Taking advantage of this opportunity that gives me this manuscript, I would like to express my sincere and special gratitude to everyone who has helped me during my four years of PhD study in Toulouse, France.

Firstly, I would like to express my special appreciations and thanks to my supervisor, Dr. Lucas Waltzer, who has been a tremendous mentor for me. Thank you for adopting me into CBD family and for continuous patience and support on both my research and my life in France. Your guidance helped me a lot all the time in research work and this thesis writing.

Besides my supervisor, I would like to thank my co-supervisor, Dr. Marc Haenlin, for his support and enthusiasm and the rest of my thesis committee members, Prof. Christiane Bierkamp, Dr. Bruno Lemaitre, Dr. Marie-Bérengere Troadec, Dr. Magalie Lecourtois and Dr. Anne Plessis for their insightful comments and encouragement, but also for making my defense be an enjoyable moment and hard questions which incented me to widen my research horizon.

My sincere thanks also go to Dr. Vanessa Gobert, Dr. Fernando Roch, Dr. Billel Benmimoun, Dr. Marion Miller and our technician Benoir Augé and Sandra Bernat-Fabre, those who taught me how to perform experiments, how to analyze experimental data, how to use various scientific instruments as well as always answer my questions kindly and patiently.

I also would like to thank my two Chinese colleagues, Dr. Qiongyue Xu and Lu Wei, at the lab, who helped me a lot when I had a hard time and made me feel like at home when I felt lonely.

Last but not the least, I would like to thank my family. Any words cannot express how grateful I am to my mother and my father, for all of the sacrifices that they have made on my behalf and for supporting me spiritually all the time. v

Abstract

Hematopoiesis is the process of formation of fully differentiated blood cells from hematopoietic stem cells (HSCs). This process is tightly controlled by the integration of developmental and homeostatic signals to ensure the generation of an appropriate number of each blood cell type. At the molecular level, the regulation of this developmental process is mediated by a number of transcription factors, especially by members of the RUNX family, and mutations affecting these factors are at the origin of numerous hemopathies, including leukemia. Intriguingly, many transcriptional regulators and signaling pathways controlling blood cell development are evolutionarily conserved from humans to Drosophila melanogaster. Hence, the fruit fly has become a potent and simplified model to study the mechanisms underlying the specification of blood cell lineages and the regulation of blood cell homeostasis.

Members of the Myeloid Leukemia Factor (MLF) family have been implicated in hematopoiesis and in oncogenic blood cell transformation, but their function and molecular mechanism of action remain elusive. Previous work in Drosophila showed that MLF stabilizes the RUNX transcription factor Lozenge (LZ) and controls the number of LZ + blood cells. During my PhD, I sought to further decipher the molecular mechanism of action of MLF on Lozenge during blood cell development.

Using a proteomic approach in Drosophila Kc167 cells, we identified the Hsp40 cochaperone family member DnaJ-1 and its chaperone partner Hsc70-4 as two partners of MLF. These interactions were confirmed by co-immunoprecipitations and in vitro pulldown assays. Importantly, we found that knocking down DnaJ-1 or Hsc70-4 expression in Kc167 cells caused a reduction in the level of Lozenge protein and a concomitant decrease in Lozenge transactivation activity, which were very similar to those caused by MLF knockdown. Similarly, over-expression of two DnaJ-1 mutants that are unable to stimulate the chaperone activity of Hsc70-4 also decreased Lozenge level and impaired its capacity to activate transcription. These results suggest that MLF could act within a chaperone complex composed of DnaJ-1 and Hsc70-4 to control Lozenge stability and activity. Along that line, we showed by co-immunoprecipitation that Lozenge interacts with MLF, DnaJ-1 and Hsc70-4, respectively. Using various truncated mutants of MLF or DnaJ-1, we showed that MLF and DnaJ-1 interact and together with Lozenge through their conserved MLF vi homology domain (MHD) and C-terminal region, respectively. Furthermore, in vitro GST pull-down assays suggested that the interactions between MLF, DnaJ-1 and Lozenge are direct. Thus, we propose that MLF and DnaJ-1 control Lozenge protein level by interacting with it and by promoting its folding and/or solubility via the Hsc70 chaperone machinery.

In parallel, we assessed DnaJ-1 function in Drosophila blood cells in vivo using a null allele of dnaj-1 generated by CRISPR/Cas9 technique. We found that, like mlf, dnaj-1 mutation leads to an increase in the number and size of LZ + blood cells, as well as to an over-activation of the Notch signaling pathway in these cells. Moreover, our data suggested that high levels of active Lozenge are required to control the number and size of LZ + blood cells, and to down-regulate Notch expression. We propose that the MLF/DnaJ-1 complex controls LZ + blood cell development in vivo by regulating Lozenge protein level/activity and thereby Notch pathway activation.

In sum, our results establish a functional link between MLF, the Hsp40 co-chaperone DnaJ-1 and the RUNX transcription factor Lozenge, which could be conserved in other species.

vii Résumé L'hématopoïèse est le processus de formation des cellules sanguines différenciées à partir de cellules souches hématopoïétiques. Ce processus est étroitement contrôlé par l'intégration de signaux de développementaux et homéostatiques pour assurer une production équilibrée des différents types de cellules sanguines. Au niveau moléculaire, la régulation de ce processus est médiée par un certain nombre de facteurs de transcription, en particulier par les membres de la famille RUNX. Ainsi, des mutations affectant les membres de cette famille peuvent entrainer une déréglementation du programme de différenciation hématopoïétique et causer des hémopathies, dont des leucémies. D'une manière intrigante, de nombreux régulateurs de la transcription et des voies de signalisation contrôlant le développement des cellules sanguines sont évolutivement conservés des humains à Drosophila melanogaster, qui est donc utilisée comme organisme modèle pour étudier les mécanismes sous-jacents à la spécification des lignages sanguins et au contrôle de l'homéostasie des cellules sanguines.

Les membres de la famille Myeloid Leukemia Factor (MLF) ont été impliqués dans En conclusion, nos résultats ont mis à jour un lien fonctionnel entre MLF, le cochaperon de type Hsp40 DnaJ-1 et un facteur de transcription de type RUNX, qui pourrait être conservé dans d'autres espèces.

Foreword

Cancers are a large family of diseases that involve abnormal cell growth with the potential to invade or spread to other parts of the body. They have some common hallmarks, including continuous cell growth and division absent the proper signals, limitless number of cell divisions, avoidance of programmed cell death, and invasion of tissue and formation of metastases. It is estimated that there are over 100 types of cancers that affect human health, with signs and symptoms including a lump, abnormal bleeding, prolonged cough, unexplained weight loss and a change in bowel movements. Many factors could contribute to the emergence of cancers, such as tobacco use, which accounts for about 22% of cancer deaths, and obesity, poor diet, excessive drinking of alcohol, exposure to ionizing radiation, environmental pollutants. At the molecular level of pathophysiology, cancers are driven by progressive genetic or chromosomal abnormalities and epigenetic alterations. In genetic abnormalities, two broad categories of genes that regulate cell growth and differentiation are affected, which are oncogenes and tumor suppressor genes. Malignant transformation can occur through the formation of new oncogenes, the inappropriate over-expression of normal oncogenes, or the under-expression or disabling of tumor suppressor genes. Epigenetic alterations are functionally relevant modification to the genome, such as changes in DNA methylation, histone modification or changes in chromosomal architecture. They don't change the underlying DNA sequence, but regulate the expression of some particular genes, for instance, DNA repair genes, whose reduced expression disrupts DNA repair.

Similarly, leukemia is a group of cancers that usually start in the bone marrow of human beings and other warm-blooded animals. It is characterized by an abnormal increase in the number of immature white blood cells in the tissues and often in the blood. According to how quickly the disease develops and which type of blood cells is affected, there provides a total of four most common types of leukemia, which are acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL) and chronic lymphoid leukemia (CLL). Among them, acute myeloid leukemia is the most common malignant myeloid disorder in adults, which frequently results in hematopoietic insufficiency, such as anemia, thrombocytopenia and granulocytopenia and other symptoms. Chromosomal translocations, for instance, t (8;21) translocation in core-binding factor RUNX or t (3;5) translocation in myeloid leukemia factor, have been believed to be involved in the pathogenesis of AML. The t (8;21) translocation produces a fusion protein x RUNX1-ETO, and the t (3;5) translocation produces a fusion protein NPM1-MLF. Although the properties and function of RUNX1 have been studied extensively, the function and mode of action of MLF have been remained rather elusive. Recently, it was shown that MLF is a conserved regulator of RUNX transcription factor activity in a Drosophila model, which sheds a new light on the relationship between MLF and RUNX in AML as well as in other cancers

In this PhD thesis, I first made a brief introduction to the hematopoiesis in human and in Drosophila melanogaster and its associated three families of genes, which are RUNX, MLF and DnaJ families. Then I presented the results of my PhD project that is entitled Regulation of Lozenge transcription factor activity and blood cell development by MLF and its partner DnaJ-1. Finally, I discussed the significance of this study and made a perspective about the future research on this topic.

List of figures

List of tables

. Ontogeny of the mammalian hematopoiesis

Blood is a bodily fluid in animals that delivers necessary substances to the cells and transports metabolic waste products away from those same cells, which is considered as one of the most highly regenerative tissues with approximately one trillion cells producing daily in adult bone marrow. It performs many important functions within the body, including supply of oxygen and nutrients to tissues, removal of carbon dioxide, immunological functions such as detection of foreign materials by antibodies, regulation of body pH and temperature, coagulation and so on. In vertebrates, blood is mainly composed of blood cells suspended in blood plasma. The cellular components of the blood can be roughly classified into three types, which are red blood cells (RBCs), white blood cells (WBCs) and platelets (Figure 1). These three highly specialized cell types are involved in gas transport, immune responses, and blood clotting, respectively. In a word, blood and its cellular components are essentially vital for human health and normal life, so it is of much significance to determine where these blood cells come from and how they are generated.

To answer these important questions, the concept of hematopoiesis would be introduced formally: hematopoiesis is the process of the generation and formation of appropriate numbers of fully differentiated blood cells, which is controlled by the integration of developmental and homeostatic signals [START_REF] Orkin | Hematopoiesis: an evolving paradigm for stem cell biology[END_REF]. In mammals,

A B Figure 1. The cell types in blood

(A) Blood is composed of red blood cells, white blood cells and platelets suspended in blood plasma. (B) A scanning electron microscope (SEM) image of a normal red blood cell, a platelet, and a white blood cell.

diverse types of blood cells are produced from rare hematopoietic stem cells (HSCs) that reside in adult bone marrow. This blood cell development occurs in at least two distinct waves in mammals, which are called primitive hematopoiesis for the first wave and definitive hematopoiesis for the second wave (Jagannathan-Bogdan et al., 2013). The primitive hematopoiesis takes place within the blood islands of the yolk sac (YC) at embryonic day 7.5 in the mouse (Figure 2.A) and gives rise to primitive erythrocytes, A B Figure 2. A summary of the process of hematopoiesis in the mouse (A) Hematopoiesis occurs first in blood islands of the yolk sac (YS) and later at the aorta-gonadmesonephros (AGM) region, and fetal liver (FL) in the mouse. (B) The hierarchical tree model for the lineage commitment of hematopoietic stem cells (From Orkin et al., 2008).

megakaryocytes and macrophages [START_REF] Dzierzak | Of lineage and legacy: the development of mammalian hematopoietic stem cells[END_REF]. The primary purpose of this primitive hematopoiesis is the production of erythrocytes that can facilitate tissue oxygenation as the embryos grow very rapidly [START_REF] Orkin | Diversification of haematopoietic stem cells to specific lineages[END_REF]. This primitive hematopoietic wave is transient and it is rapidly replaced by the second adult-type wave of hematopoiesis.

By contrast, the definitive hematopoiesis first occurs in the aorta-gonad-mesonephros (AGM) region during later embryonic development (Figure 2.A) with multipotent hematopoietic stem cells (HSCs) arising [START_REF] Dzierzak | Embryonic beginnings of definitive hematopoietic stem cells[END_REF]. These definitive HSCs can give rise to all blood cell lineages of the adult organism and subsequently they migrate to the fetal liver and thymus as the embryo develops. At the end of fetal development, hematopoietic stem cells migrate to the bone marrow, which is the major site of postnatal hematopoiesis in adults. Of note, it is now established that some of the adult macrophages are derived from the primitive wave of hematopoiesis [START_REF] Perdiguero | The development and maintenance of resident macrophages[END_REF].

HSCs in the bone marrow ensure continuous hematopoietic cell production throughout life thanks to their dual capacities: they have the capacity to self-renew to maintain their number, and the potential to differentiate into all cell lineages of the blood and immune system. HSC differentiation is a complex and dynamic hierarchical process (Figure 2.B) [START_REF] Orkin | Hematopoiesis: an evolving paradigm for stem cell biology[END_REF]. The hematopoietic stem cells first give rise to two distinct groups of multipotent progenitors: the common myeloid progenitors (CMPs) and the common lymphoid progenitors (CLPs). Then common myeloid progenitors further differentiate into megakaryocyte/erythrocyte progenitors (MEPs) and granulocyte/macrophage progenitors (GMPs). From these progenitors, committed precursors for the various lineages arise and can be further specified into diverse mature myeloid blood cells with different specific functions, including erythrocytes, megakaryocytes, granulocytes, monocytes/macrophages.

Similarly, common lymphoid progenitors will give rise to B lymphocytes, T lymphocytes, and natural killer cells.

This hierarchical tree model assumes that hematopoiesis is usually governed by binary cell fate choices, in which the lymphoid system is completely separated from the myeloid system [START_REF] Ceredig | Models of haematopoiesis: seeing the wood for the trees[END_REF]. However, some evidence suggests that under some special conditions, lymphoid progenitors retain their ability to give rise to myeloid lineage cells, and thymic progenitors have both lymphoid and myeloid potentials. So this separation may not be as absolute as thought earlier, and a need for new models is in demand. Some years ago, a simple pairwise relationships model of hematopoiesis emerged, which depicts hematopoiesis as a continuum of lineage relationships between hematopoietic stem cells and their oligopotent progeny [START_REF] Ceredig | Models of haematopoiesis: seeing the wood for the trees[END_REF]. For instance, dendritic cells could be derived from both megakaryocyte-monocyte progenitors and B-cell-T-cell progenitors. This reflects that a final cell fate could be reached through more than one type of intermediate progenitor as well as the high complexity of the hematopoietic system. More recently, single cell-based gene expression profiling analyses have suggested a much revised version of the hematopoietic tree and have questioned the existence of several previously described 'common progenitors" [START_REF] Moignard | Dissecting stem cell differentiation using single cell expression profiling[END_REF]. Thus, although mammalian blood cell differentiation has been extensively studied and considered as a paradigmatic differentiation process, much remains to be discovered in this field.

In addition, a close relationship between vascular endothelium and hematopoietic stem cells during ontogeny provides us more insights into the origin of blood cells. It has been proposed that during primitive hematopoiesis, blood cell progenitors arise from hemangioblast, a common mesodermal progenitor for both endothelial and hematopoietic cells (Figure 3.). However, most recent studies rather suggest that it is a hemogenic endothelium (i.e. an endothelial cell that has the potential to give blood cells) that give rise to blood cell progenitors in the yolk sac and to definitive HSCs [START_REF] Lacaud | Hemangioblast, hemogenic endothelium, and primitive versus definitive hematopoiesis[END_REF]. For instance, in the aorta-gonad-mesonephros (AGM) region, the endothelium in the ventral wall of the aorta undergoes an endothelial to hematopoietic transition to produce HSCs. All together, the origin of hematopoietic development can be depicted as a single linear developmental process, which originates from mesoderm, through stages of hemangioblast and/or hemogenic endothelium, hematopoietic stem cells, multipotent progenitors, committed precursors, and finally mature blood cells [START_REF] Cumano | Ontogeny of the hematopoietic system[END_REF].

Functions of mature blood cells

The different mature blood cell types produced in the bone marrow then leave this hematopoietic organ and enter into the blood circulatory system, performing several specialized functions [START_REF] Hartenstein | Blood cells and blood cell development in the animal kingdom[END_REF][START_REF] Orkin | Diversification of haematopoietic stem cells to specific lineages[END_REF].

Erythrocytes (also called red blood cells) are the most common blood cell type and occupy 40% to 45% of the blood volume. In humans, mature erythrocytes look like small flexible and oval biconcave disks lacking nucleus and most organelles, but are rich in hemoglobin, an iron-containing protein that reversibly binds oxygen and greatly increases its solubility in blood. They take up oxygen in the lungs and deliver it to the body tissue via blood flow through the circulatory system.

Thrombocytes (also called platelets) are cytoplasmic fragments without nucleus that are derived from the megakaryocytes, giant and polyploid cells from the bone marrow.

Their main function is to stop bleeding at the site of interrupted endothelium by clumping and clotting blood vessel injuries through the three steps of adhesion, activation and aggregation. Based on numerous studies on hematopoiesis in mammals, including the successful detection of hematopoietic stem cells in the bone marrow of live mice using real-time imaging technology [START_REF] Xie | Detection of functional haematopoietic stem cell niche using real-time imaging[END_REF], it has been showed that there are two subpopulations of hematopoietic stem cells, one that is quiescent and the other that is more active. The decisions of hematopoietic stem cells to become quiescent or differentiate further are tightly controlled by their microenvironment that is termed as niche. The concept of the niche was first proposed in the late 1970s by Schofield who proposed that the fate of a blood stem cell itself is controlled by its interaction with other cells/a particular microenvironment [START_REF] Schofield | The relationship between the spleen colony-forming cell and the haemopoietic stem cell[END_REF]. The niche is composed of subsets of cells and extracellular substrates that can provide structural and trophic support as well as appropriate signals to regulate stem cell functions. This model gained solid experimental and conceptual support from Drosophila germ-line stem cell studies. Over the years, a body of experimental evidence revealed that there are three main niches regulating hematopoietic stem cells: the osteoblastic niche, the vascular niche and the perivascular niche [START_REF] Nakamura-Ishizu | Hematopoietic stem cell niche: An interplay among a repertoire of multiple functional niches[END_REF]. Osteoblasts that align bone surfaces (the osteoblastic niche) interact with hematopoietic stem cells in vivo and influence their functions notably through the activation of the Notch signalling pathway to maintain their long-term quiescence. The sinusoidal endothelial cells that align the lumen of sinusoid in the central bone marrow (the vascular niche) can express Notch ligand Jagged-1 and Jagged-2 to control the proliferation of hematopoietic stem cells, which is similar to the osteoblasts. Besides, two perivascular cell groups that possess mesenchymal cells properties also function as niche cells (the perivascular niche). A study on deletion of a chemokine Cxcl12 in mice revealed that hematopoietic stem cells occupy a perivascular niche whereas early lymphoid progenitors occupy an endosteal niche, suggesting different stem and progenitor cells reside in distinct cellular niches in the bone marrow [START_REF] Morrison | The bone marrow niche for haematopoietic stem cells[END_REF].

Granulocytes

The osteoblastic niche and the vascular niche have different roles in the maintenance and differentiation of hematopoietic stem cells in mouse bone marrow, which might be related to oxygen availability. The osteoblastic niche is hypoxic, a low oxygen microenvironment essential for the maintenance and survival of hematopoietic stem cells and their protection from the harmful reactive oxygen species (ROS) accumulation [START_REF] Crozatier | Drosophila: a model for studying genetic and molecular aspects of haematopoiesis and associated leukaemias[END_REF]. Meanwhile, the distinct metabolic profile of hematopoietic stem cells also reflects their location in a hypoxic niche, which is revealed by the observation that long-term hematopoietic stem cells utilize glycolysis instead of mitochondrial oxidative phosphorylation to meet their energy demands. Notably, by regulating the response to physiological oxidative stress, the ataxia telangiectasia mutated (ATM) gene and the forkhead O (FoxO) gene play pivotal roles in the maintenance and survival of hematopoietic stem cells in bone marrow [START_REF] Morrison | The bone marrow niche for haematopoietic stem cells[END_REF]. Furthermore, similar to normal tissue stem cells, cancer stem cells (CSCs) in some tumours also contain low ROS levels and enhanced ROS defences, which may contribute to the resistance of tumours to radiation therapy. Therefore, the link between the survival of hematopoietic stem cells and ROS activates the myeloid program and represses the erythroid cell fate [START_REF] Cantor | Transcriptional regulation of erythropoiesis: an affair involving multiple partners[END_REF].

Interestingly, most of these transcription factors and signaling pathways are conserved through evolution and also control blood cell fate in other organisms, as shown by studies in Zebrafish or Drosophila [START_REF] Hartenstein | Blood cells and blood cell development in the animal kingdom[END_REF]. Thus, studies in model organisms like

Drosophila, which exhibit a simpler hematopoietic system and less genetic redundancy, can help to understand some important conserved aspects of the control of blood cell development.

Importantly, the deregulation of these transcription factors and signaling pathways that control normal blood cell development is often at the origin of blood cell cancer (and other hemopathies) in human [START_REF] Orkin | Hematopoiesis: an evolving paradigm for stem cell biology[END_REF]. Accordingly, a number of transcription factors or signaling pathways implicated in hematopoiesis are the targets of recurrent chromosomal translocations or of point mutations that alter (inhibit or activate) their functions. A better understanding of the normal functions and mode of action of these genes is thus of utmost importance.

Leukemia in human

Leukemia is a group of cancers that usually start in the bone marrow of human beings and other warm-blooded animals, which is characterized by an abnormal increase in the number of white blood cells in the tissues and often in the blood. These white blood cells are not fully developed and are called leukemic cells. Unlike normal blood cells, leukemic cells don't die when they become old or damaged, so they can build up and crowd out normal blood cells. Due to the low level of normal blood cells, some typical symptoms will appear, including bleeding, bruising problems, feeling tired, fever and an increased risk of infections. So far, the etiology of leukemia remains unknown, but both inherited and environmental factors are believed to be involved in these malignancies. Some risk factors, including smoking, ionizing radiation, chemicals (such as benzene), prior chemotherapy, could contribute to leukemia.

Classification of leukemia

Based on different criteria, leukemia can be subdivided into a variety of large groups.

According to how quickly this disease develops, leukemia can be classified into acute leukemia and chronic leukemia clinically and pathologically. Acute leukemia usually develops quickly and is characterized by a rapid increase in the number of leukemic cells.

This rapid progression and accumulation of leukemic cells, which then spill over into the bloodstream and spread to other organs of the body, out compete normal blood cells, making the bone marrow unable to produce healthy normal blood cells. Chronic leukemia usually develops slowly and is characterized by the excessive build up of relatively mature but still abnormal white blood cells. Typically, these cells are produced at a higher rate than normal blood cells, resulting in many abnormal white blood cells over months or years. It is often asymptomatic but if not treated, chronic leukemia can progress into a blast phase, which resembles acute leukemia.

In addition, according to which type of blood cells is affected, leukemia can be classified into myeloid leukemia and lymphoid leukemia, in which the leukemic cells are either derived from a myeloid or a lymphoid cell lineage, respectively. Combining these two classifications, one can distinguish four most common types of leukemia: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL)

and chronic lymphoid leukemia (CLL). And within each of these four main types, there are typically several subtypes.

Acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic progenitor cells, which is the most common malignant myeloid disorder in adults. These hematopoietic progenitor cells are designated as AML blasts that have lost the ability to differentiate normally and to respond to normal proliferation regulators. AML is characterized by the infiltration of the bone marrow, blood and other tissues by clonal, proliferative, abnormally differentiated AML blasts of the hematopoietic system and the interference with the production of normal blood cells, frequently resulting in hematopoietic insufficiency, such as anemia, thrombocytopenia and granulocytopenia, and other symptoms including fatigue, shortness of breath and increased risk of infections.

Classification of acute myeloid leukemia

As studies on AML progress, it is revealed that AML has several subtypes based on the diagnostic procedures, such as morphologic assessment of bone marrow specimens and blood smears, analysis of the expression of cell-surface or cytoplasmic markers, identification of chromosomal abnormalities or screening for selected molecular genetic lesions; subsequently, treatment and prognosis vary among these subtypes. There are two common classification systems for AML according to different criteria, which are the French-American-British (FAB) classification and the World Health Organization (WHO) classification. The FAB classification system was introduced in 1976 and defines AML into eight subtypes that are from M0 to M7 based on the morphological and cyto-chemical characteristics of the leukemic cells [START_REF] Löwenberg | Acute myeloid leukemia[END_REF]. The WHO classification system was introduced in 2001 and revised in 2008 and in 2016 and defines AML into six major subtypes (Table 1.) by incorporating genetic information with morphology, immunophenotype and clinical presentation [START_REF] Kouchkovsky | Acute myeloid leukemia: a comprehensive review and 2016 update[END_REF]. These AML classifications provide useful information about the biology and clinical features of this malignancy, also are helpful for assessing treatment options to the patients with AML.

Pathophysiology of acute myeloid leukemia

AML is a highly heterogeneous disease that appears as a de novo malignancy. The abnormal proliferation and differentiation of a clonal population of myeloid stem cells are believed to be involved in the pathogenesis of AML. Recurrent chromosomal translocations, such as t (8;21) translocation in AML or t (15;17) translocation in acute promyelocytic leukemia (APL) as well as point mutations affecting key genes that control normal blood cell development, have been implicated in the development of AML (Rowley, 2009).

The most common targets of AML-associated chromosomal translocations are the genes encoding DNA-binding transcription factors or the regulatory components of transcriptional complexes, and the fusion proteins generated from these chromosomal translocations interfere with the functions of the wild-type proteins in a dominant manner.

For instance, the RUNX transcription factor AML1 (Acute Myeloid Leukemia 1/RUNX1) and its heterodimerisation partner CBFβ, which regulate many hematopoietic-specific genes and are essential for the normal development of the hematopoietic system, are the target of the t (8;21) and inv [START_REF] Ohno | Characterization of a Drosophila homologue of the human myelodysplasia/myeloid leukemia factor (MLF)[END_REF] translocations, respectively, making this complex the most frequent target of chromosomal rearrangements in human leukemia (see below, 3.2.5) [START_REF] Speck | Core-binding factors in haematopoiesis and leukaemia[END_REF]. These mutations or translocations impair blood cell differentiation, but are not Table 1. WHO classification of AML and related neoplasms (From De Kouchkovsky et al., 2016) sufficient to cause leukemia. It is usually considered that leukemia development requires a second mutation, which will give a growth advantage to the blood cells. These mutations often target components of signaling pathways controlling blood cell proliferation or survival such as c-Kit, Flt3 or Ras [START_REF] Kelly | Genetics of myeloid leukemias[END_REF]. The cooperation between these two classes of mutations eventually leads to AML.

Of note, AML is often preceded by a pre-leukemic condition called "myelodysplastic syndrome" (MDS), a chronic disease that progresses to AML upon acquisition of new mutations.

Current therapy of acute myeloid leukemia

The primary purpose of treating patients with AML is to induce remission and thereafter prevent relapse. Initially, intensive chemotherapy is the main treatment for patients with AML who can tolerate it, and additional chemotherapy or allogeneic hematopoietic stem cell transplantation could follow. The drugs used in treatments include cytarabine, anthracycline, gemtuzumab ozogamicin, and novel agents under test, such as sarafenib, midostaurin, quizartinib, crenolanib.

While much progress has been made in our understanding of the molecular nature of the events leading to AML and in the refinement of therapeutic strategies, this disease remains of bad prognosis. Thus a better characterization of the function and mode of action of the genes mutated in MDS and AML is of prime interest to be able to develop innovative therapeutic approaches. Besides the study of human blood samples or mouse models, simple genetic model organisms such as Drosophila can help not only to decipher the basic principles underlying blood cell development across species but also to better understand the mode of action of conserved genes implicated in AML.

Drosophila: a simplified model to study the mammalian hematopoiesis

Drosophila melanogaster is a species in the family Drosophilidae of the order Diptera, which has multifaceted brick red eyes, a tan thorax studded with arched black bristles, a striped abdomen and a pair of translucent wings (Figure 5 over several days and embryonic development lasts for ~21hr. 1 st instar larvae take 2 days to molt into 2 nd then 3 rd instar larvae. 3 rd instar larvae continue feeding for one more day and eventually pupariate (prepupa then pupa). 10 days after egg-laying, adult flies emerge from the pupal case.

Ontogeny of Drosophila hematopoietic system

Over the last several decades, due to its apparent simplicity, such as an open circulatory system, fewer blood cell types and significant similarities in blood cell development shared with mammals, Drosophila melanogaster has emerged as a powerful genetic model for studying the molecular process that controls hematopoiesis under normal conditions or in pathological situations (Letourneau et al., 2016). These studies notably highlighted how intricate cell communication networks and microenvironment cues regulate blood cell homeostasis and helped revealed the mode of action of key conserved regulators of hematopoiesis.

Embryonic hematopoiesis

Similar to the two waves of the vertebrate hematopoiesis, Drosophila hematopoiesis takes place in two spatially and temporally distinct waves (Figure 6.A). The first wave, which is often compared to the primitive hematopoiesis in vertebrates, occurs in the early embryonic development, when prohemocytes (or hemocyte/blood cell precursors) emerge from the head mesoderm (Figure 6.B). These cells are first identified during embryonic stage 5 by the expression of Serpent (Srp), a GATA transcription factor required for hematopoietic development [START_REF] Rehorn | A molecular aspect of hematopoiesis and endoderm development common to vertebrates and Drosophila[END_REF]. After four rounds of division, these hemocyte precursors stop proliferation and differentiate into either plasmatocytes or crystal cells. Upon maturation in the head mesoderm, the majority of plasmatocytes that account for 95% of all embryonic hemocytes migrate out of the head region under the influence of chemo-attracting signals along stereotypical routes (Letourneau et al., 2016). In contrast, crystal cells that account for 5% of all embryonic hemocytes generally remain localized as two groups of cells around the anterior part of the gut, which is near their point of origin in the embryo, and they will disperse subsequently during the larval stages. Totally, by the end of embryogenesis, the prohemocytes in the head mesoderm give rise to 600-700 plasmatocytes and approximately 36 crystal cells.

These embryo-derived hemocytes populate the larvae where they can be found in two destinations. One fraction persists as patches of cells attached to the inner epidermis of the body cavity, which is designated as sessile hemocytes; the other fraction circulates in the hemolymph (the circulating fluid in the body cavity of Arthropods), which is designated as circulating hemocytes.

The molecular control of this embryonic hematopoiesis has been well characterized.

Briefly, prohemocytes generation requires the pan-hematopoietic Serpent (Srp), a

Drosophila homolog of human GATA transcription factors, which are also implicated in hematopoiesis. The activity of Serpent in the hemocytes is notably controlled by its corepressor U-shaped (Ush), a Drosophila homolog of human Friend of GATA (FOG) cofactor, which directly binds Srp to inhibit crystal cell fate choice and regulate plasmatocytes differentiation (Waltzer et al., 2003;[START_REF] Fossett | Combinatorial interactions of Serpent, Lozenge, and U-shaped regulate crystal cell lineage commitment during Drosophila hematopoiesis[END_REF]. The lineage commitment of prohemocytes to plasmatocytes or crystal cells depends on the expression of the lineage-specific transcription factors Glide/Glial cell missing (Gcm and Gcm2) and the RUNX transcription factor Lozenge (Lz), respectively. Specifically, the expression of Gcm factors, together with Srp and Ush, leads to differentiation of prohemocytes into plasmatocytes. In contrast, Lz, a runt-domain protein that resembles human AML1/RUNX1, is required for the differentiation of hemocyte precursors into crystal cells. When Lz is expressed, the expression of Ush is suppressed and Srp and Lz cooperate to induce the differentiation of prohemocytes into crystal cells, similar to the cooperation of GATA and RUNX in vertebrates that controls several steps of blood cell proliferation and differentiation. How Gcm expression is controlled is unknown, but it has been suggested

that Notch signalling pathway participates in the induction of Lz expression in the embryonic crystal cell lineage (Lebestky et al., 2003).

Larval hematopoiesis

The second wave of Drosophila hematopoiesis, is initiated during the larval stages in a specialized organ, the lymph gland, which forms during embryogenesis and persists through the onset of metamorphosis. During the last two decades, the lymph gland has been a

prevailing model to investigate the hematopoiesis process under normal conditions or in response to immune stress, since it is the dedicated Drosophila larval hematopoietic organ (Letourneau et al., 2016). Similar to the hematopoietic stem cell emergence in vertebrates, lymph gland cells derive from hemangioblast precursors specified in the late embryo. At that stage, the lymph gland precursors form a single pair of lobes that are localized along the dorsal vessel. This single pair of lobes only contains about 20 cells each, and they are designated as the primary lobes (or the anterior lobes). At the end of the first larval instar, additional pairs of posterior lobes also emerge along the dorsal vessel, which are designated as the secondary lobes (or the posterior lobes). So in third instar larvae, lymph gland is composed of a large pair of primary anterior lobes followed by several small pairs of posterior lobes, each separated by a pair of pericardial cells. The posterior lobes are mainly composed of prohemocytes, however, the primary lobes are organized into three domains:

the cortical zone (CZ), the medullary zone (MZ) and the posterior signalling centre (PSC) (Figure 6.C). The peripheral cortical zone contains mature and differentiated hemocytes, whereas the central medullary zone contains quiescent prohemocytes. At the posterior tip of each primary lobe, a cluster of about 30 cells constitutes the posterior signalling centre (PSC), which acts as a niche-like structure that controls prohemocyte maintenance and lymph gland homeostasis. There is also a population of "intermediate progenitors" between the cortical zone and the medullary zone that express both some progenitors and early differentiation markers. During the first two larval stages, lymph gland progenitors divide actively before entering quiescence and giving rise to plasmatocytes and crystal cells under normal conditions and to lamellocytes in response to immune challenges, such as wasp parasitism. During pupariation, the lymph gland disrupts and releases all hemocytes into the circulation.

Besides the lymph gland, there is also another site of hematopoiesis during the larval stages, which is called hematopoietic pockets or subepidermal and muscular pockets and has only been identified recently. In third instar larvae, sessile hemocytes, which are derived from the differentiated hemocytes of the embryo, are prevalent along the posterior region of the dorsal vessel and in close association with oenocytes on the lateral sides, where they are sandwiched between the epidermal and muscular layers. These so-called hematopoietic pockets, provide a specific microenvironment that attracts plasmatocytes and supports their survival, proliferation and differentiation [START_REF] Makhijani | The peripheral nervous system supports blood cell homing and survival in the Drosophila larva[END_REF]. These sessile hemocytes can expand through self-renewal in differentiated state in third instar stage and increase their accumulation at the site of injury or in circulation under certain circumstances, such as starvation, wound inflammation.

Hematopoiesis in adult Drosophila

Although the embryonic and larval hematopoiesis in Drosophila have been studied

extensively, the current knowledge about adult blood cells is much more limited. Adult blood cells are present mostly as sessile hemocytes dispersed under the cuticle and associated with different tissues/organs. It has been shown that they are derived from the embryonic and larval waves of hematopoiesis and it has long been thought that Drosophila adults lack a hematopoietic organ. Hence, it is generally assumed that adult flies only rely on a fixed pool of differentiated plasmatocytes and that no blood cell proliferation or differentiation takes place during adulthood. However, recently, a study demonstrated the presence of active hematopoietic sites in the abdomen of adult flies, which are called hematopoietic hubs and can give rise to new blood cells (Figure 6.D) [START_REF] Ghosh | Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response[END_REF].

It was shown that these hematopoietic hubs contain some hemocyte progenitors, which originate from the posterior lobes of lymph gland, and it was proposed that these cells can give rise to plasmatocytes and crystal cells. In addition, whereas the total number of hemocytes declines with age in adult flies [START_REF] Horn | Phagocytic ability declines with age in adult Drosophila hemocytes[END_REF], it was shown that adult blood cells can proliferate in response to an immune infection. These exciting observations suggest that active hematopoiesis could take place in the hematopoietic hubs of adult flies and project this hematopoietic hub as a simple version of the vertebrate bone marrow.

However, these findings need to be confirmed and the extend as well as the functional importance of adult blood cell production/differentiation remains unclear.

Functions of Drosophila hemocytes

As mentioned above, Drosophila hemocyte progenitors can differentiate into three mature blood cell types: plasmatocytes and crystal cells (during embryonic, larval and adult stages) as well as lamellocytes (during larval stage, in response to specific immune challenges).

Prohemocytes

Prohemocytes have been described in the embryonic head mesoderm, in embryoderived larval hemocytes, in the larval lymph gland and in adult hematopoietic hubs. They are generally described as small cells (4-6 um in diameter) with a high nuclear/cytoplasmic ratio but few defined characteristics. Although the GATA factor Srp is expressed in all prohemocytes, this factor is also expressed in differentiated blood cells and so far there is no known specific marker universally labelling Drosophila prohemocytes. For instance, in the lymph gland, prohemocytes express tep4 and the reporter gene dome-meso, but these two markers do not label embryonic prohemocytes and it is not known if they label adult blood cell progenitors. Therefore, further molecular characterization of the prohemocytes is needed

Plasmatocytes

Plasmatocytes are relatively round cells with a diameter of 8-10 um and contain abundant lysosomes and endoplasmic reticulum (Figure 7.A). Their differentiation from prohemocytes requires the expression of transcription factor Glial/Glide cells missing (Gcm) in the embryo, and that of the GATA transcription factor Pannier in the lymph gland [START_REF] Minakhina | Zfrp8, the Drosophila ortholog of PDCD2, functions in lymph gland development and controls cell proliferation[END_REF]. They are the professional phagocytes of the immune system, related to mammalian macrophages, which mediate the cellular immune defence.

Phagocytosis is an evolutionarily conserved process that is critical for the removal of invading pathogens and apoptotic cells. In Drosophila, plasmatocytes are able to engulf dead cells and debris as well as invading pathogens, and they are important for bacterial clearance and for the resistance to systemic infection. The phagocytic ability of plasmatocytes depends on the expression of scavenger and pattern recognition receptors on their surfaces. Croquemort (Crq), a member of the CD36 family of receptors, mediates the recognition of apoptotic cells [START_REF] Franc | Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells[END_REF]. Eater and NimC1, two members of the Nimrod family of cell-surface receptors, recognize both Gram-positive and Gram-negative bacteria, leading to rapid engulfment of invading microorganisms [START_REF] Kocks | Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila[END_REF][START_REF] Kurucz | Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes[END_REF]. Draper, the Ced-1 homologue, recognizes lipoteichic acid from

Staphylococcus aureus and mediates the uptake of this bacterium in adult flies [START_REF] Hashimoto | Identification of lipoteichoic acid as a ligand for Draper in the phagocytosis of Staphylococcus aureus by Drosophila hemocytes[END_REF]. Additionally, plasmatocytes sculpt various developing tissues and organs through the phagocytic removal of cells. For example, the embryonic nervous system doesn't condense properly if the plasmatocyte-mediated phagocytosis is absent.

Plasmatocytes are also highly motile cells, providing a powerful model to study cell migration and chemotaxis in response to developmental cues or tissue damage [START_REF] Evans | Drosophila blood cell chemotaxis[END_REF][START_REF] Ratheesh | Drosophila immune cell migration and adhesion during embryonic development and larval immune responses[END_REF]. This motility requires the expression of PVR (PDGF/VEFGrelated) receptor, Rho GTPase and fascin, and it is essential to ensure the homeostatic function of plasmatocytes. Additional functions of plasmatocytes include secretion of extracellular matrix (ECM) components required for proper tissue morphogenesis [START_REF] Bunt | Hemocyte-secreted type IV collagen enhances BMP signaling to guide renal tubule morphogenesis in Drosophila[END_REF] and for the maintenance of ovarian stem cells [START_REF] Van De Bor | Companion blood cells control ovarian stem cell niche microenvironment and homeostasis[END_REF], controlling the activity of intestinal stem cells during injury-induced regeneration [START_REF] Ayyaz | Haemocytes control stem cell activity in the Drosophila intestine[END_REF], regulation of glucose metabolism to modulate life span [START_REF] Woodcock | Macrophage-derived upd3 cytokine causes impaired glucose homeostasis and reduced lifespan in Drosophila fed a lipid-rich diet[END_REF], and mediating apoptosis-induced proliferation in the imaginal disks [START_REF] Fogarty | Extracellular reactive oxygen species drive apoptosis-induced proliferation via Drosophila macrophages[END_REF].

Similar to the mammalian macrophages that have a plethora of functions in addition to their role in immunity, Drosophila plasmatocytes constitute a very helpful model to understand how macrophage diversity of function is generated and regulated.

Crystal cells

Crystal cells are megakaryocyte-like cells with the diameter of 10-12 um (Figure 7.B).

They derive their name owing to the presence of paracrystalline inclusions composed of prophenoloxidases (PPO) in the cytoplasm. They are non-phagocytic and function in melanization, an important immune response related to clotting and wound healing in Arthropods [START_REF] Evans | Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis[END_REF]. Melanization leads to the blackening of wound sites or the surface of the invading pathogens, due to the local production and deposition of melanin.

As described above, crystal cells contain paracrystalline inclusions consisting of mass quantities of one or more components of the melanisation enzymatic cascade, which are prophenoloxidase enzymes that play a key role in melanin biosynthesis. There are three PPOs in Drosophila, which are PPO1 (Black Cell), PPO2 and PPO3. Upon injury, activation of the JNK pathway and the TNF homolog Eiger leads to crystal cells rupture and release of PPO zymogens into the hemolymph [START_REF] Bidla | Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog Eiger[END_REF]. PPOs are then cleaved into active phenoloxidase (PO) by a proteolytic cascade [START_REF] Dudzic | Drosophila innate immunity: regional and functional specialization of prophenoloxidases[END_REF], and PO in turn catalyses the oxidation of mono-and di-phenols to ortho-quinones, which subsequently polymerize into melanin and produce reactive oxygen species (ROS) as by-products [START_REF] Eleftherianos | Role and importance of phenoloxidase in insect hemostasis[END_REF]. Although crystal cells are dispensable for fly viability, melanization participates in the innate immune response, and the resistance to infection.

Crystal cell differentiation absolutely requires the induction of Lozenge expression both in the embryos and in the larvae (Lebestky et al., 2000;[START_REF] Fossett | Combinatorial interactions of Serpent, Lozenge, and U-shaped regulate crystal cell lineage commitment during Drosophila hematopoiesis[END_REF]. The processes regulating crystal cell production are described in detail below.

Lamellocytes

Lamellocytes are large, flat and adherent cells (Figure 7.C). They are scarcely present in healthy larvae, but their differentiation is massively induced in response to some specific immune challenges, such as parasitization by the wasp Leptopilina boulardi [START_REF] Lanot | Postembryonic hematopoiesis in Drosophila[END_REF]. Lamellocytes participate both in the cellular response and in melanisation, and they primarily function in the encapsulation and neutralization of pathogens or bodies that are too large to be phagocytosed. The encapsulation of parasitic wasp eggs requires the cooperation of all three types of larval hemocytes [START_REF] Mortimer | Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity[END_REF]. At the early stage of parasitization, circulating plasmatocytes recognize the injected eggs and attach to them, and subsequently form septate junctions to separate the eggs from the hemolymph circulation. This detection induces the proliferation and differentiation of sessile hemocytes as well as lymph gland hemocytes into lamellocytes. Lamellocytes adhere to the wasp eggs, activate the melanisation cascade, and eventually kill the parasites.

Genetic control of Drosophila hematopoiesis

PSC in Drosophila hematopoiesis

Similar to the mammalian bone marrow niche controlling the balance between hematopoietic stem cells (HSCs) self-renewal and differentiation, the posterior signalling centre (PSC) within Drosophila lymph gland, which controls the maintenance and differentiation of prohemocytes non-cell-autonomously and could function as a hematopoietic niche, represents a helpful and genetically tractable model to study signal integration and crosstalk during hemocyte development [START_REF] Crozatier | Drosophila: a model for studying genetic and molecular aspects of haematopoiesis and associated leukaemias[END_REF]. It has been

shown that the specification of PSC cells in the embryo critically requires the expression of two transcription factors, which are the homeotic protein Antennapedia (Antp) [START_REF] Mandal | A Hedgehogand Antennapedia-dependent niche maintains Drosophila haematopoietic precursors[END_REF] and the Drosophila orthologue of early B-cell factors (EBFs), Collier (Col) [START_REF] Crozatier | Cellular immune response to parasitization in Drosophila requires the EBF orthologue Collier[END_REF]. Once the Collier activity is absent, the PSC cells are not specified and larval prohemocytes differentiate prematurely, which indicates the critical requirement for Collier activity in PSC cells specification. Besides transcription factors, the Wingless (Wg) signalling pathway also controls both the number of PSC cells and the maintenance of prohemocytes [START_REF] Sinenko | Dual role of Wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila[END_REF]. In addition, PSC cells express the signalling molecule Hedgehog (Hh), and this Hh signalling pathway is required to maintain hemocyte homeostasis in the lymph gland [START_REF] Mandal | A Hedgehogand Antennapedia-dependent niche maintains Drosophila haematopoietic precursors[END_REF]. Furthermore, Serrate-mediated Notch signalling from the PSC is required to maintain normal levels of Collier transcription [START_REF] Krzemień | Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre[END_REF]. All together, these signalling pathways, including Wg, Hh, Notch and other signalling pathways, are integrated into PSC cells to control JAK/STAT signalling activity in prohemocytes, preventing their premature differentiation.

ROS in Drosophila hematopoiesis

As many transcription regulators and signalling pathways involved in hematopoiesis are conserved from humans to Drosophila, a model for reactive oxygen species (ROS) has also been established in Drosophila [START_REF] Owusu-Ansah | Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation[END_REF]. This study showed that the accumulation of ROS in the lymph gland is tightly controlled during development.

Specifically, the multipotent hematopoietic progenitors have an increased level of ROS under in vivo physiological conditions. Conversely, once the ROS in the hematopoietic progenitors is increased beyond its basal level, it will trigger precocious differentiation of these progenitors into all three mature hemocyte types, through a signalling pathway that involves JNK and FoxO activation as well as Polycomb down-regulation. In sum, this study provides a helpful model that could be extended to reveal the possible role of ROS in the differentiation of common myeloid progenitors (CMPs) in the mammalian hematopoiesis and oxidative stress response.

Genetic control of blood cell fate

A number of conserved signalling pathways and transcription factors controlling Drosophila hematopoiesis have been identified (Letourneau et al., 2016). In particular, Here, I will focus my description on the mechanisms of regulation of crystal cell fate, as this is the blood cell lineage in which the gene that I studied during my PhD (MLF, see below), which was shown to be required (Bras et al., 2012). As detailed below, the RUNX transcription factor Lozenge (LZ) is specifically expressed in the crystal cell lineage and it is absolutely required for crystal cell development (Lebestky et al., 2000). The expression of Lozenge, and thus the induction of crystal cell fate is dependent on the Notch signaling pathway both in circulating larval cells and in lymph gland (Mukherjee et al, 2011;Lebestky et al, 2003;Duvic et al., 2002). The activation of the Notch pathway is mediated by its ligand Serrate (Ser) and requires the transcription factor Suppressor of Hairless (Su(H)). Accordingly, decreasing Notch activity (using for instance a thermo-sensitive allele or over-expression of a dominant negative form of Notch) leads to a decrease in the number of crystal cells, while over-expression of Notch induces a strong increase in their number (Duvic et al., 2002).

many
In the lymph gland, the posterior signaling center (PSC) was first characterized thanks to its specific expression of Ser (Lebestky et al., 2003). It was thus initially proposed that the PSC is necessary for the induction of crystal cell fate by contacting the neighboring hemocytes. However, it was then shown that the PSC is not required for crystal cell development, and that it was proposed that some other cells expressing Ser and dispersed in the cortical zone could be responsible for the signal that induces crystal cell fate [START_REF] Crozatier | Cellular immune response to parasitization in Drosophila requires the EBF orthologue Collier[END_REF]. Consistent with this hypothesis, a recent study demonstrated that the expression of Yorkie and Scalloped, two components of the Hippo pathway, promotes the expression of Ser in these cells of the cortical zone, and is required for the specification of the adjacent cells into crystal cells (Ferguson et al., 2014a;2014b). Another related study suggests that the Hippo pathway controls the development of crystal cells in a non-cellautonomous manner via Ser and the Notch pathway but also in a cell-autonomous manner by directly activating the transcription of lz [START_REF] Milton | The Hippo pathway regulates hematopoiesis in Drosophila melanogaster[END_REF]. In addition, the identification of Notch/Su(H) direct targets in the Kc cells showed that Notch/Su(H) cooperates with LZ in the crystal cells to activate the transcription of certain genes, such as pebbled/hindsight, which are involved in the endo-replication of crystal cells, and klumpfuss, which represses the alternate (plasmatocytes) fate (Terriente-Felix et al., 2013).

In addition, another study showed both in the lymph gland and in circulating larval blood cells that Notch also contributes to crystal cell survival (Mukherjee et al., 2011).

Interestingly, this study showed that this function of Notch in LZ + cells does not depend on its ligand. Indeed, in LZ + cells, high level of the HIF-α homolog Sima stabilizes the internalisation of activated Notch in the endosome even in the absence of activation by its ligand Ser or Delta. This leads to a non-canonical, ligand-independent activation of Notch, which acts with Su(H) to permit crystal cell maturation and to prevent crystal cell rupture.

In addition, the relocation and accumulation of Notch in early endosomes was shown to coincide with an increase in the number of crystal cells in the lymph gland in the absence of Asrij, a conserved protein involved in endocytosis. Therefore, it was suggested that Asrij restricts the number of crystal cells by controlling the intracellular trafficking of Notch [START_REF] Kulkarni | Asrij maintains the stem cell niche and controls differentiation during Drosophila lymph gland hematopoiesis[END_REF]. Finally, it has recently been shown that crystal cells can be produced by trans-differentiation of plasmatocytes in the sessile islets in response to the activation of the Ser/Notch pathway [START_REF] Leitão | Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation[END_REF]. In sum, it appears that the Notch pathway acts at multiple levels in the development of crystal cells. First, Ser is required to activate Notch in blood cell progenitors or plasmatocytes to induce the expression of LZ and thus crystal cell fate; then, activated Notch cooperates with LZ to lock crystal cell fate and finally ligand independent activation of Notch increases crystal cell survival.

RUNX family of transcription factors in hematopoiesis and leukemia

The Core-binding factors family

As described above, many transcription factors are involved in the regulation of the mammalian hematopoiesis and its associated leukemia. Among these transcriptional regulators, core-binding factors (CBFs) are a class of transcription factors, which are essential for hematopoietic development and are also the frequent targets of mutations or gene rearrangements in human leukemia [START_REF] Speck | Core-binding factors in haematopoiesis and leukaemia[END_REF]. They are heterodimeric transcription factor complexes composed of α and β subunits (Figure 8.A) [START_REF] Hart | Core binding factor genes and human leukemia[END_REF]. The α subunit (CBFα) is the DNA-binding element of the complex, which is able to bind to a specific nucleotide sequence motif in vitro even without the β subunit. The β subunit (CBFβ) stabilizes the binding of CBFα to the specific DNA sequence motif without direct DNA contract (Ogawa et al., 1993a) and can protect the α subunit from proteolysis [START_REF] Huang | Dimerization with PEBP2beta protects RUNX1/AML1 from ubiquitin-proteasome-mediated degradation[END_REF]. Structural analyses have shown that the DNA recognition and binding by CBFα is mediated by an allosteric transition in the runt domain, which is further stabilized by CBFβ [START_REF] Tahirov | Structural analyses of DNA recognition by the AML1/Runx-1 runt domain and its allosteric control by CBFβ[END_REF], as well as by the bent-helical conformation of the free DNA target [START_REF] Bartfeld | DNA recognition by the RUNX1 transcription factor is mediated by an allosteric transition in the RUNT domain and by DNA bending[END_REF] (Figure 8.A). All CBFα subunits contain an evolutionarily conserved 128 amino acid domain called the Runt domain, in reference to Drosophila CBFα protein Runt, the founding member of this family. This domain is responsible both for DNA binding and heterodimerization with CBFβ [START_REF] Nimmo | Worming out the biology of Runx[END_REF]Ogawa et al., 1993a). Runt domain proteins are found throughout the metazoan kingdom [START_REF] Rennert | The evolution of Runx genes I. A comparative study of sequences from phylogenetically diverse model organisms[END_REF] and recognize the PyGPyGGT consensus sequence, which seems to be shared by all members of the CBF family. In addition, most RUNX proteins contain a Cterminal VWRPY motif that functions as a binding site for transcriptional co-repressor of the Groucho/TLE family [START_REF] Ito | Oncogenic potential of the RUNX gene family: 'Overview[END_REF]) (Figure 8.B).

In mammals, there are three CBFα subunits encoded by three corresponding genes, RUNX1, RUNX2 and RUNX3 [START_REF] Ito | Oncogenic potential of the RUNX gene family: 'Overview[END_REF], and a single ubiquitous ß subunit encoded by CBFß. The three RUNX genes are required for the development of various tissues. Because they bind the same DNA sequence, their specific role is largely due to their respective spatio-temporal expression pattern. In particular, RUNX1 is required for hematopoietic stem cell emergence and controls several steps of blood cell maturation, whereas RUNX2 plays a key role in osteogenesis and RUNX3 is important for neurogenesis [START_REF] Bae | Phosphorylation, acetylation and ubiquitination: the molecular basis of RUNX regulation[END_REF]de Bruijn et al., 2017;[START_REF] Blyth | Runx2 in normal tissues and cancer cells: A developing story[END_REF][START_REF] Lotem | Runx3 at the interface of immunity, inflammation and cancer[END_REF]. However, these genes also play a critical role in other organs, notably RUNX2 and RUNX3 participate in T cell development in the hematopoietic system. Of note, because of a series of aliases for CBFα subunits generated in many independent laboratories during the past decades, a unified nomenclature for this exciting class of transcription factors has been established [START_REF] Van Wijnen | Nomenclature for Runt-related (RUNX) proteins[END_REF], as shown in Table 2.

Identification of RUNX1 in mammals

It has been known that specific chromosomal translocations are closely associated with a large number of human blood malignancies. The isolation and subsequent study of many

RUNX1 CBFA2 AML1 PEBP2αB 21q22 RUNX2 CBFA1 AML2 PEBP2αA 6p21 RUNX3 CBFA3 AML3 PEBP2αC 1p36
genes located at the breakpoint regions revealed that they often play critical roles in the regulation of proliferation and differentiation of various blood cell lineages. The t(8;21)(q22;q22) translocation is one of the most common and frequent translocations in acute myeloid leukemia (AML), occurring in 12-15% of all cases, especially in the M2 AML subtype according to the French-American-British (FAB) classification system [START_REF] Sangle | Core-binding factor acute myeloid leukemia[END_REF]. After the successful isolation of important genes involved in the t (8;14) Burkitt lymphoma (c-myc), t(9;22) chronic myeloid leukemia (c-abl), t (15;17) acute promyelocytic leukemia (c-erbA), RUNX1/AML1, the first identified mammalian CBF gene, was isolated successfully by virtue of its location on human chromosome 21 at the t(8;21)(q22;q22) breakpoint [START_REF] Miyoshi | t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1[END_REF], meanwhile its murine homolog, PEBP2α/PEA2, was isolated and cloned successfully very soon after thanks to its capacity to bind the enhancer core sites of the polyomavirus (Ogawa et al., 1993b).

Structure and isoforms of RUNX1 transcription factor

Based on the sequence analyses of various forms of RUNX1 cDNA that reflects a complex pattern of mRNA species, it was revealed that RUNX1 gene has 12 exons, and two alternative promoters: the distal promoter or P1, and the proximal promoter or P2 (Figure 9.B), which are juxtaposed with their corresponding and specific first coding exons [START_REF] Sroczynska | The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis[END_REF][START_REF] Bee | Alternative Runx1 promoter usage in mouse developmental hematopoiesis[END_REF][START_REF] Challen | Runx1 isoforms show differential expression patterns during hematopoietic development but have similar functional effects in adult hematopoietic stem cells[END_REF]. This organization is conserved in mammals and the usage of two alternative promoters is also observed in other mammalian RUNX genes. So RUNX1 gene could generate more than 12 different mRNA isoforms and 3 main protein isoforms (RUNX1a, RUNX1b, RUNX1c) by alternative splicing and alternative promoter usage [START_REF] Miyoshi | Alternative splicing and genomic structure of the AML1 gene involved in acute myeloid leukemia[END_REF][START_REF] Levanon | Architecture and anatomy of the genomic locus encoding the human leukemia-associated transcription factor RUNX1/AML1[END_REF].

Systematic analyses of RUNX1 P1 and P2 promoter usage during mouse hematopoietic development showed that the proximal P2-mediated RUNX1 isoform marks a hemogenic endothelium cell population and primitive erythrocytes, whereas the distal P1-mediated RUNX1 defines fully committed definitive hematopoietic progenitors [START_REF] Bee | Alternative Runx1 promoter usage in mouse developmental hematopoiesis[END_REF].

This demonstrates that the differential activities of these 2 RUNX1 promoters define milestones of hematopoietic development, and suggests that the proximal RUNX1 isoform is a key regulator in the generation of hematopoietic cells from hemogenic endothelium [START_REF] Sroczynska | The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis[END_REF].

As mentioned above, RUNX1 codes for 3 protein isoforms. The P2 promoter regulates the expression of RUNX1a and RUNX1b, whereas the P1 promoter controls RUNX1c expression (Figure 9.B). The two long isoforms, RUNX1b and RUNX1c, contain both the DNA-binding Runt domain at their N-terminus followed by a transactivation domain (Figure 9.A), a nuclear-matrix attachment motif, two putative transcriptional repression domains and finally the VWRPY motif. They are generally considered similar in functions [START_REF] Challen | Runx1 isoforms show differential expression patterns during hematopoietic development but have similar functional effects in adult hematopoietic stem cells[END_REF], although RUNX1c has extra 27 amino acids at the N-terminus as compared to RUNX1b. In contrast, RUNX1a lacks the C-terminal transactivation domain and VWRPY motif, and it could act as a competitive inhibitor for RUNX1b [START_REF] Miyoshi | Alternative splicing and genomic structure of the AML1 gene involved in acute myeloid leukemia[END_REF][START_REF] Zhang | CCAAT enhancer-binding protein (C/EBP) and AML1 (CBFα2) synergistically activate the macrophage colony-stimulating factor receptor promoter[END_REF].

Expression pattern analyses of these RUNX1 isoforms showed that they have differential expression profiles during hematopoietic differentiation in mouse embryos and in human embryonic stem cells (hESCs) [START_REF] Challen | Runx1 isoforms show differential expression patterns during hematopoietic development but have similar functional effects in adult hematopoietic stem cells[END_REF]. RUNX1a and RUNX1b isoforms were expressed consistently throughout hematopoietic differentiation, whereas RUNX1c isoform was only expressed at the onset of the emergence of definitive HSCs as well as in the AGM region of E10.5 to E11.5 mouse embryo, which suggested that RUNX1c isoform could be essential for the specification or function of definitive HSCs.

However, several other studies indicated that RUNX1a isoform could have more diverse roles than the other two isoforms. Enforced expression of RUNX1a in the mouse primitive hematopoietic cells resulted in enhanced engraftment upon transplantation, which demonstrated that RUNX1a isoform has the capacity to potentiate stem and progenitor cell engraftment [START_REF] Tsuzuki | Isoform-specific potentiation of stem and progenitor cell engraftment by AML1/RUNX1[END_REF]. Another study revealed that ectopic expression of RUNX1a isoform in mouse HSCs facilitates their expansion [START_REF] Tsuzuki | Expansion of functionally defined mouse hematopoietic stem and progenitor cells by a short isoform of RUNX1/AML1[END_REF] and positively regulates the expression of mesoderm and hematopoietic differentiation-related factors. Similarly, RUNX1a over-expression favoured hematopoietic lineage commitment in human pluripotent stem cells [START_REF] Ran | RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells[END_REF]. In addition, a higher expression level of RUNX1a was found in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML)-M2 patients and RUNX1a antagonized with RUNX1b, which indicated that RUNX1a over-expression could promote leukemogenesis [START_REF] Liu | Overexpression of an isoform of AML1 in acute leukemia and its potential role in leukemogenesis[END_REF]). Yet, the respective functions of the different RUNX1 isoforms in hematopoiesis and leukemia remain to be firmly established.

RUNX1: a master transcriptional regulator of hematopoietic development

RUNX1 is a master transcription factor that plays a critical role in the development and differentiation of specific cell lineages from hematopoietic stem cells. Initial studies showed that it directly controls the expression of various genes that are essential for blood cell survival and differentiation, such the granulocyte/macrophage colony-stimulating factor (GM-CSF) or the macrophage colony-stimulating factor receptor (M-CSFR) [START_REF] Asou | The role of a Runt domain transcription factor AML1/RUNX1 in leukemogenesis and its clinical implications[END_REF].

It acts as an activator or repressor of target gene expression depending on the large number of transcription factors as well as transcriptional co-activators (Figure 10.A) or corepressors (Figure 10.B) that interact with it. For instance, RUNX1 interacts with CCAAT enhancer-binding protein (C/EBP) to activate synergistically M-CSFR expression [START_REF] Zhang | CCAAT enhancer-binding protein (C/EBP) and AML1 (CBFα2) synergistically activate the macrophage colony-stimulating factor receptor promoter[END_REF]. It also binds PU.1 [START_REF] Petrovick | Multiple functional domains of AML1: PU.1 and C/EBPα synergize with different regions of AML1[END_REF], p300 [START_REF] Kitabayashi | Interaction and functional cooperation of the leukemia-associated factors AML1 and p300 in myeloid cell differentiation[END_REF], Ets-1 [START_REF] Kim | Mutual activation of Ets-1 and AML1 DNA binding by direct interaction of their autoinhibitory domains[END_REF], MOZ [START_REF] Kitabayashi | Activation of AML1mediated transcription by MOZ and inhibition by the MOZ-CBP fusion protein[END_REF] or GATA-1 [START_REF] Elagib | RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation[END_REF] to activate transcription. In contrast, RUNX1 interacts with transducing-like enhancer of split (TLE1), a human homolog of the Groucho family of co-repressors, to repress gene expression [START_REF] Imai | TLE, the human homolog of Groucho, interacts with AML1 and acts as a repressor of AML1-induced transactivation[END_REF]. Other RUNX1-associated co-repressors include mSin3A [START_REF] Imai | The corepressor mSin3A regulates phosphorylation-induced activation, intranuclear location, and stability of AML1[END_REF] and histone deacetylase (HDAC) [START_REF] Reed-Inderbitzin | RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription[END_REF]. Hence, RUNX1 functions as an organizing protein that facilitates the assembly of transcriptional activation or repression complexes, which can be described that it recruits non-DNAbinding co-activators or co-repressors to initiate the activation or repression of target genes transcription under different circumstances. The effect of RUNX1 on target gene expression is thus highly context dependent, determined by the composition of the transcriptional complexes in which RUNX1 functions at a particular gene. RUNX1 target gene repertoire has now been established at the genome-wide level in different cell types [START_REF] Lichtinger | RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis[END_REF][START_REF] Lie-A-Ling | RUNX1 positively regulates a cell adhesion and migration program in murine hemogenic endothelium prior to blood emergence[END_REF][START_REF] Bevington | Inducible chromatin priming is associated with the establishment of immunological memory in T cells[END_REF][START_REF] Umansky | Runx1 transcription factor is required for myoblasts proliferation during muscle regeneration[END_REF][START_REF] Van Riel | A novel complex, RUNX1-MYEF2, represses hematopoietic genes in erythroid cells[END_REF][START_REF] Tijssen | Genome-wide analysis of simultaneous GATA1/2, RUNX1, FLI1, and SCL binding in megakaryocytes identifies hematopoietic regulators[END_REF][START_REF] Pencovich | Dynamic combinatorial interactions of RUNX1 and cooperating partners regulates megakaryocytic differentiation in cell line models[END_REF]. These studies notably highlighted the role of RUNX1 in remodelling the epigenetic landscape and promoting the association of other transcription factors with new set of target genes to induce the hematopoietic fate [START_REF] Lichtinger | RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis[END_REF].

Post-translational modifications of RUNX1

Besides the physical interactions with various co-activators or co-repressors, RUNX1 can be modified by multiple post-translational modifications (PTMs), including phosphorylation, methylation, acetylation, ubiquitination and sumoylation (Figure 9.A),

which will impact on its activity.

RUNX1 phosphorylation has been studied extensively, since it can promote the transcriptional activation or the degradation of RUNX1. For instance, the extracellular signal-regulated kinase (ERK), which is activated by several hematopoietic cytokines or phorbol ester treatment, phosphorylates RUNX1 at serine (S)249 and S266, thereby potentiating the transactivation and transforming capacities of RUNX1 in fibroblast cells [START_REF] Tanaka | The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability[END_REF][START_REF] Zhang | Phorbol ester treatment of K562 cells regulates the transcriptional activity of AML1c through phosphorylation[END_REF]. ERK-dependent phosphorylation of RUNX1 disrupts RUNX1 association with the transcriptional co-repressor mSin3A and enhances RUNX1-mediated transactivation but also results in the degradation of RUNX1 by the proteasome [START_REF] Imai | The corepressor mSin3A regulates phosphorylation-induced activation, intranuclear location, and stability of AML1[END_REF]. In addition, RUNX1 is phosphorylated on other residues by homeodomain-interacting protein kinase 2 (HIPK2), Pim-I kinase [START_REF] Aho | Pim-1 kinase phosphorylates RUNX family transcription factors and enhances their activity[END_REF], cyclindependent kinases [START_REF] Biggs | AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex[END_REF][START_REF] Zhang | Cyclin-dependent kinase phosphorylation of RUNX1/AML1 on 3 sites increases transactivation potency and stimulates cell proliferation[END_REF][START_REF] Guo | Phosphorylation of RUNX1 by cyclin-dependent kinase reduces direct interaction with HDAC1 and HDAC3[END_REF] or Src family kinase Shp2 [START_REF] Huang | A Src family kinase-Shp2 axis controls RUNX1 activity in megakaryocyte and T-lymphocyte differentiation[END_REF], resulting in a tight regulation of its activity. RUNX1 is also modified post-translationally by methylation on arginine residues. For example, the protein arginine methyltransferase PRMT1 methylates RUNX1 [START_REF] Zhao | Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity[END_REF][START_REF] Shia | PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential[END_REF], and this methylation on two arginines abrogates the interactions between RUNX1 and SIN3A to potentiate the transcriptional activity of RUNX1.

Conversely, RUNX1 methylation by PRMT4 promotes the assembly of a DPF2-containing co-repressive complex that blocks myeloid differentiation by repressing miR-233 expression in human cord blood cells [START_REF] Vu | PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex[END_REF]. Similarly, RUNX1 is acetylated on lysine residues by the histone acetyltransferase p300, which causes an increase in its DNAbinding activity and enhances its transcriptional activity [START_REF] Yamaguchi | AML1 is functionally regulated through p300-mediated acetylation on specific lysine residues[END_REF][START_REF] Kitabayashi | Interaction and functional cooperation of the leukemia-associated factors AML1 and p300 in myeloid cell differentiation[END_REF]. Moreover, RUNX1 (as well as other RUNX proteins) was found to be sumoylated by PIAS-1, but the functional impact of this modification remains to be determined [START_REF] Kim | RUNX family members are covalently modified and regulated by PIAS1-mediated sumoylation[END_REF].

Finally, it is important to note that the stability of RUNX1 is controlled by ubiquitination, notably by the E3 ubiquitin ligase CHIP [START_REF] Shang | CHIP functions an E3 ubiquitin ligase of Runx1[END_REF] and the SCF/ APC/C complex [START_REF] Biggs | AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex[END_REF], which target RUNX1 to the proteasome degradation pathway. Also CBFß was shown to stabilise RUNX1 by preventing its ubiquitination [START_REF] Huang | Dimerization with PEBP2beta protects RUNX1/AML1 from ubiquitin-proteasome-mediated degradation[END_REF] whereas RUNX1 phosphorylation promotes its ubiquitination and degradation [START_REF] Biggs | Phosphorylation of AML1/RUNX1 regulates its degradation and nuclear matrix association[END_REF][START_REF] Biggs | AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex[END_REF][START_REF] Imai | The corepressor mSin3A regulates phosphorylation-induced activation, intranuclear location, and stability of AML1[END_REF]. In sum, RUNX1 posttranslational modifications have a profound impact on its activity by controlling its interactions with different partner proteins and by regulating its level.

Role of RUNX1 in hematopoiesis

RUNX1 plays a key role in the development of the hematopoietic system.

Homozygous disruption of RUNX1 in mouse embryo resulted in the developmental defects, including lack of fetal liver hematopoiesis and mid-gestation embryonic lethality around embryonic day (E) 12.5 [START_REF] Okuda | AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis[END_REF]. These RUNX1-deficient embryos still had normal morphogenesis and yolk sac-derived erythropoiesis, but they lacked other yolk-sac derived blood cell types as well as definitive hematopoietic progenitors [START_REF] Okuda | AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis[END_REF] and no hematopoietic progenitor/stem cell cluster emerged from their aorta-gonadmesonephros (AGM) region [START_REF] Mukouyama | The AML1 transcription factor functions to develop and maintain hematogenic precursor cells in the embryonic aorta-gonad-mesonephros region[END_REF] (Figure 11). So RUNX1 is required for the emergence of all definitive hematopoietic cells, but it is not essential for the formation of primitive erythrocytes. Of note, primitive erythrocytes in null RUNX1 mice displayed abnormal morphology and reduced expression of Ter119 and GATA, which indicates that RUNX1 is also involved in the development of the primitive erythrocytes [START_REF] Yokomizo | Runx1 is involved in primitive erythropoiesis in the mouse[END_REF]. Interestingly, it was shown that RUNX1 haploinsufficiency resulted in the precocious appearance of HSCs in the AGM region and in the yolk sac, which suggests that RUNX1 dosage is important for the spatio-temporal control of mouse hematopoiesis [START_REF] Cai | Haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo[END_REF].

Expression pattern analyses showed that RUNX1 is expressed in definitive hematopoietic progenitor cells as well as in endothelial cells that reside in the yolk sac, the vitelline and umbilical arteries, which contributes directly to the generation of hematopoietic cells through the formation of intra-arterial clusters [START_REF] North | Cbfa2 is required for the formation of intra-aortic hematopoietic clusters[END_REF]. By conditional deletion study, it was shown that RUNX1 function in endothelial cells is essential for the formation of hematopoietic stem/progenitor cells (HSPCs) from the vasculature [START_REF] Yokomizo | Requirement of Runx1/AML1/PEBP2αB for the generation of haematopoietic cells from endothelial cells[END_REF][START_REF] Chen | Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter[END_REF]. Furthermore, this transition is tightly controlled by the sub-aortic mesenchyme, and RUNX1 and the Notch signalling pathway are involved in this process [START_REF] Richard | Endothelio-mesenchymal interaction controls runx1 expression and modulates the notch pathway to initiate aortic hematopoiesis[END_REF]. Altogether, RUNX1 is required for the definitive hematopoiesis and the endothelial to hematopoietic cell transition in the embryo, as well as involved in the primitive erythropoiesis. In adult mice, RUNX1 is expressed in functional hematopoietic stem cells as well as in the majority of myeloid cells and a smaller proportion of lymphoid cells, but its expression decreases substantially during erythroid differentiation [START_REF] North | Runx1 is expressed in adult mouse hematopoietic stem cells and differentiating myeloid and lymphoid cells, but not in maturing erythroid cells[END_REF]. Specifically, RUNX1 is expressed in myeloid, B-lymphoid and T-lymphoid cells, and its expression is regulated in a cell type-and maturation-specific manner in RUNX1-IRES-GFP knock-in mice [START_REF] Lorsbach | Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression[END_REF]. Using conditional knock-out mice, it was shown that RUNX1deficient bone marrow exhibited inhibition of megakaryocytic maturation, defective T-and B-lymphocyte development and increased hematopoietic progenitor cells, which demonstrated that RUNX1 is required for maturation of megakaryocytes and differentiation of T-and B-lymphocytes [START_REF] Ichikawa | AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis[END_REF]. RUNX1 transcriptionally regulates megakaryocyte development in a cell-autonomous manner [START_REF] Pencovich | Cellautonomous function of Runx1 transcriptionally regulates mouse megakaryocytic maturation[END_REF] in collaboration with the GATA transcription factor GATA1 [START_REF] Elagib | RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation[END_REF]. Notably, it promotes the megakaryocyte fate in bi-potent erythroid/megakaryocytic precursors by repressing the expression of the erythroid transcription factor KLF1 [START_REF] Kuvardina | RUNX1 represses the erythroid gene expression program during megakaryocytic differentiation[END_REF] or the erythroid miR144/451 cluster [START_REF] Kohrs | MiR144/451 expression is repressed by RUNX1 furing megakaryopoiesis and disturbed by RUNX1/ETO[END_REF], as well as by activating the expression of megakaryocyte specific genes [START_REF] Pencovich | Cellautonomous function of Runx1 transcriptionally regulates mouse megakaryocytic maturation[END_REF][START_REF] Pencovich | Dynamic combinatorial interactions of RUNX1 and cooperating partners regulates megakaryocytic differentiation in cell line models[END_REF]. And as for T-lymphocyte development, RUNX1 is required for active repression in CD4 -CD8 -thymocytes [START_REF] Taniuchi | Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development[END_REF] and regulates the two transitions of developing thymocytes from the CD4 -CD8 -double-negative stage to the CD4 + CD8 + doublepositive stage and from the double-positive stage to the mature single-positive stage [START_REF] Egawa | The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells[END_REF], which indicates that RUNX1 has critical roles at multiple stages of Tlymphocyte development. Meanwhile, during early B-lymphocyte development, loss of RUNX1 resulted in a developmental block that was bypassed following retroviral transduction of Ebf1, a key transcription factor regulating early B-lymphocyte development, demonstrating that RUNX1 is essential for B-lymphocyte lineage specification in part through the epigenetic activation of Ebf1 gene [START_REF] Seo | Runx1-Cbfβ facilitates early B lymphocyte development by regulating expression of Ebf1[END_REF]. In addition, RUNX1 also regulates the development of Flt3 + dendritic cell progenitors to facilitate multi-lineage hematopoietic differentiation [START_REF] Satpathy | Runx1 and Cbfβ regulate the development of Flt3 + dendritic cell progenitors and restrict myeloproliferative disorder[END_REF].

In sum, RUNX1 is not only required for the emergence of the HSCs, but also for the differentiation of several blood cell lineages in mice. Of note, the findings obtained in mice have been corroborated by studies in human blood cells and RUNX1 is also a key regulator of hematopoiesis in Xenopus or zebrafish, suggesting that it plays a conserved role in blood cell development in vertebrates.

RUNX1 in leukemia

The importance of RUNX1 in hematopoiesis is further exemplified by the high number of mutations found in RUNX1 in patients with hematological malignancies. Indeed, as mentioned above, RUNX1 was originally identified as the target of the t(8;21) translocation in human 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, AML1[END_REF]. Since then, a number of translocations or point mutations affecting RUNX1 have been identified in diverse blood cell cancers, making RUNX1 one of the most frequently altered genes in these malignancies.

Thus far, more than 50 different chromosomal translocations that involve RUNX1 have been discovered in acute leukemia cases [START_REF] Braekeleer | RUNX1 translocations and fusion genes in malignant hemopathies[END_REF], among which the best studied are the t(8;21)(q22;q22) [START_REF] Downing | An AML1/ETO fusion transcript is consistently detected by RNA-based polymerase chain reaction in acute myelogenous leukemia containing the (8;21)(q22;q22) translocation[END_REF][START_REF] Erickson | Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt[END_REF], t(12;21)(p13;q22) [START_REF] Golub | Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia[END_REF][START_REF] Romana | The t(12;21) of acute lymphoblastic leukemia results in a tel-AML1 gene fusion[END_REF] and t(3;21)(q26;q21) [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][START_REF] Nucifora | Consistent intergenic splicing and production of multiple transcripts between AML1 at 21q22 and unrelated genes at 3q26 in (3;21)(q26;q22) translocations[END_REF] translocations (Figure 12). These translocations are associated with the development of either myeloid or lymphoid leukemia. For instance, the t(8;21)(q22;q22) is the most frequent chromosomal translocation in AML patients (±12% of all cases) whereas t(12;21)(p13;q22) is the most frequent one in ALL patients (±17% of all cases). In addition, the inv(16)(p13;q22) that affects RUNX1 dimerization partner CBFβ accounts for approximately 15% of all AML cases. Thus modification of RUNX1 activity is implicated in a large fraction of human acute leukemia. Generally, these chromosomal translocations lead to the production of RUNX1 or CBFβ fusion proteins that act as dominant inhibitors of the wild-type RUNX1 to promote blood cell transformation.

In most cases, the translocations affecting RUNX1 led to the production of a fusion protein between RUNX1 DNA-binding domain and (part of) another protein that brings along a transcriptional repressor domain. For instance, in the t(8;21) translocation, RUNX1

C-terminal transactivation domain is replaced by the almost entire ETO (Eight Twenty One)

protein, which provides an oligomerisation domain as well as a binding interface for different co-repressors such as N-CoR [START_REF] Gelmetti | Aberrant recruitment of the nuclear receptor corepressor-histone deacetylase complex by the acute myeloid leukemia fusion partner ETO[END_REF], SMRT (Hildebrand et al., 2001), Sin3 [START_REF] Lutterbach | ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors[END_REF] and numerous HDACs [START_REF] Wang | ETO, fusion partner in t(8;21) acute myeloid leukemia, represses transcription by interaction with the human N-CoR/mSin3/HDAC1 complex[END_REF]. The resulting RUNX1-ETO fusion protein can repress transcription from RUNX1-responsive genes by competing with wild type RUNX1 [START_REF] Miyoshi | The t(8;21) translocation in acute myeloid leukemia results in production of an AML1-MTG8 fusion transcript[END_REF][START_REF] Ptasinska | Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding[END_REF]).

Yet, RUNX1-ETO mode of actions is more complex: it was shown that it also forms a complex with wild-type RUNX1 on chromatin and it was proposed that the relative binding signals of RUNX1 and RUNX1-ETO determine whether RUNX1-ETO activates or represses its targets [START_REF] Li | Genome-wide studies identify a novel interplay between AML1 and AML1/ETO in t(8;21) acute myeloid leukemia[END_REF][START_REF] Minucci | DNA binding modes of leukemia oncoproteins[END_REF]. In addition, RUNX1-ETO also binds and regulates other transcription factors such as the myeloid factors C/EBPα or PU.1 [START_REF] Pabst | AML1-ETO downregulates the granulocytic differentiation factor C/EBP-alpha in t(8;21) myeloid leukemia[END_REF][START_REF] Vangala | The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia[END_REF]. Thus, RUNX1-ETO expression has a profound impact on blood cell development by repressing myeloid differentiation and promoting leukemic cell self-renewal [START_REF] Ptasinska | Identification of a dynamic core transcriptional network in t(8;21) AML that regulates differentiation block and self-renewal[END_REF]. However, it is not sufficient to induce malignant blood cell transformation and, as for other translocations affecting RUNX1, other mutations are required for the progression towards leukemia. Thus, an important issue is to identify the genes that cooperate or interfere with RUNX1 or its oncogenic derivatives during hematopoiesis and leukemia.

Besides translocations, a number of point mutations affecting RUNX1 are associated with human blood cell diseases. Notably, rare cases of haploinsufficient germ-line mutations in RUNX1 lead to the development of familial platelet disorder with predisposition to acute myeloid leukemia [START_REF] Song | Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia[END_REF]. There are also numerous examples of somatic point mutations of RUNX1 in patients with myelodysplastic syndromes, acute myeloid leukemia but also in acute lymphoid leukemia and chronic myelomonocytic leukemia (Sood et al., 2017). Rare cases of bi-allelic "null" mutations have been found in AML-M0 subtypes, but mutations affecting RUNX1 are mostly mono-allelic and could affect RUNX1 activity by different mechanisms that remain to be established. Nonetheless, these findings highlight the critical role of RUNX1 in normal blood cell development in humans and indicate that a tight regulation of its activity and expression level is crucial for human health.

Lozenge in Drosophila hematopoiesis

Drosophila genome encodes four RUNX genes: runt, lozenge, RunxA and RunxB.

Lozenge is known to be expressed in Drosophila blood cells and I will thus focus on this factor. As a member of the RUNX family, Lozenge (Lz) harbors a highly conserved runt domain that is essential for the DNA-binding and protein-protein interactions, and a Cterminal motif VWRPY that is capable of recruiting Groucho family of co-repressors. It is worth mentioning that Drosophila has two CBFß homologs: Brother and Big-Brother that can interact with Lozenge (Li et al., 1999;[START_REF] Kaminker | Redundant function of Runt domain binding partners, Big brother and Brother, during Drosophila development[END_REF], but their function in hematopoiesis has not been investigated.

lozenge gene is located on the X chromosome and it is transcribed from a single promoter into two different mRNAs that are generated by alternative splicing of the exon 5.

There are thus two protein isoforms (Figure 13): Lz (826 amino acids long) and Lz 5 (705 amino acids long), which contain or not an interaction domain for ETS family of transcription factors just after the runt homology domain (RHD) (Jackson [START_REF] Behan | Alternative splicing removes an Ets interaction domain from Lozenge during Drosophila eye development[END_REF]. As for mammalian RUNX1, the relative contribution of these two isoforms to Drosophila hematopoiesis remains to be determined. Lozenge is involved in several different developmental processes, including the development of eye, antennal and tarsal claw, female fertility and hematopoiesis [START_REF] Canon | Runt and Lozenge function in Drosophila development[END_REF]. It is known to have a major role in cell fate determination, and the most characterized example of this function of lozenge is during eye and crystal cell development.

lozenge mutant alleles were first identified by Morgan and Bridges in 1920 thanks to their effects on the morphology of the Drosophila eye (Figure 14).

Drosophila has a compound eye composed of 800 identical units called ommatidia. In each ommatidium, there are eight light-absorbing photoreceptor neurons (R1-R8) and four non-neuronal cone cells, which produce the lens [START_REF] Batterham | Genetic analysis of the lozenge gene complex in Drosophila melanogaster: adult visual system phenotypes[END_REF]. A number of transcription factors are involved in the cell-specific patterning in the eye, which marks the identity of individual cell types. Among such transcription factors are Seven-up (Svp), a member of the steroid hormone receptor superfamily expressed in R1, R3, R4 and R6 [START_REF] Mlodzik | The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates[END_REF], Bar, a homeobox protein required in R1 and R6 [START_REF] Higashijima | Dual Bar homeo box genes of Drosophila required in two photoreceptor cells, R1 and R6, and primary pigment cells for normal eye development[END_REF], and DPax-2, the Drosophila Pax-2 homolog required for the development of the cone cells [START_REF] Fu | The Pax2 homolog sparkling is required for development of cone and pigment cells in the Drosophila eye[END_REF]. Genetic analyses have revealed that lozenge positively or negatively regulates all of the known transcription factors required for the cell lineage specification. It plays a crucial role in governing the fate of two groups of cells that are born in a single round of mitosis in the larval fly eye disc [START_REF] Daga | Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domains with AML1[END_REF]. By negatively regulating seven-up (svp), Lozenge helps to define a subset of these cells as an equipotential group that is competent to respond to the EGFR/Sevenless developmental signal. In contrast, by positively regulating Bar, Lozenge confers proper photoreceptor identity in a second group of cells [START_REF] Daga | Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domains with AML1[END_REF]. Another study showed that Lozenge directly activates argos (which encodes an inhibitor of EGFR) and klumpfuss (the homolog of apoptotic regulator WT1), to regulate programmed cell death in Drosophila eye (Wildonger et al., 2005). Therefore, Lozenge patterns multiple cell types in the Drosophila eye through the control of cell-specific transcription factors [START_REF] Flores | Lozenge is expressed in pluripotent precursor cells and patterns multiple cell types in the Drosophila eye through the control of cellspecific transcription factors[END_REF]. It is important to note that Lozenge can act both as a transcriptional activator and as a transcriptional repressor in the same cell. Its capacity to repress transcription of deadpan not only requires the corepressor Groucho but also the transcription factor Cut, whose expression is activated by Lozenge, and which binds next to Lozenge on deadpan regulatory region to stabilize the formation of a repressor complex [START_REF] Canon | In vivo analysis of a developmental circuit for direct transcriptional activation and repression in the same cell by a Runx protein[END_REF]. Conversely, it was proposed that Lozenge cooperates with the ETS factor Pointed to activate the expression of prospero and D-Pax2 (Jackson [START_REF] Behan | Alternative splicing removes an Ets interaction domain from Lozenge during Drosophila eye development[END_REF]. This duality of Lozenge function in the eye was actually used to show that the human RUNX1-ETO oncogenic fusion protein behaves as a constitutive repressor: when RUNX1-ETO is expressed in the fly eye, it represses both deadpan and D-Pax2 transcription (Wildonger et al., 2005).

Lozenge is also absolutely required for crystal cell differentiation both in the embryo and in the larva during Drosophila hematopoiesis (Lebestky et al., 2000). It is specifically expressed in the crystal cell lineage, and in a lozenge null background no crystal cells is formed. Moreover, using a thermo-sensitive allele of lozenge, it was shown that its function is continuously required for crystal cell development.

In the embryo, lozenge is expressed first in the anterior-most row of prohemocytes slightly after the onset of Serpent expression [START_REF] Bataillé | Resolving embryonic blood cell fate choice in Drosophila: interplay of GCM and RUNX factors[END_REF]. A fraction of these cells maintains lozenge expression and differentiates into crystal cells, whereas the remaining progenitors give rise to plasmatocytes [START_REF] Bataillé | Resolving embryonic blood cell fate choice in Drosophila: interplay of GCM and RUNX factors[END_REF]. In the larvae, lozenge expression is activated in scattered cells in response to the Serrate/Notch signaling pathway both in the lymph gland and in the sessile hematopoietic pockets (Duvic et al., 2002;Lebestky et al., 2003). Recently, it was proposed that the Serrate/Notch signaling in the lymph gland leads to the up-regulation of the transcriptional co-factor Yorkie, which acts together with its DNA-binding partner Scalloped to directly activate lozenge transcription in the crystal cell lineage [START_REF] Milton | The Hippo pathway regulates hematopoiesis in Drosophila melanogaster[END_REF]. Whether Scalloped and Yorkie are also involved in the activation of lozenge transcription in the embryo and in the larval sessile hematopoietic pocket is not known. So, it is still unknown how lozenge expression is initiated in the crystal cell precursors. However, it has been shown that Lozenge can activate its own transcription, suggesting that its expression in the crystal cell lineage could be maintained via an auto-activation loop (Ferjoux et al., 2007).

At the molecular level, Lozenge can trigger the expression of crystal cell specific markers in any tissues that express the GATA factor Serpent, indicating that Lozenge synergizes with Serpent to induce crystal cell differentiation (Waltzer et al., 2003;[START_REF] Fossett | Combinatorial interactions of Serpent, Lozenge, and U-shaped regulate crystal cell lineage commitment during Drosophila hematopoiesis[END_REF]. This functional cooperation is mediated by a direct interaction between Serpent and Lozenge (Waltzer et al., 2003) as well as a conserved Serpent/Lozenge-responsive cisregulatory module present in many crystal cell specific genes (such as the prophenoloxidases PPO1 and PPO2 or lozenge itself), which is composed of at least one Serpent and one Lozenge binding site in close association (Ferjoux et al., 2007).

Furthermore, the interaction between Serpent and Lozenge has been conserved through evolution (Waltzer et al., 2003), so the cooperation between Serpent and Lozenge might be used as a paradigm to study how GATA/RUNX complexes regulate transcription and blood cell development from Drosophila to vertebrates. The Serpent/Lozenge complex notably acts with several subunits of the mediator transcription complex to activate gene expression (Gobert et al., 2010). In addition, it was shown that Lozenge (and potentially Serpent)

facilitates the recruitment of the Notch/Su(H) complex to some of its target genes and thus collaborates with the Notch signaling pathway during crystal cell differentiation (Terriente-Felix et al., 2013;[START_REF] Skalska | Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation[END_REF].

Myeloid Leukemia Factor (MLF) family

Myeloid leukemia factors are a novel family of proteins in the course of evolution, and MLF1, the founding member, is associated with the emergence of blood cell cancers in human (Yoneda-Kato et al., 1996). However, their functions and molecular mechanisms of action remain still largely unknown. Myeloid leukemia factor 1 (MLF1), the founding member of the MLF family, was identified by cloning the breakpoints of the t(3;5)(q25.1;q34) translocation, which is a chromosomal rearrangement found in rare (less than 0.5%) cases of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) in human (Yoneda-Kato et al., 1996) (Figure 15). It exhibits marked variability in expression, with high levels in testis, ovary, skeletal and cardiac muscle, colon and kidney, but low or apparently absent transcription in other tissues. Soon after the discovery of MLF1, MLF2 cDNA was cloned, which encodes a protein highly homologous to MLF1 [START_REF] Kuefer | cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/myeloid leukemia factor 2 (MLF2)[END_REF]. The MLF2 gene locus was mapped to chromosome 12q13 in human, which is a chromosomal region involved frequently in the translocations and deletions in acute myeloid leukemia or acute lymphoid leukemia, although no evidence indicates so far that MLF2 directly participates in blood cell cancers and/or hematopoiesis.

The members of the MLF family appear to be present in all metazoans (Martin- et al., 2006). In human, the MLF family comprises two members, hMLF1 and hMLF2, which share nearly 40% of identity between them. At the molecular level, hMLF1 and hMLF2 are relatively small-sized proteins (about 270 amino acids). hMLF1 has an Nterminal nuclear export signal (NES) and two C-terminal nuclear localization signals (NLS) (Figure 15), which allow hMLF1 to shuttle between the nucleus and the cytoplasm [START_REF] Yoneda-Kato | Shuttling imbalance of MLF1 results in p53 instability and increases susceptibility to oncogenic transformation[END_REF]. hMLF1 and hMLF2 do not have specific known functional domains that could help ascribe their biochemical activity, and they only have a central domain preserved within the MLF family and a 14-3-3 protein binding domain (Gobert et al., 2012).

Lannerée

In addition to the adaptor protein 14-3-3ζ, which can bind MLF1 [START_REF] Lim | MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1[END_REF][START_REF] Molzan | Structural insights of the MLF1/14-3-3 interaction[END_REF] and regulate its interaction with the apoptotic regulator Bcl-XL (Sun et al., 2015), only a few proteins are known to interact with mammalian MLF proteins, including the COP9 signalosome subunit CSN3 which is required for MLF1-induced degradation of p53 (Yoneda-Kato et al., 2005), the HOP survival complex components Hax1 and HtrA2 (Sun et al., 2017) (see below), the centromere protein CENPU/MLF1IP [START_REF] Hanissian | cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP[END_REF], the adaptor protein MADM [START_REF] Lim | MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1[END_REF], and the hnRNP-U like protein MANP (Winteringham et al., 2006). Also, in many cases, the physiological relevance of these interactions has not been known, so we only have a limited understanding of MLF protein possible mode of action.

Roles of hMLF1 in hematopoiesis

So far, the roles of hMLF1 in hematopoiesis in human have still not been characterized. However, its expression profile suggests that it regulates blood cell progenitor fate. Indeed, hMLF1 is strongly expressed in CD34 + progenitor cells and its expression decreases as soon as the specification of these cells towards myeloid and erythroid lineage progresses (Matsumoto et al., 2000). The percentages of identity between MLF proteins or their MHD regions are indicated. (From Gobert et al., 2012) In a mouse model, MLF1/HLS7 (Hematopoietic Lineage Switch 7) was identified during a screening for genes controlling the transition of erythroleukemic cells (J2E)/ immature myeloid cells (J2E-m2) [START_REF] Williams | HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1[END_REF]. Indeed, the ectopic expression of MLF1 in J2E cells reduces their ability to differentiate into mature erythrocytes in response to erythropoietin and causes the acquisition of immature monocyte-type phenotypes.

Therefore, MLF1 can induce the reprogramming of erythroleukemic cells to monocytes, which is called "lineage switching". In addition, over-expression of MLF1 in primary hematopoietic cells of the fetal liver, as well as in different blood cell lines, confirmed that MLF1 promotes the differentiation of myeloid cells and inhibits erythropoietin-induced cell differentiation [START_REF] Williams | HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1[END_REF]Winteringham et al., 2004). While the consequences of MLF1 invalidation on mice hematopoiesis remains to be established, it was shown that MLF1 knock-out lymphocytes are more resistant to apoptotic stimulation than wild-type cells, suggesting that MLF1 controls lymphocyte fate (Sun et al., 2015). However, further analyses will be required to better characterize MLF1 function and mode of action during mammalian blood cell development.

Roles of hMLF in cancer, cell proliferation and apoptosis.

As mentioned above, hMLF1 was identified by cloning the breakpoints of the chromosomal translocation t(3;5)(q25.1;q34) associated with MDS and AML (Yoneda-Kato et al., 1996). This rare translocation results in the expression of the NPM-MLF1 fusion protein, which is composed of the N-terminal portion of nucleophosmin (NPM) containing a nuclear localization signal and a dimerization domain, fused to the almost entire protein sequence of hMLF1 (Figure 15). NPM is a nucleolar protein with many functions, such as control of ribosomal protein transport, regulation of cell cycle progression, maintenance of genome stability or assembly/disassembly of nucleosomes [START_REF] Colombo | Nucleophosmin and its complex network: a possible therapeutic target in hematological diseases[END_REF].

Many evidences indicate that the NPM-MLF1 fusion protein plays a direct role in malignant transformation. In particular, NPM-MLF1 expression increases the proliferative potential of hematopoietic progenitors in vitro and facilitates the oncogenic transformation induced by RasV12 in murine embryonic fibroblast [START_REF] Lee | NPM-MLF1 synergizes with Npm haploinsufficiency to enhance myeloid progenitor activity[END_REF]. Moreover, NPM is very frequently mutated in AML and it is targeted by two other chromosomal translocations found in leukemia, to generate fusion proteins with ALK or RAR [START_REF] Falini | Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias[END_REF]. It has been proposed that NPM converts its partners into onco-proteins by providing an oligomerization domain as well as a nuclear localization signal (NLS) in the case of translocations. In fact, MLF1 is mostly localized in the cytoplasm, but NPM-MLF1 is nuclear in cell culture after transfection [START_REF] Falini | Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias[END_REF]Ohno et al., 2000). It also should be noted that the expression of MLF1 is deregulated because it is under the control of the regulatory regions of NPM1 in the chromosomal translocation t (3;5). Furthermore, it has been observed that the expression of MLF1 increases during the progression of MDS to AML and that high expression levels of MLF1 are associated with a poor prognosis in MDS patients without the t(3;5) translocation. Thus, deregulation of MLF1 expression could participate in the malignant transformation of myeloid cells (Matsumoto et al., 2000). All together, the roles and the molecular mechanisms of action of MLF1 in leukemic transformation remain rather elusive.

In addition, MLF1 may have an oncogenic role in other tissues, for instance, MLF1 is over-expressed in squamous lung carcinomas (Sun et al., 2004) and esophageal carcinomas (Chen et al., 2008). Moreover, MLF2 was shown to contribute to cancer cell metastasis and potential gain of function point mutations in MLF2 have been identified in lung and breast metastatic cells [START_REF] Granados-Principal | Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling[END_REF]. Strikingly, a recent study identified a bi-allelic null mutation of MLF1 in infants with T-cell acute lymphoid leukemia (Mansur et al., 2015).

Therefore, MLF family members could play a role as a tumor suppressor or as an oncogene depending on the cell types.

Along that line, it was shown that MLF1 overexpression impairs cell cycle exit in erythrocytes by promoting the degradation of the cell cycle regulator p27 Kip1 (Winteringham et al., 2004), but inhibits the proliferation of fibroblasts through preventing p53 degradation (Yoneda-Kato et al., 2005). More recently, a study showed that MLF1 is a novel modulator of neonatal rat cardiomyocyte proliferation (Rangrez et al., 2017). MLF1 overexpression in cardiomyocytes inhibited their proliferation and promoted apoptosis, whereas MLF1 knockdown protected them from apoptosis and hypoxia-induced cell death and promoted proliferation. Interestingly, MLF1 is highly expressed in the heart in human, but at significantly reduced levels in patients with dilated cardiomyopathy and it is profoundly down-regulated in an in vivo mouse model of cardiomyopathy, suggesting that MLF1 could be implicated in this pathology. Finally, another recent study showed that MLF1 is a proapoptotic antagonist of HOP complex-mediated survival (Sun et al., 2017). By interacting with HAX1 and HtrA2, two components of the HAX1/HtrA2-OMI/PARL (HOP) mitochondrial protein complex, MLF1 inhibits HtrA2 cleavage and activation, which leads to apoptotic cell death. Interestingly, mlf1 deletion reverses lymphopenia and significantly ameliorates the progressive neurodegeneration observed in Hax null mutant mice. Thus, MLF1 could control cell survival in the hematopoietic and nervous system by regulating HOP function. However, only a small fraction of MLF1 is present in the mitochondria (Sun et al., 2017) and thus its function is probably not restricted to that cell compartment.

4.2.

Drosophila myeloid leukemia factor (dMLF)

Isoforms and structure of dMLF

In Drosophila, there is a single mlf gene, which is located on chromosome II (Ohno et al., 2000). dmlf codes for four distinct isoforms that are generated by alternative splicing, which are dMLF-A, dMLF-B, dMLF-C and dMLF-D, and these MLF isoforms that range from 273 to 376 amino acids long, differ by their N-terminal or C-terminal region (Martin-Lannerée et al., 2006) (Figure 16). dMLF-A, the most abundant isoform of dMLF, has 28% identity with hMLF1 and hMLF2 (Gobert et al., 2012). The central region of dMLF-A (amino acids 96 to 202), which corresponds to the MLF homology domain (MHD), has about 50% identity with the corresponding domain of hMLF1 or hMLF2 (Figure 15).

dMLF contains a nuclear export signal (NES) and two nuclear localization signals (NLS), and a study showed that these two NLS are required to enable nuclear localization of dMLF-A in Drosophila cell culture [START_REF] Sugano | Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor[END_REF]. MLF-C and D also carry these NLS but MLF-B does not, and it was reported to be located both in the cytoplasm and in the nucleus [START_REF] Martin-Lannerée | Characterization of the Drosophila myeloid leukemia factor[END_REF]. Finally, except a binding site for the 14-3-3 family of proteins at the C-terminus, dMLF does not possess any homology with other proteins.

Expression profile and mutants of dMLF

The expression profile of mlf gene reveals that it has a strong maternal contribution and it is ubiquitously expressed during the early embryonic stage. At later stage, its expression level increases in the central nervous system, gonads, digestive tract and crystal cells [START_REF] Martin-Lannerée | Characterization of the Drosophila myeloid leukemia factor[END_REF]Bras et al., 2012). Furthermore, mlf gene is expressed ubiquitously at different larval stages in the imaginal discs. dMLF is localized in both the nucleus and the cytoplasm in different ratios depending on the tissues and stages of development [START_REF] Martin-Lannerée | Characterization of the Drosophila myeloid leukemia factor[END_REF]. In the larval hemocytes, dMLF is expressed predominantly in the crystal cell lineage where it is mainly localized in the nucleus (Bras et al., 2012).

dMLF partners and involvement in apoptosis

Drosophila MLF was first identified in a two-hybrid screen in yeast as a partner of the Drosophila transcription factor DREF (DNA Replication Enhancer Factor), which regulates the expression of genes involved in DNA replication and cell proliferation (Ohno et al., 2000). Moreover, dMLF over-expression was found to induce a reduction in eye and wing size [START_REF] Sugano | Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor[END_REF]Yanai et al., 2014). Importantly, a recent publication showed that MLF is recruited to chromatin on a DREF-responsive enhancer in basket, a gene coding for the Jun N-terminal Kinase (JNK) whose activation promotes apoptosis, and genetic experiments indicate that gain of MLF causes a reduction in wing size by activating the JNK pathway and apoptosis (Yanai et al., 2014). Together with previous findings

showing that MLF is associated with polytene chromosomes in Drosophila (Fouix et al., 2003) and that mouse Mlf1 can bind DNA (Winteringham et al., 2006), this report demonstrated for the first time that MLF could act directly on chromatin to regulate gene expression. This was very recently confirmed at the genome-wide level in S2 cells by another study (Dyer et al., 2016) (see Discussion).

Independently, another two-hybrid screen showed that MLF is a partner of Suppressor of Fused, a negative regulator of the Hedgehog signalling pathway, and gain of function experiments showed that dMLF genetically interacts with Su(fu) in the eye disc (Fouix et al., 2003). In addition, dMLF was shown to physically interact with another negative regulator of Hedgehog, dCostal2 (Fouix et al., 2003), and as its mammalian counterpart, with the COP9 subunit CSN3 [START_REF] Sugano | Identification of novel nuclear localization signals of Drosophila myeloid leukemia factor[END_REF]. However, the importance of MLF interaction with these proteins in vivo remains largely unknown.

Roles of dMLF in hematopoiesis

In Drosophila, dMLF is strongly expressed in crystal cells, one of the three main blood cell lineages, at the embryonic stage and the larval stage [START_REF] Martin-Lannerée | Characterization of the Drosophila myeloid leukemia factor[END_REF]Bras et al., 2012). Actually, mlf expression in the crystal cell lineage seems to be directly activated by the SRP/LZ complex (Bras et al., 2012;Ferjoux et al., 2007). In addition, mlf was identified as a positive regulator of the activity of the SRP/LZ complex in a genomewide RNA interference (RNAi) screen in cell culture. Indeed, the down-regulation of dMLF expression by RNAi technology in Kc167 cells resulted in a decrease in the transactivation activity of a target reporter gene activated by the SRP/LZ complex (Gobert et al., 2010). It has been shown that MLF regulates the activity of this complex through ensuring LZ stable expression both in Kc167 cells and in crystal cells in vivo (Bras et al., 2012).

Thanks to its capacity to control transcription factor LZ stability, dMLF also regulates the development of the embryonic and the larval LZ + cell lineage in Drosophila (Bras et al., 2012). During embryonic hematopoiesis, dMLF is required for the maintenance of LZ + cells. These cells are specified normally, but their number decreases gradually in absence of dMLF. This is probably due to a lack of LZ accumulation/maintenance, which prevents the self-regulation of LZ expression and the maintenance of embryonic crystal cells. Indeed, the enforced expression of LZ in the LZ + cells can restore crystal cell number in mlf mutant embryos. On the contrary, an increase in the number of circulating LZ + cells is observed in the mlf mutant larvae. This phenotype in the larvae is also associated with a decrease in LZ protein level and is rescued by the enforced expression of LZ, suggesting that this unexpected increase in larval crystal cell number caused by the loss of dMLF is due to a decrease in LZ level. However, the precise mechanism by which dMLF regulates LZ stability and why there is an increase in larval crystal cell number still remain unknown.

During my PhD, I tried to tackle these two important questions to better understand MLF function and molecular mechanism of action during hematopoiesis.

It is worth mentioning that the defects in crystal cell number observed in mlf mutants were rescued by re-expressing dMLF specifically in the LZ + cells using the lz-GAL4 driver, demonstrating that these defects are cell autonomous and caused after the induction of LZ expression (Bras et al., 2012). In addition, hMLF1 expression also rescued mlf mutant This could be particularly relevant for hMLF1 function in the development of MDS and AML in human.

Roles of dMLF in suppression of polyglutamine aggregates and neurodegeneration

Huntington's disease (HD), an autosomal dominant neurodegenerative disease, is caused by the expansion of normally polymorphic polyglutamine (polyQ) stretches at the Nterminus of the protein Huntingtin (HTT). If the polyQ stretches in Huntingtin is expanded beyond 36, it will lead to misfolding of the protein, which causes the formation of cytoplasmic and nuclear/perinuclear aggregates that are also known as intracellular inclusions. It has been known that Huntingtin interacts with several proteins and these proteins are recruited to the aggregates. However, the contributions of these proteins to the pathogenesis of Huntington's disease have not been identified fully. Several Drosophila models have been developed to model Huntington's disease as well as other polyQ-associated neurodegenerative diseases [START_REF] Mcgurk | Drosophila as an in vivo model for human neurodegenerative disease[END_REF]. Indeed, the expression of polyQ in the Drosophila eye is toxic and produces phenotypes (eye depigmentation and cell degeneration) that can be used as readout to screen for enhancers or suppressors of polyQassociated neurodegeneration. Thereby, it was shown that over-expression of dMLF can ameliorate the cellular toxicity of the polyQ proteins expressed in the eye and in the central nervous system (Kazemi-Esfarjani et al., 2000). In particular, the endogenously or ectopically expressed dMLF co-localizes with polyQ aggregates in the retina, suggesting that dMLF alone or through an intermediary molecular partner sequesters polyQ and/or its aggregates to suppress toxicity. Moreover, in transfected primary rat neuronal culture, dMLF also co-localizes with the polyQ inclusions and suppresses their toxicity, reducing the morphological phenotypes and inclusions (Kim et al., 2005). Interestingly, similar suppression was observed with hMLF1 or hMLF2, suggesting that suppression of polyQ toxicity is a conserved function of MLF proteins. At the molecular level, dMLF was found to reduce the recruitment of the histone acetyltransferase CBP and the chaperone Hsp70 into the inclusions, two essential proteins trapped in polyQ aggregates. In addition, hMLF1 and hMLF2 were recently found to preferentially interact with the mutated N-terminal Huntingtin. Both of them significantly reduced the number of cells containing mutant Huntingtin aggregates and subsequent apoptosis in Neuro2A cells model (Banerjee et al., 2017). In presence of hMLF1 and hMLF2, the mobile fraction of mutant Huntingtin aggregates was increased, and hMLF1 could release some transcription factors from mutant Huntingtin aggregates. These data suggest that MLF proteins could modulate the formation of aggregates as well as the induction of apoptosis, resulting in a decrease in polyQ associated toxicity. Thus, beyond their role in hematopoiesis and leukemia, MLF proteins could be important regulators in neurodegenerative diseases.

The Hsp40/DnaJ chaperone family

The 40kDa heat shock protein (Hsp40/DnaJ) co-chaperones are the largest and the most diverse sub-group of the heat shock protein (HSP) family. They are widely accepted as regulators of the Hsp70 chaperones, but also have roles as co-chaperones in the Hsp90 chaperone machine. However, a growing number of evidences show that their biological functions may be independent of either of these two chaperone machines.

General description of molecular chaperones

The successful execution of cellular processes is dependent on the coordinated interactions of proteins. After synthesis as linear sequence of amino acids on ribosomes, the large majority of proteins must fold into well-defined three-dimensional structures to obtain their functions. Although some newly translated proteins are capable of folding spontaneously, a large number of proteins are less efficient to fold properly and easy to misfold, leading to the formation of protein aggregates, which can be toxic and cause diseases.

To settle these problems, cells have a network of molecular chaperones that assist in de novo folding and maintain preexisting proteins in their native states [START_REF] Hartl | Molecular chaperones in protein folding and proteostasis[END_REF].

Molecular chaperones are any proteins that interact with and aid in the folding or assembly of another protein without being part of its final structure (Kim et al., 2013). They are found in bacteria, plants, insects and other animals, and represent the most preserved system of the living kingdom. They are main players of protein homeostasis in cells under physiological or stress conditions [START_REF] Saibil | Chaperone machines for protein folding, unfolding and disaggregation[END_REF], modulating the integrity or activity of their protein substrates (called clients) with different mechanisms. They participate into many biological processes, ranging from folding of newly synthesized proteins or refolding of mis-folded protein aggregates to the regulation of their stability and subcellular location (Figure 17). Under physiological conditions, they recognize the hydrophobic regions of proteins during synthesis, and thus preventing the specific interactions with other proteins as well as the formation of lethal insoluble aggregates in cells. Under cellular stress conditions, the chaperones interact with denatured or poorly folded proteins and then try to refold their substrates properly. If the client protein cannot be refolded properly, it is usually delivered to the degradation pathways. As main cyto-protective players, molecular chaperones protect cells against different types of stresses and ensure organism survival under adverse conditions.

Heat shock proteins

Up to now, the largest and best-characterized group of molecular chaperones is composed of heat shock proteins (Hsps). The heat shock response (HSR) is an ancient and highly conserved molecular response to disruption of protein homeostasis [START_REF] Morimoto | The heat shock response: systems biology of proteotoxic stress in aging and disease[END_REF], which was discovered in 1962 and characterized with an abnormal transcription of certain loci on polytene chromosomes of salivary glands in Drosophila following an elevation of temperature [START_REF] Jamrich | Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster[END_REF]. Since then, many studies showed that other stresses, including endoplasmic reticulum stress, nutrient deficiencies or viral infections, can induce or increase the expression of Hsps. In addition, some Hsps are constitutively expressed and have household functions [START_REF] Vos | Structural and functional diversities between members of the human HSPB, HSPH, HSPA, and DNAJ chaperone families[END_REF]. Notably, many HSPs are chaperones and play an important role in protein homeostasis by binding to newly

Figure 17. Protein fates in the proteostasis network

The proteostasis network integrates chaperone pathways for the folding of newly synthesized proteins, for the remodeling of misfolded states and for disaggregation with the protein degradation mediated by the ubiquitin-proteasome-system (UPS) and the autophagy system.

(From Kim et al., 2013) synthesized polypeptides, catalyzing their conformational maturation or participating in protein quality control.

In mammals, Hsps are grouped into six main families depending on their molecular weight: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40 and the family of small stress proteins (small Hsps, or sHsps) [START_REF] Hartl | Molecular chaperones in protein folding and proteostasis[END_REF]. These families have some common specificity with respect to their subcellular localization (cytoplasm, mitochondria, endoplasmic reticulum), their mechanisms of action, their dependence on ATP. Here, it should be noted that, historically, the inducible chaperones are denoted as Hsp (Heat shock protein), while the constitutively expressed chaperones are denoted as Hsc (Heat shock cognate protein). These chaperones form a vast network of molecular chaperones that can be schematically subdivided into four families of ATP-dependent chaperones (Hsp60, Hsp70, Hsp90 and Hsp100), whose activities are regulated by their respective co-chaperones, for instance, Hsp40/DnaJ for the Hsp70 chaperone system.

The DnaJ proteins family

The Hsp70/DnaJ chaperone system

Generally, there are two common chaperone families, the Hsp70 system and the Hsp90 system, which participate broadly in de novo protein folding and refolding (Figure 18).

They are multicomponent molecular chaperone machines that promote protein folding through ATP-and co-factor-regulated binding and release cycles.

The founding member of the Hsp40 family, DnaJ, was identified as a regulator of the ATPase activity of DnaK, the Hsp70 homolog in E.coli [START_REF] Yochem | Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication[END_REF]. In this (B) The three dimensional structure of J-domain (E.coli J-domain; 1BQ0) that is currently used to define the DNAJ family. It illustrates that the J-domain structure resembles a "protruding finger" (helix 2 and 3) containing the highly conserved HPD (His-Pro-Asp) motif located on the loop between helix 2 and 3. This HPD motif is important for stimulation of Hsp70 ATPase activity (From Sterrenberg et al., 2011).

The Hsp40/DnaJ proteins family in human is composed of 49 members and 44 members in Drosophila. These proteins are classified into three types according to the presence or absence of two other domains in addition to the N-terminal J-domain and the conserved C-terminal domain [START_REF] Cheetham | Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function[END_REF] expression may be ubiquitous or restricted to a particular tissue. Some Hsp40/DnaJ proteins of type I and II could interact with unfolded polypeptides and have a chaperone function independently of Hsp70 proteins (Hageman et al., 2010). Besides the domains described above, some Hsp40/DnaJ proteins possess additional regions that enable them to exert a specific function [START_REF] Lu | Tid1 isoforms are mitochondrial DnaJ-like chaperones with unique carboxyl termini that determine cytosolic fate[END_REF][START_REF] Cunnea | ERdj5, an edoplasmic reticulum (ER)-resident protein containing DnaJ and thioredoxin domains, is expressed in secretory cells or following ER stress[END_REF][START_REF] Shi | Contributions of cysteine residues in Zn2 to zinc fingers and thiol-disulfide oxidoreductase activities of chaperone DnaJ[END_REF].

Finally, DnaJ-1, the protein that we identified in our proteomic screen as a partner of dMLF, belongs to type II (or type B) and its homologs in human are DnaJB1, DnaJB4 and DnaJB5. It was first identified in an yeast two-hybrid screen as a partner of the transcription factor HSF and it was shown to be a nuclear protein that delays the onset of the heat shock response in SL2 cells (Marchler et al., 2001). However, its function during Drosophila development and hematopoiesis still remained unknown.

Hsp70/DnaJ and hematopoiesis

So far, the role of DnaJ proteins in hematopoiesis has barely been studied. However, some studies revealed the major role of the Hsp70 chaperones family in erythropoiesis [START_REF] Weiss | Chaperoning erythropoiesis[END_REF]. Indeed, it has been shown that Hsp70 regulates erythroblast viability by preventing their death induced by apoptosis. On the one hand, erythropoietin (Epo) treatment induces the translocation of Hsp70 to the nucleus where it binds GATA-1 and performs essential functions in erythroid differentiation, since Hsp70 inhibits the cleavage of GATA-1 by Caspase 3 [START_REF] Ribeil | Hsp70 regulates erythropoiesis by preventing caspase-3-mediated cleavage of GATA-1[END_REF]. Conversely, during Epo deprivation, Hsp70 is excluded from the nucleus and GATA-1 is cleaved by Caspase 3. In fact, it has been shown that during erythropoiesis, caspases activation results in GATA-1 cleavage and cessation of erythrocyte maturation or apoptosis [START_REF] Maria | Negative regulation of erythropoiesis by caspase-mediated cleavage of GATA-1[END_REF]. Moreover, the phosphorylation of Hsp70 in response to Epo is necessary for its relocation in the nucleus and for erythroblast differentiation. On the other hand, AIF (Apoptosis-Inducing Factor) is also involved in erythropoiesis. Following Epo treatment, transient mitochondrial depolarization is observed, which leads to the release of procaspases, the caspase activator cytochrome C and AIF. AIF can then translocate to the nucleus and induce DNA fragmentation independently of the caspases to trigger full-blown apoptotic death [START_REF] Lui | Heat shock protein 70 inhibits the nuclear import of apoptosisinducing factor to avoid DNA fragmentation in TF-1 cells during erythropoiesis[END_REF][START_REF] Zermati | Caspase activation is required for terminal erythroid differentiation[END_REF]. In this case, it has been proposed that Hsp70 limits the apoptotic activity of AIF by sequestering it in the cytoplasm [START_REF] Lui | Heat shock protein 70 inhibits the nuclear import of apoptosisinducing factor to avoid DNA fragmentation in TF-1 cells during erythropoiesis[END_REF]. In addition, a study carried out in zebrafish showed the involvement of a conserved mitochondrial matrix chaperone Hspa9b during the development of erythrocytes. Indeed, in this model, the loss of the Hspa9b chaperone induces a myelodysplastic phenotype, producing oxidative stress and apoptosis in blood cells [START_REF] Craven | Loss of Hspa9b in zebrafish recapitulates the ineffective hematopoiesis of the myelodysplastic syndrome[END_REF]. These animals are anemic, and blood cells do not differentiate into mature erythrocytes and die by apoptosis.

The chaperone proteins are also involved at different stages of the hemoglobin production [START_REF] Arlet | HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in β-thalassaemia[END_REF]. In particular, it has recently been shown that the formation of hemoglobin aggregates in patients with ß-thalassemia major (ß-TM) causes Hsp70 sequestration in the cytoplasm, where it binds to the α-chain of the globin. This prevents Hsp70 from protecting GATA-1, and leads to a maturation termination of erythrocytes and their death by apoptosis [START_REF] Arlet | HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in β-thalassaemia[END_REF].

The role of DnaJ proteins in hematopoiesis remains largely unknown. However, DnaJB9/hTid1 was shown to interact with the transcription factor STAT5b and to inhibit its expression, thereby suppressing STAT5b-induced hematopoietic cell growth [START_REF] Dhennin-Duthille | The tumor suppressor hTid1 inhibits STAT5b activity via functional interaction[END_REF]. In addition, a recent study showed that over-expression of the cochaperone ERDJ4 (also called DNAJB9), by enhancing the folding of an endoplasmic reticulum protein, promotes hematopoietic stem cell survival (van Galen et al., 2014).

Molecular chaperones and polyglutamine aggregation

In mammals, the importance of molecular chaperones is underlined by the consequences of the toxicity of the specific aggregations of poorly folded proteins, which is at the origin of large numbers of neurodegenerative diseases [START_REF] Borrell-Pagès | Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase[END_REF][START_REF] Muchowski | Modulation of neurodegeneration by molecular chaperones[END_REF]. Due to their roles in the fight against aggregated proteins, molecular chaperones play a central role in the prevention of neurodegenerative diseases that are caused by the aggregation of polyglutamine (polyQ) proteins. Indeed, many studies have demonstrated the involvement of the protein quality control systems, like the Hsp70 chaperones system and the co-chaperones Hsp40/DnaJ, in different neurodegenerative diseases [START_REF] Bilen | Genome-wide screen for modifiers of ataxin-3 neurodegeneration in Drosophila[END_REF]Fernandez-Funez et al., 2000). Moreover, molecular chaperones are also involved in some other neurodegenerative diseases, such as Alzheimer's disease [START_REF] Dou | Chaperones increase association of tau protein with microtubules[END_REF] and Parkinson's disease [START_REF] Huang | Heat shock protein 70 inhibits α-synuclein fibril formation via interactions with diverse intermediates[END_REF]. Notably, over-expression of DnaJ-1 can suppress the polyQ cytotoxicity in different Drosophila neurodegeneration models [START_REF] Fayazi | A Drosophila ortholog of the human MRJ modulates polyglutamine toxicity and aggregation[END_REF]Kazemi-Esfarjani et al., 2000;Chan et al., 2000), and DnaJ-1 cooperates with Hsp70 in this process (Chan et al., 2000). In addition, mutations in different genes encoding for DnaJ-like proteins are at the origin of pathologies associated with proteostasis problems in human, such as neuropathy, muscular dystrophy or Parkinson's disease [START_REF] Koutras | J protein mutations and resulting proteostasis collapse[END_REF].

Objective of this project

MLF factors constitute a small family of poorly characterized proteins and they have been implicated in hematopoiesis and in oncogenic blood cell transformation. However,

their

Introduction

Proper blood cell development requires a fine-tuned regulation of transcription factors and signaling pathways activity. Consequently mutations affecting key regulators of hematopoiesis such as members of the RUNX transcription factor family or components of the Notch signaling pathway are associated with several blood cell disorders including leukemia [START_REF] Sood | Role of RUNX1 in hematological malignancies[END_REF][START_REF] Gu | Notch signaling: its roles and therapeutic potential in hematological malignancies[END_REF]. Also, leukemic cells often present recurrent chromosomal rearrangements that participate in malignant transformation by altering the function of these factors [START_REF] Rowley | Chromosomes in leukemia and beyond: from irrelevant to central players[END_REF]. The functional characterization of these genes is thus of importance not only to uncover the molecular basis of leukemogenesis but also to decipher the regulatory mechanisms controlling normal blood cell development. Myeloid leukemia factor 1 (MLF1) was identified as a target of the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) more than 20 years ago [START_REF] Yoneda-Kato | The t(3;5)(q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1[END_REF]. Further findings suggested that MLF1 could act as an oncogene [START_REF] Matsumoto | Elevated MLF1 expression correlates with malignant progression from myelodysplastic syndrome[END_REF][START_REF] Sun | Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization[END_REF][START_REF] Chen | Genomic profiling of 766 cancer-related genes in archived esophageal normal and carcinoma tissues[END_REF][START_REF] Lim | Detection of t(3;5) and NPM1/MLF1 rearrangement in an elderly patient with acute myeloid leukemia: clinical and laboratory study with review of the literature[END_REF] or a tumor suppressor [START_REF] Mansur | Distinctive genotypes in infants with T-cell acute lymphoblastic leukaemia[END_REF] depending on the cell context and it was shown that MLF1 over-expression either impairs cell cycle exit and differentiation [START_REF] Winteringham | Myeloid Leukemia Factor 1 inhibits erythropoietin-induced differentiation, cell cycle exit and p27Kip1 accumulation[END_REF], promotes apoptosis [START_REF] Sun | Myeloid leukemia factor 1 interfered with Bcl-XL to promote apoptosis and its function was regulated by 14-3-3[END_REF][START_REF] Sun | MLF1 is a proapoptotic antagonist of HOP complex-mediated survival[END_REF] or inhibits proliferation [START_REF] Yoneda-Kato | Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3[END_REF][START_REF] Rangrez | Myeloid leukemia factor-1 is a novel modulator of neonatal rat cardiomyocyte proliferation[END_REF] in different cell types ex vivo. Yet, its function and mechanism of action remains largely unknown.

MLF1 is the founding member of a small evolutionarily conserved family of nucleocytoplasmic proteins present in all metazoans but lacking recognizable domains that could help ascribe their biochemical activity [START_REF] Gobert | Myeloid leukemia factor: a return ticket from human leukemia to fly hematopoiesis[END_REF]. Whereas vertebrates have two closely related MLF paralogs, Drosophila has a single mlf gene encoding a protein that presents around 50% of identity with human MLF in the central conserved domain [START_REF] Ohno | Characterization of a Drosophila homologue of the human myelodysplasia/myeloid leukemia factor (MLF)[END_REF][START_REF] Martin-Lanneree | Characterization of the Drosophila myeloid leukemia factor[END_REF]. In fly, MLF was identified as a partner of the transcription factor DREF (DNA replication-related elementbinding factor) [START_REF] Ohno | Characterization of a Drosophila homologue of the human myelodysplasia/myeloid leukemia factor (MLF)[END_REF], for which it acts as a co-activator to stimulate the JNK pathway and cell death in the wing disc [START_REF] Yanai | Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway[END_REF]. MLF has been shown to bind chromatin [START_REF] Yanai | Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway[END_REF][START_REF] Fouix | Overexpression of a novel nuclear interactor of Suppressor of fused, the Drosophila myelodysplasia/myeloid leukaemia factor, induces abnormal morphogenesis associated with increased apoptosis and DNA synthesis[END_REF][START_REF] Dyer | Myeloid Leukemia Factor acts in a chaperone complex to regulate transcription factor stability and gene expression[END_REF], as its mouse homolog [START_REF] Winteringham | Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule[END_REF], and it can either activate or repress gene expression by a still unknown mechanism [START_REF] Yanai | Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway[END_REF][START_REF] Dyer | Myeloid Leukemia Factor acts in a chaperone complex to regulate transcription factor stability and gene expression[END_REF]. MLF also interacts with Suppressor of Fused, a negative regulator of the Hedgehog signaling pathway [START_REF] Fouix | Overexpression of a novel nuclear interactor of Suppressor of fused, the Drosophila myelodysplasia/myeloid leukaemia factor, induces abnormal morphogenesis associated with increased apoptosis and DNA synthesis[END_REF], and, like its mammalian counterpart [START_REF] Yoneda-Kato | Myeloid leukemia factor 1 regulates p53 by suppressing COP1 via COP9 signalosome subunit 3[END_REF], with CSN3, a component of the COP9 signalosome [START_REF] Sugano | The myeloid leukemia factor interacts with COP9 signalosome subunit 3 in Drosophila melanogaster[END_REF], but the functional consequences of these interactions remain elusive. Interestingly, the overexpression of Drosophila MLF or that of its mammalian counterparts can suppress polyglutamine-induced cytotoxicity in fly and in cellular models of neurodegenerative diseases [START_REF] Martin-Lanneree | Characterization of the Drosophila myeloid leukemia factor[END_REF][START_REF] Kazemi-Esfarjani | Suppression of polyglutamine toxicity by a Drosophila homolog of myeloid leukemia factor 1[END_REF][START_REF] Kim | Evidence for sequestration of polyglutamine inclusions by Drosophila myeloid leukemia factor[END_REF][START_REF] Banerjee | Modulation of mutant Huntingtin aggregates and toxicity by human myeloid leukemia factors[END_REF].

Moreover phenotypic defects associated with MLF loss in Drosophila can be rescued by human MLF1 [START_REF] Martin-Lanneree | Characterization of the Drosophila myeloid leukemia factor[END_REF][START_REF] Bras | Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis[END_REF]. Thus MLF function seems conserved through evolution and Drosophila appears as a genuine model organism to characterize MLF proteins [START_REF] Gobert | Myeloid leukemia factor: a return ticket from human leukemia to fly hematopoiesis[END_REF].

Along this line, we recently analyzed the role of MLF during Drosophila hematopoiesis [START_REF] Bras | Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis[END_REF]. Indeed, a number of proteins regulating blood cell development in human, such as RUNX and Notch, also controls Drosophila blood cell development [START_REF] Letourneau | Drosophila hematopoiesis under normal conditions and in response to immune stress[END_REF]. In Drosophila, the RUNX factor Lozenge (LZ) is specifically expressed in and required for the development of the crystal cells [START_REF] Lebestky | Specification of Drosophila hematopoietic lineage by conserved transcription factors[END_REF], one of the three hematopoietic lineages, which accounts for ±4% of the circulating larval blood cells [START_REF] Letourneau | Drosophila hematopoiesis under normal conditions and in response to immune stress[END_REF]. The Notch pathway also controls the development of this lineage: it is required for the induction of LZ expression and it contributes then to LZ + cell survival and differentiation [START_REF] Lebestky | Specification of Drosophila hematopoietic lineage by conserved transcription factors[END_REF][START_REF] Duvic | Notch signaling controls lineage specification during Drosophila larval hematopoiesis[END_REF][START_REF] Mukherjee | Interaction between Notch and Hif alpha in development and survival of Drosophila blood cells[END_REF][START_REF] Terriente-Felix | Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme[END_REF]. Interestingly, our previous analysis revealed a functional and conserved link between MLF and RUNX factors [START_REF] Bras | Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis[END_REF].

Indeed MLF controls LZ activity and prevents its degradation in cell culture and the stabilization of LZ by MLF appears to be critical to control crystal cell number in vivo [START_REF] Bras | Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis[END_REF].

Intriguingly, though while LZ is required for crystal cell development, mlf mutation caused a decrease in LZ expression and an increase in crystal cell number. In humans, deregulation of RUNX protein level is associated with several pathologies. For instance, haploinsufficient mutations in RUNX1 are associated with MDS/AML in the case of somatic mutations, and with familial platelet disorders with associated myeloid malignancy for germline mutations [START_REF] Sood | Role of RUNX1 in hematological malignancies[END_REF]. In the opposite, RUNX1 overexpression can promote lymphoid leukemia [START_REF] Blyth | Runx1 promotes Bcell survival and lymphoma development[END_REF][START_REF] Wotton | Proviral insertion indicates a dominant oncogenic role for Runx1/AML-1 in T-cell lymphoma[END_REF]. Understanding how RUNX protein level is regulated and how this affects specific developmental processes is thus of particular importance.

Here, we further studied MLF function and mode of action in Drosophila blood cells by using proteomic, transcriptomic and genetic approaches. We show that MLF interacts with the Hsp40 co-chaperone protein DnaJ-1 to stabilize LZ as well as to control LZ + cell number and differentiation. In addition, consistent with the analysis of mlf or dnaj-1 mutant, it appears that high levels of LZ are required to tune-down Notch expression and signaling to prevent aberrant blood cell accumulation. These findings thus establish a functional link between the MLF/DnaJ-1 complex and the regulation of a RUNX-Notch axis required for blood cell homeostasis in vivo.

Results

MLF interacts with DnaJ-1

To better characterize MLF molecular mode of action, we sought to identify its partners.

Accordingly As DnaJ-1 was the strongest hit in our proteomic approach, we focused our analysis on this candidate. To confirm the interaction between MLF and DnaJ-1, we performed coimmunoprecipitation experiments in Kc167 cells transfected with expression plasmids for HA-DnaJ-1, GFP or GFP-MLF. We found that GFP-MLF (but not GFP alone) coprecipitated HA-DnaJ-1 and that conversely HA-DnaJ-1 co-precipitated GFP-MLF as well as endogenous MLF (Fig 1C and 1D), indicating that these proteins specifically interacted.

Moreover, in vitro translated MLF and DnaJ-1interacted with GST-DnaJ-1 and GST-MLF respectively but not with GST alone in pull-down assays (Fig 1E and 1F). Of note, immunostaining also showed that DnaJ-1 and MLF co-localized in the nuclei of Kc167 transfected cells (S1A Fig) . We then mapped the domains required for their interactions.

Hsp40/DnaJ co-chaperones play a crucial role in the regulation of protein folding and degradation; they chiefly act by delivering client proteins to Hsp70/DnaK chaperones and stimulating their ATPase activity [START_REF] Kim | Molecular chaperone functions in protein folding and proteostasis[END_REF][START_REF] Kampinga | The HSP70 chaperone machinery: J proteins as drivers of functional specificity[END_REF]. DnaJ-1 belongs to the DnaJB/class II subfamily of Hsp40/DnaJ proteins, which are characterized by an N-terminal J domain required to stimulate Hsp70 ATPase activity (amino acids 4 to 57 in DnaJ-1), a central glycine/phenylalanine (G/F)-rich region (amino acids 64 to 144 in DnaJ-1), and a conserved C-terminal region (amino acids 157 to 320 in DnaJ-1) that contains the client binding domain followed by a dimerization interface [START_REF] Kampinga | The HSP70 chaperone machinery: J proteins as drivers of functional specificity[END_REF]. Immunoprecipitations of GFP-MLF expressed with different HA-tagged DnaJ-1 variants indicated that DnaJ-1 C-terminal region mediates MLF binding (Fig 1G). In contrast, a point mutation in the highly conserved HPD loop (P32S) crucial for Hsc70 activation [START_REF] Kampinga | The HSP70 chaperone machinery: J proteins as drivers of functional specificity[END_REF], deletion of the J-domain or deletion of the J and G/F domains did not affect DnaJ-1 binding to GFP-MLF. MLF does not harbor characteristic domains besides a central "MLF homology domain" (MHD, amino acids 96 to 202) conserved between MLF family members [START_REF] Gobert | Myeloid leukemia factor: a return ticket from human leukemia to fly hematopoiesis[END_REF]. Using GFP-DnaJ-1 as bait and deletion mutants in MLF as preys, we found that the MHD was sufficient for binding to

MLF acts in a chaperone complex with DnaJ-1 to control LZ activity and stability

We have previously shown that MLF is required for LZ-induced transactivation and stable expression [START_REF] Bras | Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis[END_REF]. We thus asked whether DnaJ-1 also controlled LZ function. As shown in Reminiscent of our previous results with MLF [START_REF] Bras | Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis[END_REF], we observed that DnaJ-1 overexpression was associated with an increase in LZ-induced transactivation and LZ expression In sum, our results support the idea that MLF acts in a chaperone complex with DnaJ-1 and Hsc70-4 to control LZ stability and activity.

DnaJ-1 cell-autonomously controls LZ + cell number and differentiation in vivo

Since DnaJ-1 interacts with MLF and controls LZ level ex vivo, we sought to analyze DnaJ-1 function during larval crystal cell development, using the lz-GAL4/+ driver and the UAS-mCD8GFP reporter to monitor this LZ + blood cell lineage. Given that no mutant for dnaj-1, we used a CRIPR/Cas9 strategy to generate dnaj-1 null alleles (See Materials and Methods and S4 Fig) [START_REF] Port | Creating Heritable Mutations in Drosophila with CRISPR-Cas9[END_REF]. In the following experiments, we used an allelic combination between two mutant lines obtained from independent founder flies (dnaj-1 A and dnaj-1 C ), which harbor a complete deletion of dnaj-1 coding sequence (S4 Fig) . Around 65% of the dnaj-1 A/C mutants reached larval stage and 15% emerged as adult flies, but they did not show obvious morphological defect. Notwithstanding and reminiscent of mlf phenotypes, bleeding of third instar larvae revealed that dnaj-1 mutants exhibited a ±1.8-fold increase in circulating lz>GFP + cells as compared to wild type (Fig 3A). In addition, as in mlf mutants, crystal cells from dnaj-1 mutant larvae still expressed the differentiation marker PPO1 and were capable of melanization upon heat treatment (Fig 3C -H). A closer examination also revealed the presence of unusually large lz>GFP + cells in dnaj-1 mutants and quantitative analyses confirmed that dnaj-1 loss caused a significant increase in lz>GFP + cell size whereas lz>GFP -cells were unaffected (Fig 3B). Interestingly, a similar phenotype is observed in mlf mutant larvae, suggesting that both genes not only control crystal cell number but also their differentiation (see below). Importantly, lz>GFP + cell number and size was restored to wild-type when DnaJ-1 was re-expressed in the crystal cell lineage of dnaj-1 A/C mutant larvae using the lz-GAL4 driver (Fig 3A and3B), demonstrating that these phenotypes are specifically caused by dnaj-1 mutation and that DnaJ-1 acts cell autonomously after the onset of lz expression in the crystal cell lineage. Furthermore, the increase in crystal cell number and size was also observed when we monitored crystal cell presence by immunostaining against PPO1 in larvae carrying dnaj-1 A or dnaj-1 C homozygous mutation or over a deficiency covering dnaj-1 locus (Def(3L)BSC844) (S4 Fig) . Overall, these results demonstrate that, like mlf, dnaj-1 controls larval crystal cell number and size.

We then assessed whether DnaJ-1 affected LZ stability in vivo as it does in cell culture.

Unexpectedly, immunostaining against LZ did not reveal a decrease in LZ expression in dnaj-1 mutant crystal cells, while LZ level was clearly lower in mlf mutant (Fig 3I -K).

Actually, quantitative analyses revealed a slight (30%) but significant (p=0.006) increase in LZ level in dnaj-1 mutant as compared to wild type, whereas LZ level dropped by more than 2 folds in mlf mutant (Fig 3L). Thus, unlike mlf, dnaj-1 loss is not sufficient to destabilize LZ in vivo.

DnaJ-1 and MLF act together to control LZ accumulation and crystal cell development

One potentially important difference between Kc167 cells, in which DnaJ-1 is required to stabilize LZ, and crystal cells, in which it is not, is MLF expression. Indeed, in Kc167 cells, MLF is chiefly detected in the cytoplasm and is expressed at low levels in the nucleus (S5A To test this hypothesis, we designed two complementary experiments. On the one hand, we assessed whether MLF over-expression in Kc167 cells could protect LZ from degradation following dnaj-1 knockdown. On the other hand, we asked whether LZ protein would still be stable in dnaj-1 mutant crystal cells if MLF level is decreased. As shown in Figure 4, LZ expression was reduced when Kc167 cells were treated with a dsRNA targeting dnaj-1 and increased upon over-expression of MLF. Strikingly though, and reminiscent of the above observations in dnaj-1 mutant crystal cells, LZ level was not reduced but further increased when dnaj-1 was knocked down in MLF overexpressing cells (Fig 4D and4E). Conversely, in vivo, the expression of a dsRNA against mlf in lz>GFP + cells caused a drop in LZ expression that was significantly enhanced in dnaj-1 deficient larvae, while dnaj-1 mutation alone increased LZ level (Fig 4F -J). Hence, it appears that high levels of MLF can prevent LZ degradation in the absence of DnaJ-1.

Then, since chaperones are important for proper protein folding [START_REF] Kim | Molecular chaperone functions in protein folding and proteostasis[END_REF][START_REF] Kampinga | The HSP70 chaperone machinery: J proteins as drivers of functional specificity[END_REF], we postulated that LZ protein accumulating in crystal cells in the absence of DnaJ-1 might be less active.

Thus increasing LZ expression should be sufficient to rescue lz>GFP + cell number and size. Consistent with this hypothesis, and as observed in mlf mutant larvae, lz>GFP + cell number and size was restored to wild-type when we enforced LZ expression in this lineage (Fig 5A and 5B). Finally, since MLF and DnaJ-1 bind to each other and jointly control LZ stability, we tested whether they genetically interacted to regulate crystal cell development.

While heterozygous mutation in either mlf or dnaj-1 did not efficiently alter circulating lz>GFP + cell number or size, mlf ΔC1 /+, dnaj-1 A /+ transheterozygote larvae displayed a significant increase of both parameters (Fig 5C and5D). We thus conclude that DnaJ-1 and MLF act together to control LZ expression and crystal cell development in vivo.

MLF and DnaJ-1 control crystal cell differentiation

In parallel, to gain further insights into the function of MLF in the control of crystal cell development, we established the transcriptome of circulating LZ + blood cells in wild type and mlf mutant larvae. Heterozygous lz-GAL4, UAS-mCD8GFP L3 larvae carrying or not mlf null mutation were bled, lz>GFP + cells were collected by FACS and their gene expression profile was determined by RNA sequencing (RNAseq) from biological triplicates (see M&M for details). Using Drosophila reference genome dm3, we detected the expression of 7399 genes (47% of the total fly genes) in each of the 6 samples (Fig 6B and S6 Table). Consistent with the role of the crystal cells as the main source of phenoloxidases [START_REF] Neyen | The Black cells phenotype is caused by a point mutation in the Drosophila pro-phenoloxidase 1 gene that triggers melanization and hematopoietic defects[END_REF], the two most strongly expressed genes were PPO1 and PPO2. In addition, lz expression as well as that of several other crystal cell markers (see below) was readily detected. It was recently shown that larval circulating LZ + cells derive from plasmatocytes, which express Hemolectin (Hml) and Nimrod C1 (NimC1) and transdifferentiate into mature crystal cells [START_REF] Leitao | Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation[END_REF]. Accordingly, we detected the expression of these genes as well as other "plasmatocytes" markers such peroxidasin and croquemort (which were actually shown to be also expressed in crystal cells [START_REF] Jung | The Drosophila lymph gland as a developmental model of hematopoiesis[END_REF][START_REF] Waltzer | Cooperation between the GATA and RUNX factors Serpent and Lozenge during Drosophila hematopoiesis[END_REF]) in lz>GFP + cells.

Using DESeq2 to identify differentially expressed genes between wild type and mlf mutant lz>GFP + cells, we found 779 genes with significantly altered expression (adjusted p-value <0.01): the transcript level of 469 genes was decreased and that of 310 genes was increased in the absence of MLF (Fig 6A,6B and S7 Table). In line with our previous in situ hybridization results [START_REF] Bras | Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis[END_REF], RNAseq analysis did not reveal a significant modification of PPO1 or PPO2 expression in the absence of mlf. However, lz transcript level was reduced by ±2 folds (p-value=0.0018), which could be due to defective maintenance of lz autoactivation loop [START_REF] Ferjoux | A GATA/RUNX cis-regulatory module couples Drosophila blood cell commitment and differentiation into crystal cells[END_REF]. To assess whether other crystal cell markers were affected by mlf loss, we established a compilation of genes expressed in (embryonic or larval) crystal cells based on Flybase data mining and re-examination of Berkeley Drosophila Genome Project in situ hybridizations (http://insitu.fruitfly.org/cgi303bin/ex/insitu.pl) (S8 Table ). Among these 129 genes (i.e. excluding mlf itself), 44 (34%) were differentially expressed in the absence of mlf To substantiate these results, we assessed by in situ hybridization the expression of 4 genes that were either down-regulated (CG7860 and Oscillin) or up-regulated (CG6733 and Jafrac1) in mlf mutant. CG7860 and Oscillin were specifically expressed in lz>GFP + but not in the surrounding lz>GFP -hemocytes in wild-type conditions (Fig 6D and6G).

Consistent with our RNAseq data, the expression of CG7860 and Oscillin was strongly reduced in mlf mutant larvae. Although it is expressed in embryonic crystal cells [START_REF] Ferjoux | A GATA/RUNX cis-regulatory module couples Drosophila blood cell commitment and differentiation into crystal cells[END_REF],

CG6733 was not detectably expressed by in situ hybridization in circulating hemocytes of wild-type larvae, but it was expressed in the lz>GFP + lineage in mlf mutant larvae (Fig 6J and6K). Finally, Jafrac1 expression increased in lz>GFP + cells of mlf mutant larvae as compared to wild-type, whereas its (lower) expression in lz>GFP -blood cells seemed similar (Fig 6M and 6N). These data thus confirm the RNAseq results and demonstrate that MLF controls the expression of several crystal cell markers. Since MLF acts together with DnaJ-1, we also tested whether these four genes were deregulated in dnaj-1 mutant larvae.

As for mlf, we observed that dnaj-1 mutation caused a down-regulation of CG7860 and Oscillin expression and an up-regulation of CG6733 and Jafrac1 expression in lz>GFP + blood cells (Fig 6F,6I,6L and 6O). Therefore, the loss of mlf or dnaj-1 leads to the deregulation of crystal cell gene marker expression, indicating that both genes are required for proper differentiation of the LZ + blood cell lineage.

MLF and DnaJ-1 repress Notch signaling

Interestingly, the levels of Notch receptor transcripts were significantly higher in mlf mutant (p=1.3x10 -6 ) (Fig 6C). Notch signaling plays a key role in crystal cell development [START_REF] Letourneau | Drosophila hematopoiesis under normal conditions and in response to immune stress[END_REF]:

Notch is first activated by its ligand Serrate to specify crystal cell precursors and subsequently Notch activation is maintained in LZ + cells in a ligand independent manner to promote crystal cell growth and survival [29-31, 40, 44]. The rise in lz>GFP + cell number and size observed in mlf and dnaj-1 mutants could thus be due to increased ligandindependent Notch signaling [START_REF] Mukherjee | Interaction between Notch and Hif alpha in development and survival of Drosophila blood cells[END_REF][START_REF] Terriente-Felix | Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme[END_REF]. Hence, we further investigated the level of Notch expression and activation. Immunostaining using an antibody against Notch extracellular domain (NECD) showed that Notch was expressed at higher levels in lz>GFP + cells of mlf and dnaj-1 mutant larvae than in wild-type condition (Fig 7A -C). Quantitative analyses confirmed that mlf loss caused a significant increase of Notch expression in lz>GFP + cell, whereas the (lower) expression of Notch in lz>GFP -blood cells was not affected (Fig 7D ).

Similar results were obtained when we measured Notch protein levels using an antibody directed against its intra-cellular domain (NICD) (Fig 7E). These data strongly suggest that Notch expression is specifically increased in lz>GFP + cells of mlf and dnaj-1 mutants. We then tested whether this resulted in increased signaling by monitoring the expression of two Notch signaling pathway reporter genes expressed in larval crystal cells: Klumpfuss-Cherry [START_REF] Terriente-Felix | Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme[END_REF] and NRE-GFP [START_REF] Saj | A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network[END_REF]. Both mlf and dnaj-1 loss were associated with a strong increase in the expression of these reporters (Fig 7F -J). Thus mlf and dnaj-1 are required to tune down Notch signaling in the crystal cell lineage.

Crystal cells tend to increase their size as they mature in response to Notch signaling [START_REF] Terriente-Felix | Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme[END_REF][START_REF] Leitao | Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation[END_REF]. To better characterize the defects associated with mlf or dnaj-1 loss, we analyzed the distribution of lz>GFP + cells as well as Notch expression level according to lz>GFP + cell size category. Whereas cells more than 1.3-fold larger than the mean wild-type cell size represented a small fraction (±10%) of the lz>GFP + population in wild-type larvae, they constituted the prevalent population in mlf or dnaj-1 mutant (respectively 49.6% and 37%) (Fig7K). In addition, while Notch expression was maximum in the population of lz>GFP + cells of mean cell size and lower in larger cells of wild-type larvae, it was maintained at higher levels in the larger cell populations of mlf or dnaj-1 mutants (Fig 7L -N).

All together, these results show that MLF/DnaJ-1 loss causes the accumulation of large and mis-differentiated crystal cells exhibiting abnormal Notch signaling activation.

High levels of LZ are required to prevent accumulation of lz>GFP + cells and to repress

Notch expression/signaling

LZ is absolutely required for crystal cell formation and differentiation [START_REF] Letourneau | Drosophila hematopoiesis under normal conditions and in response to immune stress[END_REF]. The above data

show that the increase in crystal cell number and size observed in mlf or dnaj-1 mutant is rescued by enforcing LZ expression. This is intriguing since it suggests that decreasing LZ and the non-muscular myosin heavy chain SMMHC [START_REF] Li | Differential interactions between Brother proteins and Runt domain proteins in the Drosophila embryo and eye[END_REF]. This chimera mimics the CBFß-MYH11 fusion protein generated by the inv( 16) translocation in human AML and can sequester RUNX factors in the cytoplasm [START_REF] Sood | Role of RUNX1 in hematological malignancies[END_REF][START_REF] Adya | The leukemic protein core binding factor beta (CBFbeta)-smooth-muscle myosin heavy chain sequesters CBFalpha2 into cytoskeletal filaments and aggregates[END_REF]. Bro-SMMHC expression in lz>GFP + cells titrated LZ from the nucleus and also caused an increase in lz>GFP + cell number and suggesting that Notch might be a direct target gene of LZ. We thus tested the effect of LZ dosage manipulation on the activity of this enhancer-GAL4 line. Strikingly, a hypomorphic lozenge mutation (lz g /Y) [START_REF] Behan | Yan regulates Lozenge during Drosophila eye development[END_REF] or the expression of Bro-SMMHC caused an increase in the expression of this enhancer, whereas the over-expression of LZ resulted in its downregulation (Fig 9G -K). These findings strongly argue that LZ directly represses Notch expression All together, these results demonstrate that high levels of LZ are required to prevent the accumulation of over-grown lz>GFP + cells as well as over-activation of the Notch pathway and we propose that LZ-mediated repression of Notch transcription is critical for this process.

Discussion

Members of the RUNX and MLF families have been implicated in the control of blood cell development in mammals and Drosophila and deregulation of their expression is associated with human hemopathies including leukemia [START_REF] Sood | Role of RUNX1 in hematological malignancies[END_REF][START_REF] Mansur | Distinctive genotypes in infants with T-cell acute lymphoblastic leukaemia[END_REF][START_REF] Gobert | Myeloid leukemia factor: a return ticket from human leukemia to fly hematopoiesis[END_REF][START_REF] De Bruijn | Runx transcription factors in the development and function of the definitive hematopoietic system[END_REF]. Our results establish the first link between MLF, DnaJ-1 and the regulation of RUNX transcription factor in vivo. In addition, our data suggest that the stabilization of LZ by the MLF/DnaJ-1 complex is critical to control Notch signaling and thus blood cell growth and survival. These findings pinpoint the specific function of the Hsp40 chaperone DnaJ-1 in hematopoiesis, reveal a potentially conserved mechanism of regulation of RUNX activity and highlight a new layer of control of Notch signaling at the transcriptional level.

In line with results published as this manuscript was in preparation [START_REF] Dyer | Myeloid Leukemia Factor acts in a chaperone complex to regulate transcription factor stability and gene expression[END_REF], we found that MLF binds DnaJ-1 and Hsc70-4 and that these two proteins, like MLF, are required for LZ stable expression in Kc cells. In addition, we show that MLF and DnaJ-1 bind to each other via evolutionarily conserved domains and also interact with LZ, suggesting that LZ is a direct target of a chaperone complex formed by MLF, DnaJ-1 and Hsc70-4. Of note, a systematic characterization of Hsp70 chaperone complexes in human cells identified MLF1 and MLF2 as potential partners of DnaJ-1 homologs, DNAJB1, B4 and B6 [START_REF] Taipale | A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways[END_REF], a finding corroborated by Dyer et al. [20]. Therefore, the MLF/DnaJ-1/Hsc70 complex could play a conserved role in mammals, notably in the regulation of RUNX transcription factors stability. How MLF acts within this chaperone complex remains to be determined. In vivo,

we demonstrate that dnaj-1 mutation leads to defects in crystal cell development strikingly similar to those observed in mlf mutant larvae and we show that these two genes act together to control LZ + cell development by impinging on LZ activity. Our data suggest that in the absence of DnaJ-1, high levels of MLF lead to the accumulation of defective LZ proteins, whereas lower levels of MLF allow its degradation. We thus propose that MLF stabilizes LZ and, together with DnaJ-1, promotes its proper folding/conformation. In human, DnaJB4 stabilizes wild-type E-cadherin but induces the degradation of mutant E-cadherin variants associated with hereditary diffuse gastric cancer [START_REF] Simoes-Correia | DNAJB4 molecular chaperone distinguishes WT from mutant E-cadherin, determining their fate in vitro and in vivo[END_REF]. Thus the fate of DnaJ client proteins is controlled at different levels and MLF might be an important regulator in this process.

In this work, we present the first null mutant for a gene of the DnaJB family in metazoans and our results demonstrate that a DnaJ protein is required in vivo to control hematopoiesis. There are 16 DnaJB and in total 49 DnaJ encoding genes in mammals and the expansion of this family has likely played an important role in the diversification of their functions [START_REF] Qiu | The diversity of the DnaJ/Hsp40 family, the crucial partners for Hsp70 chaperones[END_REF][START_REF] Hageman | Computational analysis of the human HSPH/HSPA/DNAJ family and cloning of a human HSPH/HSPA/DNAJ expression library[END_REF]. DnaJB9 overexpression was found to increase hematopoietic stem cell repopulation capacity [START_REF] Van Galen | The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress[END_REF] and Hsp70 inhibitors have anti-leukemic activity [START_REF] Reikvam | Heat shock protein 70-the next chaperone to target in the treatment of human acute myelogenous leukemia?[END_REF], but the participation of other DnaJ proteins in hematopoiesis or leukemia has not been explored.

Actually DnaJ molecular mechanism of action has been fairly well studied but we only have limited insights as to their role in vivo. Interestingly though both DnaJ-1 and MLF suppress polyglutamine protein aggregation and cytotoxicity in Drosophila models of neurodegenerative diseases [START_REF] Martin-Lanneree | Characterization of the Drosophila myeloid leukemia factor[END_REF][START_REF] Kazemi-Esfarjani | Suppression of polyglutamine toxicity by a Drosophila homolog of myeloid leukemia factor 1[END_REF][START_REF] Kim | Evidence for sequestration of polyglutamine inclusions by Drosophila myeloid leukemia factor[END_REF][START_REF] Wotton | Proviral insertion indicates a dominant oncogenic role for Runx1/AML-1 in T-cell lymphoma[END_REF][START_REF] Kazemi-Esfarjani | Genetic suppression of polyglutamine toxicity in Drosophila[END_REF][START_REF] Fernandez-Funez | Identification of genes that modify ataxin-1-induced neurodegeneration[END_REF][START_REF] Chan | Mechanisms of chaperone suppression of polyglutamine disease: selectivity, synergy and modulation of protein solubility in Drosophila[END_REF][START_REF] Kuo | Suppression of polyglutamine protein toxicity by co-expression of a heat-shock protein 40 and a heat-shock protein 110[END_REF][START_REF] Tsou | The deubiquitinase ataxin-3 requires Rad23 and DnaJ-1 for its neuroprotective role in Drosophila melanogaster[END_REF], and this function is conserved in mammals [START_REF] Kim | Evidence for sequestration of polyglutamine inclusions by Drosophila myeloid leukemia factor[END_REF][START_REF] Banerjee | Modulation of mutant Huntingtin aggregates and toxicity by human myeloid leukemia factors[END_REF][START_REF] Gibbs | Emerging roles of J proteins in neurodegenerative disorders[END_REF][START_REF] Hageman | A DNAJB chaperone subfamily with HDAC-dependent activities suppresses toxic protein aggregation[END_REF]. It is tempting to speculate that MLF and DnaJB proteins act together in this process as well as in leukemogenesis and thus a better characterization of their mechanism of action may help develop new therapeutic approaches for these diseases.

As shown here, mlf or dnaj-1 mutant larvae harbor more crystal cells than wild type larvae and display a higher fraction of the largest lz>GFP + cell population, which likely corresponds to the more mature crystal cells [START_REF] Terriente-Felix | Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme[END_REF][START_REF] Leitao | Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation[END_REF]. It is thus tempting to speculate that mlf or dnaj-1 loss promotes the survival of fully differentiated crystal cells. Our RNAseq data demonstrate that mlf is critical for expression of crystal cell associated genes, but we observed both up-regulation and down-regulation of crystal cell differentiation markers in mlf or dnaj-1 mutant LZ + cells. In addition, our transcriptome did not reveal a particular bias toward decreased expression for "plasmatocyte" markers in LZ + cells from mlf mutant larvae (for instance, hml, peroxidasin, viking or Cg25C expression was unaffected, croquemort was down-regulated and NimC1 was up-regulated). Thus, it appears that MLF and DnaJ-1 loss do lead to the abnormal accumulation of mis-differentiated crystal cells.

Our data support a model whereby MLF and DnaJ-1 act together to promote LZ expression, which in turn represses Notch transcription and signaling pathway to control crystal cell size and number. Indeed, we observe an over-activation of the Notch pathway in LZ + blood cells of mlf or dnaj-1 mutant larvae as well as when we interfere with LZ activity,

and it has been shown that Notch activation increases crystal cell growth and survival [START_REF] Mukherjee | Interaction between Notch and Hif alpha in development and survival of Drosophila blood cells[END_REF][START_REF] Terriente-Felix | Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme[END_REF][START_REF] Leitao | Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation[END_REF]. Our results suggest that LZ directly represses Notch transcription as we identified a LZ-responsive Notch cis-element that contains a conserved RUNX binding site. Activation of Notch pathway in circulating LZ + cells is ligand-independent and mediated through stabilization of the full length Notch protein in endocytic vesicles [START_REF] Mukherjee | Interaction between Notch and Hif alpha in development and survival of Drosophila blood cells[END_REF][START_REF] Palmer | Ligand-independent mechanisms of Notch activity[END_REF]. Hence, a tight control of Notch expression is of particular importance to keep Notch pathway activation in check and prevent abnormal development of the LZ + blood cell lineage. By stabilizing LZ, MLF and DnaJ-1 thus provide a cell-autonomous mechanism to inhibit Notch signaling.

Further experiments will now be required to establish how LZ represses Notch transcription.

RUNX factors can act as transcriptional repressors by recruiting co-repressors such as members of the Groucho family [START_REF] Chuang | RUNX family: Regulation and diversification of roles through interacting proteins[END_REF]. Whether MLF and DnaJ-1 directly contribute to LZinduced repression in addition to regulating its stability is an open question. MLF and DnaJ-1 were recently found to bind and regulate a common set of genes in cell culture [START_REF] Dyer | Myeloid Leukemia Factor acts in a chaperone complex to regulate transcription factor stability and gene expression[END_REF]. They may thus provide a favorable chromatin environment for LZ binding or be recruited with LZ and/or favor a conformational change in LZ that allows its interaction with co-repressors.

The scarcity of lz>GFP + cells precludes a biochemical characterization of LZ, MLF and DnaJ-1 mode of action notably at the chromatin level, but further genetic studies should help decipher their mode of action. While the post-translational control of Notch has been extensively studied, its transcriptional regulation seems largely overlooked [START_REF] Bray | Notch signalling in context[END_REF]. Our findings indicate that this is nonetheless an alternative entry point to control the activity of this pathway. Given the importance of RUNX transcription factor and Notch signaling in hematopoiesis and blood cell malignancy [START_REF] Sood | Role of RUNX1 in hematological malignancies[END_REF][START_REF] Gu | Notch signaling: its roles and therapeutic potential in hematological malignancies[END_REF], it will be of particular interest to further study whether RUNX factors can regulate Notch expression and signaling during these processes in mammals.

In conclusion, our study shows that MLF and DnaJ-1 act together to regulate RUNX transcription factor activity, which in turn controls Notch signaling during hematopoiesis in vivo. We anticipate that the extraordinary genetic toolbox available in Drosophila will help shed new light on the mechanism of action of these evolutionarily conserved proteins and will bring valuable insights into the control of protein homeostasis by MLF and DnaJ-1 during normal or pathological situations.

Materials and Methods

Fly strains

The following Drosophila melanogaster lines were used: mlf ΔC1 , UAS-mlf [START_REF] Martin-Lanneree | Characterization of the Drosophila myeloid leukemia factor[END_REF], UAS-ds-mlf (National Institute of Genetics, NIG), UAS-lz, lz GAL4 , UAS-mCD8-GFP, lz g , lz r1 , P{EPgy2}DnaJ-1EY04359, UAS-dnaj-1, Def(3L)BSC884, vas-Cas9, UAS-GFPnls, NRE-GFP, GMR30C06, GMR30A01 (Bloomington Drosophila Stock Center), Bc-GFP [START_REF] Tokusumi | New hemocyte specific enhancer-reporter transgenes for the analysis of hematopoiesis in Drosophila[END_REF], Klu-mCherry [START_REF] Terriente-Felix | Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme[END_REF], UAS-Bro-SMMHC [START_REF] Li | Differential interactions between Brother proteins and Runt domain proteins in the Drosophila embryo and eye[END_REF]. were cloned in pCFD3 (Addgene, [START_REF] Port | Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila[END_REF]). vas-cas9 females were crossed to P{EPgy2}DnaJ-1EY04359 males and the resulting embryos were injected using standard procedures with both pCFD3-g2 and pCFD3-g3 plasmids (500ng/μl). Deletion of the P{EPgy2}EY04359 transposon, as revealed by loss of the w + marker, was screened for at the F2 generation, and deletion of dnaj-1 locus was assessed by PCR and sequencing. All crosses were conducted on standard food medium as described in [START_REF] Benmimoun | The EBF transcription factor Collier directly promotes Drosophila blood cell progenitor maintenance independently of the niche[END_REF].

Immunostainings and in situ hybridizations

For each sample, four third instar larvae were bled (or 5.0x10 [START_REF] Fouix | Overexpression of a novel nuclear interactor of Suppressor of fused, the Drosophila myelodysplasia/myeloid leukaemia factor, induces abnormal morphogenesis associated with increased apoptosis and DNA synthesis[END_REF], anti-PPO1 [START_REF] Li | Properties of Drosophila melanogaster prophenoloxidases expressed in Escherichia coli[END_REF], anti-GFP (Fisher Scientific), anti-HA (Sigma).

For in situ hybridizations: after fixation, the cells were washed and permeabilized in PBS-0.1% Tween20 (PBSTw), pre-incubated for 1h at 65°C in HB buffer (50% formamide, 2xSSC, 1 mg/ml Torula RNA, 0.05 mg/ml Heparin, 2% Roche blocking reagent, 0.1% CHAPS, 5 mM EDTA, 0.1% Tween 20) and 535 incubated over-night with anti-sense DIGlabeled RNA probes (against CG6733, CG7860, Jafrac or Oscillin) diluted in HB. The samples were washed in HB for 1h at 65°C, in 50% HB-50% PBSTw for 30 min at 65°C and three times in PBSTw for 20 min at room temperature. Then the cells were incubated for 30 min in PBSTw-1% BSA before being incubated with anti-DIG antibody conjugated to alkaline phosphatase (Roche, 1/2000 in PBSTw) for 3h. After 4 washes in PBSTw, in situ hybridization signals were revealed with FastRed (Roche). The cells were then processed for immunostaining against GFP as described above, incubated in Topro3, washed in PBS and mounted in Vectashield medium for analysis.

Experiments were performed using at least biological triplicates. Samples were imaged with laser scanning confocal microscopes (Leica) and images were analyzed with ImageJ.

Cell size and protein expression levels were measured on maximal intensity projections of Z-sections through the whole cell on a minimum of 25 cells per genotype. Crystal cell counts were performed as described in [START_REF] Banerjee | Modulation of mutant Huntingtin aggregates and toxicity by human myeloid leukemia factors[END_REF]. Most statistical tests and graphs were performed using Prism v5 (GraphPad Software).

Plasmids

The following previously described plasmids were used: pAc-LZ-V5, 4xPPO2-Firefly luciferase (originally named 4xPO45-Fluc, [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]), pET-3c-LZ [START_REF] Wildonger | Lozenge directly activates argos and klumpfuss to regulate programmed cell death[END_REF], pAc-MLF [START_REF] Martin-Lanneree | Characterization of the Drosophila myeloid leukemia factor[END_REF]. We supplemented with 5% fetal bovine serum (FBS) and 50 μg/ml of penicillin/streptomycin (Invitrogen). For RNAi experiments, double stranded RNA duplexes (dsRNA) corresponding to 400-600bp exonic regions were produced using T7 promoter containing primers and MEGAscript T7 transcription kit (Ambion). After an annealing step, dsRNA probes were purified using the RNeasy clean-up protocol (Qiagen). Independent dsRNA targeting different regions of dnaj-1 and hsc70-4 were produced. Cells were seeded at 2x10 6 /ml on dsRNA (16 μg/well for 6-well-plate, 8 μg for 12-well plate and 1 ug for 96well-plate) and incubated in Schneider medium without FBS for 40min before being transferred to 5% FBS containing medium. 24h later, cells were transfected with the plasmids of interest using Effectene (Qiagen) and they were collected 72h later for subsequent analyses.

Luciferase reporter assays

For luciferase assays, 50 ng of 4xPPO2-Firefly luciferase reporter plasmid [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] were cotransfected with 20 ng of pAc-Renilla luciferase plasmid, 10 ng of pAc-LZ-V5 and/or 10 ng of pAc expression plasmid for the protein of interest in 96-well plate. Firefly and Renilla luciferases activities were measured 72h after transfection using Promege Dual luciferase reporter assay. Three biological replicates were performed for each transfection assay.

Real-time quantitative PCR

For RT-qPCR, RNA was from Kc167 cells using RNeasy kit (Qiagen) with an additional on-column DNAse treatment step. 1 μg of total RNA was used for reverse transcription using Superscript II and random primers (Invitrogen). 10 μl of a 1/300 dilution of cDNA was used as template for real time PCR using HOT Pol Evagreen qPCR mix (Bio-rad) to analyse dnaj-1, mlf, lz, PPO2, renilla luciferase and rp49 expression. All experiments were performed using biological triplicates or quadruplicates. Sepharose beads for 4h at 4°C. The beads were spun down and washed in IP buffer and immunoprecipitated proteins were processed for SDS-PAGE and Western Blot analyses.

In vitro

Western blots were performed using standard techniques and the blots were developed by photoluminescence procedure using Lumi-Light PLUS Western Blotting Substrate (Roche)

and Amersham Hyperfilm TM ECL (GE Healthcare) or Chemidoc TM Touch Imaging System (BioRad). The following antibodies were used for western blots: anti-V5 (Invitrogen), anti-HA (BioLegend), anti-GFP, anti-tubulin (Sigma-Aldrich), anti-Renilla luciferase (MBL)

and anti-MLF [START_REF] Fouix | Overexpression of a novel nuclear interactor of Suppressor of fused, the Drosophila myelodysplasia/myeloid leukaemia factor, induces abnormal morphogenesis associated with increased apoptosis and DNA synthesis[END_REF].

Affinity purification and mass spectrometry analysis

Stable Kc167 cells carrying an inducible expression vector for MLF were obtained by co- Quantification of proteins was performed using the label-free module of the MFPaQ software, where a protein abundance index based on the average of peak area values for the three most intense tryptic peptides of the protein was calculated [START_REF] Muntel | An exclusion list based label-free proteome quantification approach using an LTQ Orbitrap[END_REF]. Triplicate injections were performed.

RNA-seq experiments

RNAseq experiments were performed using independent biological triplicates. For each sample, around 150 third instar larvae of control (lz-GAL4, UAS-mCD8GFP/+) or mlf mutant (lz-GAL4, UAS-mCD8GFP/+, mlf ΔC1 /mlf ΔC1 ) genotypes were bled in ice-cold PBS. central glycine/phenylalanine (G/F) rich region, and a conserved C-terminal region that contains a client binding domain followed by a dimerization interface (Kampinga et al., 2010). Interestingly, when we performed in vitro GST pull-down assays, we found that in vitro translated 35 S-labeled DnaJ-1 weakly interacted with GST-DnaJ-1, but not with GST alone (Figure 22.B), suggesting that DnaJ-1 can directly interact with itself, probably due to the existence of a dimerization interface in its conserved C-terminal region. Similarly, we also found that in vitro translated MLF strongly interacted with GST-MLF, but not with GST alone (Figure 22.A), indicating that MLF interacts with itself.

Hsc70-5 interacts with MLF and DnaJ-1

Previous work has revealed that Hsc70-4 interacts specifically with MLF and DnaJ-1 (see above and Dyer et al., 2016). Hsc70-4 belongs to the family of constitutively expressed Hsp70 chaperones, which are composed of 5 paralogs in Drosophila melanogaster (Hsc70-1 to Hsc70-5). Considering the similarity in protein structure of the various Hsc70s (the 5

Hsc70 proteins exhibit ±60% of identity), it is interesting to know whether the interaction between Hsc70-4 and MLF or between Hsc70-4 and DnaJ-1 is conserved among other Hsc70s. Actually, we also identified Hsc70-3 as a potential partner of MLF in our proteomic analysis (see above; are conserved across members of the Hsc70 family and support the idea that MLF acts within the Hsp70 chaperone system.

Hsp83 does not interact with MLF or DnaJ-1

There are two main chaperone systems, the Hsp70 system and the Hsp90 system, both of which participate broadly in de novo protein folding and refolding [START_REF] Hartl | Molecular chaperones in protein folding and proteostasis[END_REF].

Our data show that MLF can act through DnaJ-1 and the Hsp70 system to control Lozenge stability and activity. However, it is still unknown whether MLF or DnaJ-1 might also interact with the Hsp90 chaperone machinery. To answer this question, we cloned Hsp83, a Drosophila homolog of human Hsp90 chaperones, and performed immunoprecipitation experiments between Hsp83 and MLF or DnaJ-1 after co-transfection of the corresponding expression plasmids in Kc167 cells. As Western blotting results showed, Hsp83 coprecipitated neither MLF (Figure 24.A) nor DnaJ-1 (Figure 24.B), suggesting that MLF and DnaJ-1 do not act in the Hsp90 chaperone system. These data also further confirm the specificity of the interactions that we observed between MLF, DnaJ-1 and Hsc70 proteins.

MLF, DnaJ-1 and Hsc70-4 control human RUNX1 stability

As we know, members of the RUNX family of proteins contain a highly evolutionarily conserved 128 amino acid domain at their N-terminus, which is designated as the runt homology domain (RHD) and mediates the interactions between RUNX family proteins with DNA but also with some other proteins. We have demonstrated that Lozenge, a Drosophila homolog of human RUNX1 transcription factor, interacts with MLF, DnaJ-1 and Hsc70-4, and that its activity and stability are regulated by these three proteins to control Drosophila blood cell development. Here, we asked that whether the MLF/DnaJ-1/Hsc70-4 chaperone complex might also regulate other RUNX factors. First, we tested whether human RUNX1 interacts with MLF, DnaJ-1 or Hsc70-4. Accordingly, we expressed RUNX1 in Kc167 cells together with GFP-tagged versions of MLF, DnaJ-1 or Hsc70-4 and we performed co-immunoprecipitation experiments. Our results showed that MLF is known to be a conserved regulator of RUNX transcription factor Lozenge (LZ) stability and activity, but the molecular mechanism of action of MLF on LZ stability has not been well understood (Bras et al., 2012). To answer this key question, an affinity purification approach followed by mass spectrometry was applied to search for MLF interacting partners, which could provide some valuable clues to this mystery. This approach allowed us to discover that MLF interacts with DnaJ-1 in Kc167 cells, which is in accordance with the results published by Dyer et al. who used a similar strategy in Drosophila S2 cells. We confirmed these results by co-immunoprecipitation experiments in Kc167 cells and our in vitro GST pull-down assay results further suggest that the interaction between MLF and DnaJ-1 is direct, which is consistent with the findings using purified recombinant proteins (Dyer et al., 2016). Furthermore, our domain mapping experiments show that MLF homology domain (MHD) interacts with DnaJ-1's conserved C-terminus, suggesting that the interaction between MLF and DnaJ-1 is conserved. In line with this idea, Dyer et al. showed that over-expressed hMLF1 or hMLF2 interacts with DnaJB6, a homolog of DnaJ-1, in human 293T cells. This provides a possible mechanism for MLFdirected transcription factor stabilization, since DnaJ-1 co-chaperone plays a crucial role in the regulation of protein folding and degradation, and MLF is likely to participate in the chaperone complex and exerts its function in the regulation process of a particular molecular chaperone complex. These findings open a new direction to decipher MLF molecular mode of action in the Drosophila model organism. However, it is still difficult to assess the interaction between MLF and DnaJ-1 in vivo, due to the small amount of crystal cells and the technical difficulties of their successful isolation from Drosophila.

Technologies like BiFc (Bimolecular Fluorescence Complementation) [START_REF] Hudry | Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay[END_REF] using transgenically expressed MLF and DnaJ-1 proteins fused to the N-or C-terminal domain of GFP, or Proximity Ligation Assays [START_REF] Weibrecht | Proximity ligation assays: a recent addition to the proteomics toolbox[END_REF] using antibody directed against each protein, could be an alternative option to try to validate MLF/DnaJ-1 physical interaction in vivo.

MLF/Hsc70-4 complex

DnaJ-1, a member of the Hsp40/DnaJ co-chaperone family, chiefly acts by delivering client proteins to the Hsp70 chaperones and stimulating the Hsp70 ATPase activity by interacting with them via their N-terminal J-domain. Interestingly, we also recovered Hsc70-4 and Hsc70-3 as potential MLF partners in our proteomic approach, two Hsp70 paralogs in Drosophila. Our co-immunoprecipitation experiments confirmed that MLF and DnaJ-1 interact with Hsc70-4, respectively. Similarly, Dyer et al. also found that MLF interacts with Hsc70-4. In addition, our co-immunoprecipitation results showed that MLF (and DnaJ-1) can also interact with Hsc70-5, which is only ±50% identical to Hsc70-4, suggesting that MLF very probably interacts with several Hsc70 proteins and participates in diverse Hsp70 chaperone complexes in association with DnaJ-1. Yet, whether MLF directly interacts with Hsc70 proteins or indirectly interacts with them via DnaJ-1 remains to be determined. In in vitro GST pull-down assays, I only observed a weak interaction between in vitro translated Hsc70-4 and GST-MLF, but a strong interaction with GST-DnaJ-1 (data not shown). However, these results need to be confirmed. In addition, there is another common chaperone complex, the Hsp90 system, which participates broadly in de novo protein folding and refolding [START_REF] Hartl | Molecular chaperones in protein folding and proteostasis[END_REF]. So we tested whether MLF or DnaJ-1 could also interact with this Hsp90 chaperone. Our co-immunoprecipitation results showed that neither MLF nor DnaJ-1 interacts with Hsp83, a Drosophila homologue of human Hsp90 chaperones, indicating that MLF and DnaJ-1 are not part of the Hsp90 chaperone system.

Of note, Dyer et al. also identified the nucleotide exchange factor (NEF) BAG2, which stimulates the release of client proteins from the Hsp70 chaperones, as one of the main partners of MLF. However, we did not retrieve this protein in our proteomic approach, which could be due to its weak association with the MLF/DnaJ-1/Hsc70-4 complex.

Furthermore, a systematic characterization of Hsp70 chaperone complexes in human cells has identified hMLF1 and hMLF2 as potential partners of DnaJ-1 homologs, DnaJB1, DnaJB4 and DnaJB6 (Taipale et al., 2014). All together, these publications and our work strongly support the conclusion that MLF is a conserved component of the Hsp70 chaperone system that interacts with DnaJB co-chaperones.

MLF/DnaJ-1/Hsc70-4 complex and LZ

Results from our team suggested that MLF proteins act as a conserved regulator of RUNX transcription factor stability and activity. In particular, Drosophila MLF controls LZ activity and prevents its degradation by the proteasome in cell culture (Bras et al., 2012).

However, it was unknown whether MLF physically interacts with LZ to control its stability.

123

Our co-immunoprecipitation results showed that MLF interacts specifically with LZ and our in vitro GST pull-down assays suggested that the interaction between MLF and LZ is direct.

In addition, we demonstrated that DnaJ-1 and Hsc70-4 also interact specifically with LZ, respectively, suggesting that LZ is a direct target of the chaperone complex formed by MLF, DnaJ-1 and Hsc70-4. Importantly, we found that knockdown of DnaJ-1 or Hsc70-4 expression by RNAi in Kc167 cells leads to a reduction in LZ protein level and activity without affecting its mRNA level, similar to what was observed following MLF knockdown. Dyer et al. also observed similar results on LZ protein level in S2 cells. In addition, we found that over-expression of DnaJ-1 mutants unable to stimulate Hsp70 chaperone activity also causes a reduction in LZ level and activity, suggesting that the effect of DnaJ-1 on LZ is strictly dependent on its capacity to activate Hsc70-4. Thus, the MLF/DnaJ-1/Hsc70-4 chaperone complex is required for the stable expression of LZ in cell culture. Yet, how each component acts within this chaperone complex still remains to be determined.

MLF in the Hsp70 chaperone complex: a chaperone or a co-chaperone?

Many studies have shown that over-expression of MLF, DnaJ-1 and some Hsp70s can suppress the cytotoxicity associated with polyglutamine (polyQ) protein aggregations in Drosophila (Kuo et al., 2013;Kim et al., 2005). In view of these publications and our results, it is tempting to speculate that the chaperone complex formed by MLF, DnaJ-1 and Hsc70-4 could play a role in the progression of neurodegenerative pathologies due to protein aggregation. This also further provides clues concerning the possible mode of action of MLF.

DnaJ-1 is known to act as a co-chaperone to assist the Hsp70 chaperones by delivering client proteins to their C-terminal substrate binding domain and stimulating their ATPase activity, but also as a chaperone by itself to mediate protein folding and refolding (Kampinga et al., 2010). On one hand, given the strong interaction between MLF and DnaJ-1, MLF could act as a chaperone when DnaJ-1 acts as a co-chaperone, or MLF could act as a co-chaperone when DnaJ-1 acts as a chaperone by itself. On the other hand, MLF interacts with Hsc70-4 (directly or via DnaJ-1), an Hsp70 chaperone involved in a wide range of protein quality control functions, including de novo protein folding and protein degradation.

So it would be interesting to test whether (1) MLF holds an intrinsic chaperone activity, ( 2)

MLF modulates DnaJ-1 chaperone activity, (3) MLF modulates the activity of the DnaJ/Hsc70 complexes. In that sense, we tried to produce highly purified MLF, DnaJ-1 and Hsc70-4 recombinant proteins in E. coli to assess the effects of MLF on the stimulation of Hsc70-4 ATPase activity and on Hsc70-4 dependent refolding of a denatured model substrate protein (i.e., firefly luciferase), as well as the effects of MLF on DnaJ-1 intrinsic chaperone activity in an assay based on the prevention of luciferase aggregation [START_REF] Perrody | Bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor σ 32 of Escherichia coli[END_REF]. However, MLF was highly insoluble and we could not produce enough proteins to perform these experiments. MLF could also act by regulating the client specificity of DnaJ proteins, and it would be interesting to identify other factors whose stability is regulated by MLF and/or DnaJ-1. According to the reaction cycle of the Hsp70 chaperone system, MLF could bind to LZ to prevent it from forming aggregates and then, together with DnaJ-1, present LZ to Hsc70-4 for proper protein folding. Our preliminary data suggest that an important fraction of LZ is not soluble when expressed in Kc167 cells and might form aggregates. In this model, MLF could act as a co-chaperone to prevent LZ but also polyglutamine (polyQ) protein aggregations. It would be interesting to further investigate the impacts of MLF, DnaJ-1 or Hsc70-4 knockdown on LZ soluble and insoluble fractions.

Regulation of LZ stability and activity by MLF and DnaJ-1

It has been demonstrated that MLF stabilizes RUNX transcription factor Lozenge, as the endogenous nuclear Lozenge is degraded in LZ + cells from mlf null mutant larvae and the Lozenge expressed in Kc167 cells is decreased when the endogenous MLF is knocked down (Bras et al., 2012). Due to loss of Lozenge, a key transcription factor for crystal cell development, phenotypic defects in LZ + cells emerge, such as changes in the number and the size of LZ + cells, indicating that the regulation of Lozenge stability by MLF is vital for the normal development of LZ + cells (Bras et al., 2012). However, how MLF regulates Lozenge stability remains largely unknown. Understanding this process might provide valuable information as to MLF functions and mode of action but also concerning the regulation of RUNX transcription factors. Both MLF and RUNX are implicated in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) in human, and thus our findings might be of interest in terms of human health. Based on our findings and those of Dyer et al., the prevailing hypothesis is that MLF acts together with the chaperone complex DnaJ-1/Hsc70-4 to control Lozenge level post-translationally by preventing its degradation.

The human homolog of Lozenge, RUNX1 is essential for hematopoiesis and it is subjected to proteolytic degradation by the ubiquitin-proteasome pathway in absence of CBFβ subunit [START_REF] Huang | Dimerization with PEBP2beta protects RUNX1/AML1 from ubiquitin-proteasome-mediated degradation[END_REF], suggesting that RUNX1 could be unstable in the nucleus without its β subunit and this instability could be an intrinsic property of RUNX1. It has been shown that RUNX1 is modified by a chain of ubiquitin and then the 70S proteasome can recognize this ubiquitylated RUNX1 to degrade it [START_REF] Huang | Dimerization with PEBP2beta protects RUNX1/AML1 from ubiquitin-proteasome-mediated degradation[END_REF].

However, when we tested whether Lozenge could be ubiquitylated, we didn't detect ubiquitylation of Lozenge in Kc167 cells (data not shown). Nonetheless, Lozenge is degraded in the absence of mlf in LZ + cells as well as in Kc167 cells and its degradation in Kc167 cells is partly inhibited by MG132 treatment, suggesting that the proteasome is implicated in Lozenge degradation (Bras et al., 2012). There is also a proteasome pathway independent of ubiquitylation [START_REF] Erales | Ubiquitin-independent proteasomal degradation[END_REF], and it could be interesting to check whether Lozenge is degraded by this pathway. Besides degradation mediated by proteasome, some specific proteases and certain lysosomes also cause the degradation of particular proteins. We propose that MLF might act at three different levels. Firstly, MLF binding to Lozenge could block its interaction with the proteolytic chamber of the 20S proteasome or interfere with its recognition by the 70S proteasome. Secondly, MLF could block Lozenge ubiquitylation and its subsequent targeting to the proteasome, even though we have not obtained direct evidence that Lozenge can be ubiquitylated. Thirdly, MLF could inhibit the proteasome-independent degradation of Lozenge. An important challenge will be to further define MLF mode of action and the pathway that mediates Lozenge degradation.

We have demonstrated that MLF is involved in the Hsp70 chaperone system, and Hsp70 chaperones can also mediate the degradation of some proteins that are not refolded by Hsp70 chaperones through chaperone mediated autophagy (CMA) or chaperone-assistant selective autophagy (CASA), both of which are dependent or independent on ubiquitylation [START_REF] Kettern | Chaperone-assisted degradation: multiple paths to destruction[END_REF]. So, even though our results argue that Hsc70-4 is required for Lozenge stability, it could be interesting to test whether Lozenge degradation is caused by Hsp70 chaperones-mediated autophagy in the absence of MLF.

Intriguingly, we found that in the presence of high levels of MLF (LZ + larval blood Finally, since DnaJ-1 and MLF can also bind chromatin and regulate transcription (Dyer et al., 2016), it would be interesting to test whether Lozenge recruits these proteins to chromatin or whether these two proteins are also involved in the recruitment of Lozenge to chromatin. Indeed, even though our data show that the defects caused by MLF or DnaJ-1 loss can be rescued by the re-expression of Lozenge in vivo, we can not exclude the possibility that these two protein somehow participate directly in the regulation of Lozenge capacity to regulate gene expression. However, the limited amount of crystal cells present in the larva precludes our possibility to perform ChIP experiments in vivo to test these hypotheses.

MLF, DnaJ-1, LZ and the control of crystal cell size and number

Based on the data collected from the dnaj-1 -/-mutant larvae, we showed that this gene is required for the normal development of the circulating crystal cells and that it genetically interacts with mlf during this process. The loss of DnaJ-1 notably caused an increase in the number and the size of LZ + blood cells, which was rescued by the re-expression of DnaJ-1 in this lineage. This demonstrates that these phenotypes are specifically due to the absence of DnaJ-1 and that DnaJ-1 acts after the induction of crystal cell fate and in a cellautonomous way. So far, the function of DnaJB proteins in hematopoiesis has barely been studied and thus our results provide the first evidence that this conserved family of cochaperones controls blood cell development in vivo. Besides the circulating larval blood cells, it would be interesting now to study the role of DnaJ-1 in the development of the crystal cells in the embryo, the lymph gland and the adult but also to assess its function in other blood cell types during Drosophila hematopoiesis.

In addition, like in mlf -/-mutant larvae, we found that the increase in LZ + blood cell size and number observed in dnaj-1 -/-larvae is rescued when we enforced the expression of Lozenge in this lineage. Together with our results in Kc167 cells, this strongly suggests that MLF and DnaJ-1 to control LZ + cell development by promoting Lozenge activity.

Consistent with this hypothesis, we found that reducing Lozenge expression or interfering with its activity causes an increase in the number and the size of LZ + cells, a phenotype that could be described as "preleukemic". This not only reinforces our conclusion but also establishes an interesting parallel with the situation in mammals where a reduction in RUNX1 activity has been associated with oncogenic blood cell transformation.

Moreover, we could link the decrease in Lozenge level/activity with an over-activation of the Notch signaling pathway and our data suggest that high levels of Lozenge directly repress Notch expression and thus activation of the Notch pathway to control LZ + blood cells number and size. It will be interesting to decipher how Lozenge represses Notch transcription and to explore further how the Notch pathway controls LZ + cells growth and survival. Again, this could be particularly interesting to study the role of Notch pathway activation in human leukemia.

Conservation of regulation of RUNX transcription factor stability and activity by MLF/DnaJ-1

Given the conservation of the RUNX, MLF, DnaJ and Hsp70 families through evolution, it will be particularly interesting to study the relationships between the MLF/DnaJ/Hsc70 complex and RUNX factors in other species. RUNX1 and Lozenge share a highly conserved runt domain, and we showed that RUNX1 stability is regulated by MLF, DnaJ-1 as well as Hsc70-4 in Kc167 cells. In addition, we found that MLF and Hsc70-4

(but not DnaJ-1) bind to RUNX1. Moreover, it was shown previously in the lab that RUNX1-ETO protein level is also regulated by MLF both in Drosophila and in human leukemia cells (Bras et al., 2012). Thus, the MLF/DnaJ/Hsc70 complex could play a conserved role in the regulation of RUNX transcription factors stability and it would be worth investigating whether homologs of MLF, DnaJ-1 and Hsc70-4 also control the level and/or the activity of RUNX transcription factors in human, particularly in normal and leukemic blood cells. One limitation of these study is that mammals could have the high level of gene redundancy between members of the DnaJ family, Thus, studies in simpler model organisms like Drosophila still offer a good opportunity to study in vivo the function of these factors.

Perspectives

We have demonstrated that MLF, DnaJ-1 and Hsc70-4 interact with Lozenge, and regulate the stability as well as the activity of this RUNX transcription factor. Yet, we still don't know whether all these proteins can be in the same complex and how they organize topologically. Therefore, more biochemical approaches are needed to gain insights into the relationships among these proteins and to decipher how MLF and DnaJ-1/Hsc70-4 control Lozenge stability.

In our study, biochemical experiments were carried out in Kc167 cells, an embryonic blood cell line. These cells have lower levels of endogenous MLF than crystal cells, which lead to differences in Lozenge expression and regulation between these two cell types.

Notably, a reduction in Lozenge expression was observed in Kc167 cells when DnaJ-1 was knocked down, while Lozenge accumulation was present (and stronger) in LZ + cells in dnaj-1 null mutant larvae. In addition, we cannot exclude the possibility that other important proteins implicated in Lozenge regulation by the MLF/DnaJ/Hsc70 complex are differentially expressed between Kc167 cells and LZ + larval blood cells. Thus Kc167 cells are not ideal to characterize the regulation of Lozenge stability by MLF and DnaJ-1. It would be best if we could isolate and purify sufficient amounts of LZ + cells from Drosophila embryos or larvae to perform these experiments, or if we could obtain a cell line that contains endogenous MLF level equivalent to those observed in vivo in crystal cells.

We have revealed that MLF and DnaJ-1 interact with each other via their highly evolutionarily conserved domains and that Lozenge also associates with each one of them, establishing a functional link between RUNX transcription factors and the Hsp70 chaperone complex formed by MLF, DnaJ-1 and Hsc70-4. Based on our study of RUNX1 regulation in Kc167 cells, we propose that a complex homologous to the MLF/DnaJB/Hsc70 complex could regulate the stability of RUNX in human. More generally, it seems that MLF1 and MLF2 could regulate the Hsp70 chaperone machinery in human. Hence, besides their impact on RUNX transcription factors, it will be interesting to identify other proteins whose stability/activity is regulated by MLF and DnaJ and that could be implicated for instance in neurodegenerative diseases, in which these two families of proteins are involved. This would provide valuable insights into the molecular mechanism of action of MLF and DnaJ protein families in normal development and in pathological situations.

  l'hématopoïèse et dans la transformation oncogénique des cellules sanguines, mais leur fonction et leur mécanisme d'action moléculaire restent insaisissables. Des travaux précédents chez la Drosophile ont montré que MLF stabilise le facteur de transcription de type RUNX Lozenge (LZ) et contrôle le nombre de cellules sanguines LZ + . Au cours de ma thèse, j'ai cherché à déchiffrer le mécanisme moléculaire d'action de MLF sur Lozenge dans les cellules sanguines. Par une approche protéomique puis par des expériences de co-immunoprécipitation dans les cellules de Drosophile Kc167, nous avons identifié le co-chaperon de type Hsp40 DnaJ-1, et son partenaire le chaperon Hsc70-4, comme deux partenaires de MLF. De façon importante, nous avons montré que l'inhibition de l'expression de DnaJ-1 ou de Hsc70-4 dans les cellules Kc167 induit une réduction du niveau de protéine Lozenge et une diminution de sa capacité à activer la transcription très semblable à celles observées suite à l'inhibition de l'expression de MLF. De plus, la sur-expression de mutants de DnaJ-1 incapables d'activer le chaperon Hsc70-4 entraîne aussi une réduction du niveau de Lozenge et de sa capacité de transactivation et des expériences de coimmunoprécipitation montrent que Lozenge interagit avec MLF, DnaJ-1 et Hsc70-4. Nos résultats suggèrent donc que MLF agit au sein d'un complexe chaperon composé de DnaJ-1 et Hsc70-4 pour contrôler le viii niveau de Lozenge. En utilisant différents mutants de MLF ou DnaJ-1, nous avons montré que MLF et DnaJ-1 interagissent ensemble et avec Lozenge via des domaines phylogénétiquement conservés. D'autre part, des expériences de GST « pull down » in vitro suggèrent que ces trois protéines peuvent interagir ensemble directement. Nous proposons donc que MLF et DnaJ-1 contrôlent le niveau de protéine Lozenge en interagissant avec elle et en favorisant son repliement et/ou sa solubilité via l'activité chaperon de Hsc70-4. En parallèle, nous avons étudié la fonction de DnaJ-1 in vivo dans le développement des cellules sanguines de la Drosophile. Nos résultats montrent que, comme mlf, la perte de dnaj-1 s'accompagne d'une augmentation de la taille et du nombre des cellules sanguines LZ + , ainsi que d'une hyperactivation de la voie de signalisation Notch dans ces cellules. Nos résultats suggèrent que des hauts niveaux de Lozenge sont nécessaires pour contrôler le nombre et la taille des cellules LZ + et pour inhiber l'expression de Notch. Nous proposons que le complexe MLF/DnaJ-1 contrôle le développement du lignage LZ + en régulant le niveau de protéine Lozenge, et ainsi le niveau d'activité de la voie Notch.

Figure 1 .Figure 3 .

 13 Figure 1. The cell types in blood Figure 2. A summary of the process of hematopoiesis in the mouse Figure 3. Schematic representation of the progressive evolution of blood island mesodermal cells to a functional vascular network and primitive erythroid cells

Figure 4 .Figure 5 . 6 .Figure 8 .Figure 10 .Figure 13 .Figure 16 .Figure 17 .Figure 19 . 1 Figure 22 . 1 Figure 24 . 1 Figure 25 .Figure 26 .Figure 27 .Figure 28 .Figure 29 .

 4568101316171912212412526272829 Figure 4. The model of ecological hematopoietic stem cell niche Figure 5. Drosophila melanogaster and its life cycle Figure 6. Drosophila hematopoiesis during development Figure 7. Three types of hemocytes in Drosophila melanogaster and their functions Figure 8. Structural representation of the RUNX1-CBFβ and diagrammatic representation of three RUNX protein subtypes Figure 9. RUNX1b structure, its post-translational modifications and two promoters of RUNX1 genes Figure 10. Proposed mechanisms to explain the ability of RUNX1 to both activate and repress transcription Figure 11. RUNX1 expression in hematopoiesis sites in the E10.5 embryo Figure 12. Structures of CBF fusion genes that are associated with leukemia Figure 13. Genomic region and two isoforms of lozenge gene Figure 14. Scanning electron micrographs of Drosophila adult eyes Figure 15. Schematic representation of the members of the MLF family in human and in Drosophila Figure 16. mlf transcripts and its mutants in Drosophila Figure 17. Protein fates in the proteostasis network Figure 18. The proteostasis network for the Hsp70 complex and the Hsp90 complex in human cells characterized by mass spectrometry (MS) Figure 19. Structure and reaction cycle of Hsp70 Figure 20. Classification and functional domains of DNAJ Figure 21. Endogenous MLF interacts with DnaJ-1 Figure 22. MLF or DnaJ-1 forms a dimer Figure 23. Hsc70-5 interacts with MLF and DnaJ-1 Figure 24. Hsp83 does not interact with MLF or DnaJ-1 Figure 25. High levels of MLF rescue Lozenge stability and activity when DnaJ-1 is knocked down Figure 26. High levels of DnaJ-1 do not rescue Lozenge stability or activity when MLF is knocked down

Figure 3 .

 3 Figure 3. Schematic representation of the progressive evolution of blood island mesodermal cells to a functional vascular network and primitive erythroid cells(From Cumano et al., 2007)

  are a category of white blood cells that have a segmented nucleus with varying shapes and are packed with granules filled with a variety of enzymes in their cytoplasm. Distinguished by their appearance under Wright's staining, granulocytes are classified into three principal types, which are neutrophils, eosinophils and basophils. These cells are professional phagocytes that are dedicated to the ingestion and destruction of bacteria and other pathogens invading the body, and they release their granule contents to these pathogens by exocytosis to help fight infection and inflammation.Monocytes are mononuclear professional phagocytes like granulocytes that invade the tissue at the infection sites. They undergo further differentiation into macrophages that can divide and multiply at the needed sites. Macrophages respond to foreign materials (such as bacteria, protozoa or tumour cells) and phagocytose them. Besides their role in phagocytosis and thus in the innate immune response, macrophages process proteins of the pathogens to present them to T lymphocytes that help initiate the adaptive immunity.Lymphocytes are one of the subtypes of white blood cells in a vertebrate immune system, including B-lymphocytes, T-lymphocytes and natural killer cells (NK cells). Blymphocytes and T-lymphocytes constitute the major cellular components of the adaptive immunity while natural killer cells play a role in the cell-mediated cytotoxic innate immunity. B-lymphocytes are primarily responsible for humoral immunity and they respond to a specific pathogen by producing large quantities of antibodies that then neutralize these foreign substances. T-lymphocytes are involved in cell-mediated immunity and they recognize antigen/major histocompatibility complex (MHC) complexes presented by macrophages with the receptors on their surfaces, which triggers direct cytotoxic effects on the recognized infected cells.

Figure 4 . 1 . 1 . 3 . 2 .

 41132 Figure 4. The model of ecological hematopoietic stem cell nicheHSC homing, maintenance, and differentiation depend on their specific microenvironment (or niche). In the bone marrow, there are at least two major HSC niches, the vascular niche and the osteoblastic niche. In addition, there are growing indications that other types of cells. (From[START_REF] Shiozawa | Getting blood from bone: An emerging understanding of the role that osteoblasts play in regulating hematopoietic stem cells within their niche[END_REF]

Figure 5 .

 5 Figure 5. Drosophila melanogaster and its life cycle (A) An adult Drosophila melanogaster. (B) At 25°C, fertilized females lay hundreds of eggs

Figure 6 .

 6 Figure 6. Drosophila hematopoiesis during development (A) Overview of Drosophila hematopoiesis during development. (B) Embryonic hematopoiesis. (C) Larval hematopoiesis. (D) Adult hematopoiesis. (From Letourneau et al., 2016 (A, C); Bataillé et al., 2005 (B); Ghosh et al., 2015 (D))

Figure 7 .

 7 Figure 7. Three types of hemocytes in Drosophila melanogaster and their functions (A) Plasmatocyte (B) Crystal cell (C) Lamellocyte (From Krzemień PhD thesis)

  publications have focused on the regulation of blood cell progenitor fate in the larval lymph gland, showing for instance that the Hedgehog, JAK/STAT, Wnt, Insulin/Target of Rapamycin, or Notch signalling pathways, the level of reactive oxygen species (ROS), as well as transcription factors, such as the EBF factor Collier, or the GATA factor Serpent and Pannier, control lymph gland homeostasis during normal development. In contrast, there were fewer studies about the fate of embryo-derived hemocytes that populate the larval hemocoel or of the adult blood cells, for which we only have very limited information. Similarly, while we have a relatively good understanding of crystal cell fate development, how plasmatocyte and lamellocyte differentiation is controlled is less well understood.

Figure 8 .

 8 Figure 8. Structural representation of the RUNX1-CBFβ and diagrammatic representation of three RUNX protein subtypes (A). Structures of the RUNX1 runt domain (green) and the CBFβ heterodimerization domain (brown) (From Speck et al., 2002). (B). A diagrammatic representation of RUNX1, RUNX2 and RUNX3 together with Drosophila Runt. Conserved runt domain and VWRPY sequence at the C-terminus of the proteins are indicated(From Ito, 2004). The breakpoints for two major translocations affecting RUNX1 are shown.

Figure 9 .

 9 Figure 9. RUNX1b structure, its post-translational modifications and two promoters of RUNX1 gene (A) Structure and post-translational modifications of human RUNX1 (From Goyama et al., 2015). (B) Two promoters (P1 and P2) of the RUNX gene family in human (From Challen et al., 2010).

Figure 10 .

 10 Figure 10. Proposed mechanisms to explain the ability of RUNX1 to both activate and repress transcription (A) RUNX1/AML1 cooperates with activator proteins such as p300/CBP, Ets, Myb on C/EBP to activate transcription. (B) RUNX1/AML1 binds to repressor proteins such as mSin3 and TLE to repress transcription.(From Lutterbach et al., 2000)

Figure 11 .

 11 Figure 11. RUNX1 expression in hematopoiesis sites in the E10.5 embryo (A) RUNX1 is expressed (blue) in a small population of endothelial cells and hematopoietic cells that are scattered throughout the yolk sac (ys), in endothelial cells lining the vitelline (v) and umbilical (u) arteries, and in endothelial cells, mesenchymal cells and intra-aortic hematopoietic cluster in the ventral portions of the dorsal aorta within the aorta/gonad/mesonephros (agm) region. (B) Detailed view of RUNX1 expression in endothelial cells (e), mesenchymal cells (m) and a hematopoietic cluster (hc) in the ventral AGM region. (From Speck et al., 2002)

Figure 12 .

 12 Figure 12. Structures of CBF fusion genes that are associated with leukemia These structures of CBF fusion genes shown here include RUNX1-ETO (t(8;21) translocations), CBFβ-SMMHC (inv(16) translocations), TEL-RUNX1 (t(12;21) translocations), RUNX1-EVI1 (t(3;21) translocations)(From Speck et al., 2002)

Figure 13 .

 13 Figure 13. Genomic region and two isoforms of lozenge gene (A) lozenge genomic region is shown as well as the two lozenge mRNAs (grey: un-translated region, pink: coding sequences). (B) Schematic representation of two Lozenge isoforms. The runt domain (RD) is shown in grey, the VWRPY motif is in red and the sequence coded by exon 5 (E5) is in blue.

Figure 14 .

 14 Figure 14. Scanning electron micrographs of Drosophila adult eyes (A) The wild-type Drosophila eye has a regular array of ordered facets. (B) lz ts1 flies, when reared at 25°C, have wild-type eyes. (C) When lz ts1 flies are reared at 29°C, the eye appears rough and disorganized. (D) The lz R1 null gives rise to severe eye phenotype. (From[START_REF] Kaminker | Redundant function of Runt domain binding partners, Big brother and Brother, during Drosophila development[END_REF]

Figure 15 .

 15 Figure 15. Schematic representation of the members of the MLF family in human and in Drosophila The fusion protein NPM-MLF1 produced by the t(3;5) chromosomal translocation consists of the N-terminal region of NPM (amino acids 1 to 175) fused to the almost entire hMLF1 (amino acids 16 to 268). The various domains identified in these proteins are shown. Abbreviations: NES (nuclear export signal), NLS (nuclear localization signal), OLIGOM (NPM oligomerization domain), 14-3-3 (consensus binding motif for 14-3-3 proteins), MHD (MLF homology domain).

  The imprecise mobilization of a transposable element located in the first intron of the mlf gene allowed to generate two mlf null mutant alleles[START_REF] Martin-Lannerée | Characterization of the Drosophila myeloid leukemia factor[END_REF], and both alleles resulted in a deletion of almost entire mlf coding region (Figure16.A). mlf null mutation is associated with a strong lethality during development. Some mlf mutant individuals can survive to the adult stage, but they do not exhibit obvious morphological defects apart from the loss of some interocellar bristles, frequent shortening of head macrochaetes and ectopic vein formation in the wings (Figure16.B)[START_REF] Martin-Lannerée | Characterization of the Drosophila myeloid leukemia factor[END_REF]. Therefore, the phenotypes of these mlf mutants do not provide strong hints concerning mlf functions in vivo.

Figure 16 .

 16 Figure 16. mlf transcripts and its mutants in Drosophila (A) Structures of dmlf transcripts. The four isoforms are depicted and coding exons are shown in color, untranslated regions in grey. The extent of dmlf deletion in the two mlf alleles is shown. (B) Phenotypes of the adult dmlf mutant flies. Views of the head (A-C) and wings (A'-C') of dmlf R2 /dmlf R2 (A and A'; wild type derived from precise excision of the P element),dmlf ΔC1 /dmlf ΔC1 , act5C-Gal4/+ (B and B'), dmlf ΔC1 /dmlf ΔC1 , act5C-Gal4/UAS-hMLF1(22.1). The ubiquitous expression of hMLF1 partially rescues mlf mutant phenotypes (C and C').(From Martin-Lannerée et al., 2006)

  defects in crystal cell number, indicating a conservation of MLF function. Finally, RUNX1-ETO accumulation in Kc167 cells as well as in Drosophila crystal cells or in human leukemic cells was also dependent on dMLF or hMLF1, respectively, suggesting that MLF factors could regulate the stability of different Runt-domain containing transcription factors.

Figure 18 .

 18 Figure 18. The proteostasis network for the Hsp70 complex and the Hsp90 complex in human cells characterized by mass spectrometry (MS) Protein-protein interactions were identified by AP-MS. Proteins are shown as rectangles, and lines represent interactions between the proteins (From Taipale et al., 2014).

5 . 3 . 2 .Figure 20 .

 53220 Figure 20. Classification and functional domains of DNAJ (A) DNAJ proteins are classified into three families. DNAJ may be classified according to the presence or absence of three domains, namely the J domain, a glycine/phenylalanine rich region (G/F) and the cysteine repeat motif (Cys-repeat) together with a C-terminal domain. (B) The three dimensional structure of J-domain (E.coli J-domain; 1BQ0) that is currently used to define the DNAJ family. It illustrates that the J-domain structure resembles a "protruding

  : Type I (or Type A) has a glycine/phenylalanine (G/F) rich domain as well as a cysteine repeat domain, Type II (or Type B) has a glycine/phenylalanine (G/F) rich domain and type III (or Type C) does not have any of these extra domains (Figure 20.A). Hsp40/DnaJ proteins are found in the cytosol, mitochondria, nucleus, endoplasmic reticulum, endosomes or ribosomes, and their

1 :

 1 function and molecular mechanism of action still remain elusive. Previous work in Drosophila melanogaster showed that dMLF controls the number of crystal cells, one blood cell lineage related to megakaryocyte, and stabilizes the RUNX transcription factor Lozenge that is indispensable for the development of crystal cells. Moreover, our results suggested that regulation of RUNX transcription factor stability and activity is a conserved characteristic of MLF factors, which could therefore play a role in many cancers caused by a change in RUNX activity in human. During my PhD study, I used Drosophila melanogaster as a model to decipher the molecular mechanism of action of MLF on Lozenge stability and activity during blood cell development by searching for MLF interacting partners and characterizing the role of these MLF partners in the regulation of the homeostasis of the hematopoietic system and particularly in the control of proliferation and differentiation of LZ + cell lineage. Centre de Biologie du Développement (CBD), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, 118 route de Narbonne, F-31062 Toulouse, France 2: Institut de Pharmacologie et de Biologie Structurale, Université de Toulouse, CNRS, UPS, France # Corresponding authors, E-mail: lucas.waltzer@univ-tlse3.fr ; marc.haenlin@univ-tlse3.fr * These authors contributed equally to this work.AbstractA tight regulation of transcription factor activity is critical for proper development. For instance, modifications of RUNX transcription factor dosage are associated with several diseases, including hematopoietic malignancies. In Drosophila, myeloid leukemia factor (MLF) has been shown to control blood cell development by stabilizing the RUNX transcription factor Lozenge (LZ). However, the mechanism of action of this conserved family of proteins involved in leukemia remains largely unknown. Here, we further characterized MLF mode of action in Drosophila blood cells using proteomic, transcriptomic and genetic approaches. Our results show that the Hsp40 co-chaperone family member DnaJ-1 is a partner of MLF and demonstrate that like MLF, DnaJ-1 binds LZ and promotes its expression in cell culture, suggesting that MLF and DnaJ-1 form a chaperone complex to regulate LZ level and activity. Importantly, dnaj-1 loss causes an accumulation of LZ + blood cells similar as in mlf mutant larvae, and we find that dnaj-1 genetically interacts with mlf to control LZ level and LZ + blood cell development in vivo. In addition, we show that mlf or dnaj-1 loss alters LZ + cell differentiation and causes increased Notch expression and over-activation of the Notch signaling pathway. Finally, using different conditions to manipulate LZ activity, we show that high levels of LZ are required to repress Notch transcription and signaling. Our findings thus establish a functional link between MLF and the co-chaperone DnaJ-1 to control RUNX transcription factor activity and Notch signaling during blood cell development in vivo. We propose that MLF/DnaJ-1dependent increase in RUNX level allows the repression of Notch expression and signaling to prevent aberrant blood cell development.

  , we established a Drosophila Kc167 cell line expressing a V5-tagged version of MLF close to endogenous level of MLF in a copper-inducible manner (Fig 1A). After anti-V5 affinity purification from whole cell extracts of control or MLF-V5-expressing cells, isolated proteins were identified by mass spectrometry (see M&M for details). Five proteins reproducibly co-purified with MLF and were either absent or present at more than 4 folds lower levels in control purifications (Fig 1B): the Hsp40 co-chaperone DnaJ-1 (also known as DROJ1, [34]), the constitutively expressed Hsp70 chaperones Hsc70-4 and Hsc70-3, the RNA binding protein Squid (Sqd), and the retrotransposon-encoded protein Copia.

DnaJ- 1 ,

 1 while MLF N and C-terminal regions were dispensable (Fig 1H). Finally, consistent with the above results, the C terminal region (amino acids 157 to 334) of DnaJ-1 fused to GFP but not the GFP moiety alone co-precipitated the HA-tagged MHD (S1B Fig). In sum, these data indicate that MLF and DnaJ-1 specifically bind to each other through their conserved central and C-terminal region, respectively.

Fig

  Fig 2A, transfection of LZ expression plasmid in Kc167 cells induced a robust activation of the 4xPPO2-Fluc reporter gene[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], which was significantly decreased when either MLF or DnaJ-1 expression was knocked down by dsRNA treatment. Furthermore, Western blot analyses revealed that, like mlf, dnaj-1 knockdown caused a drop in LZ protein expression(Fig 2B). Importantly RT-qPCR experiments showed that mlf and dnaj-1 knockdown did not affect the expression of each other or decrease lz transcript level, while they caused a significant reduction in the expression of LZ target gene ppo2 (Fig 2C-F). Hence, like MLF, DnaJ-1 controls LZ protein activity by regulating its stability.

  level(Fig 2I and 2J). The over-expression of C-terminus-truncated DnaJ-1 proteins did not affect LZ-induced transcription or LZ expression. In contrast, the over-expression of DnaJ-1 carrying the P32S point mutation or deleted of its J domain caused a decrease in LZ-induced transactivation and a drop in LZ level (Fig.2Iand 2J), suggesting that Hsc70 activation by DnaJ-1 is required for LZ stable expression. Since we identified Hsc70-4 as a potential partner of MLF, we further tested this hypothesis. Immunoprecipitation experiments confirmed that MLF and DnaJ-1 interacted with Hsc70-4 (S3A and S3B Fig). Moreover, knocking down hsc70-4 by dsRNA caused a strong decrease in LZ-induced transactivation of the 4xPPO2-Fluc reporter gene and a concomitant reduction in LZ expression (S3C and S3D Fig).

Fig) .

 . Fig). In contrast, MLF is present at high levels in the nucleus of larval crystal cells (S5B Fig). Moreover, its expression in this lineage is not affected by dnaj-1 loss (S5C and 5D Fig). We thus supposed that the presence of high levels of nuclear MLF might prevent LZ degradation in the absence of DnaJ-1.

(

  19 repressed and 25 activated) (Fig 6C), indicating a strong over-representation of deregulated genes in the "crystal cell" gene set as compared to all expressed genes (p-value=2.6x10 -13 , hypergeometric test) and showing that mlf plays a crucial role in proper crystal cell differentiation.

  activity causes an expansion of the crystal cell population associated with aberrant differentiation. We thus tested what happens when LZ activity is reduced. Accordingly, we introduced the lz r1 null allele in the lz GAL4 context. This hypomorphic allelic combination caused a decrease in LZ expression(Fig 8B) and resulted in an increase in lz>GFP + cell number and size (Fig 8Eand 8F). Interestingly, lz GAL4 /Y hemizygote larvae displayed similar phenotypes(Fig 8C, 8E and 8F), indicating that this P{GAL4} insertion in lz alters its expression in the crystal cell lineage. As an alternate strategy, we interfered with LZ activity by expressing a fusion protein between LZ partner Brother (Drosophila CBFß homolog)

  Fig). Interestingly, the regulatory elements carried by these two lines (GMR30A01 and GMR30C06) overlapped on a 668bp DNA segment that contains two consensus binding sites for RUNX transcription factors conserved in other Drosophila species (S10A Fig),

  generated the following Drosophila expression plasmids for C-terminally tagged or Nterminally tagged proteins using standard cloning techniques: pAc-LZ-EGFP, pAc-MLF-EGFP, pMT-MLF-V5-His, pAc-DnaJ-1-EGFP, pAc-Hsc70-4-EGFP, pAc-3xHA-DnaJ-1(2-334), pAc-3xHA-DnaJ-1(P32S), pAc-3xHA-DnaJ-1(58-334), pAc-3xHA-DnaJ-1(2-156), pAc-3xHA-DnaJ-1(2-191), pAc-3xHA-DnaJ-1(2-269), pAc-3xHA-DnaJ-1(157-334), pAc-3xHA-MLF(2-309), pAc-3xHA-MLF(2-147), pAc-3xHA-MLF(2-202), pAc-3xHA-MLF(202-309), pAc-3xHA-MLF(148-309), pAc-3xHA-MLF(96-309), pAc-3xHA-MLF (96-202). DnaJ-1 and MLF cDNA were also cloned into pBlueScript II to generate pBS-DnaJ-1 and pBS-MLF and in pGEX-2T to generate pGEX-DnaJ-1 and pGEX-MLF. All constructs were verified by sequencing. Cell culture, dsRNA treatments and transfections Drosophila Kc167 cells were grown at 25°C in Schneider medium (Invitrogen)

  pull down assays pET-3c-LZ, pBS-MLF and pBS-DnaJ-1 plasmids were used as template to produce 35Smethionine-labeled proteins in vitro using Rabbit Reticulocyte Lysate coupled transcriptiontranslation system (Promega). pGEX-2T, pGEX-MLF and pGEX-DnaJ-1 were used to produce GST, GST-MLF and GST-DnaJ-1 in Escherichia coli (BL21). Equivalent amounts of GST purified proteins immobilized on Glutathione-Sepharose beads were used to pull down LZ, MLF or DnaJ-1. Proteins were incubated for 2h at 4°C in buffer A (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM KCl, 1 mM EDTA, 0.1 mg/ml BSA, 1 mM DTT, 0.05% NP40). After extensive washing in buffer B (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.05% NP40), bound proteins were eluted in SDS-loading buffer, separated by SDS-PAGE and visualized by autoradiography.Protein extraction, immunoprecipitations and western blotsKc167 cells were collected, washed in PBS and incubated for 30 min on IP buffer (150 mM NaCl, 0.5% NP40, 50 mM Tris-HCl, pH8.0, 1mM EGTA) supplemented with protease inhibitor cocktail (Roche). The extracts were cleared by centrifugation at 13 000g for 15 min at 4°C and subjected to SDS-PAGE (50 μg of proteins par lane) or immunoprecipitation (1 mg per point). For immunoprecipitation, proteins were pre-absorbed with 100 μl of Sepharose beads slurry for 1h at 4°C before being incubated with 20 μl of anti-GFP (Chromotek), anti-V5 (Sigma-Aldrich) or anti-HA (Covance) antibody coupled to

  transfecting pMT-MLF-V5-His and pCoBlast (Thermo Fisher Scientific) expression plasmids and selecting individual clones with 25μg/ml blasticidin. For affinity purification, MLF-inducible or parental Kc167 cells were seeded at 10 6 /ml and cultivated for 24h in the presence of 50 mM CuSO 4 to induce MLF expression. 20 mg of proteins extracted in IP buffer were then incubated on 200 μl of anti-V5 coupled Sepharose beads (Sigma-Aldrich) or 400 μl of anti-V5 coupled magnetic beads (MBL). After several washes in IP buffer, affinity purified proteins were eluted in Laemmli buffer, reduced in 30 mM DTT and alkylated with 90 mM Iodoacetamide before being loaded on 12% SDS-PAGE. The single band of proteins was cut and digested overnight at 37°C with 1 μg of Trypsin (Promega) in 50 mM NH 4 CO 3 . Digested peptides were extracted from the gel by incubating 15 min at 37°C in 50 mM NH 4 CO 3 and twice for 15 min at 37°C in 5% formic acid/acetonitrile (1:1).The dried peptide extracts were dissolved in 17 μl of 2% acetonitrile, 0.05% trifluoroacetic acid and the peptide mixtures were analysed by nanoLC-MS/MS using an Ultimate3000-RS system (Dionex) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). 5μl of each peptide extract were loaded on a 300 μm ID x 5 mm PepMap C18 precolumn (LC Packings, Dionex,) at 20 μl/min in 5% acetonitrile, 0.05% trifluoroacetic acid. After 5 minutes desalting, peptides were online separated on a 75μm IDx50 cm C18 Reprosil C18 column. The flow rate was set at 300 nl/min. Peptides were eluted using a 0 to 50% linear gradient of solvent B (solvent A: 0.2% formic acid in 5% acetonitrile, solvent B: 0.2% formic acid in 80% acetonitrile) for 80 min at 300 nl/min. The LTQ Orbitrap was operated in data-dependent acquisition mode with the XCalibur software (version 2.0 SR2, Thermo Fisher Scientific), on the 350-1800 m/z mass range with the resolution set to a value of 60 000. The twenty most intense ions per survey scan were selected for CID-MS/MS fragmentation and the resulting fragments were analyzed in the linear ion trap (parallel mode). A 60 s dynamic exclusion window was used to prevent repetitive selection of the same peptide. The Mascot Daemon software (version 2.2.0, Matrix Science, London, UK) was used for protein identification against a non-redundant SwissProt database. Mascot results were parsed with Mascot File Parsing and Quantification (MFPaQ) version 4.0[START_REF] Bouyssié | Mascot file parsing and quantification (MFPaQ), a new software to parse, validate, and quantify proteomics data generated by ICAT and SILAC mass spectrometric analyses: application to the proteomics study of membrane proteins from primary human endothelial cells[END_REF].

FIG 1 .

 1 FIG 1. MLF interacts with the co-chaperone DnaJ-1. (A) Western blots showing MLF and MLF-V5 expression in Kc167 cells stably transfected with the copper-inducible pMT-MLF-V5 expression vector and treated or not with 50 μm CuSO4 for 24h. Tubulin (Tub) was used as an internal loading control. (B) Proteins identified by mass spectrometry from CuSO4-induced Kc167-pMT-MLF-V5 cells using anti-V5 antibody coupled to Sepharose (IP1) or magnetic (IP2) beads for purification. The number of quantified peptides (#Qpep), sequence coverage and fold enrichment in comparison to control (parental Kc167 cells) are indicated for each experiment. Spe IP: not detected in control condition. (C, D) Western blots showing the results of immunoprecipitation experiments against GFP (C) or HA (D) performed in Kc167 cells transfected with expression vectors for the indicated proteins. (E, F) Autoradiograms showing the results of pull down assays between in vitro translated 35 S methionine labeled MLF (E) or DnaJ-1 (F) and the indicated GST fusion proteins produced in E. coli. (G, H) Schematic representation of DnaJ-1 (G) and MLF protein domains (H) and western blots showing the results of immunoprecipitation experiments against GFP performed in Kc167 cells transfected with expression vectors for GFP-MLF and various HA-DnaJ-1 mutants (G) or GFP-DnaJ-1 and different HA-MLF mutants (H). Conserved domains are highlighted in grey. J: J domain. G/F: glycine/phenylalanine-rich region. C-ter: C-terminal domain. MHD: MLF homology domain.

FIG 5 .

 5 FIG 5. DnaJ-1 and MLF interfere with LZ activity and act together to control LZ + blood cell development Relative lz>GFP + blood cell number (A, C) and size (B, D) in lz-GAL4, UAS-mCD8GFP/+ third instar larvae of the indicated genotypes. *: p-value<0.05, ***: p-value<0.001.

Fig 9 .

 9 Fig 9. LZ represses Notch expression. (A-D) Immunostainings against NECD (Notch extracellular domain) in blood cells from lz-GAL4, UAS-mCD8GFP/+ (A), lz-GAL4, UAS-mCD8GFP/Y (B), lz-GAL4, UAS-mCD8GFP/Y; UAS-lz (C) and lz-GAL4, UAS-mCD8GFP/+; UAS-lz (D) third instar larvae. The expression of Notch protein only is shown in the lower panels. Nuclei were stained with Topro3. (E) Corresponding quantification of NECD level in lz>GFP + blood cells. (F-F''') Immunostaining against LZ in circulating blood cells from Notch GMR30A01-GAL4, UAS-GFPnls third instar larvae. Nuclei were stained with Topro3. (F'-F"'): single channel images. (G-J) Notch GMR30A01-GAL4-driven expression of GFP in circulating blood cells from larvae of the indicated genotypes. (K) Corresponding quantification of GFP expression level. (A-D, F-J) Scale bar: 10μm. (E, K) *: p-value<0.05, *** p-value<0.001.

Figure 1 .Figure 22 .

 122 Figure 22. MLF or DnaJ-1 forms a dimer (A, B) Autoradiograms of pull-down assays between in vitro translated 35 S-methionine labeled MLF (A) or DnaJ-1 (B) and the indicated GST fusion proteins produced in E. coli.

Figure 24 . 2 . 6 .Figure 25 .

 242625 Figure 23. Hsc70-5 interacts with MLF and DnaJ-1 Western blotting results of immunoprecipitation experiments against GFP performed in Kc167 cells co-transfected with GFP or Hsc70-5-GFP and 3HA-MLF (A) or 3HA-DnaJ-1 (B) expression plasmids, as indicated in the upper part of each panel.

Figure 26 .

 26 Figure 26. High levels of DnaJ-1 do not rescue Lozenge stability or activity when MLF is knocked down (A) Western blotting results of LZ-V5, DnaJ-1, and Tubulin (Tub) expression in Kc167 cells treated with the indicated dsRNA. (B) Luciferase assays in Kc167 cells treated with the indicated dsRNA and transfected with 4xPPO2-Fluc reporter plasmid in the presence or not of pAc-LZ-V5 expression plasmid with or without DnaJ-1 expression. pAc-Rluc was used as an internal normalization control.

  cells or Kc167 cells over-expressing MLF), the absence or the knockdown of DnaJ-1 does not result in a decrease in Lozenge level, in contrast to what we observed in the presence of low level of MLF (naive Kc167 cells or LZ + larval blood cells with MLF knockdown) (Figure2and 4 in the manuscript). It is possible that MLF and DnaJ-1 somehow compete for binding to Lozenge or, alternatively, that the association of MLF with DnaJ-1 changes the fate of Lozenge protein. MLF could act first to stabilize Lozenge (prevent its degradation), and then the MLF/DnaJ-1 complex could favor proper Lozenge folding and thus its activity. In view of our results, we propose that in the absence of MLF, Lozenge is degraded whereas in the absence of DnaJ-1, Lozenge is not properly folded and either it is degraded if there is a low level of MLF or it accumulates in a unfolded/inactive state if there is enough MLF to prevent its degradation (Figure29).Our preliminary results indicate that an important fraction of Lozenge is insolublewhen it is expressed in Kc167 cells (data not shown). This suggests that Lozenge is prone to form aggregates and that MLF or DnaJ-1, which has been involved in the regulation of polyQ aggregates, might participate in the solubilization of Lozenge. It would be interesting to further test this hypothesis by systemically assessing the effects of MLF, DnaJ-1 and Hsc70-4 knockdown or over-expression on the soluble and insoluble fraction of Lozenge as well as by testing in vitro whether these factors modify the solubility of purified recombinant Lozenge protein.

Figure 29 .

 29 Figure 29. Model for regulation of Lozenge protein fate by MLF and DnaJ-1

Table 1 .

 1 WHO classification of AML and related neoplasms

Table 2 .

 2 Synonyms for mammalian RUNX gene subtypes and locus

xviii 1.

A general description of hematopoiesis and leukemia 1.1. Hematopoiesis in mammals 1.1.1

Table 2 . Synonyms for mammalian RUNX gene subtypes and locus

 2

(CBFA: Core Binding Factor Alpha; AML: Acute Myeloid Leukemia; PEBP2: Polyomavirus Enhancer Binding Protein 2)

  3 Kc167 cells were dispensed) in 1ml of PBS in 24-well-plate containing a glass coverslip. Unless mentioned otherwise,

	only female larvae were used. The hemocytes were centrifuged for 2 min at 900g, fixed for
	20 min with 4% paraformaldehyde in PBS and washed twice in PBS. For immunostainings:
	cells were permeabilized in PBS-0.3% Triton (PBST) and blocked in PBST-1% Bovine
	Serum Albumin (BSA). The cells were incubated with primary antibodies at 4°C over-night
	in PBST-BSA, washed in PBST, incubated for 2h 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 Topro3
	(ThermoFisher). The following antibodies were used: anti-LZ, anti-Notch intracellular
	domain, anti-Notch extracellular domain (Developmental Studies Hybridoma Bank,
	DSHB), anti-MLF

Acknowledgements Acknowledgments

We thank the Toulouse RIO imaging platform for assistance with confocal microscopy and FACS analysis and S. Bernat-Fabre for technical assistance with in vitro pull down assays.

We are grateful to A. Plessis, M. Yamaguchi, E. Ling, the Bloomington and NIG Drosophila stock centers as well as the DSHB for providing reagents and fly stocks. We thank members of our team and P. Genevaux for discussions. This work was supported by grants from the Agence Nationale de la Recherche, Fondation ARC, Ligue Midi Pyrénée contre le Cancer and Fédération de Recherche en Biologie de Toulouse to LW and in part by grants from the Région Midi-Pyrénées, Fonds Européen de Développement Régional (FEDER), Toulouse Métropole, and the French Ministry of Research for the Programme Investissement d'Avenir Infrastructures Nationales en Biologie et Santé (PIA, ANR 10-INBS-08, French Proteomics Infrastructure, ProFI) to OBS. MM was supported by fellowships from Université Paul Sabatier and Fondation pour la Recherche Médicale. AC was supported by a fellowship from the China Scholarship Council (CSC).

List of abbreviations

AGM: Aorta-Gonad-Mesonephros AIF: Apoptosis-Inducing Factor

Chapter I Introduction

renature the denatured luciferase in vitro [START_REF] Fink | Chaperone-mediated protein folding[END_REF]. In addition, some DnaJ proteins could be direct or indirect regulators of the Hsp90 chaperones [START_REF] Sterrenberg | Human DNAJ in cancer and stem cells[END_REF]. The open state has fast on and off rates for substrate peptide. Hsp40 stimulates ATP hydrolysis on Hsp70, resulting in the closing of the SBD α-helical lid over the bound substrate peptide. The closed state has slow on and off rates for substrate peptide. NEFs stimulate the release of ADP from the NBD, and ATP binding causes substrate release (From Kim et al., 2013).

Chapter II Results

During my PhD, I focused my work on the characterization of MLF mode of action in relation with the RUNX transcription factor stability and hematopoiesis. To gain insights into the possible mechanism of action of MLF proteins, we carried out a proteomic approach to identify Drosophila MLF partners in the Drosophila Kc167 blood cell line. This allowed us to identify the small chaperone DnaJ-1 as a partner of MLF. We then proceeded with the characterization of Drosophila MLF/DnaJ-1 interactions and the functional analysis of DnaJ-1 in cell culture and in vivo, as described thereafter.

Author Summary

A tight regulation of protein expression level is required for proper development. Notably, the abnormal expression of key transcription factors or signaling pathway components controlling blood cell development contributes to hematological diseases, such as leukemia.

In this report, we used Drosophila as a model to study the function and mode of action of a family of conserved but poorly characterized proteins implicated in leukemia called myeloid leukemia factors (MLF). By combining proteomic, transcriptomic and genetic approaches, we show that MLF acts in concert with an Hsp40 co-chaperone to control the level and activity of a RUNX transcription factor and therefore RUNX + blood cell number and differentiation. Furthermore, we show that RUNX dosage directly impinges on the activity of Notch signaling pathway, which is critical for RUNX + cell survival and differentiation, by regulating the transcription of the Notch receptor. These findings shed light on a new mode of regulation of RUNX level and Notch activity to prevent abnormal blood cell accumulation, which could be involved in leukemogenesis.

Supplementary results (not present in the article)

Endogenous MLF interacts with DnaJ-1

Our results show that MLF and DnaJ-1 interact with each other specifically in coimmunoprecipitation experiments following co-transfection of epitope-tagged version of these two proteins as well as in in vitro GST pull-down assays, suggesting that the interaction between them is direct. In addition, we found that 3HA-DnaJ-1 co-precipitated GFP-MLF as well as the endogenous MLF (see above; Figure 1.D of the submitted manuscript). To strengthen our conclusions, we asked whether the endogenous MLF could also co-precipitate 3HA-DnaJ-1 in Kc167 cells. So we performed a co-immunoprecipitation experiment with extracts of Kc167 cells transfected with pAc-3HA-DnaJ-1 expression plasmid by using rabbit anti-MLF antibody to precipitate the endogenous MLF and rabbit anti-GFP antibody as a negative control. Western blot analysis showed that MLF antibody precipitated the endogenous MLF and co-precipitated 3HA-DnaJ-1, while GFP antibody did not precipitate either protein (Figure 21), which further confirmed the interaction between MLF and DnaJ-1. Of note, since we do not have the antibody against DnaJ-1, we could not test the interaction between both endogenous DnaJ-1 and MLF.

MLF or DnaJ-1 can form a dimer

DnaJ-1 is a member of the DnaJB/class II subfamily of Hsp40/DnaJ proteins, which contains an N-terminal J-domain required for the stimulation of Hsp70 ATPase activity, a MLF and Hsc70-4 co-precipitated RUNX1. However, we did not observe a coimmunoprecipiation of RUNX1 with GFP-DnaJ-1 (Figure 27.A). Then we asked whether these proteins are required for the stable expression of RUNX1 in Kc167 cells.

Interestingly, the knockdown of MLF, or DnaJ-1 or Hsc70-4 caused a decrease in the level of RUNX1 (Figure 27.B). Thus, even though we could not detect yet a physical interaction between DnaJ-1 and RUNX1 in our assays, these data indicate that RUNX1 stability can be regulated by MLF, DnaJ-1 and Hsc70-4 from Drosophila, and we propose that RUNX1 stability might also be controlled by the homologs of these proteins in human. These data also suggest that the runt homology domain, which is conserved between Lozenge and RUNX1, is an important determinant of the regulation by the MLF/DnaJ/Hsc70 chaperone machinery. In line with this hypothesis, co-immunoprecipitation results showed that Lozenge RHD is sufficient for the interaction between Lozenge and MLF as MLF coprecipitated the runt domain, but DnaJ-1 didn't (Figure 28). Thus, other RUNX proteins might also be regulated by these factors.