reference sample.

Figure 3 .

 3 Figure 3. enok and Br140 are required independently of the ING5 complex for crystal cell differentiation. (A) Melanization assay on larvae of the indicated genotypes (Df-B263: abbreviation for Df(2R)BSC263, Df-B387: abbreviation for Df(3L)BSC387). (B) Quantification of the proportions of NRE-GFP + cells expressing Lz as revealed by immunofluorescent staining. Each dot represents the proportion of Lz + cells in an individual larva. Complete genotypes: NRE-GFP/+ (control), enok 1 , NRE-GFP/Df(2R)BSC155 (enok 1 /Df-B155), Br140 S781 , NRE-GFP/Df(2R)BSC263 (Br140 S781 /Df-B263) and enok KAT , NRE-GFP/Df(2R)BSC155 (enok KAT /Df-B155). Statistics: control genotype was used as a reference sample; **** indicate a p-value≤0.0001, ns: not significantly different from reference sample.

Figure 4 .

 4 Figure 4. Lz precedes Yki expression during plasmatocyte transdifferentiation into crystal cells. (A) Immunofluorescent staining on circulating cells from larvae of the indicated genotypes; cells were stained with DAPI and a-Yki. (B) Quantification of the proportions of NRE-GFP + cells expressing Yki as revealed by immunofluorescent staining. Complete genotypes: NRE-GFP/+ (control) and enok 1 ,NRE-GFP/Df(2R)BSC155 (enok 1 /Df-B155). (C) Relative fluorescence intensity of the NRE-GFP transgene plotted against the relative fluorescence intensity of Yki staining in total hemocytes of control larvae; blue dots are cells negative for Lz expression and red dots are cells positive for Lz expression. The vertical dashed line represents the threshold for NRE-GFP positive cells (upper part) and the horizontal dashed line represents the threshold for Yki positive cells (right part). (D) Relative fluorescence intensity of the NRE-GFP transgene plotted against the relative fluorescence intensity of Lz staining in total hemocytes of control larvae; blue dots are cells negative for Yki expression and red dots are cells positive for Yki expression. The vertical dashed line represents the threshold for NRE-GFP positive cells (upper part) and the horizontal dashed line represents the threshold for Lz positive cells (right part). The dashed ellipse encloses an unexpected population of cells that express both the NRE-GFP reporter and Lz, but that do not express Yki. (E) Fluorescence intensity of Yki staining in NRE-GFP + cells (relative to the mean Yki intensity measured in all NRE-GFP + cells of control larvae) in larvae of the following genotypes: w 1118 /Y; NRE-GFP/+ (control) and lz R1 /Y; NRE-GFP/+ (lz R1 ). (F) Overexpression of UAS-lz, UAS-yki or UAS-yki* using the hml D -Gal4 driver. Left panel: proportions of total circulating hemocytes expressing Lz. Right panel: proportions of total circulating hemocytes expressing Yki. Complete genotypes are as follows: w/w; hml D -Gal4/+ (control), w/UAS-lz; hml D -Gal4/+ (UAS-lz), w/w; hml D -Gal4/UAS-yki-GFP (UAS-yki) and w/w; hml D -Gal4/+; UAS-yki S168A -GFP/+ (UAS-yki*). (B,F) Each dot represents the proportion observed in an individual larva. (C,D,E) Each dot represents the values attributed to a single cell. (B,E,F) Statistics: control genotype was used as a reference sample; **** indicate a p-value≤0.0001; *** indicate a p-value≤0.0010, ns: not significantly different from reference sample.

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Figure 5 .

 5 Figure 5. Enok binds a hematopoietic enhancer in lz third intron. (A) Immunofluorescent staining on circulating cells from NRE-GFP/+; lz VT059215 -Gal4,UAS-RedStinger/+ larvae; cells were stained with DAPI and a-Lz antibody. (B) Proportions of the different categories of NRE-GFP + cells quantified in NRE-GFP/+; lz VT059215 -RedStinger/+ larvae. Each dot represents the proportion of the indicated cell type observed in an individual larva. (C) Immunofluorescent staining on circulating cells from larvae of the indicated genotype; cells were stained with DAPI and a-Lz antibody. (D) Quantification of the proportion of NRE-GFP + cells expressing Lz in lz int3 and control male larvae, as revealed by immunofluorescent staining. Each dot represents the proportion of Lz + cells in an individual larva. (E) Melanization assay on larvae of the indicated genotypes. (F) Quantification of the proportion of total hemocytes expressing Lz as revealed by immunofluorescent staining. Each dot represents the proportion of Lz + cells in an individual larva. Statistics: w/Y; lz VT059215 -Gal4/+ genotype was used as a reference sample. (G) Top: schematic representation the lz locus (not to scale); in purple are represented the amplicons used in Chromatin Immunoprecipitation (ChIP). Bottom: results of ChIP experiments performed on tub-Gal4/UAS-3HA (3HA, orange histograms) and tub-Gal4/UAS-3HA-enok whole embryos (3HA-Enok, blue histograms) using an a-HA antibody. (D,F,G) Statistics: *** indicate a p-value≤0.0001, * indicate a p-value≤0.0500, ns: not significantly different from reference sample.

Figure 6 .

 6 Figure 6. Model of stepwise plasmatocyte to crystal cell transdifferentiation in larval Hematopoietic Pockets. We hypothesize that in the sessile microenvironment, similar to what has been described in the lymph gland, a Serrate-expressing inducer cell activates the Notch signaling pathway in a neighboring plasmatocyte. This activated plasmatocyte (crystal cell precursor) responds by expressing the NRE-GFP reporter gene. Notch pathway activation then results in the expression of the crystal cell fate determinant Lz, which in turn ensures downregulation of the plasmatocyte specific marker P1 and promotes expression of yki as well as that of the PPO1 and PPO2 terminal differentiation markers.

Development 140 :

 140 926-937 Ullah M, Pelletier N, Xiao L, Zhao SP, Wang K, Degerny C, Tahmasebi S, Cayrou C, Doyon Y, Goh S-L, Champagne N, Côté J & Yang X-J (2008) Molecular architecture of quartet MOZ/MORF histone acetyltransferase complexes. Mol. Cell. Biol. 28: 6828-6843 Voss AK, Collin C, Dixon MP & Thomas T (2009) Moz and retinoic acid coordinately regulate H3K9 acetylation, Hox gene expression, and segment identity. Dev. Cell 17: 674-686 Waltzer L, Ferjoux G, Bataillé L & Haenlin M (2003) Cooperation between the GATA and RUNX factors Serpent and Lozenge during Drosophila hematopoiesis. EMBO J. 22: 6516-6525 Xin T, Xuan T, Tan J, Li M, Zhao G & Li M (2013) The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche. Dev. Biol. 384: 1-12 B. Complementary results

Figure 11 .

 11 Figure 11. Structure function study of the Enok protein A. In the lower panel is represented the result for the I931E mutation which aims at disabling DNA binding ability of Enok. The sequenced individual was heterozygous, which results in double peaks at the site of interest corresponding to the modified and wild type alleles. Additional silent mutations were introduced for screening purposes. B. Top: schematic representation of the Enok protein. Here are represented at scale the N-terminal part of Enok MOZ MORF (NEMM), the tandem Plant Homeodomains (PHD), the MYST and Neurofilament-like domains. Red arrows represent the location of the sgRNA used for the directed excision. Bottom: representation of all mutant alleles recovered during the screen. Left: allele names / Right: result on translated proteins. In frame deletions are represented in red, and new stop codons by red crosses.

Figure 13 .

 13 Figure 13. The lz 2AB enhancer is crucial for lz expression in circulating crystal cell precursors.(A) Immunofluorescent staining on circulating cells from larvae of the indicated genotype; cells were stained with DAPI and a-Lz antibody.

  first one is trimethylation of histone H3 on lysine 27 (H3K27me3), which promotes transcriptional repression, and the second is trimethylation of histone H3 on lysine 4 (H3K4me3), which promotes transcriptional activation. The authors propose a model where, in response to developmental cues, this bivalency is resolved, and the corresponding gene is either transcribed or repressed. My work demonstrated that both Enok and Br140 participate in the same process that is the control of lz expression during crystal cell differentiation. In addition, I showed that Enok is able to directly bind the lz gene in a region that is essential for its expression in circulating crystal cell precursors. Interestingly, in the Drosophila S2 cell line, data from MODencode project reveal that the lz locus lies in a H3K27me3 chromatin domain(Kharchenko et al. 2011) and a previous study byKang et al show that it is heavily bound by Polycomb. According to the model proposed by Kang et al, it is then possible that, in response to Notch activation in plasmatocytes, Enok/Br140 would allow the recruitment of activating factors triggering a switch in chromatin state at the lz locus from a repressed to an active state, thereby promoting lz expression.

  during development raises an interrogation: is this differential requirement for catalytic and non-catalytic functions specific of Enok/MOZ ? The high level of conservation of the MYST domains allows us to introduce the same mutation that I used to abrogate the catalytic activity of Enok in other Drosophila MYST proteins in vivo. Systematic comparison of the phenotypes induced by complete loss of function and specific catalytic abrogation should allow us to establish whether non-catalytic functions are a common feature of all Drosophila MYST proteins or not. Given the versatility of CRISPR/Cas9 techniques, similar approaches could be conducted in vertebrate models like Zebrafish, in order to establish whether all MYSTs lysine acetyltransferases display this dual mode of action.D. Drosophila as a model to study MOZ-related diseasesIt is well established that in mammals MOZ is a crucial player during hematopoietic development and this work demonstrated that Enok also plays an essential role during

Table of Contents

 of

I. Preamble ............................................................................................................ II. Introduction ........................................................................................................

  Those mutations have been classified into two categories (De Kouchkovsky et

	Abdul-Hay 2016). Class I mutations affect genes implicated in proliferative pathways (such as
	c-Kit and STAT3; The Cancer Genome Atlas Research Network 2013; Yamada et Kawauchi
	2013), and class II mutations affect genes implicated in normal blood cell formation (such as
	NMP1 and CEBPA; The Cancer Genome Atlas Research Network 2013); according to the
	classical double hit model of leukemogenesis, class I and II mutations have to occur
	concomitantly to trigger the pathology. However, over the past few years, a third type of

mutation has emerged, which corresponds to mutations targeting epigenetic regulators (such as DNMT3A and TET2;

[START_REF] Patel | Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia[END_REF]

) involved both in proliferation and differentiation. This double hit model is based on punctual mutations, but it does not take into account bigger genomic instabilities such as chromosomal rearrangements (translocations, duplications…), which in some cases can be sufficient to initiate a leukemic state.

Table 1 :

 1 Expression pattern of lz-related Vienna Tile and Janelia lines

Table 1 :

 1 Expression pattern of lz-related Vienna Tile and Janelia lines of MOZ reveals a regulatory mechanism for HOXA9 transcription. Genes Dev. 26: 1376-1391 Scott EK, Lee T & Luo L (2001) enok encodes a Drosophila putative histone acetyltransferase required for mushroom body neuroblast proliferation. Curr. Biol. 11: 99-104

	Strübbe G, Popp C, Schmidt A, Pauli A, Ringrose L, Beisel C & Paro R (2011) Polycomb
	purification by in vivo biotinylation tagging reveals cohesin and Trithorax group proteins as
	interaction partners. Proc. Natl. Acad. Sci. U.S.A. 108: 5572-5577
	Terriente-Felix A, Li J, Collins S, Mulligan A, Reekie I, Bernard F, Krejci A & Bray S (2013)
	Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme.

Table 3 :

 3 Antibody stock list

	antibody raised against	type	source	use	reference
	PPO1	rabbit, polyclonal	kind gift of Dr Erjun Ling	immunofluorescent staining	Li et al., 2012
	Lz	mouse, monoclonal Developmental Studies Hybridoma Bank immunofluorescent staining	Lebestky et al., 2000
	P1	mouse, monoclonal kind gift of Dr istvan Ando	immunofluorescent staining	Kurucz et al., 2007
	H3K23ac	rabbit, polyclonal	Merck Millipore (07-355)	immunofluorescent staining	Suka et al., 2001
	Yki	rabbit, polyclonal	kind gift of Dr Matthew Gibson	immunofluorescent staining	Ikmi et al., 2014
	HA	mouse, monoclonal Biolegend	chromatin immunoprecipitations -

Table 4 :

 4 Primer and CRISPR reagent listDuring my PhD work I described for the first time that Enok, the Drosophila counterpart of MOZ, also plays a critical role during Drosophila larval hematopoiesis. Enok is indeed required for the formation of one of the three blood cell lineages, the crystal cell lineage. It controls the expression of lz, which encodes a RUNX transcription factor specific of the crystal cell lineage. In addition, I showed that Enok plays its role in a cell-autonomous manner in crystal cell precursors that are plasmatocytes activated by the Notch signaling pathway. Also, I demonstrated that, Enok acts independently of its catalytic activity together with only one of its canonic partners, Br140, to control lz expression. Furthermore, I showed that Enok is able to bind specifically the third intron of lz, on a genomic region that drives expression in the crystal cell progenitors and, more importantly, which is able to rescue lz loss of function phenotype by overexpressing lz itself. In addition, excision of this third intron leads to a hematopoietic specific loss of lz function, since mutant larvae are deprived of crystal cells in circulation. Then, I will discuss about the catalytic independent roles and mode of action of Enok throughout development and more particularly during hematopoiesis. Finally, I will discuss the possibility that Drosophila could be a valuable model to study the molecular mechanisms involved during MOZ-related diseases in mammals.B. A possible role for Yki in Hematopoietic PocketsI demonstrated that the regulation of lz expression in circulating Notch-activated hemocytes is not the same as what has been shown by Ferguson et al in the lymph gland(Ferguson et Martinez-Agosto 2014). Indeed, the Hippo pathway effector Yorkie is not initiating lz expression in circulating crystal cell precursors, since in this compartment lz expression precedes that of yki. There are major differences between the sessile/circulating and lymph gland compartments, and thus crystal cell differentiation is likely to rely on different mechanisms depending on the cellular context. Indeed, it is reported that in the lymph gland crystal cells are formed directly from the de novo differentiation of naïve progenitors, whereas in Hematopoietic Pockets crystal cells are generated by the transdifferentiation of mature, fully differentiated plasmatocytes

		Cloning primers
	3xHANotI-Enok	GCTGCTCATGCGGCCGCTATGAGGGAATCGGCGCATGATATCAAC
	KpnI-STOP-Enok	AAAGATCCTCTAGAGGTACCCGCATCTATTATCTGCGAATAGAACCGTTGAG
	VT059215 RedStinger F	CCCGGTCTCCAAATGCACTGACCTTTTGGCCG
	VT059215 RedStinger R	CCCGGTCTCCCGTAACTCAATCCCGAAATGTCTTCA
		Chromatin Immunoprecipitation primers
	A1 F	GCTGACGTTCTTCTCACGCT
	A1 R	TCCGACTTATCACATCGAAGGG
	A2 F	TTCGACCACAAACCAAGGCT
	A2 R	AGCGAAGGAAAGGCGTGATA
	A3 F	AACAGCACTGCGTGGAATGG
	A3 R	GCTCTTCTGTAAATGCATGGCC
	A4 F	AGTAAACAAAAAGCGCGCCA
	A4 R	AGTTGGTGACGAAATTCGAGTT
	A5 F	GACTTAGTGGTCGCATGGGC
	A5 R	TTCCGTGGAAAACTGCCTGT
	A6 F	GGATGCTCCACATTTTGCGG
	A6 R	ATAGGCCTGGCTCCTTTTCG
		CRISPR guide RNA
	guide K807R	CGTCGTAGTAGAGTGTTTTG
	guide ING5 5'	CTGTAAAGTAATCGATAACT
	guide ING5 3'	ATCCACGCGTGCCAAATCCG
	guide EAF6 5'	ACTGGCTTCAAAATGCAAAG
	guide EAF6 3'	AGTGCGACAGCGAAGAAAAG
	guide lzint3 3'	AGCGGCACGTCATTAGAGAG
	guide neuro 1 5'	CATCGGCACCCATTCCCATT
	guide neuro 1 3'	GTCCAGCCAAGGAAATAACT
	guide neuro 2 5'	AGTTGGATCTTGTCCCGAAT
	guide neuro 2 3'	TCCCTAGTTATTTCCTTGGC
	guide I931E	AGTTCAATAACTCGAAGGCC
	guide lzint3 5'	GTGCTGTCCGAACGGTCACT
		ssODN sequence
	ssODN K807R	ATTTATTGCCAGAACCTGTGCCTGCTGGCCAAGTTCTTTCTCGACCACAGGACGTTATACTACGACGTAGAACCCTTTCTGTTCTATATTCTCACTAAGAATGAT CAAAGCGGGTGTCATTTGGTTGGCTACTTCTCAAAGGAGAAGCACTGCACCCAAAAGTACAATGTGTCCGCTATCCTGACGATGCCGCAGTAC
	ssODN I931E	GTCGTGTTGGAGTATTTGTACAAGCATCGCAACTACACAAAGATCACCTTCAAGGACATCGCCATTAAAACAGGTCTGGCTATTTCCGACGAGGCTTTAGCCT TCGAGTTATTGAACTTCATCAAGCTAAGAAAGAACGATGGCGACATTAGGTATCAGATTAATGTGAAAATTGAGTGGAAAAAGGTTTTGGCCCAC
		CRISPR genotyping primers
	Geno Ing5 F	TGCCCAATTGTTTTGGGTTA
	Geno Ing5 R	TCCGAATTAAAGGGCATTGA
	Geno Eaf6 F	TGACGCGATGCTGAAAGTAG
	Geno Eaf6 R	CCACGGGGAGATCATTAGAA
	Geno lzint3 F	CGCAACTTCACCACCCAGAT
	Geno lzint3 R	TGATGGCTTTGGCGTAGGTT
	Geno enok F	GGATGGCAATGTCAACAAGA
	Geno enok R	TCCAACACGACTGATTTCCA
	K807R F	TTCTCGACCACAGGACGTTA
	geno I931E F	GCTTCCTGATCGACTTCAGC
	geno I931E R	TGGTTGCTCAAATATTCACGAT
	I931E F	GCTATTTCCGACGAGGCTTTA
	geno ΔNeuro F	TCCGAGGAGCCTCAGTTCA
	geno ΔNeuro R	TGCGTCCAGGTGAGAGAGA

In the last part of my training, I generated specific deletions in the enok gene using the CRISPR/Cas9 system. While the Neurofilament-like domain of Enok appears dispensable for its functions, deletion of the C-terminus of enok induces lethality without affecting hematopoiesis or its ability to acetylate H3K23. This phenotype indicates an essential role of Enok for survival, which relies on its C-terminal part.

during my PhD work. I showed that lz expression in Hematopoietic Pockets is not induced by Yki. Since Yki expression is detected in NRE-GFP positive cells, I will first discuss about a potential role of Yki during crystal cell differentiation.

  complex, this third intron itself is a good candidate, as it could be required for the maintenance of lz expression in differentiating crystal cells.YAP interacting domain on RUNX1 has been identified, thus if the amino acid sequence homology is sufficient between RUNX1 and Lz, it would be possible to mutate the identified Yki-interacting domain in Lz, and see if Lz/Yki interaction is required for correct lz expression during crystal cell differentiation in vivo.Catalytically inactive enok mutants are viable and I observed that homozygous adult females are sterile. This may be due to a requirement of this KAT activity in the germline, as it has been shown that Enok plays an essential role both in the maintenance of germline stem cells[START_REF] Xin | The Drosophila Putative Histone Acetyltransferase Enok Maintains Female Germline Stem Cells through Regulating Bruno and the Niche[END_REF]) and oocyte polarization[START_REF] Huang | Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire[END_REF]) in females. Since catalytically inactive mutant females do not display any obvious defect in egg-laying, the sterility is likely due to an embryonic development failure. Huang et al demonstrated that enok mutant germline clone embryos (lacking maternal contribution) die during embryonic development due to a segmentation defect[START_REF] Huang | Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire[END_REF]. It could be interesting to look at the early stages of development in enok KAT mutant embryos to determine if the arrest in embryonic development is caused by the same segmentation defect.Thanks to the strong enok maternal contribution, mutant phenotypes only appear in the larval stages, thus preventing us from characterizing easily a potential function of enok in embryonic hematopoiesis. However, enok mutant germline clone embryos develop until at least embryonic stage 12. Since srp expression is detected in prohemocytes at stage 5, and crystal cells specific markers PPO1 and PPO2 are expressed at stage 11, enok mutant germline clone embryos can be used to address the question of enok function in embryonic hematopoiesis. If hematopoietic defects are observed in germline clone embryos, the comparison with embryos laid by enok KAT mutant females should establish whether they depend on the catalytic activity of Enok or not. Alternatively, it could be possible to investigate enok function during embryonic hematopoiesis by using targeted RNA interference. In such experiments it would be possible to investigate specifically enok involvement at different steps of the hematopoietic process, such as prohemocyte emergence or differentiation.

C. Study of Enok catalytic-independant activity during Drosophila development 1. enok KAT activity during embryogenesis

Acknowledgments

We are grateful to Leila Abraham, Camille Calvel, Maxime Fayel and Delhia Gigan for their participation in mutant fly strain engineering, Amélie Destenabes and Julien Favier at the CBD fly facilities for transgenesis and CRISPR/Cas9 injections, Caroline Monod for critical reading of the manuscript and all our colleagues at the CBD for valuable comments at various steps of the project. We thank Brice Ronsin, Stéphanie Bosch, and Jessie Bourdeaux at the Toulouse RIO imaging platform for assistance with confocal microscopy and Operetta imaging and Benjamin Tritschler for invaluable help with logistics. We are also deeply grateful to Drs Erjun Ling, Takashi Suzuki, Matthew Gibson, Steven Hou, Konrad Basler and Jean-Philippe Combier for sharing resources, and to the Bloomington stock center, the Vienna Drosophila Ressource Center, the Kyoto stock center and the Developmental Studies Hybridoma Bank for providing us with antibodies and fly stocks. T.G was supported by a doctoral grant from La Ligue Nationale contre le Cancer, and the project benefitted from the support of the Centre National de Recherche Scientifique (CNRS) and the Université Toulouse III -Paul Sabatier (UPS).

68

V. Material and methods

A. Thesis article in preparation

Engineering mutants carrying an excision of the Neurofilament domain

In addition to the MYST domain, there are a few identified domains in Enok, and the largest one is called the Neurofilament-like domain. Its function is not described in the literature and thus, using CRISPR/Cas9-mediated deletions, I undertook to excise the Neurofilament-like domain and look at its requirement for larval crystal cell formation (Figure 11B). Two of these deletions will be described below. In order to assess the role of the Neurofilament-like domain, I needed to generate a precise in-frame deletion and recovered the enok F61.8 allele, whose sequence corresponds to the desired modification. I also included the enok M31.1 allele in my analysis, because this deletion generates a stop codon after the MYST domain and removes the entire C-terminal part of the protein. Analysis of the established stocks showed that the enok F61.8 allele is homozygous viable, whereas the enok M31.1 allele is lethal, as is the case for all other enok alleles [START_REF] Scott | Enok Encodes a Drosophila Putative Histone Acetyltransferase Required for Mushroom Body Neuroblast Proliferation[END_REF][START_REF] Berger | Systematic identification of genes that regulate neuronal wiring in the Drosophila visual system[END_REF]. Quantification of the H3K23 acetylation levels in these two new mutant contexts showed that neither excision of the Neurofilament-like domain, nor truncation of the Cterminus of Enok, affects its catalytic activity, since circulating hemocytes exhibit a wild-type level of acetylated H3K23 (Figure 12A). I then quantified the proportion of Lz-expressing cells in circulating crystal cell precursors. I observed that enok F61.8 and enok M31.1 alleles displayed a wild-type proportion of differentiating crystal cells (Figure 12B). Altogether these results

indicate that the Neurofilament-like domain is not required for Drosophila viability, for the catalytic activity of Enok, or for crystal cell development during the larval stages.

The enok M31.1 allele does not impact the ability of the protein to acetylate its substrates, and homozygous individuals display pupal lethality; together with my previous observations that the catalytic activity of Enok is dispensable for viability, these results suggest that a domain located in the most C-terminal part of Enok is essential for viability. I therefore examined more in detail the sequences corresponding to the C-terminal parts of MOZ and Enok, seeking a domain whose loss would be responsible for the lethality of enok M31.1 in mutant pupae. I identified some sequence homology between the C-terminal part of Enok and the Proline-rich domains of MOZ (Figure 5), which contain p53 interaction domains that are Drosophila stocks, genetics and melanization assays.

Stocks and crosses were maintained at 25°C on standard yeast-agar-cornmeal medium. The complete list of stocks used in this study is provided as supplemental material (Table 2). Larvae collected for phenotypic analysis were raised in controlled density conditions: in brief, for each cross, 10 females were fertilized by 4 males and left to lay eggs for 16 hours at 25°C. Third instar wandering larvae of the appropriate genotypes were collected 5 days after egg laying. Female larvae were used, unless otherwise specified. For melanization assays, two batches of six larvae were collected in two 1.5 mL eppendorf tubes containing 200 uL of Phosphate Buffered Saline (PBS) each, incubated in a 65°C waterbath for 30 minutes, stored on ice for 30 minutes and imaged on a SMZ18 stereomicroscope (Nikon).

Each experiment was reproduced at least three times (minimum of total larvae observed for each phenotype, n=36).

Immunofluorescent staining and Operetta quantifications on circulating cells.

Four female third instar larvae were bled in 1ml of PBS in 24-well-plate containing a glass coverslip. Hemocytes were centrifuged for 2 min at 900 g, fixed for 20 minutes with 4% paraformaldehyde in PBS and washed twice in PBS. Cells were then permeabilized in PBS-0.3% Triton (PBST), blocked in PBST-1% Bovine Serum Albumin (BSA) and incubated with primary antibodies at 4°C over night in PBST-BSA. The complete list of primary antibodies used in this study is provided as supplemental material (Table 3). Next, cells were washed in PBST, incubated for 2 hours at room temperature with corresponding Alexa Fluor-labeled secondary antibodies (Molecular Probes), washed in PBST and mounted in Vectashield medium (Eurobio-Vector) following incubation with DAPI. Imaging was performed on a Leica SP8 confocal microscope. For fluorescence quantification, single female larvae were bled individually in 96well-plate and samples were processed as described above; imaging was performed on an Operetta microscope (Perkin-Elmer); for each well 30 fields were captured at 20X magnification. Statistical analyses were performed with GraphPad Prism (GraphPad Software, Inc.); two-tailed Student t-Test were used for comparisons of two samples and one-way ANOVA followed by Dunnett test for comparisons of more than two samples.

Generation of UAS-3HA-Enok and lz VT059215 -Red-Stinger fly strains.

-cloning of UAS-3HA-Enok: a three HA-tags encoding sequence was directionally inserted in the pUAStAttB vector (Addgene) polylinker using EcoRI and NotI restriction enzymes, yielding a pUAStAttb-3HA plasmid. A p-BlueScript containing the full-length enok cDNA (kind gift from Takashi Suzuki) was then used as a template to PCR-amplify the enok coding sequence (Phusion high-fidelity polymerase, Thermofisher scientific). The amplified fragment was cloned in frame with the 3HA sequence, between the NotI and KpnI restriction sites of the pUAStAttB-3HA vector, using the In-fusion HD cloning kit (Clontech) according to supplier's instructions.

-cloning of lz VT059215 -Red-Stinger: this cloning was performed in two steps. We first generated a phiC31-based transformation vector containing a red fluorescent reporter gene compatible with golden gate cloning. Briefly, the pAttB vector [START_REF] Bischof | A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila[END_REF](kind gift of K Basler) was modified to remove the existing BsaI restriction sites and we then inserted a polylinker containing BsaI restriction sites on each sides of a lacZ gene from the pCambia2200 vector (kind gift of Jean-Philippe Combier), the hsp70 promoter and the DsRed. T4-NLS coding sequence from the pRed-H-Stinger vector [START_REF] Barolo | New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP[END_REF])(DGRC). In a second step, we cloned the VT059215 sequence into the pAttB-Red-H-Stinger vector with BsaI restriction enzyme following a golden gate cloning protocol.

All constructs were checked by sequencing for polymorphisms/mutations prior to injection for phiC31-mediated insertion in an AttP2 platform containing fly line. All plasmid sequences and detailed cloning procedures are available on request.

Chromatin Immunoprecipitation on embryos.

Four independent chromatin immunoprecipitation experiments were performed as follows: embryos were collected twice a day for 2-3 days, washed, frozen dry and kept at -80°C. They were then processed as described in [START_REF] Loubiere | Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs[END_REF]. The immunoprecipitated chromatin was subsequently used in RealTimePCR using the SsoFast Evagreen chemistry (Biorad) in a CFX96 thermocycler (Biorad). 100 mg of embryos were used for each replicate. The complete list of primers used in this study is provided as supplemental material (Table 4).