AML-Associated Mutations of the Transcription Factor C/Ebpα: Studies in a Neutrophil Differentiation Model

AML-associated mutations of the transcription factor C/EBPα: studies in a neutrophil differentiation model

Inaugural-Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)

an der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau

vorgelegt von

Isabel Mölter geboren in Pforzheim

Freiburg im Breisgau Juli 2017

Die Untersuchungen zur vorliegenden Arbeit wurden von August 2012 bis Juli 2017 am Institut für Medizinische Mikrobiologie und Hygiene des Universitätsklinikums der Albert-Ludwigs-Universität Freiburg unter der Leitung von Prof. Dr. Georg Häcker durchgeführt.

Dekan der Fakultät für Biologie: Prof. Dr. Bettina Warscheid

Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner

Betreuer der Arbeit: Prof. Dr. Georg Häcker

Referent: Prof. Dr. Georg Häcker

Koreferentin: Prof. Dr. Heike Pahl

Drittprüfer: Prof. Dr. Tilman Brummer

Datum der mündlichen Prüfung: 27.09.2017

I. Table of contents

I. Summary ...... 8

II. Zusammenfassung...... 9

III. Introduction ...... 11

Neutrophil granulocytes ...... 11

C/EBPα, the master transcription factor in granulopoiesis ...... 14

Lymphoid-enhancer binding factor 1 (Lef-1) and severe congenital neutropenia .. 18

Neutrophil granulocytes in inflammation ...... 18

Granules ...... 20

NETs ...... 22

Neutrophil cell death ...... 23

Acute myeloid leukaemia (AML) ...... 24

The Hoxb8 system ...... 27

IV. Objectives ...... 29

V. Results ...... 30

Generation of Hoxb8 neutrophils ...... 30

Conditional C/EBPα-/- ER-Hoxb8 neutrophils do not differentiate but are lost over time ...... 31

Lef-1-/- Hoxb8 neutrophils have heterogeneous phenotypes ...... 32

Retroviral overexpression of C/EBPα mutants in ER-Hoxb8 neutrophils as a model to study their implication in AML ...... 35

Proliferation of Hoxb8 neutrophils expressing AML-associated C/EBPα mutations is not significantly altered ...... 38

Proliferation in suspension ...... 38

Proliferation in semi-solid media: methylcellulose colony assay ...... 40

ER-Hoxb8 neutrophils expressing N- and C-terminal mutations of C/EBPα display differentiation defects ...... 41

Effector functions of Hoxb8 neutrophils expressing C-terminal mutations of C/EBPα are reduced ...... 48

Expression of the granule protein neutrophil elastase is reduced in C/EBPα K313 and C/EBPα BRM2 expressing ER-Hoxb8 neutrophils ...... 48

Secretion of the pro-inflammatory cytokines TNF and IL-6 by C/EBPα K313 expressing Hoxb8 neutrophils ...... 49

C/EBPα K313 retains transactivation capacity: luciferase reporter assay ...... 50

Cell death of Hoxb8 neutrophils ...... 53

C/EBPα expression on mRNA and protein levels ...... 55

C/EBPα mRNA-levels are slightly higher in wt C/EBPα expressing Hoxb8 neutrophils than in C/EBPα K313 expressing cells ...... 56

C/EBPα protein levels of C/EBPα K313/BRM2 expressing ER-Hoxb8 cells are strongly increased ...... 56

Protein stability of C/EBPα K313 is not significantly increased compared to wt C/EBPα ...... 58

C/EBPα K313 protein levels are decreased in HEK293FT cells upon transient transfection ...... 61

In vivo differentiation of ER-Hoxb8 neutrophils expressing C/EBPα wt/K313 ...... 61

Upon adoptive transfer, Hoxb8 neutrophils expressing C/EBPα K313 are detectable for longer times in the bone marrow ...... 62

Hoxb8 neutrophils expressing C/EBPα K313 showing delayed maturation are able to differentiate in vivo ...... 65

C/EBPα protein levels are increased in AML patient samples ...... 67

VI. Discussion ...... 68

Regulation of C/EBPα expression ...... 68

C/EBPα and its influence on proliferation of Hoxb8 neutrophil progenitor cells ..... 69

Differentiation of ER-Hoxb8 cells is impaired upon expression of N- or C-terminally mutated C/EBPα...... 71

Hoxb8 neutrophil effector functions are altered dependent on different C/EBPα mutations ...... 75

Cell death of Hoxb8 neutrophils ...... 78

Conclusion...... 78

VII. Material and Methods ...... 80

1. Material ...... 80

1.1. Mice...... 80

1.2. Cell lines ...... 80

1.3. Culture media and amendments ...... 81

1.4. Viral Constructs ...... 82

1.5. Inhibitors, cytokines and other reagents ...... 84

1.6. Blocking solutions and antibodies ...... 85

1.7. Buffers and solutions ...... 87

1.8. Electronic Devices ...... 91

2. Methods ...... 92

2.1. Cell Culture ...... 92

2.2. Generation of Hoxb8 neutrophil progenitor cells ...... 92

2.3. Retrovirus production and transduction of Hoxb8 neutrophil progenitor cell lines ...... 93

2.4. Transfection of HEK293FT cells ...... 94

2.5. Proliferation of Hoxb8 neutrophil progenitors in liquid culture ...... 94

2.6. Proliferation of Hoxb8 neutrophil progenitors in semi-solid medium ...... 94

2.7. In vitro differentiation of Hoxb8 neutrophil progenitor cells ...... 94

2.8. FACS staining of cell surface markers ...... 95

2.9. Cell death staining ...... 95

2.10. Giemsa staining ...... 95

2.11. Lysate preparation, SDS-Page and Western Blotting ...... 95

2.12. Inhibition of translation using cycloheximide ...... 96

2.13. RNA extraction and cDNA synthesis ...... 96

2.14. Quantitative real-time PCR (qRT-PCR)...... 97

2.15. Luciferase reporter assay...... 98

2.16. Determination of IL-6 and TNF levels by ELISA ...... 98

2.17. Adoptive transfer of Hoxb8 neutrophil progenitor and BM cells into mice 99

VIII. References ...... 100

IX. Appendix ...... 114

Index of figures ...... 114

Index of tables ...... 115

Abbreviations...... 116

X. Acknowledgements ...... 121

Summary

I. Summary0B The study of neutrophil granulocytes is difficult as they have a very short life-span and are not easy to manipulate genetically. This pertains to the study of normal neutrophil development as well as of malignant granulopoiesis. Here we used the Hoxb8 system to obtain large numbers of neutrophils that can be kept in a progenitor state as long as β-estradiol is added to the culture. This enabled us to grow neutrophil precursor cells in unlimited quantities and to genetically engineer them to express the transcription factor CCAAT/enhancer binding protein α (C/EBPα), which is crucial for proliferation, differentiation and survival of neutrophil granulocytes. The C/EBPα gene is mutated in 9-11 % of cases of acute myeloid leukaemia (AML) but the molecular function of such mutations has been unclear. We expressed typical N- and C-terminal C/EBPα mutations found in AML patients (K313 and BRM2 are C-terminal mutations; N-terminal mutations typically result in the expression of a p30 form of C/EBPα). We did not observe increased proliferation rates of Hoxb8 neutrophil progenitor cells. The most striking effect of AML-associated C/EBPα mutations was the inability of C/EBPα K313 expressing neutrophils to differentiate in vitro. The same defect was observed upon expression of p30 C/EBPα. Although C/EBPα K313 expressing neutrophils displayed higher levels of anti-apoptotic proteins (Mcl-1, Bcl-2) and lower levels of the pro-apoptotic protein Bim, they did not show increased survival compared to control cells. Despite a clear differentiation defect, the ability to secrete cytokines (IL-6, TNF) and activate a reporter gene was increased in C/EBPα K313 Hoxb8 cells. Those observations are linked to strongly increased protein levels of C-terminally mutated C/EBPα, as cells expressing C/EBPα K313 or C/EBPα BRM2 showed much higher C/EBPα levels than cells expressing wt protein. This higher expression of C/EBPα K313 was confirmed in samples from AML patients. Elevated protein amounts were not due to increased mRNA levels or protein stability but probably result from increased translation. Interestingly, C/EBPα K313 expressing neutrophils showed delayed but still possible maturation in a mouse model. Which factors are responsible to overcome the maturation defect in vivo and how exactly translation is increased upon K313 mutation of C/EBPα needs to be elaborated in future studies.

Zusammenfassung

II. Zusammenfassung1B Das Untersuchen neutrophiler Granulozyten ist schwierig, da sie lediglich über eine sehr kurze Lebensdauer verfügen und zudem genetisch schwer zu manipulieren sind. Dies betrifft sowohl die Analyse der normalen Neutrophilenentwicklung sowie der malignen Granulopoese. Hier haben wir das Hoxb8 System verwendet, um große Mengen an Neutrophilen zu gewinnen, die in einem Vorläuferstadium gehalten werden können, solange β-Östradiol im Kulturmedium vorhanden ist. Dies ermöglichte es uns, neutrophile Vorläuferzellen in unbegrenzter Menge zu kultivieren und sie genetisch zu manipulieren, sodass sie den Transkriptionsfaktor CCAAT/Enhancer-bindendes Protein α (C/EBPα) exprimieren, welcher essentiell für Proliferation, Differenzierung und Überleben von neutrophilen Granulozyten ist. Das C/EBPα-Gen ist bei 9-11 % der Fälle von akuter myeloischer Leukämie (AML) mutiert, aber die molekulare Funktion solcher Mutationen war unklar. Wir haben typische N- und C-terminale C/EBPα Mutationen (K313 und BRM2 sind C-terminale Mutationen, N-terminale Mutationen resultieren typischerweise in der Expression einer p30 Isoform von C/EBPα), die bei AML-Patienten entdeckt wurden, exprimiert. Wir konnten keine erhöhten Proliferationsraten von Hoxb8 neutrophilen Vorläuferzellen beobachten. Der auffälligste Effekt AML-assoziierter C/EBPα Mutationen war das Unvermögen von C/EBPα K313 exprimierenden Neutrophilen in vitro zu differenzieren. Der gleiche Defekt konnte bei Expression von p30 C/EBPα beobachtet werden. Obwohl C/EBPα K313 exprimierende Neutrophile größere Mengen an anti-apoptotischen Proteinen (Mcl-1, Bcl-2) und geringere Mengen des pro-apoptotischen Proteins Bim aufwiesen, zeigten sie keine gesteigerte Überlebensfähigkeit verglichen mit Kontrollzellen. Trotz eines eindeutigen Differenzierungsdefektes war die Fähigkeit Zytokine (IL-6, TNF) zu sezernieren und ein Reportergen zu aktivieren in C/EBPα K313 Hoxb8 Zellen erhöht. Diese Beobachtungen sind mit stark erhöhten Proteinmengen von carboxyterminal mutiertem C/EBPα verbunden, da Zellen mit Expression von K313- als auch BRM2- Mutation viel höhere C/EBPα-Spiegel aufwiesen als Zellen, die wildtypisches Protein exprimierten. Eine erhöhte C/EBPα K313 Proteinmenge konnte mithilfe von Proben von AML-Patienten bestätigt werden. Erhöhte Proteinspiegel waren nicht auf gesteigerte mRNA-Menge oder Proteinstabilität zurückzuführen, sondern resultieren womöglich aus verstärkter Translation. Interessanterweise zeigten C/EBPα K313

Zusammenfassung exprimierende Neutrophile eine verzögerte, aber dennoch mögliche Reifung in einem Mausmodell. Welche Faktoren verantwortlich dafür sind, den Reifungsdefekt in vivo zu überwinden und wie genau die Translation bei Mutation von C/EBPα K313 erhöht ist, bleibt in weiteren Studien zu untersuchen.

Introduction

III. Introduction2B

Neutrophil10B granulocytes Neutrophil granulocytes are the most abundant of all leukocyte cell types in human peripheral blood. Neutrophils are also called polymorphonuclear leukocytes (PMNs) because of their variously shaped nuclei. Normally, neutrophils and their progenitor cells comprise around 60 % of all nucleated cells in human bone marrow and blood. The neutrophil’s best known function is the surveillance of tissues for invading microorganisms. Circulating mature neutrophils in the blood have a half-life of 6-8 hours (Amulic et al., 2012) and die by apoptosis if they do not receive stimuli from the host or pathogens . Neutrophils are “programmed to die”, their life span can only be extended if they encounter inflammatory mediators of pathogens (for instance lipopolysaccharide, LPS) or the host (like TNF, GM-CSF, IL-3)(Akgul et al., 2001; Luo and Loison, 2008). During infections, large numbers of neutrophils can be recruited to the site of infection and during systemic infections increased numbers of (partly immature) neutrophils are released from the bone marrow. Upon encountering stimuli from pathogens or macrophages, neutrophils are able to use a wide repertoire of antimicrobial effector functions and are considered the first cellular defense against invading pathogens.

Granulocytes originate from hematopoietic stem cells (HSCs) in the bone marrow. These HSCs possess the ability of self-renewal and multi-potentiality and give rise to multipotent progenitors (MPP), which exhibit a more narrow differentiation potential. MPPs are highly proliferative cells expressing receptors for specific growth and survival factors (colony-stimulating factors, CSFs) and will give rise to the lymphoid, erythroid, megakaryocytic and myeloid compartments of the hematopoietic system.

The common myeloid progenitor (CMP) gives rise to all different types of myeloid cells, while the granulocyte-monocyte progenitor (GMP) has a limited potential to differentiate into granulocytes and macrophages. The GMP gives rise to the eosinophil lineage-committed progenitor (EoP) (Iwasaki et al., 2005) and the basophil/mast cell progenitor (BMCP), which in turn gives rise to the mast cell progenitor (MCP) and the basophil progenitor (BaP) (Arinobu et al., 2005). Regarding neutrophils, no committed progenitor has as yet been described.

Introduction

Figure 1: Hematopoietic progenitors

This scheme of hematopoietic progenitors is based on results generated by isolation and characterization of different mouse progenitor cells. HSC, hematopoietic stem cell; MPP, multipotent progenitor; LMPP, lymphoid-primed multipotent progenitor; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte-erythrocyte progenitor; GMP, granulocyte-macrophage progenitor; MDP, monocyte-dendritic cell progenitor; EoP, eosinophil progenitor; BMCP, basophil-mast cell progenitor; MCP, mast cell progenitor; BaP, basophil progenitor. Figure from (Fiedler and Brunner, 2012)

Differentiation from the HSC into lineage-committed and finally mature effector cells involves activation of lineage-specific genes as well as repression of lineage-foreign genes in a specified order. This is mediated by “master” transcription factors. For example, expression of GATA-1 will lead to development towards the erythroid, megakaryocyte, mast and eosinophil lineage (Bresnick et al., 2010; Morceau et al., 2004), while expression of PU.1 will favour monocyte and B-lymphoid development (Gangenahalli et al., 2005; Sharrocks, 2001). High levels of PU.1 will lead to myeloid lineage restriction and upregulation of C/EBPα, which is needed for the transition from CMP to GMP. Lineage choice between monocytes and granulocytes depends

Introduction on the expression levels of PU.1 and C/EBPα (Dahl et al., 2003; Reddy et al., 2002). High levels of PU.1 will lead to monocytic differentiation, whereas strong expression of C/EBPα will lead to granulopoiesis (Radomska et al., 1998; Zhang et al., 1997). In absence of C/EBPα, C/EBPβ may rescue granulopoiesis if IL-3 and GM-CSF (granulocyte-monocyte colony stimulating factor) are present (Hirai et al., 2006).

Terminal granulopoiesis starts with the switch from proliferation to differentiation at the myeloblast and promyelocyte stage. At the promyelocyte stage, the formation of primary or azurophilic granules begins. The most important transcription factors at this stage are C/EBPα and Gfi-1 (growth factor independent-1), which suppress monocyte development and proliferation but are also necessary for transcriptional activation of granulocyte-specific genes like neutrophil elastase, myeloperoxidase and other C/EBP proteins (Borregaard, 2010; Theilgaard-Monch et al., 2005). Ongoing differentiation will lead to myelocytes and metamyelocytes, which can be defined by beginning nuclear segmentation and the formation of secondary, specific granules. These granules contain high amounts of lactoferrin, cathelicidin and collagenase. In the matrix, they exhibit lysozyme and leukolysin (Borregaard and Cowland, 1997). Expression of these secondary granule proteins is regulated by C/EBPε (Bjerregaard et al., 2003), which is also necessary for the formation of tertiary granule proteins (Verbeek et al., 1999; Yamanaka et al., 1997) and cell cycle exit (Gery et al., 2004). The final step of granulopoiesis is the differentiation into band cells and segmented neutrophils. Their name is based on the segmented shape of their nuclei and they contain tertiary as well as secretory granules. Tertiary granules contain high amounts of gelatinase, lysozyme and leukolysin, while secretory granules hold plasma proteins in their matrix.

Introduction

Figure 2: Granulopoiesis

During granulopoiesis, a series of morphologically distinct cells arises from immature myeloblasts. They can be identified by their granule content and characteristic nuclear shapes. The figure shows their occurrence among the granulocytic compartment as percent of total nucleated BM cells as well as the expression pattern of C/EBP proteins. Figure from (Nerlov, 2004).

In the course of terminal differentiation, expression of C/EBPα decreases successively during the myeloblast stage. The myelocyte and metamyelocyte stages are mainly characterised by C/EBPε expression, while expression of the transcription factors C/EBPβ, C/EBPδ, C/EBPγ, C/EBPζ and PU.1 increases constantly during development to the metamyelocyte stage (Bjerregaard et al., 2003). The formation of homo- and heterodimers of the C/EBP proteins and their regulation by phosphorylation (Chumakov et al., 2007) enables the highly selective expression of granule proteins during terminal differentiation.

C/EBP11B α, the master transcription factor in granulopoiesis The intronless C/EBPα gene encodes a transcription factor essential for controlling proliferation and differentiation of myeloid progenitor cells. It belongs to the family of basic leucine zipper proteins (bzip) able to form homo- and heterodimers with other C/EBP proteins to activate transcription of target genes. It is highly expressed in

Introduction granulocytes, hepatocytes and adipocytes and to a smaller extent also in monocytes, eosinophils (Scott et al., 1992) and type II pneumocytes (Birkenmeier et al., 1989). A number of reports indicate that C/EBPα is an important factor regulating the balance between cell proliferation and differentiation. Like many transcription factors, C/EBP proteins are built in a modular way, consisting of an activation domain, a DNA- binding basic region and a leucine-rich dimerization domain (Figure 3). This dimerization domain consists of a periodic repetition of leucine residues at every seventh position interdigitating with repeats of the dimerization partner (Landschulz et al., 1988) and is also called leucine-zipper. Together the dimerization partners form a coiled coil of α-helices in parallel orientation. Dimerization of C/EBP proteins is a prerequisite to DNA binding (Landschulz et al., 1989).

The basic region (approximately 20 amino acids) mediates DNA binding specificity (Johnson, 1993), in this case it recognizes the CCAAT motif. As soon as C/EBPα is bound to DNA, it activates transcription of target genes via its two N-terminal activation domains (Friedman and McKnight, 1990). Even without DNA binding, C/EBPα is able to affect gene expression. Interaction of the non-DNA binding residues of the BR with E2F1 reduces the transcription rate of c-Myc (Johansen et al., 2001; Porse et al., 2001), and C/EBPα may displace HDAC1/3 from chromatin- bound NF-κB p50 in order to activate gene transcription of Bcl-2, Flip or Nfkb1 (Paz- Priel et al., 2005; Paz-Priel et al., 2009; Paz-Priel et al., 2011).

Figure 3: Scheme illustrating C/EBPα functional domains

Two different translational products can be made from C/EBPα mRNA by using different start codons within the same open reading frame. The short isoform, known as p30, lacks the N terminal 117 amino acids but contains the same carboxyl terminus as the long (p42) isoform. The basic region leucine zipper (BR LZ) has been shown to mediate protein-protein interactions with other transcription factors (like GATA 1, PU.1, RUNX1 and E2F) and DNA- binding. Interactions with the transcriptional machinery are mediated by transactivation elements (TE I, TE II and TE III). p30 C/EBPα lacks transactivation element TE I and TE II. The K313 mutation carries a duplication of lysine at position 313 in humans, a conserved

Introduction region between basic region and leucine zipper. The BRM2 mutation carries two amino acid substitutions in the basic region. Adapted from (Nerlov, 2004).

The C/EBPα mRNA contains two start codons giving rise to two different translation products: a short 30 kDa and a long 42 kDa version of C/EBPα. The shorter form, also known as p30, comprises the same carboxyl terminus as the full-length form, also known as p42, but lacks the amino-terminal part (117 amino acids)(Lin et al., 1993). Therefore, p30 is missing two transactivation elements and hence the ability to block cell proliferation and to induce differentiation of granulocytes and adipocytes (Calkhoven et al., 2000). Nevertheless, it retains the ability to bind DNA and hetero- dimerize with p42 C/EBPα, thereby dominantly inhibiting activation of target genes by p42 C/EBPα. Additionally, p30 C/EBPα induces expression of the E2 conjugating enzyme Ubc9, which sumoylates C/EBPα lysine 161 (Geletu et al., 2007). This sumoylated C/EBPα exhibits a reduced capacity to slow down proliferation or activate transcription of target genes (Geletu et al., 2007; Sato et al., 2006).

The p30/p42 ratio in a cell is regulated via the target of rapamycin (TOR) and protein kinase R signalling pathway. Proliferation is promoted if growth factors and nutrients are abundant by increasing the activity of the translation initiation factors EIF2α/EIF4E (Rosenwald et al., 1993). High activity of these factors leads to increased p30 production at the expense of p42 because they promote the translation of the smaller open reading frame, bypassing the p42 initiation codon. Regulation of the p30/p42 provides a mechanism for the cells to respond to extracellular conditions. p30 C/EBPα molecules are still able to induce eosinophil lineage commitment, but not terminal differentiation. This had been suggested as early eosinophil markers EOS47 and C/EBPβ were present, but granules of these immature eosinophils were missing (Nerlov et al., 1998). Similarly, early adipocyte differentiation can be carried out by p30 C/EBPα, but terminal differentiation and cell-cycle arrest fail (Calkhoven et al., 2000). Commitment to the monocyte lineage is regulated by PU.1 activity, which can be affected negatively by protein-protein interactions with the BR-LZ domain of C/EBPα (Radomska et al., 1998; Reddy et al., 2002). This indicates that lineage choice is mediated by interactions with other transcription factors and remodeling of the chromatin of lineage-specific genes, which both can still be executed by p30

Introduction

C/EBPα. Terminal differentiation however requires the N-terminal transactivation domains missing in p30 C/EBPα.

In addition to its function as transcription factor, C/EBPα is also able to mediate growth control. This was originally published by McKnight and colleagues (Umek et al., 1991), who found out that C/EBPα exhibits the ability to arrest cell proliferation. Terminal differentiation and cell-cycle arrest usually are linked processes, and here C/EBPα works as a master switch between proliferating, uncommitted and cell-cycle arrested, differentiated cells.

C/EBPα exerts its function in cell-cycle arrest by repressing the E2F complex. E2F is a transcription factor complex regulating genes that are important for cell-cycle progression. For example, it activates expression of the c-myc oncogene (which for instance blocks granulocytic differentiation). Repression of E2F is mediated by the C/EBPα N-terminus (D'Alo' et al., 2003; Porse et al., 2001), in particular the transactivation element 1 (TE-I), and non-DNA binding residues of the basic region α-helix (Porse et al., 2001). E2F-repression by C/EBPα has been shown to lead to downregulation of c-MYC, which will finally lead to granulocytic differentiation (Slomiany et al., 2000). This repression seems to be the central mechanism interconnecting growth arrest and differentiation and hence its loss might be a pivotal event in leukaemogenesis. The requirement for E2F repression by C/EBPα seems to be tissue-specific (Porse et al., 2001) and it is the most active mechanism in myeloid cells. Apart from E2F repression, C/EBPα inhibits proliferation by induction of p21 and via interaction with CDK2 and CDK4 (Timchenko et al., 1996; Wang et al., 2001).

Deletion of the C/EBPα gene causes an arrest at the CMP to GMP transition, resulting in reduced formation of granulocytes and monocytes (Zhang et al., 2004). Mice homozygous for a deletion of the entire C/EBPα-coding sequence die perinatally because of a severe defect in glucose homeostasis. These mice show decreased expression of glycogen synthase, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (Zhang et al., 1997) and therefore suffer from an inability to store glucose in the form of glycogen before birth. Their ability to synthesize and export glucose from the liver is decreased after birth and they die consequently from hypoglycaemia. Also, C/EBPα-/- mice lack white adipose tissue (Wang et al., 1995) as well as mature neutrophil and eosinophil granulocytes (Zhang

Introduction et al., 1997). There is no detectable expression of the granulocyte colony-stimulating factor (G-CSF) or interleukin-6 (IL-6) receptors, while mRNA levels for both are reduced (Zhang et al., 1998) in hematopoietic cells of fetal livers from C/EBPα deficient mice.

Lymphoid12B -enhancer binding factor 1 (Lef-1) and severe congenital neutropenia Lymphoid-enhancer binding factor 1 (Lef-1), a transcription factor of the Wnt signaling pathway, has been reported to be important for granulopoiesis (Welte et al., 2006). This was shown in bone marrow cells of patients suffering from severe congenital neutropenia (SCN) who display a differentiation block at the promyelocyte stage of neutrophils. These patients exhibited reduced expression of LEF-1 and its target genes, among them C/EBPα. This was correlated with the lack of mature neutrophils in peripheral blood and recurring life-threatening infections (Welte et al., 2006). SCN is also regarded as a pre-leukaemic syndrome, as more than 20 % of patients develop acute myeloid leukaemia (AML)(Rosenberg et al., 2006). LEF-1 is expressed at all stages of myelopoiesis in healthy individuals, with a peak in expression at the promyelocyte stage (Skokowa et al., 2006). It was proposed that it directly regulates C/EBPα and that its downregulation, leading to decreased C/EBPα expression, causes the maturation block in neutrophil granulocytes (Skokowa et al., 2006). Missing C/EBPα function can also be found in AML, but is then due to mutations rendering C/EBPα proteins (at least partially) non-functional instead of their lack due to LEF-1 dysfunction in SCN (Skokowa et al., 2006).

Neutrophil13B granulocytes in inflammation If pathogens manage to surmount the physical barriers of a host, skin and mucous membranes, and infiltrate the tissue, neutrophils become activated by signals from these pathogens and resident tissue cells, often macrophages or dendritic cells. Those macrophages release cytokines upon activation that in turn activate endothelial cells and enable neutrophils to migrate from the blood stream and enter the infected tissue. This crossing of the vascular walls takes place mainly at

Introduction postcapillary venules (Finger et al., 1996; Lawrence et al., 1997). Following arrival at the site of infection, neutrophils may exert their function in fighting pathogen by several different mechanisms. They may phagocytose microorganisms and degrade them using oxidative and non-oxidative mechanisms or trap them in so-called NETs (neutrophil extracellular traps) by extruding their DNA, coated with antimicrobial substances to immobilise and lyse them. Furthermore, neutrophils are able to recruit and activate additional neutrophils, macrophages and T-cells and thereby modulate the immune response (Amulic et al., 2012).

Neutrophils are efficient phagocytes. Upon ligation of surface receptors like Fcγ receptors, C-type lectins or complement receptors, the pathogen is engulfed (Underhill and Ozinsky, 2002). This process involves membrane lipids, intracellular signalling cascades and cytoskeletal rearrangements. Once phagocytosed, microorganisms are localized in a specialised compartment called the phagolysosome. Phagocytosis of pathogens activates the NADPH oxidase system, an enzyme system consisting of multiple cytosolic and membrane-bound subunits (Jesaitis et al., 1990). In activated neutrophils, this complex is assembled in the phagolysosome membrane where it reduces molecular oxygen to form superoxide anions (Babior et al., 1973). These oxygen radicals and their reaction products are referred to as reactive oxygen species (ROS). Superoxide anions are enzymatically converted to hydrogen peroxide, which will be further converted to hypochloric acid by myeloperoxidase (Eiserich et al., 1998; Klebanoff, 1980). Hypochloric acid is a highly toxic and microbicidal agent that may cause tissue damage if released (Klebanoff, 1980). The reactive oxygen species (ROS) produced by the NADPH oxidase system are released into phagolysosomes and represent the oxidative part of the microbicidal action of neutrophils. Intracellular sequestration of microorganisms also provokes fusion of neutrophil granules with the phagolysosome and release of their content into this specialized compartment. The release of antimicrobial peptides and proteases and their action on the engulfed microorganism constitutes the non- oxidative part of the neutrophil’s microbicidal action. Defects in the NADPH oxidase apparatus abrogate the generation of superoxide anions and their derivatives, leading to a severe immunodeficiency termed chronic granulomatous disease (CGD) (Roos and de Boer, 2014).

Introduction

Granules39B Granulocytes can be characterized by two morphological features: the segmented nucleus and the intense enrichment of storage vesicles, named granules, in the cytoplasm. Neutrophils possess at least four different types of granules: azurophil (primary) granules, specific (secondary) granules, gelatinase granules (it is under debate whether they belong to the secondary granules or constitute another type, the tertiary granules) and secretory granules (Faurschou and Borregaard, 2003). These granules are formed during different stages of neutrophil maturation in the bone marrow. They can be classified according to their protein content and their ability to be exocytosed after the neutrophil has encountered an antigen. Segregation of the granule components reflects the time during neutrophil maturation when they are produced, as the components are not tagged to be directed to one granule or the other (Arnljots et al., 1998). It might also be necessary to separate them because the different active proteases might degrade or degranulate inadvertently.

The different types of granules have different thresholds for exocytosis, starting with the lowest threshold for secretory vesicles, then gelatinase granules and specific granules (Kjeldsen et al., 1992). Azurophil granules have the highest threshold and can only partially be mobilized (Faurschou et al., 2002; Sengelov et al., 1993). They are believed to contribute mainly to degradation of microorganisms inside the phagolysosome.

Primary77B granules Granules are stores of proteins that have microbicidal, but also tissue-digestive functions. They are filled passively by high amounts of protein coming from the proximal Golgi compartments. Primary or azurophilic granules are formed when neutrophil progenitor cells reach the promyelocyte stage. They are defined by a high content of myeloperoxidase (MPO), defensins, and a family of structurally related serine proteases: cathepsin G, neutrophil elastase and proteinase 3 (Pham, 2006). Knockout models have shown that neutrophil elastase helps in intracellular clearance of Gram-negative bacteria in mice although NE-deficient mice do not appear to be immunocompromised as they are not at increased risk of infections with Escherichia coli or Klebsiella pneumoniae (Belaaouaj et al., 1998; Weinrauch et al., 2002). Cathepsin G is crucial for resistance against infection with Staphylococcus aureus

Introduction

(Reeves et al., 2002). Both of them mediate protection against fungal infections (Reeves et al., 2002; Tkalcevic et al., 2000).

Apart from their intracellular activities, neutrophil serine proteases may have extracellular effects if they are released together with DNA to form NETs (see below). These NETs enable neutrophils to increase the local concentration of serine proteases and facilitate degradation of virulence factors and bacteria (Brinkmann et al., 2004).

Neutrophil serine proteases not only have beneficial, microbicidal activities, but also play a role in non-infectious, inflammatory processes. This has been shown in animal models of arthritis (Adkison et al., 2002; Hu and Pham, 2005), glomerulonephritis (Schrijver, 1989) and bullous pemphigoid (Liu et al., 2000). Initially, the role of serine proteases in inflammatory diseases was deduced from studies using beige mice, which have reduced levels of these proteases and are protected from proteinuria in a model of nephritis (Schrijver, 1989). Schrijver and colleagues concluded that serine proteases released from neutrophils degrade the extracellular matrix (ECM) and thereby cause protein leakage. Nevertheless, more recent studies suggest that the role of neutrophil serine proteases in non-infectious, inflammatory processes is more complex than only degradation of ECM components.

Most serine protease molecules remain bound to the neutrophil cell surface when azurophil granules fuse with the plasma membrane (Campbell et al., 2000; Owen et al., 1995). This results in a high local concentration of these catalytically active enzymes and might provide neutrophils with the ability to modulate the inflammatory response (Pham, 2006) by regulating chemokine activity. Proteinase 3 and cathepsin G are able to truncate the CXC-chemokine ligands 8 and 5 (CXCL8 and CXCL5) N-terminally and thereby increase their chemotactic activity towards neutrophils compared to the full-length molecules (Nufer et al., 1999; Padrines et al., 1994). This might influence the second wave of inflammatory cells recruited to the site of inflammation and provide a link between innate and adaptive immune response.

Secondary78B granules Secondary or specific granules are formed at the myelocyte and metamyelocyte stage and are secretory vesicles, destined to fuse with the plasma membrane to

Introduction empty their content (Gallin, 1984). Regarding their protein profile, they differ substantially from primary granules (Borregaard et al., 1993). Collagenase, lactoferrin and gelatinase are the main components of specific granules. Gelatinase is involved in degradation of collageneous constituents of the extracellular matrix. It cooperates with neutrophil collagenase and elastase in degrading all major components of the ECM.

Secondary granules can further be divided into three subsets based on their content of lactoferrin and gelatinase. They may either express one of them or both. This granule heterogeneity enables the differentiated release of certain granule proteins according to the stage of inflammation (Sengelov et al., 1995). In addition, it separates proteins which cannot coexist inside the same granule (Le Cabec et al., 1997).

Secretory79B granules Secretory vesicles serve as storage for membrane-bound receptors. They are formed in band cells and segmented cells by endocytosis (Borregaard et al., 1993). Secretory vesicles are rich in membrane-associated receptors like complement receptor 1 (CR1) (Sengelov et al., 1994b), receptors for formylated bacterial peptides (fMLP) (Sengelov et al., 1994a), the LPS/lipoteichoic acid-receptor CD14 and the FcγIII receptor CD16 (Detmers et al., 1995). Secretory vesicles can be mobilized by several stimuli which are important for the interaction between neutrophils and activated epithelium, for example signaling from selectins or secreted chemokines (e.g. IL-8 and fMLP).

NETs40B Even after their life has come to an end, neutrophils are able to retain their antimicrobial activity. Instead of undergoing apoptosis or necrosis, they may perform NETosis, a process in which decondensed DNA is extruded from the cell to trap pathogens in so-called NETs (neutrophil extracellular traps). This DNA is loaded with proteins from disintegrated granules, cytosol and histones from chromatin (Brinkmann et al., 2004; Brinkmann and Zychlinsky, 2007; Fuchs et al., 2007). Among those NET proteins are bactericidal proteins like defensins, elastase, proteinase 3, lactoferrin and myeloperoxidase (Urban et al., 2009), but also the pattern recognition protein Pentraxin 3 (Jaillon et al., 2007).

Introduction

The mechanism of NET formation is not completely understood. The very dense chromatin of mature neutrophils needs to decondense, a process which is helped by elastase released from azurophil granules in synergy with myeloperoxidase (Papayannopoulos et al., 2010). Neutrophils deficient in myeloperoxidase have been reported to be unable to form NETs (Metzler et al., 2011).

Finally, NETs are cleared by DNases to minimize potential negative effects on the host. For example, NETs were shown to cause thrombosis in vitro by stimulating platelets and NET components were even found in thrombi of a baboon in vivo model (Fuchs et al., 2010). They also damaged activated human endothelial cells in culture (Gupta et al., 2010) and may serve as a target for autoantibodies of systemic lupus erythematosis (SLE) patients. All these data indicate a potential proinflammatory role of NETs. The contribution of NETs to overall antimicrobial activity of neutrophils compared to the classical microbicidal activity is still unclear.

Neutrophil14B cell death Mature neutrophils have a life-span of only a few hours, they are programmed to die as they leave the bone marrow. Cell death occurs by apoptosis and prevents the release of proteolytic and cytotoxic substances from granules and protects from tissue damage. Neutrophils have been observed to undergo nuclear decondensation (Savill et al., 1989) and cell shrinkage but not to show membrane blebbing to the extent of many other cells (Kennedy and DeLeo, 2009). Apoptotic neutrophils are finally removed by phagocytosis through macrophages (Savill, 1997).

There are two major pathways mediating apoptosis: the extrinsic or death receptor (for instance via Fas and TRAIL)(Ashkenazi and Dixit, 1998) pathway and the intrinsic or mitochondrial pathway (Adams, 2003). Although they have few mitochondria and low amounts of cytochrome c, neutrophils die via the mitochondrial pathway of apoptosis (Kirschnek et al., 2011; Maianski et al., 2004; Murphy et al., 2003). This pathway is regulated by Bcl-2 (B-cell lymphoma) family proteins. It consists of pro-apoptotic Bcl-2 homology domain (BH3) only proteins that activate the pro-apoptotic effectors Bax (Bcl-2-associated X protein) and Bak (Bcl-2 homologous antagonist/killer) and anti-apoptotic Bcl-2-like proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1

Introduction and A1), which inhibit this pathway. The pro-apoptotic effectors Bax and Bak permit release of cytochrome c and other compounds from mitochondria to promote assembly of the apoptosome. The apoptosome is an enzyme complex triggering the recruitment and activation of caspases in the intrinsic pathway of apoptosis (Zou et al., 1999).

Acute15B myeloid leukaemia (AML) Acute myeloid leukaemia (AML) involves a block in differentiation concerning one or more hematopoietic lineages (Tenen, 2003). Precursor cells of granulocytic or monocytic origin accumulate in the bone marrow. Traditionally, research in the field of AML was focused on the analysis of tumour suppressor genes and oncogenes regulating proliferation or cell death. Mutations of individual transcription factors might affect both cell cycle and differentiation and provide another concept for explaining leukaemogenesis. Mutations in such transcription factors have been found in AML cells, of which C/EBPα is the most frequently studied. It is mutated in 9-11 % of AML patients, however, the mutations are almost never loss-of-function mutations. This most likely reflects the need of cancer cells to maintain C/EBPα functions that are important for lineage identity, and to dispense with those involved in promoting terminal differentiation and growth arrest (Nerlov, 2004). Very common are mutations abolishing translation of full-length C/EBPα (p42 C/EBPα), leading to increased expression of p30 C/EBPα. Because p30 C/EBPα cannot repress E2F activity, it is only able to promote granulocytic lineage commitment but not terminal differentiation. In addition to that, p30 C/EBPα is thought to have a dominant-negative effect on p42 C/EBPα, meaning that even if only one allele would be affected by this truncation mutation and the other one stayed wild-type, C/EBPα function would still be lost (Pabst et al., 2001b).

C/EBPα mutations on both alleles are common in AML. In AML patients with biallelic C/EBPα mutations, it is frequently the case that one allele carries an N-terminal C/EBPα truncation and the other a point mutation, often situated inside the basic region leucine-zipper (BR-LZ) DNA-binding domain. Most of these point mutations are clustered in the junction between the basic region and the leucine zipper and can generate frame-shifts. Because of the α-helical structure of this region, frame-shift

Introduction mutations cause misalignment of the DNA-binding residues of the basic region with the major groove of the DNA once the leucine zipper has dimerized (Nerlov, 2004). This may lead to a failure of C/EBPα molecules in binding to their cognate DNA target or even to a dominant-negative function (Asou et al., 2003). Even if the BR-LZ mutations are in-frame insertions or deletions they may still contribute to leukaemic transformation. By induction of Bcl-2 or the caspase-8-inhibitory protein (cFLIP) inhibitory protein, mutated C/EBPα proteins can inhibit apoptosis. This is dependent on interaction of their BR with NF-κB p50, which is already bound to the DNA in the target gene promotor regions (Paz-Priel et al., 2005; Paz-Priel et al., 2009). The prevalence of the combination of N- and C-terminal C/EBPα mutations indicates that the loss of C/EBPα function alone does not account for the development of AML. Instead, the modulation of C/EBPα function seems to be the driving factor in pathogenesis.

Bilallelic mutations of C/EBPα have been found in cases of AML with good prognosis, and most AML patients carrying C/EBPα mutations have a normal karyotype and a better relapse-free and overall survival than patients with mutations in other transcription factors (Pabst and Mueller, 2007).

K313 is a recurrent C/EBPα mutation in AML patients. It consists of a duplication of lysine at position 313, a highly conserved region between the DNA-binding basic region and the leucine zipper (Figure 3). Alteration of the α-helix structure at this position might preclude the sequence-specific recognition of DNA and abrogate transactivation capacity (Pabst et al., 2001b). In HSC-resembling C/EBPα K313+/+ cells, loss of quiescence and subsequent expansion have been reported (Bereshchenko et al., 2009). There are also several basic region mutations (BRM) located in the conserved BR-LZ domain. The BRM1, -2 and -3 mutations are predicted to reside on the non DNA-binding face of the basic region α helix (Porse et al., 2001) while BRM5 is located downstream of the DNA-binding residues. Although the residues affected by these mutations are not directly involved in DNA binding, they are still highly conserved in C/EBP proteins. The mutations BRM2 and BRM5 both cause impaired E2F-repression by C/EBPα and the mutant proteins have decreased ability to suppress cell growth (Porse et al., 2001). Another group reported enhanced interaction of these mutants with E2F and its associated dimerization

Introduction partner (DP), going along with reduced affinity for DNA (Zaragoza et al., 2010). They also observed preservation of transactivation potential in BRM2 overexpressing MEF and 293T cells. The BRM2 mutation is interesting as it resembles frequently observed C/EBPα mutations in human AML patients (Nerlov, 2004; Pabst et al., 2001b; Porse et al., 2005).

AML occurs predominantly spontaneously and is only rarely associated with inherited disorders such as syndromes of trisomy 21 or defective DNA repair. A number of studies have indicated that the proliferation rate of the leukaemic stem cell in AML is not necessarily increased compared to that of normal HSCs and myeloid blasts (Guan and Hogge, 2000; Jordan, 2002). Nevertheless, it is likely that cell-cycle control abnormalities occur in leukaemic cells.

Apart from genomic mutations, alterations in C/EBPα function can be acquired by transcriptional suppression through leukemic fusion proteins (like RUNX1-ETO or FLT3-ITD). RUNX1-ETO is thought to inhibit C/EBPα autoregulation and reduce C/EBPα mRNA levels (Pabst et al., 2001a). In vitro data indicate that C/EBPα mRNA production can also be suppressed by FLT3-ITD signaling in 32Dcl3 cells (Bullinger et al., 2004; Valk et al., 2004; Zheng et al., 2004).

Post-translational modification of C/EBPα can be achieved by phosphorylation at serine 21, which is mediated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) (Radomska et al., 2006; Zheng et al., 2004). Phosphorylation at this position causes a conformational change, moving the transactivation domains of two C/EBPα molecules within a dimer further apart. This can be blocked by activation of FLT3 and might explain the differentiation block of blasts with activated FLT3 in human AML (Radomska et al., 2006). FLT3-ITD, an internal tandem duplication of the Flt3 gene, leads to an activated receptor tyrosine kinase mutant and is found in 30 % of AML cases.

Further C/EBPα phosphorylation sites have been reported in in vitro studies (Behre et al., 2002; Liu et al., 2006; Wang and Timchenko, 2005), but it is unclear whether they are directly involved in subsets of myeloid leukaemias. Other post-translational mechanisms of C/EBPα activity modulation in leukaemia need to be investigated, like alteration of the composition of heterodimers with other C/EBP family members. In

Introduction

2006, the Tribbles homolog 2 (Trib2) was shown to inactivate C/EBPα and cause AML by changing the 42/30 kDa C/EBPα isoform ratio in favour of the oncogenic 30 kDa isoform (Keeshan et al., 2006). In addition, Trib2 was shown to block the 42 kDa C/EBPα wild-type protein by physically interacting with it, resulting in its proteasome-dependent degradation.

AML is, at the molecular level, a very heterogenous type of cancer (Ley et al., 2013). New subgroups have been identified due to the observation of recurring mutations in genes encoding transcription factors in AML patients. Linking of these mutations with a prognostic outcome of AML patients might permit a more risk-adapted therapeutic approach. For example, cytogenetically normal patients carrying C/EBPα mutations are no longer candidates for allogenous transplantation as they have a good prognosis in first complete remission.

The16B Hoxb8 system The availability of large numbers of neutrophils and their progenitors is important to study terminal granulopoiesis as well as myeloid leukaemogenesis. When using primary cells, costly breeding of mice and often time-consuming purification of cells is necessary. Additionally, these cells are not easy to manipulate genetically. Also, differentiation of primary cells in vitro will give rise to a heterogeneous population of cells (Gupta et al., 2014).

The study of tumour-derived cell lines can be problematic as they might have undergone genetic alterations and lost the potential to differentiate or to undergo cell death (Goodspeed et al., 2016). Human leukemic cell lines like NB-4 and HL-60 are able to undergo myeloid development upon induction with retinoic acid but fail to upregulate expression of secondary granule protein genes (Khanna-Gupta et al., 1994; Lanotte et al., 1991). The murine myeloblastic cell line 32Dcl3 exhibits terminal differentiation and expression of neutrophil-specific genes upon stimulation with G-CSF (Graubert et al., 1993; Lawson et al., 1998), but in the early phase of differentiation many cells undergo apoptosis (Gupta et al., 2014). In this study, we used the Hoxb8 system to derive mouse neutrophils by immortalizing their

Introduction progenitors with estrogen-regulated Hoxb8 and the cytokine SCF (stem cell factor) to expand committed progenitor cells.

Hox oncoproteins belong to the class I homeodomain transcription factors, promote self-renewal of HSCs and regulate progenitor cell expansion (Argiropoulos and Humphries, 2007). The estrogen-binding domain of the estrogen receptor (ER) was fused to the N-terminus of Hoxb8, producing a retroviral expression vector for progenitor cell transduction (Wang et al., 2006). This allows conditional expression of Hoxb8. In the presence of estrogen, Hoxb8 is active and immortalised factor- dependent progenitors will proliferate, while upon estrogen withdrawal Hoxb8 is switched off and progenitor cells will start to differentiate, showing normal morphologic and functional maturation comparable to primary cells (Wang et al., 2006). They also show spontaneous apoptosis which is similar to primary neutrophils (Kirschnek et al., 2011).

Figure 4 Schematic representation of the Hoxb8 system

Mouse fetal liver or bone marrow cells were transduced with a retrovirus expressing Hoxb8 fused to the estrogen binding domain of the estrogen receptor. In the presence of β-estradiol, Hoxb8 is active and able to regulate target genes, keeping the cells in a progenitor state. Upon estrogen withdrawal, Hoxb8 can no longer regulate these target genes and the cells will start differentiating.

Advantages of the Hoxb8 system are the unlimited supply of cells that can be analysed by classical biochemistry or molecular biology approaches, but also genetically engineered, which is (also because of their very short life span) harder in primary cells.

Introduction

IV. Objectives3B Mutations of C/EBPα are associated with AML (found in 9-11 % of patients) but the molecular mechanism is unclear.

C/EBPα is expressed as a short (p30) and a long (p42) isoform. The two isoforms are likely to have different functions, the shorter one being unable to activate genes essential for terminal differentiation. In AML, typical mutations include small alterations in the C-terminal leucine-zipper region and N-terminal mutations that block expression of p42; the two mutations are frequently found in the same patient cells.

The frequent C/EBPα K313 mutation carries a duplication of lysine at position 313 while C/EBPα BRM2 harbours two amino acid substitutions.

To understand the molecular effects of C/EBPα mutations we here introduce the Hoxb8 neutrophil differentiation system. We expressed typical C/EBPα mutations found in AML in these progenitor cells and tested for the biological effects and molecular background. This system allowed us to obtain information on the influence of C/EBPα mutations on proliferation rate and differentiation of neutrophils. We also performed experiments analysing functional aspects of the cells. Molecular aspects of C/EBPα function were also analysed. Finally, we investigated whether the results observed in vitro would be reflected in a mouse model in vivo.

Results

V. Results4B

Generation17B of Hoxb8 neutrophils In order to study proliferation, differentiation and survival of neutrophil granulocytes, we generated Hoxb8 neutrophil progenitor cell lines from fetal liver or from bone marrow cells by transduction with a retrovirus driving the expression of an estrogen- regulated from of Hoxb8. Cells were then selected in the presence of SCF and β-estradiol by culture for at least three weeks. By removing β-estradiol from the medium, Hoxb8 is switched off and the cells start differentiating in the presence of stem cell factor (SCF) over four days into CD11b/Gr-1 double-positive cells (Figure 5 A) resembling primary, slightly immature neutrophils. Maturation of these cells can be shown by analysis of many parameters, for instance downregulation of c-kit during differentiation, which is only expressed at early stages of neutrophil development (Figure 5 B).

Figure 5: Hoxb8 neutrophils differentiate into mature granulocytes in vitro

Flow cytometric analysis of wt Hoxb8 neutrophils at the progenitor stage and different time points of differentiation. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. (A) Surface staining for CD11b/Gr-1. (B) Surface staining for c-kit.

Results

-/- C18B onditional C/EBPα ER-Hoxb8 neutrophils do not differentiate but are lost over time We wanted to investigate neutrophil differentiation in the absence of the transcription factor C/EBPα and to overexpress AML-associated mutations of C/EBPα in the absence of wt protein. For this, we used bone marrow cells of C/EBPαfl/fl LysMCretg mice (and C/EBPαfl/wt LysMCretg mice as corresponding control) to generate ER-Hoxb8 neutrophils. This genotype is expected to lead to a knockout of C/EBPα in neutrophil granulocytes during differentiation and should overcome the problem of perinatal lethality of C/EBPα-/- mice. In these mice, the recombinase Cre is expressed under the control of the lysozyme M-promoter, which is active during differentiation of macrophages and neutrophils (Clausen et al., 1999). Expression of Cre will delete the cebpa-gene, which should result in the loss of the protein during differentiation. Conditional C/EBPα-/- mice and their derived Hoxb8 neutrophils would be a convenient system to study C/EBPα mutations in the context of acute myeloid leukaemia.

After four weeks of selection, C/EBPαfl/fl LysMCretg Hoxb8 neutrophil progenitors were differentiated for the first time together with their corresponding wt cell line (C/EBPαfl/wt LysMCretg). On day 4 of differentiation, most of the cells expressed the early myeloid marker CD11b (encoding a myeloid integrin) and the granulocytic marker Gr-1. There was still a double-negative population left which resembled more the progenitor phenotype (Figure 6). This population was absent in the wt cell line and may represent individual cells with an efficient and probably earlier deletion of C/EBPα than in the other partly differentiating cells.

Comparing the two cell lines, more wt cells showed a mature CD11b/Gr-1 double- positive phenotype than cells of double-mutant genotype. When the differentiation process was repeated seven and nine weeks after transduction with the Hoxb8 retrovirus however, this difference between C/EBPαfl/fl LysMCretg and wt cells was smaller. It seems that selection against the non-differentiating population occurs, possibly because of proliferation or survival defects. The fact that there were still C/EBPαfl/fl LysMCretg ER-Hoxb8 neutrophils left able to differentiate could be due to relatively late expression of LysMCre on day 2 of neutrophil differentiation (data not shown). As C/EBPα levels are highest on day 1 of differentiation (see Figure 7 and

Results

Figure 8, below), the stimulus for terminal differentiation may occur before C/EBPα is knocked out on day 2.

Figure 6: C/EBPαfl/fl LysMcretg Hoxb8 neutrophils change over cultivation time

Flow cytometric analysis showing CD11b/Gr-1 expression of C/EBPαfl/fl LysMcretg and C/EBPαfl/wt LysMcretg Hoxb8 neutrophils on day 4 of differentiation. Cells were differentiated by removal of β-estradiol from the culture in the presence of SCF. The analysis was performed 4, 7 and 9 weeks after transduction with Hoxb8 retrovirus.

-/- Lef19B -1 Hoxb8 neutrophils have heterogeneous phenotypes We also generated Lef-1-/- and corresponding wt Hoxb8 neutrophils as Lef-1 is known to positively regulate C/EBPα and its loss has been reported to result in loss of C/EBPα expression (Skokowa et al., 2006). This was shown to lead to a maturation arrest and increased apoptosis of myeloid progenitor cells.

Lymphoid enhancer-binding factor 1 (Lef-1) is an important transcription factor of the Wnt signaling pathway involved in granulopoiesis. It is supposed to play a role in lineage commitment, control of cell proliferation and granulocytic differentiation. One of its target genes is C/EBPα (Skokowa et al., 2006). Lef-1 expression in granulocytes was reduced in severe congenital neutropenia (SCN), a rare disorder of myelopoiesis characterised by extremely low numbers of mature neutrophils in the

Results blood (Welte et al., 2006). This has been suggested to be due to a differentiation block at the promyelocytic stage of development (Welte et al., 2006).

Lef-1 deficient mice die in utero. We therefore used E 14.5 and E 18.5 fetal liver of Lef-1-/- and wt mice to generate Hoxb8 neutrophil progenitor cells. ER-Hoxb8 cells generated from embryonic day 14.5 fetal liver had been generated by Dr. Sanjivan Gautam. These cells had shown defects in survival as well as differentiation. Dr. Gautam had found that, upon induction of differentiation, Lef-1-/- ER-Hoxb8 cells underwent massive cell death already on the first day after estrogen withdrawal and by the second day about 70 % of the cells had died (Figure 7 A, left). To be able to study maturation of Lef-1 deficient neutrophils, the anti-apoptotic Bcl-2 family protein

Bcl-XL was expressed retrovirally. This restored survival, indicating that cell death of Lef-1 deficient ER-Hoxb8 cells was due to mitochondrial apoptosis (Figure 7 A, right). Expression of C/EBPα in Lef-1-/- ER-Hoxb8 cells also was able to restore survival during differentiation (Figure 7 A, right).

On day 4 of differentiation, all wt cells expressed the myeloid marker CD11b and 60.9 % were double-positive for CD11b/Gr-1. Most of the Lef-1 deficient cells expressed CD11b, but only a minor proportion expressed the granulocytic marker Gr-1 (Figure 7 B). This suggested a defect in maturation due to Lef-1 deficiency. Upon reconstitution with C/EBPα, Lef-1 deficient ER-Hoxb8 cells were able to differentiate at least partially.

Analysing expression of the granulocyte key transcription factor C/EBPα, Dr. Gautam observed a strong reduction of both p30 and p42 C/EBPα in Lef-1-/- ER-Hoxb8 cells (Figure 7 C). This led to the assumption that the differentiation defect observed in those cells could be due to decreased C/EBPα expression. Upon retroviral transduction with C/EBPα, survival and capability to differentiate were restored in ER-Hoxb8 cells.

Results

Figure 7: Lef1-/- Hoxb8 neutrophils are unable to mature and have lower C/EBPα levels

Unpublished data generated by Dr. Sanjivan Gautam. (A) Flow cytometric analysis of propidium-iodide (PI) stained ER-Hoxb8 cells during differentiation. Hoxb8 neutrophils were differentiated by withdrawal of β-estradiol from the medium in presence of SCF. PI-negative cells were considered alive. (B) Flow cytometric analysis of d4 differentiated Lef-1-/- and wt Hoxb8 neutrophils showing expression of CD11b/Gr-1. (C) Western blot analysis showing -/- C/EBPα expression of Lef-1 Bcl-XL and wt Bcl-XL Hoxb8 neutrophils during differentiation. Tubulin served as loading control.

When we established and investigated new, independent Lef-1-/- and wt Hoxb8 cell lines from E 18.5 fetal livers, we did not observe defects in survival or maturation. Lef-1-/- Hoxb8 neutrophils survived equally well until day 4 of differentiation as wt cells (Figure 8 A). There was no block in differentiation in Lef-1 deficient Hoxb8 cell lines as they were able to upregulate CD11b and Gr-1 (Figure 8 B). Further, both

Results

Lef-1-/- ER-Hoxb8 cell lines showed C/EBPα upregulation at the early stages of differentiation in the same way as wt cells did (Figure 8 C).

Figure 8: Lef1-/- Hoxb8 neutrophils are able to differentiate and express C/EBPα

(A) Flow cytometric analysis of propidium-iodide (PI) stained ER-Hoxb8 cells during differentiation. Hoxb8 neutrophils were differentiated by withdrawal of β-estradiol from the medium in presence of SCF. PI-negative cells were considered alive. (B) Flow cytometric analysis of d4 differentiated Lef-1-/- and wt Hoxb8 neutrophils showing expression of CD11b/Gr-1. (C) Western blot analysis showing C/EBPα expression of Lef-1-/- and wt Hoxb8 neutrophils during differentiation. Tubulin served as loading control.

Retroviral20B overexpression of C/EBPα mutants in ER-Hoxb8 neutrophils as a model to study their implication in AML In order to study AML-associated mutations of C/EBPα, we decided to use an overexpression model in wt ER-Hoxb8 neutrophils. The use of these cells for the

Results study of malignant myelopoiesis may have a number of advantages. The cells are relatively easy to manipulate retrovirally and stably express genes of interest. They differentiate very reproducibly in culture without the use of additional chemical stimuli. They can be generated in basically unlimited numbers and can in principle be established from any gene-manipulated mouse. Of importance for neutrophils is also the fact that the cells die rapidly in culture, as do primary neutrophils (or neutrophil progenitors) but not established cell lines.

Using the retroviral expression systems pMIG and pBABE, we expressed several C/EBPα mutants, listed in Table 1. Transduced cells were selected according to their expression of green fluorescent protein (GFP) and flow cytometry or by means of their resistance against puromycin. To verify our results with untagged overexpressed C/EBPα proteins, we also generated N-terminally FLAG- (p42) and HA-tagged (p30) versions of the different mutations.

ER-Hoxb8 progenitor cells expressing the various constructs exhibited similar GFP intensity (as the vector contained an IRES-GFP) up to day 4 of differentiation, suggesting similar transcription rates of the constructs (Figure 9).

Figure 9: GFP intensity of Hoxb8 neutrophils transduced with pMIG-C/EBPα wt/K313/BRM2 is comparable even upon differentiation

Flow cytometric analysis of GFP intensity of Hoxb8 neutrophil progenitors (A) and on day 4 of differentiation (B). Hoxb8 neutrophils were differentiated by withdrawal of β-estradiol from the medium in presence of SCF. Neutrophils retrovirally express pMIG-FLAG-C/EBPα wt/K313/BRM2.

Results

Table 1: Retroviral expression vectors used for transduction of Hoxb8 progenitor cell lines

Name Description Selection marker pMIG-R1 empty vector control GFP pMIG-C/EBPα wt C/EBPα GFP pMIG-p30C/EBPα short isoform of C/EBPα (30 kDa); the expression GFP construct contains the C/EBPα sequence starting from the second start codon pMIG-p42C/EBPα long isoform of C/EBPα (42 kDa); a point mutation GFP (ATGATA) was introduced into the second start codon to abolish p30 C/EBPα expression pMIG-C/EBPα K313 C/EBPα carrying a duplication of a lysine in the GFP BR-LZ domain. It is named after the human K313 duplication at aa position 313. In mice it is actually aa position 314, but for reasons of clarity we decided to keep the same denotation as the human mutation. pMIG-C/EBPα BRM2 C/EBPα carrying two point mutations: Ile294Ala, GFP Arg297Ala pMIG-FLAG-C/EBPα FLAG-tagged wt C/EBPα GFP pMIG-FLAG-C/EBPα FLAG-tagged C/EBPα K313 GFP K313 pMIG-FLAG-C/EBPα FLAG-tagged C/EBPα BRM2 GFP BRM2 pBABE-HA-p30C/EBPα HA-tagged short isoform of C/EBPα (30 kDa) Puromycin puro

Results

Proliferation21B of Hoxb8 neutrophils expressing AML-associated C/EBPα mutations is not significantly altered One characteristic of leukaemic cells is increased proliferation. For example, the N-terminal truncation mutant p30 C/EBPα has been reported to lead to a hyperproliferative phenotype in granulocytes of p30 homozygous mice (Kirstetter et al., 2008). We analysed the proliferative capacity of Hoxb8 neutrophil progenitors overexpressing AML-associated mutations both in liquid and semi-solid culture conditions. We decided to use wt ER-Hoxb8 cells as we were not successful in establishing C/EBPα knockout or knockdown cell lines.

Proliferation41B in suspension The strongest proliferation rates could be observed in Hoxb8 neutrophil progenitor cells overexpressing pMIG-R1 (empty vector) and pMIG-FLAG-C/EBPα K313 (Figure 10). There was no difference between both cell lines, indicating that C/EBPα K313 does not lead to a hyperproliferative phenotype. C/EBPα wt overexpressing cells showed 1.6-fold reduced proliferation rates compared with empty vector expressing cells. Interestingly, C/EBPα BRM2 overexpressing cells showed the slowest rate of proliferation, 2.5-fold lower than empty vector expressing and 1.5-fold lower than C/EBPα wt cells.

Figure 10: Proliferation of Hoxb8 neutrophil progenitors changes upon C/EBPα expression

Cell counts of Hoxb8 neutrophil progenitor cells expressing empty vector or FLAG-C/EBPα wt/K313/BRM2 were assessed by counting the cells each day using the Casy Cell Counter. Initially, 1x105 cells/ml were seeded and cells were re-seeded every second day to avoid limitation by the culture conditions. Theoretical proliferation rate was calculated based on the

Results dilution factor determined for re-seeding of cells. Data represent mean and SEM of three independent experiments.

We also analysed ER-Hoxb8 neutrophil progenitors expressing either p30 or p42 C/EBPα, as different functions for these isoforms have been reported. p30 C/EBPα has been reported to be important for proliferation of neutrophils while p42 is supposed to have a role in differentiation. In AML patients, N-terminal truncation mutations often abolish translation of the p42 C/EBPα protein in favour of p30 C/EBPα production. In this experiment, a different parental cell line than in the experiments using FLAG-tagged C/EBPα proteins was used. In addition, the C/EBPα proteins overexpressed were untagged. This might lead to variations which need to be considered when comparing the results of these experiments. In our model, we did not observe a proliferation-enhancing effect caused by overexpression of p30 C/EBPα (Figure 11). There was also no proliferation-reducing effect upon expression of p42 C/EBPα. Rather, p30 and p42 expressing cell lines showed similar and higher rates of proliferation than empty vector and wt C/EBPα expressing cells. It seems that enhanced expression of one of the two C/EBPα isoforms does not necessarily lead to increased proliferation.

Figure 11: Proliferation of Hoxb8 neutrophil progenitors changes upon p30/p42 C/EBPα expression

Cell counts of Hoxb8 neutrophil progenitor cells expressing p30/p42 or wt C/EBPα were assessed by counting the cells each day using the Casy Cell Counter. Initially, 1x105 cells/ml were seeded and cells were re-seeded every second day. Theoretical proliferation rate was calculated based on the dilution factor determined for re-seeding of cells. Data represent mean and SEM of three independent experiments.

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As many AML patients carry both N-terminal and C-terminal mutations of their C/EBPα gene, we decided to co-express wt/K313/BRM2 C/EBPα together with p30 C/EBPα to examine whether this leads to increased proliferation of ER-Hoxb8 neutrophil progenitors. We observed strongest proliferation in cells expressing FLAG- tagged wt and K313 C/EBPα together with HA-tagged p30 C/EBPα (Figure 12). Hoxb8 neutrophil progenitor cells expressing p30 C/EBPα alone or in combination with C/EBPα BRM2 showed similar rates of proliferation. Nevertheless, there was no increase in proliferation compared to expression of N- or C-terminal C/EBPα mutations alone.

Figure 12: Proliferation of Hoxb8 neutrophil progenitors changes upon expression of wt/K313/BRM2 C/EBPα in parallel to p30 C/EBPα

Proliferation of Hoxb8 neutrophil progenitor cells expressing HA-tagged p30 C/EBPα without or with FLAG-tagged C/EBPα wt/K313/BRM2 was assessed by counting the cells each day using the Casy Cell Counter. Initially, 1x105 cells/ml were seeded and cells were re-seeded every second day. Theoretical proliferation rate was calculated based on the dilution factor determined for re-seeding of cells. Data represent mean and SEM of three independent experiments.

Prol42B iferation in semi-solid media: methylcellulose colony assay Proliferation was also assessed in semi-solid media to investigate whether only some clones or the whole population of cells overexpressing a certain C/EBPα mutant possessed an altered proliferative capacity. This also enabled us to draw a conclusion on how many of the originally seeded cells survive. Already after 3 days of culture first differences in colony number were visible. In culture plates of C/EBPα K313 overexpressing cells more colonies were visible than in empty vector and in

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C/EBPα wt overexpressing cell lines. This difference was more prominent by day seven, when colonies were visible with the naked eye. The median of colony size of C/EBPα K313 overexpressing cells on day seven was 6.3-fold higher than in wt C/EBPα overexpressing cell lines and 1.8-fold higher than in empty vector cells (Figure 13 A). The difference in colony number that had grown out by day 7 was small. Colony numbers were 1.3-fold increased in C/EBPα K313 expressing cells compared to empty vector expressing cell colonies. Wt C/EBPα expression seemed to have a negative effect on colony formation, as colony number was decreased by 0.7-fold compared to control cells. This makes a difference of a 2.0-fold increase in colony number of C/EBPα K313 in comparison to wt C/EBPα expressing cells (Figure 13 B).

Figure 13: Proliferation in semi-solid media

Proliferation of Hoxb8 neutrophil progenitors in semi-solid media changes with C/EBPα expression. (A) Colony size of Hoxb8 progenitor cells was assessed using ImageJ. All colonies of a 3 cm dish with a circularity of 0.2-1 and a size of minimum 10 pixels were used for analysis. Box plots show median of colony size (line inside box), upper and lower quartile of colonies (box below and above the median line) and minimal and maximal size of colonies (whiskers). Means are indicated by a plus symbol (+). Data are from three independent experiments. (B) Colony number of whole 3 cm dishes was assessed using ImageJ. Data represent mean and SEM of three independent experiments (n.s.= non-significant, one-way ANOVA).

ER22B -Hoxb8 neutrophils expressing N- and C-terminal mutations of C/EBPα display differentiation defects The lack of differentiation of certain cell types is one hallmark of leukaemia. As C/EBPα is a key transcription factor in differentiation of neutrophil granulocytes, we

Results analysed the differentiation potential of cells overexpressing wt and mutant versions of this factor. Duplication of K313 has been reported to lead to a loss of the capacity to transactivate target genes (Pabst et al., 2001b), which could mean that terminal differentiation is abolished in cells overexpressing this C/EBPα mutation.

We analysed two wt Hoxb8 cell lines generated from different mice which were transduced with retroviruses coding for FLAG-tagged or untagged versions of C/EBPα. The proliferative capacity of differentiating Hoxb8 neutrophils was similar in cells expressing empty vector, wt C/EBPα and C/EBPα BRM2 (Figure 14). We observed increased proliferation rates in cells expressing C/EBPα K313 during differentiation.

Figure 14: Cell numbers of C/EBPα wt/K313/BRM2 expressing Hoxb8 neutrophils during differentiation

Cell counts of Hoxb8 neutrophils expressing empty vector or FLAG-C/EBPα wt/K313/BRM2 were assessed during differentiation by counting the cells each day using the Casy Cell Counter. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. Data represent mean and SEM of three independent experiments.

On day 4 of differentiation, we observed nearly 100 % CD11b-positive cells in two cell lines expressing empty vector, C/EBPα wt or C/EBPα BRM2 (Figure 15 A, B). C/EBPα K313 overexpressing cell lines failed to upregulate this marker of early myeloid differentiation. Expression of Gr-1 differed more among the various cell lines. Overexpression of wt C/EBPα even increased the number of CD11b/Gr-1 positive cells compared to empty vector transduced cells. C/EBPα BRM2 had a slightly

Results negative effect on maturation of Hoxb8 neutrophils, with less Gr-1 positive cells than in empty vector and C/EBPα wt expressing cell lines. The most prominent maturation defect was seen in C/EBPα K313 overexpressing cells, where only 20-35 % of cells were able to become CD11b+/Gr-1+ (Figure 15 C, D). Expression of the early myeloid marker c-kit reflected the maturation phenotype observed by staining for expression of CD11b and Gr-1. Cell lines expressing empty vector, C/EBPα wt or C/EBPα BRM2 showed strong downregulation of c-kit while C/EBPα K313 expressing ER-Hoxb8 cells still expressed c-kit on day 4 of differentiation (Figure 15 E, F).

Maturation of ER-Hoxb8 cells can also be observed microscopically. Progenitor cells are bigger than differentiated neutrophils and can be discriminated from mature neutrophils by Giemsa staining. Giemsa stains DNA phosphate groups, so nuclei will appear in a deep purple while the cytoplasm will be stained in a light pink. This facilitates the discrimination between immature cells with compact, round nuclei and mature cells with doughnut-shaped or segmented nuclei. Empty vector-transduced and wt C/EBPα or C/EBPα BRM2 expressing Hoxb8 neutrophils induced to differentiate by estrogen-withdrawal for 4 days exhibited doughnut-shaped nuclei and were distinctly smaller than progenitor cells. ER-Hoxb8 cells expressing C/EBPα K313 resembled the immature progenitor cells in size and shape of nuclei (Figure 15 G).

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Figure 15: Differentiation of Hoxb8 neutrophils upon C/EBPα expression

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Flow cytometric analysis of day 4 differentiated Hoxb8 neutrophils. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. (A) Hoxb8 neutrophils expressing FLAG-tagged versions of C/EBPα compared to empty vector cells on day 4 of differentiation stained for expression of CD11b/Gr-1. (B) Hoxb8 neutrophils expressing untagged versions of C/EBPα compared to empty vector cells on day 4 of differentiation stained for expression of CD11b/Gr-1. (C) Quantification of CD11b/Gr-1 double-positive Hoxb8 neutrophils expressing FLAG-tagged C/EBPα. (D) Quantification of CD11b/Gr-1 double-positive Hoxb8 neutrophils expressing untagged C/EBPα. (E) Quantification of c-kit positive Hoxb8 neutrophils expressing FLAG-tagged C/EBPα. (C-E) Data represent mean and SEM of three independent experiments (n.s.= non- significant, one-way ANOVA). (F) Hoxb8 neutrophils expressing FLAG-tagged versions of C/EBPα compared to empty vector cells on day 4 of differentiation stained for expression of c-kit. (G) Giemsa staining of Hoxb8 neutrophils expressing FLAG-tagged versions of C/EBPα or empty vector on day 4 of differentiation compared to Hoxb8 progenitor cells.

The two different translation products of C/EBPα, p30 and p42, have been reported to carry different functions. The shorter isoform (p30) is lacking two transactivation domains and therefore is said to be unable to arrest proliferation and induce differentiation, which can only be executed by the long (p42) isoform. Therefore we analysed the capability of Hoxb8 neutrophils to differentiate upon expression of p30, p42 and wt C/EBPα. Cells expressing p42 C/EBPα (which cannot make p30 C/EBPα) or expressing wt C/EBPα (which can synthesize both isoforms) showed differentiation that was comparable to cells transduced with empty vector (Figure 16). Interestingly, p30 expressing ER-Hoxb8 cells showed a phenotype similar to C/EBPα K313 expressing neutrophils with only partial upregulation of CD11b and Gr-1.

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Figure 16: Differentiation of Hoxb8 neutrophils upon expression of different C/EBPα isoforms

(A) Flow cytometric analysis of Hoxb8 neutrophils expressing p30, p42 and wt C/EBPα compared to empty vector tarnsduced cells on day 4 of differentiation. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. (B) Quantification of CD11b/Gr-1 double-positive Hoxb8 neutrophils expressing p30, p42 and wt C/EBPα. Data represent mean and SEM of three independent experiments (n.s.= non- significant, one-way ANOVA). (C) Scheme of the C/EBPα gene showing the two start codons and the resulting transcription products. In the p42 C/EBPα construct, the second start codon was destroyed by introducing a point mutation (ATGATA). The p30 C/EBPα construct carried the C/EBPα sequence starting at the second start codon.

We then decided to express C/EBPα proteins with C-terminal mutations (C/EBPα K313 and C/EBPα BRM2) together with the N-terminal truncation mutation p30. AML patients often carry biallelic combinations of N- and C-terminal C/EBPα mutations but almost never homozygous mutations of the same type. Mimicking this, we showed that expression of wt and p30 C/EBPα simultaneously resulted in impaired maturation (Figure 17). Hoxb8 neutrophils were only partially able to upregulate CD11b and Gr-1 and resembled the phenotype of cells expressing p30 C/EBPα only. Interestingly, cells expressing HA-tagged p30 only did not show reduced maturation. This contrasts the result of the untagged p30 cell line which showed decreased upregulation of CD11b and Gr-1 (Figure 16). These differences might be due to the use of cell lines generated from different mice, different p30 expression levels as two

Results different vectors for transduction were used (pMIG for the untagged and pBABE for the HA-tagged construct) or a side-effect of the HA-tag which might influence p30 function. When p30 C/EBPα was expressed together with C/EBPα K313, the maturation defect was even more pronounced than in cells expressing each mutation alone (p=0.0008, see Figure 15 and Figure 16). Interestingly, cells expressing C/EBPα BRM2 in combination with p30 C/EBPα did not show any failure to differentiate but rather resembled ER-Hoxb8 cells expressing C/EBPα BRM2 alone.

Figure 17 Differentiation of Hoxb8 neutrophils upon expression of different C/EBPα C-terminal mutations in combination with the N-terminal truncation mutation p30

(A) Flow cytometric analysis of Hoxb8 neutrophils expressing FLAG-tagged versions of C/EBPα with and without C-terminal mutations (K313 and BRM2) in combination with the N-terminal truncation mutation p30 (HA-tagged) on day 4 of differentiation. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. (B/C) Quantification of (B) CD11b/Gr-1 double-positive Hoxb8 neutrophils (C) c-kit positive Hoxb8 neutrophils expressing FLAG-tagged C/EBPα with and without C-terminal mutations (K313 and BRM2) in combination with the N-terminal truncation mutation p30 (HA-tagged) on day 4 of differentiation. Data represent mean and SEM of at least three independent experiments (n.s.= non-significant, one-way ANOVA).

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Effector23B functions of Hoxb8 neutrophils expressing C-terminal mutations of C/EBPα are reduced The strong differentiation defect observed upon expression of C/EBPα K313 led us to the question whether cells carrying this mutation would be able to exert neutrophil effector functions. We analysed their ability to produce the primary granule protein neutrophil elastase and their ability to secrete the cytokines TNF and IL-6 in response to LPS. Furthermore, we employed a luciferase reporter assay to analyse transactivating capacity of C/EBPα K313, as this mutation has been reported to be non-functional (Pabst et al., 2001b).

Expression43B of the granule protein neutrophil elastase is reduced in C/EBPα K313 and C/EBPα BRM2 expressing ER-Hoxb8 neutrophils Since C/EBPα K313 expressing Hoxb8 neutrophils are arrested in differentiation, we asked whether overexpression of C/EBPα K313 would alter the ability of differentiating neutrophils to produce granule proteins. We observed expression of neutrophil elastase (NE), which was strongest on day 2 of differentiation in empty vector and wt C/EBPα expressing cells (Figure 18). There was also weaker expression on day 1 and in progenitors while in C/EBPα overexpressing cells it was detectable up to day 3 of differentiation. C/EBPα K313 expressing cells showed very little detectable expression of neutrophil elastase. This could be due to the generally delayed and impaired maturation of those cells. Interestingly, C/EBPα BRM2 expressing cells also had almost no detectable expression of NE although they do not display a prominent maturation defect. It seems that although those two different mutations are located in the same region of the protein, their impact on C/EBPα functions is not the same in some respects.

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Figure 18: Expression of Neutrophil Elastase is reduced in C/EBPα K313 expressing cells

Western blot analysis showing expression of Neutrophil Elastase during differentiation of Hoxb8 neutrophils expressing empty vector or wt/K313/BRM2 C/EBPα. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. Control refers to wt Hoxb8 neutrophils. Tubulin served as loading control.

Secretion44B of the pro-inflammatory cytokines TNF and IL-6 by C/EBPα K313 expressing Hoxb8 neutrophils We analysed secretion of the pro-inflammatory cytokines TNF and IL-6. Neutrophil progenitors produced only minor amounts of TNF upon LPS stimulation, whereas day 4 differentiated cells secreted substantial amounts (Figure 19 A). wt C/EBPα expressing cells secreted higher quantities of TNF than empty vector cells and surprisingly, C/EBPα K313 expressing cells had even higher levels. IL-6 production did not change much during differentiation, both progenitor and day 4 differentiated Hoxb8 neutrophils were able to produce similar amounts of IL-6 (Figure 19 B). C/EBPα K313 expressing Hoxb8 progenitors produced detectable IL-6 even in the absence of stimuli. Upon stimulation with LPS, empty vector and wt C/EBPα expressing cells secreted IL-6, while secretion by C/EBPα K313 expressing cells increased drastically. Mature Hoxb8 neutrophils did not show any spontaneous IL-6 secretion. Stimulation with LPS led to slight secretion of IL-6 by empty vector and wt C/EBPα cells, while C/EBPα K313 expressing mature neutrophils produced higher amounts than progenitor cells.

Although some effector functions of mature neutrophils like granule protein expression are defective in C/EBPα K313 expressing ER-Hoxb8 cells, others seem to be intact. The production of cytokines is a marker of maturation in neurophils. The fact that C/EBPα K313 expressing Hoxb8 cells are able to secrete high amounts of

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IL-6 and TNF on day 4 of differentiation indicates that these cells are at least partially able to maturate.

Figure 19: Cytokine secretion of C/EBPα wt/K313 expressing Hoxb8 neutrophils

ELISA showing levels of (A) TNF and (B) IL-6 in Hoxb8 progenitors and day 4 differentiated neutrophils expressing empty vector or wt/K313 C/EBPα. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF and stimulated with 1 µg/ml LPS for 8 h. Data represent mean and SEM of three independent experiments with duplicate samples each.

C/EBP45B α K313 retains transactivation capacity: luciferase reporter assay Mutations in transcription factors may change their functions. In the case of C/EBPα, dimerisation with other proteins or binding to DNA may be disrupted. C/EBPα K313 has been reported to be unable to transactivate target genes (in CV1 monkey fibroblast cells), due to its mutation inside the basic region leucine zipper (Pabst et al., 2001b). Still, the capacity to activate cytokine production suggested some form of transcriptional activity. We next analysed the capacity of C/EBPα mutations to activate C/EBP target genes. To investigate this, we transduced the corresponding cell lines with a lentivirus expressing a C/EBPα-responsive Luciferase reporter construct. This construct contains three C/EBPα recognition sites which induce expression of firefly luciferase upon C/EBPα binding. If the C/EBPα mutant expressed in a cell line is active, it will bind to the responsive element and enable expression of the luciferase, which will then convert its substrate D-luciferin to a quantifiable light signal.

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Figure 20: C/EBPα Luciferase Reporter Activity in living cells

Luciferase reporter activity was investigated in cells expressing a C/EBPα responsive element which enabled the transcription of firefly luciferase. Luciferase activity was measured as luminescence emitted by metabolisation of D-Luciferin. (A) Total luminescence measured in wt Hoxb8 neutrophils expressing the C/EBPα reporter construct during differentiation (d0-d4). Cells were differentiated by removal of β-estradiol from the culture

Results medium in the presence of SCF. Data show mean and SEM of five independent experiments with duplicate samples each. (B) N-fold activity of the C/EBPα reporter in wt/K313 C/EBPα expressing cells (d0-d4 of differentiation) compared to cells expressing only the reporter. Data show mean and SEM of five independent experiments with duplicate samples each. (C) N-fold activity of the C/EBPα reporter in wt/p30 C/EBPα expressing cells (d0-d4 of differentiation) compared to cells expressing only the reporter. Data show mean and SEM of three independent experiments with duplicate samples each.

Analysis of wt Hoxb8 neutrophils expressing the luciferase reporter showed the strongest C/EBPα activity in progenitor cells while it was lower but stayed relatively constant during differentiation (Figure 20 A). We compared C/EBPα activity in Hoxb8 neutrophils expressing the reporter construct only and in combination with wt and K313 C/EBPα during differentiation. Upon expression of wt C/EBPα, reporter activity was increased compared to luciferase reporter expression only, nearly 25-fold on day 1 of differentiation (Figure 20 B). It dropped at later stages of differentiation but always stayed at least 5-fold higher than in the reporter only cell line. Surprisingly, luciferase activity was drastically increased in C/EBPα K313 expressing cells (up to 169-fold higher compared to cells expressing only the reporter and up to 25-fold compared to wt C/EBPα expressing cells). It also dropped after day 1 of differentiation, ending with still 18-fold increased reporter activity compared to only luciferase reporter expressing cells. The drop in luciferase reporter activity observed in C/EBPα K313 expressing cells was surprising, as these cells show incomplete maturation (Figure 15). Still, they displayed a downregulation in C/EBPα reporter activity similar to C/EBPα wt expressing Hoxb8 cells.

We also compared C/EBPα reporter activity of wt and p30 C/EBPα as the truncated form of C/EBPα lacks two transactivation domains in the N-terminus. This experiment was accomplished using a wt Hoxb8 cell line generated from fetal liver cells of a different mouse. The C/EBPα proteins expressed in these cells were not FLAG- tagged as the C/EBPα versions used for the comparison of C/EBPα wt and C/EBPα K313. This might lead to variations which make a comparison of the results between these experiments difficult. Reporter activity was also strongest in progenitor cells, with a smaller peak on day 2 of differentiation in all cell lines analysed. Wt C/EBPα expression increased reporter activity up to 2.5-fold compared to reporter expression only (Figure 20 C) on day 2 of differentiation. Interestingly, p30 C/EBPα expression increased the luciferin signal up to 5-fold on day 2 (2.4-fold compared to C/EBPα wt)

Results and up to 11-fold on day 4 of differentiation (6-fold compared to C/EBPα wt). This might be due to incomplete differentiation of p30 C/EBPα expressing cells (Figure 16).

The high reporter activity upon C/EBPα K313 and p30 expression was surprising, as we anticipated that both mutants would have no or only little transcription factor activity. The luciferase reporter assay shows a strong capacity for both in terms of activating a C/EBP-responsive element. It is conceivable that the drastic increase of luciferase activity caused by C/EBPα K313 is due to already increased protein levels of this C/EBPα mutant and not necessarily by increased affinity towards target genes.

Cell24B death of Hoxb8 neutrophils After neutrophil granulocytes have completed terminal maturation, they will die within several hours unless they encounter stimuli like pattern recognition signals, chemokines or growth factors. If they do not, they will undergo apoptosis by the intrinsic pathway and finally be cleared by phagocytic cells. Cell death by apoptosis is very similar for Hoxb8 neutrophils (Kirschnek et al., 2011).

We investigated the rate of spontaneous cell death in Hoxb8 neutrophils during differentiation. Cell lines expressing empty vector, wt C/EBPα, C/EBPα K313 and C/EBPα BRM2 started with similar levels of live cells (70-80 %) (Figure 21 A). Empty vector and C/EBPα wt expressing cells behaved very similar during differentiation, with slightly more C/EBPα wt expressing cells alive on day 4. C/EBPα BRM2 expressing cells showed more PI-positive (dead) cells during differentiation than the other cell lines. C/EBPα K313 expressing cells showed the highest proportion of live cells during differentiation up to day 4. This can only be achieved when cells are given fresh media on day 2 of differentiation, as C/EBPα K313 expressing cells keep proliferating at higher rather while differentiating and consume nutrients in the medium much faster than the other cell lines. Despite their immature state on day 4, C/EBPα K313 do not stay alive longer than the other cell lines but also die by day five of differentiation (data not shown).

Wt Hoxb8 neutrophils express high levels of the anti-apoptotic Bcl 2 family proteins Bcl 2 and Mcl 1 at the progenitor stage. They are downregulated during

Results differentiation, while the pro-apoptotic protein Bim, which is expressed at low levels at the progenitor stage, is upregulated. The difference in viability between C/EBPα K313 and C/EBPα BRM2 cell lines was surprising as both cell lines express lower levels of the pro-apoptotic Bcl-2 family protein Bim than empty vector and wt C/EBPα expressing cell lines (Figure 21 B). Those upregulated expression of Bim on day 3 and 4 of differentiation, rendering them more susceptible to apoptosis. Bim levels in C/EBPα K313 and C/EBPα BRM2 level were not only in general lower but also lacked upregulation during the last days of differentiation. Expression of the anti- apoptotic Bcl-2 family member Mcl-1 was increased in C/EBPα wt, C/EBPα K313 and C/EBPα BRM2 cell lines and dropped less at later stages of differentiation compared to empty vector expressing cells. This could explain the slightly better survival of wt C/EBPα expressing cells compared to the empty vector control. The anti-apoptotic protein Bcl-2 was expressed from the progenitor stage until day 2 of differentiation and then downregulated on day 3 and 4. However, this was not the case in C/EBPα K313 expressing cells, where Bcl-2 expression is maintained until day 4. This might be a consequence of the inability of C/EBPα K313 expressing cells to differentiate. The fact that they stay at a maturation stage between d1 and d2 of wt Hoxb8 neutrophils could explain maintained expression of anti-apoptotic proteins.

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Figure 21: Survival of Hoxb8 neutrophils and expression of Bcl-2 family proteins

(A) Survival of Hoxb8 neutrophils was assessed by propidium iodide (PI) staining during differentiation. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF and supplied with fresh media on day 2 of differentiation. Data represent mean and SEM of at least three independent experiments. (B) Western blot analysis of Hoxb8 neutrophils (progenitor stage up to day 4 of differentiation) expressing C/EBPα wt/K313/BRM2 as well as empty vector. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. Protein levels of Bim, Mcl-1 and Bcl-2 were assessed, Tubulin served as loading control.

C/EBPα25B expression on mRNA and protein levels Changes at the nucleotide level can affect transcription and translation of a certain gene. Amino acid substitutions, deletions and insertions may lead to changes in secondary and tertiary structures of RNA and proteins. To find out whether the C/EBPα K313 and C/EBPα BRM2 mutations affect the levels of the protein, we analysed mRNA- and protein levels in differentiating Hoxb8 neutrophils. Comparison of the GFP intensity of C/EBPα wt/K313/BRM2 expressing ER-Hoxb8 cells indicated similar vector carriage and transcription rates (Figure 9) as C/EBPα expression is connected to GFP expression by an IRES (inner ribosome entry site).

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C/EBPα46B mRNA-levels are slightly higher in wt C/EBPα expressing Hoxb8 neutrophils than in C/EBPα K313 expressing cells To investigate whether the amino acid duplication of the C/EBPα K313 mutation affected mRNA levels, we analysed C/EBPα mRNA levels of wt and C/EBPα wt/K313 expressing Hoxb8 neutrophils in progenitor cells and during early stages of differentiation (day 1 and 2). Upon C/EBPα overexpression, its mRNA levels were increased compared to wt Hoxb8 neutrophils as expected; in C/EBPα wt expressing cells the levels were even higher than in C/EBPα K313 expressing cells (Figure 22). This indicates that the transcription rate and mRNA stability of C/EBPα K313 are not severely altered compared to wt C/EBPα.

Figure 22: C/EBPα levels in wt Hoxb8 neutrophils as well as upon expression of FLAG- wt/K313 C/EBPα qRT-PCR showing relative mRNA levels of C/EBPα (normalised to β-Actin) in Hoxb8 neutrophil progenitor cells and in early differentiating cells (d1 and d2). Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. Control refers to wt Hoxb8 neutrophils, FLAG-C/EBPα wt and FLAG-C/EBPα K313 represent Hoxb8 neutrophils overexpressing wt/K313 C/EBPα. Data represent mean and SEM of three independent experiments with duplicate samples each.

C/EBPα47B protein levels of C/EBPα K313/BRM2 expressing ER-Hoxb8 cells are strongly increased The transcription rates were similar for C/EBPα wt and C/EBPα K313 expression, therefore we next tested whether this would be reflected by C/EBPα protein levels.

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Endogenous C/EBPα protein was transiently upregulated during differentiation (Figure 25). Strongest C/EBPα expression could be observed on day 1 of differentiation in wt cells. The same pattern was seen in ER-Hoxb8 cells expressing FLAG-tagged C/EBPα (Figure 23). Surprisingly, very strongly elevated levels of C/EBPα K313 and C/EBPα BRM2 were observed. Expression of C/EBPα K313 or C/EBPα BRM2 was already high at the progenitor stage and even increased on day 1 of differentiation. Levels of mutated C/EBPα were in general much higher than of wt protein and were not downregulated when Hoxb8 was turned off.

Figure 23: C/EBPα protein levels differ between C/EBPα wt/K313/BRM2 expressing ER- Hoxb8 cells

Western blot analysis showing protein levels of ER-Hoxb8 cells expressing C/EBPα wt/K313/BRM2 during differentiation (d0-d4). Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. Control refers to wt Hoxb8 neutrophils. Overexpressed C/EBPα protein was detected using an anti-FLAG antibody, α-Tubulin served as loading control.

We also tested C/EBPα protein levels in Hoxb8 neutrophils overexpressing the short (p30) and the long (p42) isoform separately or simultaneously. Empty vector transduced cells showed an increase in endogenous C/EBPα expression on days 1 and 2 of differentiation, however there was very little detectable endogenous p30 C/EBPα (Figure 24). Hoxb8 cells overexpressing p30 C/EBPα showed increased expression of the short C/EBPα isoform compared to empty vector, wt C/EBPα and p42 C/EBPα expressing cells. C/EBPα wt/p42 expressing Hoxb8 neutrophils showed slightly increased C/EBPα expression of both the short and the long isoform in comparison to empty vector transduced cells which was present until day 4 of differentiation in substantial amounts.

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Figure 24: C/EBPα protein levels of p30/wt/p42 C/EBPα expressing ER-Hoxb8 cells

Western blot analysis showing protein levels of ER-Hoxb8 cells expressing empty vector or C/EBPα p30/wt/p42 during differentiation (d0-d4). Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. α-tubulin served as loading control.

Protein48B stability of C/EBPα K313 is not significantly increased compared to wt C/EBPα These data show that at least one consequence of the K313 mutation in C/EBPα is a strong increase of protein levels while mRNA levels are not increased. This could be due to the additional lysine in the C/EBPα K313 mutant, as lysine residues are often sites of posttranslational modifications like ubiquitylation, which might alter proteasomal degradation. The C/EBPα BRM2 mutation carries two amino acid substitutions which might also cause differences in posttranslational modification or tertiary protein structure.

We investigated the possibility of altered protein stability by administration of cycloheximide. Cycloheximide is a translation inhibitor blocking de novo protein synthesis. Since we found out that Hoxb8 neutrophils are very sensitive to cycloheximide treatment, we further transduced them with the anti-apoptotic Bcl-2 family protein Bcl-XL. Unfortunately, the FLAG antibody used in this study was not sensitive enough to pick up a signal for FLAG-tagged wt C/EBPα for all time points studied. Therefore, we also used an anti-C/EBPα antibody which would also detect endogenous C/EBPα levels. As these are much lower than the overexpressed FLAG- tagged C/EBPα protein, they should only constitute a minor part of the total C/EBPα signal, especially at later stages of differentiation. Figure 25 shows the comparison of wt Hoxb8 neutrophils (referred to as control cells) and Hoxb8 neutrophils expressing FLAG-tagged wt C/EBPα. Both isoforms, p30 and p42, are detected by the anti-

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C/EBPα antibody, p42 being the more abundant one. Control cells showed C/EBPα upregulation on day 1 of differentiation and expression levels decreased at later stages of development (as already shown above). FLAG-C/EBPα expressing cells showed much stronger expression, already at the progenitor stage. The amount of endogenous protein picked up with this antibody is much lower than the exogenous, overexpressed protein and is therefore unlikely to affect the analysis of the much higher C/EBPα K313 levels in this experiment.

Figure 25: C/EBPα-transduced ER-Hoxb8 cells show much higher protein levels than wt Hoxb8 neutrophils

Western blot analysis showing expression of endogenous and overexpressed C/EBPα protein levels during differentiation of Hoxb8 neutrophils. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. Control refers to wt Hoxb8 neutrophils. Both isoforms (p30 and p42) of C/EBPα are recognised. α-tubulin served as loading control.

Hoxb8 neutrophil progenitors and day 1 differentiated cells expressing wt C/EBPα showed a half-life of 4 h for total C/EBPα, which was increased for C/EBPα K313 expressing cells (16 h)(Figure 26 A, B). C/EBPα half-life of C/EBPα wt and C/EBPα K313 expressing cells both was around 8 h on day 2 of differentiation and approximately 24 h on day 3 of differentiation. The difference in C/EBPα protein stability is therefore highest early during differentiation of Hoxb8 neutrophils, probably when transcription factor function of C/EBPα is needed the most. Nonetheless, C/EBPα K313 levels are increased at all stages of ER-Hoxb8 neutrophil development compared to C/EBPα wt. The difference in protein stability of C/EBPα wt and C/EBPα K313 seems to be too small to explain the disparity in protein levels. Similar mRNA

Results levels and protein stability of the C/EBPα K313 mutation indicate that elevated C/EBPα protein levels are due to increased translation.

Figure 26: Protein stability is not significantly changed in C/EBPα K313 expressing Hoxb8 neutrophils

Western blot showing C/EBPα protein levels during treatment with 10 µg/ml cycloheximide of Hoxb8 neutrophil progenitors and differentiated cells (d1-d3) expressing (A) wt C/EBPα (B) K313 C/EBPα. Cells were differentiated by removal of β-estradiol from the culture medium in the presence of SCF. Tubulin served as loading control. Numbers indicate band intensity normalised to tubulin.

Results

C/EB49B Pα K313 protein levels are decreased in HEK293FT cells upon transient transfection We then tested whether increased protein levels of mutant C/EBPα were universal or cell-type specific. For this, we transfected HEK293FT cells with a retroviral vector driving transient protein expression. While C/EBPα K313 protein levels were drastically increased over C/EBPα wt in ER-Hoxb8 neutrophils, HEK293FT did not show increased expression of C/EBPα K313 compared to C/EBPα wt protein. Rather, wt C/EBPα levels were higher than the protein levels of the C/EBPα K313 mutant. It therefore appears that factors expressed in neutrophil progenitors but not in 293 FT cells are responsible for this differential protein expression.

Figure 27: Hek293FT cells show lower C/EBPα levels of C/EBPα K313 compared to C/EBPα wt protein

Western blot analysis showing transient C/EBPα expression 24 h after transfection of HEK293FT cells with 0.25, 0.5, 1 or 2 µg of pcDNA-C/EBPα wt/K313. β-actin served as loading control.

In26B vivo differentiation of ER-Hoxb8 neutrophils expressing C/EBPα wt/K313 As shown above, cells expressing C/EBPα K313 displayed strongly reduced differentiation when Hoxb8 was turned off in vitro. However, in vitro studies cannot completely rebuild the situation during differentiation in the bone marrow, in terms of stimuli like cytokines and cell-cell contacts. We therefore tested the differentiation of Hoxb8 cells expressing C/EBPα wt or C/EBPα K313 in mice. Hoxb8 neutrophil progenitor cells expressing empty vector, C/EBPα wt or C/EBPα K313 were co- transferred with unfractionated wt bone marrow cells into lethally irradiated wt C57Bl/6 mice. This model has previously been used by our group. When wt Hoxb8 cells are injected using this protocol, mature neutrophils can be observed in peripheral blood and lymphoid organs, with the peak of Hoxb8-derived neutrophils on

Results day 6 post transfer (Gautam et al., 2013). By comparison with wt cells it is therefore possible to test the differentiation potential of modified Hoxb8 cells.

On days 6, 8 and 10 after transfer we analysed peripheral blood, bone marrow and spleen of recipient mice. We focused on later time points in the analysis since we speculated that the cells expressing C/EBPα K313 would differentiate more slowly and be detectable at later time points than wt cells.

Upon50B adoptive transfer, Hoxb8 neutrophils expressing C/EBPα K313 are detectable for longer times in the bone marrow Total leukocyte cell counts in peripheral blood on day 8 were similar in mice transplanted with empty vector, wt C/EBPα or C/EBPα K313 expressing Hoxb8 neutrophils (Figure 28 A). In the bone marrow, similar numbers of total leukocytes were found in mice grafted with empty vector and wt C/EBPα expressing cells (Figure 28 B). Leukocyte cell numbers were slightly increased in mice engrafted with cells expressing C/EBPα K313 compared to wt C/EBPα. In the spleen, more total leukocytes were detectable in mice which received C/EBPα wt (22 %) and C/EBPα K313 (70 %) expressing cells compared to mice which received empty vector transduced cells (Figure 28 C). There were 39 % more total leukocytes in mice which were grafted with C/EBPα K313 than with C/EBPα wt expressing cells. Until day 10 after transfer, total leukocyte numbers of mice transferred with C/EBPα K313 Hoxb8 cells started to decline in all organs analysed. These data show that engraftment of C57Bl/6 mice with wt BM and ER-Hoxb8 cells worked equally well for all cell lines with a slight difference in migration to the spleen.

Results

Figure 28: Leukocyte cell counts show equal engraftment of C57Bl/6 mice by BM cells and ER-Hoxb8 neutrophils with minor differences in the spleen

Cell counts of lethally irradiated C57Bl/6 mice grafted with empty vector or C/EBPα wt/K313 expressing Hoxb8 neutrophils together with unfractionated bone marrow cells at a 10:1 ratio. The analysis was performed on day 8 after cell transfer if not indicated otherwise. (A) Leukocyte cell counts per ml blood. (B) Absolute leukocyte numbers in the bone marrow. (C) Absolute leukocyte numbers in spleen. (A-C) Data represent absolute values and mean of two experiments with three mice per group (n.s.= non-significant, one-way ANOVA).

The number of Hoxb8-derived, GFP-positive cells found in the blood, bone marrow and spleen varied substantially between the different Hoxb8 cell genotypes. On day 6, 47.2 % of total leukocytes were GFP-positive in mice transplanted with empty vector Hoxb8 neutrophils, while C/EBPα wt and C/EBPα K313 expressing cells were found at higher proportions in peripheral blood (65.9 % and 68.8 %)(Figure 29 A). By day 8 after transfer, the number of GFP-positive leukocytes in the blood declined in all mice analysed (Figure 29 B). In the bone marrow, very few GFP-positive leukocytes were detectable in mice grafted with empty vector and wt C/EBPα cells on day 8 after transfer, while a substantial amount of C/EBPα K313 expressing cells was detectable (Figure 29 C). The number of GFP-positive leukocytes in C/EBPα K313 grafted mice declined by day 10 after transfer, indicating that although C/EBPα K313 cells seemed to be able to stay for longer times in the bone marrow niche, they were also supplanted by wt bone marrow cells which were co-transferred with the genetically modified cells over time. This indicates that ER-Hoxb8 neutrophils are not able to build a self-renewing niche in the bone marrow. On days 8 and 10, GFP- positive leukocytes in the spleen were detectable at very low numbers (Figure 29 D).

Results

Figure 29: The proportion of GFP-positive cells varies strongly upon expression of C/EBPα wt/K313

Flow cytometric analysis of the proportion of GFP-positive leukocytes in lethally irradiated C57Bl/6 mice grafted with empty vector or C/EBPα wt/K313 expressing Hoxb8 neutrophil progenitors together with unfractionated bone marrow cells at a 10:1 ratio. The analysis was performed on day 8 after cell transfer if not indicated otherwise. (A) Proportion of GFP- positive cells in the blood on day 6 after cell transfer. (B) Proportion of GFP-positive cells in the blood. (C) Proportion of GFP-positive cells in the bone marrow. (D) Proportion of GFP- positive cells in the spleen. (A-D) Data represent absolute values and mean of two experiments with three mice per group (n.s.= non-significant, one-way ANOVA).

Results

Hoxb851B neutrophils expressing C/EBPα K313 showing delayed maturation are able to differentiate in vivo We analysed the differentiation status of ER-Hoxb8 cells on day 6 after transfer in peripheral blood. Until then, wt Hoxb8 neutrophils would have reached terminal differentiation and start to perish unless they encountered pro-inflammatory stimuli. There were less CD11b/Gr-1 double-positive neutrophils expressing empty vector (45.92 %) than wt C/EBPα (68.7 %) and C/EBPα K313 cells (88.79 %)(Figure 30 A), probably because they had already accomplished complete maturation and started to die off. On day 8 after adoptive cell transfer, the number of mature Hoxb8 neutrophils detectable in the blood had dropped to 20-25 % in mice grafted with empty vector or wt C/EBPα expressing cells, only mature C/EBPα K313 cells were found at higher numbers (58.6 %)(Figure 30 B). By day 10, CD11b/Gr-1 double-positive C/EBPα K313 cell numbers declined to similar levels as empty vector and wt C/EBPα expressing cells earlier. It is surprising that C/EBPα K313 expressing cells become fully mature in vivo, as they exhibit a strong maturation defect in vitro. This can probably be explained by the accessory cytokines and interactions with other cell types in a mouse model compared to the limited conditions of in vitro culture. Nevertheless, they still show delayed maturation compared to empty vector and wt C/EBPα expressing Hoxb8 neutrophils. In the bone marrow, similar numbers of empty vector and C/EBPα wt expressing mature Hoxb8 neutrophils were found while the number of CD11b/Gr-1 positive C/EBPα K313 expressing neutrophils was substantially increased (Figure 30 C). The number of mature C/EBPα K313 expressing cells declined by day 10, suggesting they migrated to the periphery and started to perish. Similar numbers of mature Hoxb8 neutrophils expressing empty vector or wt C/EBPα were also found in the spleen, while the number of mature C/EBPα K313 expressing Hoxb8 neutrophils was increased and stayed relatively constant until day 10 after transfer (Figure 30 D). These data show that C/EBPα K313 expressing Hoxb8 neutrophils are able to overcome the differentiation block observed in vitro in an in vivo model, although they still show delayed maturation.

Results

Figure 30: More CD11b/Gr-1 double-positive Hoxb8 neutrophils expressing C/EBPα K313 are detectable on day 8 and 10 after transfer

Flow cytometric analysis of the proportion of CD11b/Gr-1 double-positive leukocytes of GFP- positive (Hoxb8) cells in lethally irradiated C57Bl/6 mice grafted with empty vector or C/EBPα wt/K313 expressing Hoxb8 neutrophil progenitors together with unfractionated bone marrow cells at a 10:1 ratio. The analysis was performed on day 8 after cell transfer if not indicated otherwise. (A) Proportion of CD11b/Gr-1 double-positive Hoxb8 neutrophils in the blood on day 6 after cell transfer. (B) Proportion of CD11b/Gr-1 double-positive Hoxb8 neutrophils in the blood. (C) Proportion of CD11b/Gr-1 double-positive Hoxb8 neutrophils in the bone marrow. (D) Proportion of CD11b/Gr-1 double-positive Hoxb8 neutrophils in the spleen. (A- D) Data represent absolute values and mean of two experiments with three mice per group (n.s.= non-significant, one-way ANOVA).

Results

C/EBPα27B protein levels are increased in AML patient samples The highly elevated C/EBPα protein levels of Hoxb8 neutrophils expressing the K313 mutation led us to the question whether AML patients showed a similar phenotype. We analysed AML patient samples carrying mutations at position K313 of C/EBPα. Four patients carried K313 duplications (CEBPα K313 mutant samples 1, 2, 4 and 5) and one carried a deletion of amino acid K313 (CEBPα K313 mutant sample 3). In addition, four patients carried N-terminal stop codons on the second C/EBPα allele (CEBPα K313 mutant samples 1, 2, 3 and 5). We compared the C/EBPα protein levels of C/EBPα K313 mutant AML samples to patient samples with wt C/EBPα. Interestingly, increased C/EBPα protein levels were found in samples with duplications of Lysine 313 but also in one patient who carried a deletion of this codon (Figure 31). This suggests that not necessarily the addition of another lysine but the alteration of the amino acid sequence in the BR-LZ is causing the increased expression levels observed with this mutation.

Figure 31: C/EBPα protein levels are drastically increased in human AML samples with C/EBPα K313 mutations

Western blot analysis showing protein levels of patients bearing C/EBPα K313 mutations compared to C/EBPα wt samples. C/EBPα K313 mutated samples are: 1, 2, 4, 5= C/EBPα K313 duplication, 3= C/EBPα K313 deletion; 1, 2, 3, 5= N-terminal stop codon. α-tubulin and β-actin served as loading control.

Discussion

VI. Discussion5B

Regulation28B of C/EBPα expression Alterations in structurally important protein regions are likely to cause differences in protein stability and might already influence translation rates. The insertion at lysine K313 could affect the leucine zipper structure and in addition is a possible target for ubiquitylation, which could affect protein levels. The two amino acid substitutions of the BRM2 mutation may also alter folding or already affect mRNA secondary structure.

GFP intensity suggested equal mRNA expression of C/EBPα wt/K313/BRM2 (Figure 9) and RT-PCR confirmed similar RNA-levels of C/EBPα wt/K313 (Figure 22). It is possible that the C/EBPα K313 RNA structure is changed and therefore interacts with ribosomes in a different way from wt C/EBPα, increasing protein levels. We observed dramatically increased protein levels of C/EBPα K313 and C/EBPα BRM2 in comparison to C/EBPα wt (Figure 23). However, this was only the case in ER-Hoxb8 neutrophils. HEK293FT cells transfected with a retroviral expression vector expressed even lower protein levels of C/EBPα K313 compared to C/EBPα wt. This suggests that the increased amount of mutated C/EBPα in ER-Hoxb8 cells underlies neutrophil- or lineage-specific regulation. Different RNA-binding proteins might be interacting with C/EBPα mRNA in neutrophils and other cell types and thereby cause the differences in protein expression. Protein stability of C/EBPα K313 was not significantly enhanced compared to wt C/EBPα and can also not explain the differences in protein levels observed in ER-Hoxb8 neutrophils (Figure 26).

We conclude that translation accounts for the increased protein levels of C/EBPα K313, as both transcription and protein stability are not significantly altered. Unfortunately, neither in vitro translation nor tracking of de-novo synthesized protein have so far been able to give information on this due to technical issues.

Discussion

C/EBPα29B and its influence on proliferation of Hoxb8 neutrophil progenitor cells In addition to its role as a key regulator of neutrophil granulocyte development, C/EBPα has been reported to be an important suppressor of cell proliferation. Several mechanisms for cell cycle regulation have been proposed, among them induction of p21/cdki (Timchenko et al., 1996) and interaction with cdk2/4 (Wang et al., 2001). Repression of the E2F complex by C/EBPα seems to be the most important way of regulating proliferation in neutrophils (Johansen et al., 2001; Porse et al., 2001; Slomiany et al., 2000) while cdk2/4 is dispensable (D'Alo' et al., 2003; Porse et al., 2001; Wang et al., 2003). This would suggest that mutations of C/EBPα regions important for binding to the E2F complex lead to increased proliferation of cells bearing those mutations.

Expression of wt C/EBPα did not have a strong effect on the proliferation rates of ER Hoxb8 neutrophil progenitor cells in our system (Figure 10, Figure 11). We observed a slight decrease and a minor increase in cell numbers of two cell lines analysed, expressing FLAG-tagged and untagged C/EBPα wt protein. However, different parental cell lines were used to generate C/EBPα wt overexpressing cell lines. Therefore, the difference observed between those cell lines might be explained by variance. Both empty vector and C/EBPα K313 expressing Hoxb8 neutrophil progenitor cells showed a similar rate of proliferation which was higher than the one of wt C/EBPα expressing cells. Empty vector transduced cells hardly express any endogenous C/EBPα at the progenitor stage and therefore may exhibit less E2F repression. Knowing that C/EBPα K313 levels are drastically increased already in progenitor cells, the rate of proliferation leads to the assumption that this C/EBPα mutation is unable to restrict proliferation. This could be due to defective repression of the E2F complex. Although the N-terminal transactivation element essential for this interaction is intact, the BR-LZ region needed for DNA binding (amongst others to E2F) is altered and might not be able to exercise its function. Nevertheless, we observed increased proliferation rates of Hoxb8 neutrophils expressing C/EBPα K313 upon withdrawal of estrogen, which can probably explained by the maturation defect seen in these cells (Figure 14).

Discussion

In mice carrying the BRM2 mutation in the cebpa-locus, a high proliferative potential has been described for myeloid progenitors (Porse et al., 2005). It was also reported that C/EBPα BRM2 was unable to repress E2F transcription (Porse et al., 2005; Porse et al., 2001). We did not observe increased proliferation by cells expressing the C/EBPα BRM2 mutation. Rather, the opposite was the case, with substantially reduced proliferation of ER Hoxb8 neutrophil progenitor cells. Progenitor cells proliferated even at a slower rate than cells expressing wt C/EBPα.

Endogenous C/EBPα protein levels are very low at the Hoxb8 neutrophil progenitor stage. Therefore, the effect of endogenous C/EBPα protein on cell numbers of our Hoxb8 proliferation studies is likely to be very small. In addition, it is unclear whether C/EBPα K313 is able to form functionally active heterodimers with wt C/EBPα proteins. The lysine duplication at position 313 alters the distance between basic region and leucine zipper of C/EBPα proteins. It has been reported that alteration of this distance affects the orientation of the amino acid residues of the basic region involved in DNA binding (Pu and Struhl, 1991). Therefore, a heterodimer composed of C/EBPα wt and C/EBPα K313 might not be able to recognize its target DNA. However in the C/EBPα BRM2 mutation, the distance between basic region and leucine zipper is not affected. Hence, heterodimers composed of wt C/EBPα and C/EBPα BRM2 should be able to form a functional DNA-binding domain. Still, the amino acid substitutions of C/EBPα BRM2 proteins have been reported to affect residues involved in DNA recognition (Porse et al., 2001) and might reduce binding specificity of C/EBPα dimers to target DNA.

The results obtained from liquid culture proliferation studies were confirmed by colony assays, showing smaller and fewer colonies upon wt C/EBPα expression and slightly bigger and more colonies upon C/EBPα K313 expression compared to vector control cells (Figure 13). A hyperproliferative phenotype of C/EBPαK313/K313 HSC-like cells was reported by Bereshchenko et al., although they did not observe increased proliferation in a heteroallelic setting (C/EBPαK313/wt) and concluded that mutation of both C/EBPα alleles was necessary for C-terminal mutations to take effect (Bereshchenko et al., 2009). However, in our setting with endogenous C/EBPα wt and overexpressed C/EBPα K313, we observed a mild difference in proliferation rates.

Discussion

For the two different isoforms of C/EBPα, different functions have been suggested. p30 C/EBPα has been suggested to be incapable of arresting cell cycle while p42 was proposed to lead to cell cycle exit and terminal differentiation (Calkhoven et al., 2000; Lin et al., 1993; Umek et al., 1991). However, all these studies were performed in different, non-haematopoietic cell types (3T3-L1 fibroblast-like cells which differentiate into adipocyte-resembling cells). We did not observe a difference in proliferation rates of p30 and p42 C/EBPα (Figure 11). In a model using C/EBPαp30/p30 mice, there was no increase in proliferation of HSC-resembling cells (Bereshchenko et al., 2009). Although these cells have a different lineage-generating potential than our ER-Hoxb8 progenitor cells, they are closer to our system than adipocyte-generating fibroblast cells.

Co-expression of N- and C-terminal C/EBPα mutations did not result in increased total cell numbers of Hoxb8 neutrophil progenitors, although C/EBPα wt/p30 and C/EBPα K313/p30 expressing cell lines showed enhanced proliferation compared to p30 C/EBPα and C/EBPα BRM2/p30 expressing lines. HSC-resembling C/EBPαK313/p30 mouse cells were shown to escape homeostatic control of cell numbers (Bereshchenko et al., 2009) but again, they still have a higher self-renewing potential than Hoxb8 neutrophil progenitors.

The effects of C/EBPα mutations on proliferation seem to be very specific among cell types and their differentiation status. Therefore, differences in fibroblast proliferation need not be reproducible in granulocytes. They might be due to different sets of transcription factors interacting with C/EBPα.

Diffe30B rentiation of ER-Hoxb8 cells is impaired upon expression of N- or C-terminally mutated C/EBPα AML-associated mutations of C/EBPα affect either the C-terminal BR-LZ or abolish translation of the full-length (42 kDa) protein, leading to increased expression of the N-terminally truncated 30 kDa isoform. We analysed the differentiation potential of Hoxb8 neutrophils expressing N-terminal and C-terminal mutations alone or in combination.

Discussion

The striking maturation defect of neutrophils we observed upon expression of C/EBPα K313 has not been reported so far. A lineage commitment defect was observed in a mouse model with cells carrying a C/EBPα allele with the K313 mutation (Bereshchenko et al., 2009). Fetal liver-derived HSC-resembling cells homozygous for the C/EBPα K313 mutation were unable to generate cells with granulocytic maturation. Downregulation of myeloid-specific gene expression in C/EBPαK313/K313 and C/EBPαK313/p30 hematopoietic stem cells was observed, accompanied by upregulation of erythroid-specific genes. The ER-Hoxb8 cells used in this study were more committed than the knockin cells used by Bereshchenko et al. They were able to start differentiating but did not complete terminal maturation (Figure 15). The additional lysine at position 313 might alter the ability of leucine zipper formation or interaction of the basic region with its target genes. Both would result in a failure to activate downstream effectors essential for differentiation and explain the maturation defect observed in this study.

The slightly reduced differentiation observed upon C/EBPα BRM2 expression might also be due to defective interaction with maturation-associated target genes. The two affected amino acids have been reported to reside on the non-DNA binding surface of the BR but to contribute to binding specificity (Porse et al., 2001). It has been published that these residues account partially for E2F repression (Porse et al., 2001), which is necessary for granulocytic differentiation (Slomiany et al., 2000). It has been reported that C/EBPα BRM2 knockin mice display defects in adipocyte and neutrophil maturation (Porse et al., 2001). Nevertheless, it was published that adult C/EBPα BRM2 mice might recover granulopoiesis if the balance between E2F and C/EBPα was readjusted (Porse et al., 2005). Keeshan et al. reported a reduced mobility of C/EBPα BRM2-DNA complexes compared to those with wt C/EBPα (Keeshan 2003). They suggested this might be due to an altered protein complex binding to the promotor region of target genes. In addition to its DNA-binding defect, the BRM2 mutation has been reported to show augmented binding to the E2F dimerization partner DP (Zaragoza et al., 2010). A balancing mechanism was suggested in which C/EBPα switches from a transcriptional active, DNA-bound state to a DP-bound transcriptional inactive state. This would render C/EBPα BRM2 more susceptible to transcriptional inactivation and could explain the slightly delayed maturation observed in our model.

Discussion

N-terminal truncation of C/EBPα in our model likewise resulted in a decrease of CD11b/Gr-1 double-positive, mature neutrophils when Hoxb8 was turned off. This is consistent with the observation that p30 C/EBPα, lacking the N-terminal transactivation elements TE-I and TE-II, fails to repress E2F activity (Johansen et al., 2001) and is therefore unable to execute terminal granulopoiesis. Studies on NIH3T3 fibroblasts transduced with a C/EBPα encoding retrovirus carrying mutations in the transactivation elements I-III have shown that TE-I and non-DNA binding residues of the BR α-helix are required for E2F repression (Porse et al., 2001). If C/EBPα TE-I function is inhibited, fibroblasts are unable to form mature adipocytes (Calkhoven et al., 2000). ER-Hoxb8 neutrophils expressing p30 C/EBPα kept on proliferating during differentiation more than control, C/EBPα wt and p42 expressing cell lines, which was also reported for 3T3-L1 fibroblasts expressing p30 C/EBPα (Calkhoven et al., 2000).

A dominant-negative effect of p30 C/EBPα on the p42 isoform has been reported earlier (Gombart et al., 2002; Pabst et al., 2001b) and can also observed upon expression of wt and p30 C/EBPα in our ER-Hoxb8 cell model. These cells showed reduced expression of the neutrophil maturation markers CD11b and Gr-1 on day 4 of differentiation. AML cells also showed reduced wt C/EBPα activity on target genes when a monoallelic N-terminal truncation occurred (Pabst et al., 2001b). Fetal liver-derived HSC-like cells carrying C/EBPαK313/p30 mutations transplanted into mice have been reported to lead to lethal leukaemia with faster progression than C/EBPα K313 or p30 homozygous mutations (Bereshchenko et al., 2009). We observed a very immature state of Hoxb8 cells expressing these mutations. Interestingly, we did not observe a dominant-negative effect on differentiation of p30 C/EBPα in BRM2- expressing ER-Hoxb8 cells. This could be due to altered binding affinities of C/EBPα BRM2 to its target genes.

It remains unclear whether C/EBPα proteins carrying C-terminal mutations (like C/EBPα K313 and C/EBPα BRM2) are able to form dimers, either homo- or heterodimers. Loss of dimerization capability and therefore a loss-of-function was suggested for these mutations, which could even be extended to other C/EBP proteins (Gombart et al., 2002). Studies on CEBP and GCN4 (a member of bzip family proteins in yeast) indicated that the leucine zipper is important for correct orientation of the basic region for DNA binding. They showed formation of fully

Discussion functional mutant homodimers (as long as insertion mutations met an integral number of α-helical turns), but heterodimers of wt and mutated proteins (their existence was proved by crosslinking experiments) were unable to bind to DNA (Pu and Struhl, 1991). It remains to be clarified whether the dominant-negative effect reported for p30 C/EBPα on p42 is based on the formation of heterodimers.

Having observed substantial differences in maturation of C/EBPα wt/K313 expressing ER-Hoxb8 neutrophils in vitro, we were surprised to note that these effects were not seen to this extent in an in vivo model. We observed similar overall cell counts of mice engrafted with empty vector, C/EBPα wt and C/EBPα K313 expressing ER-Hoxb8 neutrophils in peripheral blood and bone marrow. However in the spleen, leukocyte cell counts of C/EBPα K313 mice were slightly increased. Taken together, this shows that engraftment of all three cell lines analysed worked similarly well. Looking at GFP-positive cells only (ER-Hoxb8 neutrophil progenitor-derived cells) we observed a slight increase of C/EBPα wt and C/EBPα K313 expressing Hoxb8 neutrophils in the peripheral blood on day 6 after transfer. This could be due to increased survival of these cells, which is plausible since they express higher levels of anti-apoptotic proteins (Mcl-1 and Bcl-2) than empty vector expressing cells.

Cells released into the blood are mature and will die by apoptosis soon unless they encounter a pro-inflammatory stimulus. Two days later, the proportion of GFP- positive cells in the blood had dropped similarly in all cell lines. C/EBPα K313 expressing Hoxb8 neutrophils stayed at an equal proportion at least until day 10 after transfer. In the bone marrow, only C/EBPα K313 cells were detectable on day 8 after transfer. These cells had also almost completely disappeared by day 10.

It is likely that this difference in cell numbers is mainly due to delayed differentiation and that empty vector and wt C/EBPα expressing cells have already reached complete maturation and have started to die by day 8 after transfer. Hardly any GFP- positive cells were detectable in the spleen on day 8 or 10 after transfer. In another mouse model, FL-derived HSC-resembling cells heterozygous for the C/EBPα K313 duplication (C/EBPαwt/K313) did not show increased expansion 4.5 weeks after competitive transplantation together with wt BM cells (Bereshchenko et al., 2009). K313 mutation of both C/EBPα alleles resulted in elevated LSK (Lin- Sca-1+ c-kit+) cell numbers, a cell type also termed phenotypic HSCs. If C/EBPαK313/K313 or

Discussion

C/EBPαK313/p30 cells were cotransplanted together with wt BM cells, the frequency of LSK cells was increased relative to wt control cells. This was interpreted to be mainly due to decreased quiescence of C/EBPα mutant HSCs. In this model, only transplantation of the HSC-containing fraction and not a myeloid progenitor or differentiated tumour cell fraction of the mutation-bearing cells led to lethal leukaemia in the recipient mice. The ER-Hoxb8 progenitor cells transferred in our system are at a more committed stage. Therefore, they might lack the potential to induce leukaemia.

Surprisingly, C/EBPα K313 expressing Hoxb8 neutrophils became fully mature in vivo (as judged by Gr 1 expression) and did not exhibit the strong maturation defect observed in vitro. This can probably be explained by cytokines and interactions with other cell types which are present in a mouse model but not under the conditions of in vitro culture. Most C/EBPα K313 expressing ER-Hoxb8 neutrophils in the blood were CD11b/Gr-1 double-positive on day 6 after adoptive transfer, a time point when empty vector and wt C/EBPα expressing cells started to decline already. This may suggest that although C/EBPα K313 cells are able to differentiate, their maturation is still delayed. It may also mean that an element of self-renewal of the progenitor cells is present in these cells while in the bone marrow, and the cells are therefore able for a longer time to produce neutrophils. C/EBPα K313 expressing mature neutrophil numbers declined at later stages in all organs analysed, indicating that the pool of immature C/EBPα K313 ER-Hoxb8 cells in the BM cannot be maintained much longer.

In the mouse model using C/EBPαK313/K313 FL-derived cells, 25 % of the tumours found could be classified as being myeloid with maturation (Bereshchenko et al., 2009), but the majority of tumours displayed a more immature phenotype.

Hoxb831B neutrophil effector functions are altered dependent on different C/EBPα mutations The differentiation block described in AML does not only influence cell counts in the bone marrow and periphery but also the ability of cells to fulfill their effector functions.

Discussion

Neutrophils that are arrested in their development may not be able to form all different kinds of granules or phagocytose pathogens efficiently.

Unlike normal hematopoietic cells, blasts from AML patients have been shown to constitutively express cytokines like IL-8 and GM-CSF (Bradbury et al., 1990; Tobler et al., 1993). We observed expression of IL-6 by Hoxb8 progenitor cells expressing C/EBPα K313 already in the absence of stimuli. Upon stimulation with LPS, ER-Hoxb8 cells expressing C/EBPα K313 were very well able to synthesize and secrete cytokines like TNF and IL-6, even at higher levels than wt C/EBPα expressing cells. It is possible that the increased amount of mutated C/EBPα protein, despite its mutation inside the BR-LZ, is able to form dimers capable of activating target genes and therefore driving cytokine production. Nevertheless, synthesis of the primary granule protein neutrophil elastase was not detectable in ER-Hoxb8 cell expressing C/EBPα K313 and severely diminished in C/EBPα BRM2 cells. For 32Dcl3 cells (a murine myeloid cell line able to differentiate into neutrophils) transduced with C/EBPα BRM2, decreased levels of NE have been found (Keeshan et al., 2003). The lack of neutrophil elastase expression in C/EBPα K313 expressing cells might be due to delayed maturation, although this seems unlikely as NE is expressed already at early stages of differentiation. In addition, C/EBPα BRM2 expressing cells, which exhibit only a minor defect in differentiation, have severely diminished NE levels. Another possibility for reduced effector functions would be reduced dimer formation and therefore insufficient activation of target genes. C-terminal mutations of C/EBPα have often been reported to be defective in DNA- binding and transactivation of effector genes (Gombart et al., 2002; Pabst et al., 2001b). However, our luciferase reporter assay revealed strong transactivation potential of C/EBPα K313. It is not clear whether this is due to increased protein levels or altered interaction with other transcription factors. C/EBPα K313 expressing Hoxb8 neutrophils do not differentiate or downregulate C/EBPα protein levels. Therefore it was surprising that reporter activity declined during differentiation similar to C/EBPα wt expressing cells. So far it is not clear whether mutations inside the C/EBPα BR-LZ allow formation of functionally active homo- or heterodimers. Still, it is possible that wt C/EBPα and mutated C/EBPα with alterations in the BR-LZ cooperate with other transcription factors and therefore keep the ability to activate certain subsets of target genes.

Discussion

The N-terminal truncation mutant p30 exhibited transactivation potential. This is not surprising as its BR-LZ domain is intact, therefore the formation of transcriptionally active dimers should not be affected. It is also possible that heterodimers (consisting of p30 and p42 C/EBPα) with endogenous C/EBPα may be formed. We observed even stronger activation of the luciferase reporter by p30 than by wt C/EBPα, which might be linked to increased protein levels of p30 C/EBPα. This is consistent with reports on efficient DNA-binding of mouse p30 C/EBPα in liver cells (An et al., 1996; Calkhoven et al., 1994; Lin et al., 1993). In terms of DNA binding, there seems to be a difference between rodent and human p30 C/EBPα. The human N-terminal p30 truncation mutation is formed together with a truncated 20 kDa protein and binds DNA much less efficient than full-length C/EBPα. Pabst et al. reported a decrease in binding to the GSF3R-promoter by human p30 when transfected into CV1 kidney cells (Pabst et al., 2001b). Two N-terminal deletion mutations (255–279del and 263– 269del) showed around 80 % reduced transactivation of the GSF3R-promoter (Pabst et al., 2001b).

C/EBPα is a nuclear protein. Abnormal localization of mutated C/EBPα in patients suffering from AML could explain the dominant-negative effects on differentiation and effector functions of granulocytes. Immunostainings of different N-terminal mutants revealed localisation in the nuclei of transfected cells indistinguishable from wt C/EBPα (Gombart et al., 2002; Pabst et al., 2001b). The same was true upon co- transfection of wt and mutant C/EBPα plasmids. This suggests that effects of C/EBPα mutants on wt protein are not mediated by abnormal localization of mutated C/EBPα proteins.

We conclude that the capability of fulfilling effector functions is not necessarily dependent on neutrophil maturation status but on the ability of C/EBPα proteins to physically interact with each other and with target genes. While some effector functions stay intact (like cytokine production and transactivation of a reporter construct), BR-LZ mutations were shown to abolish others (like expression of neutrophil elastase).

Discussion

Cell32B death of Hoxb8 neutrophils Apoptosis is a normal process and the end of the neutrophil’s life. We monitored the rate of spontaneous cell death during differentiation of ER-Hoxb8 neutrophils. Although there were differences in survival of progenitor and mature neutrophils expressing wt and mutant C/EBPα, we did not detect drastically increased survival of cells expressing AML-associated mutations of C/EBPα. On day five of differentiation, most cells had stopped differentiating and underwent apoptosis. Expression of wt C/EBPα increased the survival rate as well as protein levels of Mcl-1 compared to empty vector expressing cells. C/EBPα has been reported to promote cell survival in cooperation with NF-κB p50 (Paz-Priel et al., 2011). Also, its dimerization partner E2F regulates genes of cell cycle progression and apoptosis (Muller et al., 2001; Ren et al., 2002). The difference in viability between C/EBPα K313 and C/EBPα BRM2 expressing cell lines was surprising as both cell lines express lower Bim levels than empty vector and wt C/EBPα expressing cell lines. They also show a similar pattern of Mcl-1 expression, the most important anti-apoptotic protein of neutrophils (Kirschnek et al., 2011). One difference between C/EBPα K313 and C/EBPα BRM2 expressing ER-Hoxb8 cells seems to be the failure to downregulate Bcl-2 at later stages of differentiation in the C/EBPα K313, but not the C/EBPα BRM2 cell line. Bcl-2 induction by C/EBPα leucine zipper oncoproteins has been reported to be dependent on the interaction of the basic region with NF-κB p50 bound to the promotor region of target genes (Paz-Priel et al., 2011). Mature granulocytes in a mouse model carrying the BRM2 mutation in the cebpa-locus were reported to have accelerated apoptosis due to increased E2F activity (Porse et al., 2001). As the mutations in the basic region interfere with complete E2F repression, free E2F might induce apoptosis. This effect has been suggested based on observations in 32Dcl3 cells, a lineage-negative mouse BM cell line able to form mature granulocytes upon treatment with G-CSF.

Conclusion33B C/EBPα is a crucial transcription factor in neutrophil granulocytes and is composed of a number of functionally important domains. N-terminal transactivation elements and

Discussion elements of the C-terminal BR-LZ collaborate to exert C/EBPα functions in cell cycle arrest and differentiation.

We conclude that increased protein levels of C-terminally mutated C/EBPα observed in our Hoxb8 model probably result from increased translation, perhaps due to mRNA secondary structure changes. C/EBPα mRNA levels and protein stability were similar and make the possibilities of expressional issues or post-translational modifications of mutated C/EBPα proteins unlikely as the cause of the observed protein level differences.

E2F repression by C/EBPα has been reported be an important mechanism for the regulation of cell cycle arrest and differentiation in neutrophils. Increased E2F repression by overexpressed C/EBPα wt protein could explain slower proliferation rates, while a failure to repress E2F may account for accelerated proliferation upon C/EBPα K313 expression. The C/EBPα BRM2 mutation leads to decreased viability and less proliferation, which could be explained by defective E2F repression, leading to free E2F which has been reported to induce apoptosis (Porse et al., 2001). The maturation potential of cells expressing the C/EBPα K313 mutation seems to be dependent on the cell’s commitment status as well as on external stimuli, as suggested by delayed maturation of C/EBPα K313 neutrophils in our in vivo model. The additional lysine increasing the distance between leucine zipper and basic region might interfere with dimerization of C/EBP proteins or with binding to target genes.

This could also explain the differences in the capability to fulfill effector functions. Slightly reduced differentiation upon C/EBPα BRM2 expression as well as the lack of neutrophil elastase expression could be explained by the mutation in the non-DNA binding residues which have been reported to confer specificity for interaction partners. Additionally, augmented binding to the E2F dimerization partner DP might render C/EBPα BRM2 molecules more susceptible to transcriptional inactivation, as has been supposed by Zaragoza et al (Zaragoza et al., 2010). It is conceivable that endogenous C/EBPα present in our Hoxb8 model is able to attenuate the effects of mutated C/EBPα proteins on differentiation. Therefore, a C/EBPα knockout cell line would be interesting to discriminate between dominant-negative and loss-of-function effects of the studied C/EBPα mutations.

Material and Methods

VII. Material6B and Methods

1. Material34B

1.1. Mice52B

Genotype Company

C57Bl/6 Janvier

C/EBPαfl/wt Fritz-Lipmann Institut, Jena

LysMCretg Prof. Dr. Marco Prinz, Universitätsklinikum Freiburg

1.2. Cell53B lines

Name Description Medium Company

Hoxb8 neutrophil Hoxb8 immortalised Optimem Established during progenitor cells murine neutrophil this study + 10 % FCS (heat progenitor cells inactivated)

+ 1 % Pen/Strep

+ 30 µM β-mercaptoethanol + 1 % SCF supernatant

+ 1 µM β-estradiol

Phoenix-ECO Human embryonic DMEM Invitrogen kidney cell line + 10 % FCS transgenic for + 1 % Pen/Strep ecotropic envelope protein used for retrovirus production

Material and Methods

HEK 293 FT Human embryonic DMEM Invitrogen kidney cell line used + 10 % FCS for lentivirus +1 % Pen/Strep production

Chinese hamster Stem cell factor Optimem ovary cells (SCF)-producing + 10 % FCS (heat transgenic cell line inactivated)

+ 1 % Pen/Strep

+ 30 µM β-mercaptoethanol

Human AML BM cells none Prof. Dr. Konstanze samples carrying Döhner, C/EBPα K313 Universitätsklinikum mutations Ulm

Human AML BM cells none Prof. Dr. Michael samples carrying Lübbert, C/EBPα wt Universitätsklinikum Freiburg

1.3. Culture54B media and amendments

Name Description Ordering Number Company

Optimem Glutamax Culture medium 51985-026 Gibco, Life Technologies Lot no: 181-5270

DMEM Culture medium 41965-039 Gibco, Life Technologies

Methocult™ SF Serum-free 03236 Stemcell M3236 methylcellulose-based Technologies medium for single-cell analysis

Dulbecco’s PBS Wash buffer for cell 14190-094 Gibco, Life culture Technologies

Material and Methods

Trypsin-EDTA Used for cell detachment 25300-054 Gibco, Life in culture Technologies

FCS Fetal calf serum (used for S0115 Biochrom Hoxb8 cell culture) Lot no: 0677 B

FCS Fetal calf serum (used for 10270-106 Gibco, Life Phoenix-ECO and Technologies Lot no: 41G3430K HEK293FT cell culture)

Pen/Strep Antibiotics in culture 15140-122 Gibco, Life medium Technologies

β-mercaptoethanol Reducing agent used in 31350010 Gibco, Life culture medium Technologies

SCF supernatant Survival factor used for SCF-producing culture of Hoxb8 CHO cell line neutrophil granulocytes from Hans Häcker, Memphis, USA

β-estradiol Needed for estrogen- E2758 Sigma regulated Hoxb8 expression

Puromycin Antibiotics used for cell ant-pr-1 InvivoGen selection

1.4. Viral55B Constructs

Name Description pENTR/SD/D- Vector of the Gateway cloning system used for ligation of PCR TOPO-GW products and mutagenesis pCLEco Packaging vector for retroviruses

PAX.2 Packaging vector for lentiviruses

MD2.G Packaging vector for lentiviruses pMIG-GW pMCSV-based bicistronic retroviral vector carrying the green

Material and Methods

fluorescent protein (GFP) DNA as a reporter gene used for cloning of PCR products and retroviral transductions pMIG-R1 Retroviral transduction vector expressing green fluorescent protein (GFP) pMIG-C/EBPα Retroviral transduction vector expressing C/EBPα pMIG-p30C/EBPα Retroviral transduction vector expressing p30C/EBPα pMIG-p42C/EBPα Retroviral transduction vector expressing p42C/EBPα pMIG-C/EBPα K313 Retroviral transduction vector expressing C/EBPα carrying a lysine duplication. It is named after the human C/EBPα K313 duplication at aa position 313. In mice it is actually aa position 314, but for reasons of clarity we decided to keep the same denotation as the human mutation. pMIG-p30C/EBPα Retroviral transduction vector expressing p30C/EBPα carrying a K313 lysine duplication at aa position 313 pMIG-p42C/EBPα Retroviral transduction vector expressing p42C/EBPα carrying a K313 lysine duplication at aa position 313 pMIG-C/EBPα Retroviral transduction vector expressing C/EBPα carrying two point BRM2 mutations: Ile294Ala, Arg297Ala pMIG-p30C/EBPα Retroviral transduction vector expressing p30C/EBPα carrying two BRM2 point mutations: Ile294Ala, Arg297Ala pMIG-p42C/EBPα Retroviral transduction vector expressing p42C/EBPα carrying two BRM2 point mutations: Ile294Ala, Arg297Ala pMIG-FLAG- Retroviral transduction vector expressing FLAG-tagged C/EBPα C/EBPα pMIG-FLAG- Retroviral transduction vector expressing FLAG-tagged C/EBPα C/EBPα K313 K313 pMIG-FLAG- Retroviral transduction vector expressing FLAG-tagged C/EBPα C/EBPα BRM2 BRM2 pBABE-GW puro Retroviral vector used for cloning of PCR products pBABE-Bcl-XL Retroviral vector used for expression of the anti-apoptotic protein puro Bcl-XL

Material and Methods pBABE-HA- Retroviral transduction vector expressing p30C/EBPα p30C/EBPα puro

Pf3xC/EBPα Lentiviral vector used for analysis of C/EBPα activity Luciferase Reporter pcDNA™6.2-DEST- Gateway destination vector suitable for constitutive expression in GW mammalian cell lines; used for cloning of PCR products pcDNA-C/EBPα Vector used for transfection of HEK293FT cells with C/EBPα pcDNA-C/EBPα Vector used for transfection of HEK293FT cells with C/EBPα K313 K313

1.5. Inhibitors,56B cytokines and other reagents

Name Description Ordering Number Company

Cycloheximide Translation inhibitor 01810 Sigma mIL-3 Hematopoietic growth 213-13 Peprotech (recombinant) factor mIL-6 Pleiotropic cytokine 216-16 Peprotech (recombinant)

FuGene HD Transfection E2312 Promega

reagent

DMSO Dimethyl sulfoxide; used D2650 Sigma for cryopreservation of cells

D-Luciferin derivative of firefly L9504 Sigma luciferin used for quantification of C/EBPα reporter luciferase expression

Ficoll-Paque PLUS Separation of bone 17-1440-02 GE Healthcare marrow components

Material and Methods

Propidium iodide Measurement of cell P4170 Sigma death

Red Blood Cell Lysis of red blood cells R7757 Sigma Lysing buffer

1.6. Blocking57B solutions and antibodies

Blocking80B agent

Name Application w/v Ordering Company Number

BSA Western Blot 5 % in TBS-T K45-001 GE Healthcare

Flow Cytometry 0.5 % in PBS

Nonfat dried Western Blot 5 % in TBS-T A0830 Applichem milk powder

West81B ern Blot

Primary Ab Host Dilution Diluent Catalogue Company (clone) number

FLAG mouse 1:1000 5 % milk in TBS-T F1804 Sigma

C/EBPα rabbit 1:1000 5 % milk in TBS-T 2295 Cell signalling

HA (3F10) rat 1:1000 5 % milk in TBS-T 11867423001 Roche

Mcl-1 rabbit 1:2000 5 % milk in TBS-T 600401394 Rockland

Bcl-2 hamster 1:2000 5 % milk in TBS-T 554218 BD

Bcl-XL rabbit 1:1000 5 % milk in TBS-T 2764 Cell signalling

Bim (C34C5) rabbit 1:5000 5 % milk in TBS-T 2933 S Cell signalling

Neutrophil goat 1:500 5 % milk in TBS-T sc-9521 Santa Cruz Elastase

Material and Methods

(M-18)

Fbxw7 rabbit 1:1000 5 % milk in TBS-T ARP47419- Aviva P050 Systems Biology

GAPDH mouse 1:50000 5 % milk in TBS-T MAB374 Millipore

α-Tubulin mouse 1:50000 5 % milk in TBS-T T9822 Sigma

β-Actin mouse 1:50000 5 % milk in TBS-T A5441 Sigma (AC-15)

Secondary Host Dilution Diluent Catalogue Company Ab (clone) number

Rabbit IgG goat 1:10000 5 % milk in TBS-T A6667 Sigma

Mouse IgG goat 1:10000 5 % milk in TBS-T 115035166 Dianova

Hamster IgG goat 1:10000 5 % milk in TBS-T 127035160 Dianova

Rat IgG goat 1:10000 5 % milk in TBS-T 112-035-062 Dianova

Goat IgG donkey 1:10000 5 % milk in TBS-T 705-495-147 Dianova

FACS82B staining

Antibody Conjugate Dilution Catalogue Number Company

Fc block 1:300 553142 BD

CD11b PE 1:300 12-0112-82 e-Bioscience

Gr-1 APC 1:300 553129 BD c-kit APC 1:300 17-1172 e-Bioscience

CXCR2R Alexa Fluor 647 1:300 129101 Biolegend

CXCR4R APC 1:300 558644 BD

Material and Methods

1.7. Buffers58B and solutions

Material83B for SDS-Page and Western Blot

Name Catalogue Company Number

DC Protein Assay 500-0116 BioRad

EZ-Run™ 12.5 % Protein Gel Solution BP7712-500 Thermo Fisher Scientific

Ammonium persulfate (APS) A3678 Sigma

Tetramethylethylenediamine (TEMED) T9281 Sigma

EZ-Run™ Running buffer 20x BP7700-500 Thermo Fisher Scientific

Amersham Hybond PVDF 0.45 μm 10600023 GE Healthcare

TWEEN® 20 P2287 Sigma

Nonfat dried milk powder A0830 Applichem

PageRuler™ Prestained Protein Ladder 26617 Thermo Fisher Scientific

SuperSignal™ West Pico 34080 Thermo Fisher Scientific Chemiluminescent Substrate

Amersham ECL Prime Western Blotting RPN 2232 GE Healthcare Detection Reagent

SuperSignal™ West Femto Maximum 34096 Thermo Fisher Scientific Sensitivity Substrate

Buffers84B for Western Blot

Buffer Components

6x SDS-sample buffer 380 mM Tris/HCl pH 6.8, 33 % Glycerol, 1 % 2-mercaptoethanol, 10 % SDS

Transfer buffer 25 mM Tris/HCl, 190 mM Glycine, 20 % Methanol

10x TBS 24 g Tris, 80 g NaCl in 1 l H2O ; pH 7.6

TBS-T 1x TBS, 0.05 % Tween 20

Material and Methods

Molecular85B biology kits and bacteria

Name Description Catalogue Company Number pENTR/SD/D-TOPO Cloning of PCR products into K242020 Thermo Fisher Cloning kit Gateway vectors Scientific

Gateway LR Subcloning of expression 11791-020 Thermo Fisher Clonase II Enzyme constructs into Gateway vectors Scientific mix

Herculase II Fusion PCR Polymerase (proof-reading) 600679 Agilent Enzyme kit Technologies

One Shot® TOP10 bacterial strain used for cloning C404003 Thermo Fisher Chemically procedures Scientific Competent E.coli

One Shot® Stbl3™ bacterial strain used for cloning C737303 Thermo Fisher Chemically procedures with Gateway vectors Scientific Competent E.coli

Pure Yield Plasmid Purification of plasmid DNA from A1222 Promega miniprep system bacterial cultures

Pure Yield Plasmid Purification of plasmid DNA from A2495 Promega midiprep system bacterial cultures

Wizard SV Gel and Purification of PCR products and A9282 Promega PCR clean-up agarose-gel separated DNA system

High Puro RNA RNA purification 11 828 665 Roche Isolation kit 001

Transcription First cDNA synthesis 04 897 030 Roche Strand cDNA 001 Synthesis kit

Light Cycler Taq qPCR 04 735 536 Roche Man Master kit 001

Universal Probe qPCR 04 683 633 Roche Library set 001

Material and Methods

Primers86B

Primer Sequence (5’ to 3’) mC/EBPα fwd (UPL Probe #67) mC/EBPα rev (UPL Probe #67) mAktin fwd (UPL Probe #64) mAktin rev (UPL Probe #64)

C/EBPα Fwd CGG CGG CGC GGT CAT ATC CGC GGG

C/EBPα Rev CCC GCG GAT ATG ACC GCG CCG CCG

C/EBPα N-terminal Fwd TCA GAA TTG GTT AAT TGG TTG TAA CAC TGG CAG AGC ATT ACG

C/EBPα N-terminal Rev GCG GAT ATG ACC GCG CCG CCG GG

C/EBPα C-terminal Fwd GCG CGG TCA TAT CCG CGG GGG CG

C/EBPα C-terminal Rev TTA GAA AAA CTC ATC GAG CAT CAA ATG AAA CTG

C/EBPα K313 duplication Fwd GAG ACG CAA CAG AAG AAG GTG CTG GAG TTG ACC

C/EBPα K313 duplication Rev GGT CAA CTC CAG CAC CTT CTT CTG TTG CGT CTC

C/EBPα BRM2 Fwd CGG GAA CGC AAC AAC GCC GCG GTG GCC AAG AGC CGA GAT AA

C/EBPα BRM2 Rev TTA TCT CGG CTC TTG GCC ACC GCG GCG TTG TTG CGT TCC CG

Q5 C/EBPα FLAG Fwd GAT GAT GAT AAA GAG TCG GCC GAC TTC TAC

Q5 C/EBPα FLAG Rev ATC TTT ATA ATC CAT GGT GAA GGG CTC CTT C

Q5 p30C/EBPα FLAG Fwd GAT GAT GAT AAA TCC GCG GGG GCG CAC GGG

Q5 p30C/EBPα FLAG Rev ATC TTT ATA ATC CAT GGT GAA GGG CTC CTT CTT AAA GTT AAA CAA GGC

Material and Methods pENTR-HA-p30C/EBPα Fw GCC GGA TTA TGC GTC CGC GGG GGC GCA CGG G pENTR-HA-p30C/EBPα Re ACA TCA TAC GGA TAC ATG GTG AAG GGC TCC TTC TTA AAG TTA AAC AAG GCG GC

C/EBPα-Luc Fwd (PacI) GCC TTT AAT TAA ATT GCG CAA TTT GAT ATC GGA

C/EBPα-Luc Rev (BamHI) GTG GGG ATC CTT ATC GAT TTT ACC ACA TT

LysMCre Fwd 139 CCC AGA AAT GCC AGA TTA CG

LysMCre Rev 140 CTT GGG CTG CCA GAA TTT CTC

1.8. Electronic59B Devices

Name Company

CASY® Cell Counter Roche

Centrifuge 5430R Eppendorf

Centrifuge Multifuge 3SR Plus Heraeus

Centrifuge Multifuge™ X3 Heraeus

Incubator BBD 6620 Thermo Fisher Scientific

PCR machine T3000 Biometra

NanoDrop® ND-2000 Thermo Fisher Scientific

Blotting chamber Trans-Blot® Cell Bio-Rad

Developing machine WB Curix 60 AGFA

Sonifier Bioruptor diagenode plus Diagenode

Thermomix comfort Eppendorf

Power Supply Power Pac™ Universal Bio-Rad

Power Supply Power Pac™ HC Bio-Rad

Shaker Polymax 2040 Heidolph

Microplate reader SpectraMax 340 PC Molecular Devices

Tecan infinite M200 Tecan Trading AG

Material and Methods

Microscope Axiovert 40C + AxioCam ERc 5S Zeiss

FACS Canto II Becton Dickinson

Light Cycler® 2.0 Roche

2. Methods35B

2.1. Cell60B Culture Hoxb8 neutrophil progenitors were cultured in Optimem including 10 % FCS (heat inactivated), 1 % Pen/Strep and 30 µM β-mercaptoethanol with freshly added stem cell factor (SCF) supernatant produced by SCF transgenic Chinese Hamster Ovary cells and 1 µM β-estradiol. They were cultured in 6-well plastic dishes and split every 2-3 days.

Cell culture was performed using standard culture conditions (37°C, 5 % CO2) in a humidified incubator and cells were replaced every 6 weeks by a freshly thawed stock of cells. For seeding of cells for experiments, they were counted either by using a Neubauer chamber or a Casy cell counter. For freezing, cells were spun and resuspended in progenitor outgrowth medium to which FCS mixed with 20 % DMSO was added to obtain a final concentration of 10 % DMSO. Cells were frozen slowly using isopropanol containing freezing boxes placed at -80°C and transferred to liquid nitrogen 4 days later.

2.2. Generation61B of Hoxb8 neutrophil progenitor cells Bone marrow cells were collected by flushing femurs and tibia of C57Bl/6 mice of corresponding genotype with RP-10 media (RPMI including 10 % FCS, 1 % Pen/Strep and 30 µM β-mercaptoethanol) into sterile petri dishes filled with 10 ml of media. 10 ml syringes fitted with 26 G needles were used. Bone marrow cells were then transferred to 50 ml conical tubes for each mouse and pelleted (1500 rpm, 5 min). Pellets were resuspended carefully in 2 ml of ACK red blood cell lysis buffer and incubated for 2 min at RT. Lysis was stopped by adding 18 ml RP-10 media. Cell suspensions were washed and resuspended in 4 ml PBS with 1 % FCS, loaded onto 3 ml Ficoll-Paque and separated by centrifugation at 450 g for 30 min. The supernatant was collected and washed with PBS 1 % FCS at 800 g for 10 min and

Material and Methods again at 400 g for 5 min. Cells were then resuspended in stem cell medium (RP-10 media supplemented with recombinant mouse cytokines IL-3 (10 ng/ml) and IL-6 (20 ng/ml) as well as 1 % SCF supernatant) and cultured for 2-3 days. Then, cells were washed and resuspended in progenitor outgrowth medium (Optimem Glutamax supplemented with 10 % FCS, 1 % Pen/Strep, 30 µM β-mercaptoethanol, 1 % SCF supernatant, 1 µM β-estradiol). 1x105 cells/250 µl progenitor outgrowth medium were infected with 500 µl Hoxb8 retrovirus supernatant supplemented with 5 µg/ml polybrene in a 12-well plate by spin infection at 1500 g for 90 min at 25-30°C. 1 ml of progenitor outgrowth medium was added to the cells after spinning. The cells were cultured for 3-4 weeks until the cell populations were stably expanding.

For generation of wt/Lef1-/- neutrophil progenitor cells, fetal liver at E18.5 were removed and smashed through nylon meshes to prepare single-cell suspensions in RP-10 media. Blood cell lysis, Ficoll-Paque and cytokine pre-stimulation were performed as with bone marrow cells.

2.3. Retrovirus62B production and transduction of Hoxb8 neutrophil progenitor cell lines Retrovirus for transduction of Hoxb8 cell lines was generated by transient transfection of the Phoenix-ECO retrovirus producer cell line with a retroviral construct (pMIG or pBABE vector, 7.5 µg) in combination with the packaging vector pCL-ECO (2.5 µg). 3.5x106 Phoenix-ECO cells were seeded the day before transfection into a 10 cm culture dish. FuGene HD (Promega) was used as a transfection reagent. 10 µg DNA in total were added to 400 µl serum-free medium (Optimem Glutamax) followed by 40 µl FuGene HD and incubation for 15 min at room temperature. Then, the transfection mixture was added dropwise onto the

Phoenix cells. After 8 h of incubation (37°C, 5 % CO2) medium was exchanged. 48 hours post transfection, virus supernatant was harvested by collecting cell supernatant and centrifugation at 1942 g (3000 rpm) for 5 min followed by filtration through a 0.45 µm filter. The retrovirus supernatant was then used to transduce Hoxb8 cells by spin infection. For this, 5x104 cells were resuspended in 500 µl retrovirus supernatant supplemented with 1 % SCF supernatant, 1 µM β-estradiol and 5 µg/ml polybrene and seeded in a 12-well plate. The plate was centrifuged at 699 g (1800 rpm) for 90 min at 25-30°C. Following spin infection, 500 µl progenitor

Material and Methods outgrowth medium was added. Selection via GFP expression or antibiotic resistence was performed 48 hours post transduction.

2.4. Transfection63B of HEK293FT cells For expression analysis of C/EBPα wt/K313, 0.5x106 HEK293FT cells were transfected in 6-well plates using the transfection reagent polyethylenimine (PEI). 100 µl Optimem were mixed with 0.25, 0.5, 1 and 2 µg of DNA (either pMIG-FLAG- C/EBPα wt/K313 and empty vector or pcDNA-C/EBPα wt/K313) and 3 µl of PEI reagent per µg of DNA. The mixture was incubated at RT for 15 min and then added to the HEK293FT cells in 3 ml of medium (DMEM supplemented with 10 % FCS, 1 % Pen/Strep). After 24 h, cell lysates were prepared and analysed by western blot.

2.5. Proliferation64B of Hoxb8 neutrophil progenitors in liquid culture To examine the proliferative capacity of Hoxb8 neutrophil progenitors, 1x105 progenitors cells were seeded in 3 ml of progenitor outgrowth medium into 6-well plates. They were counted using the Casy Cell Counter every day and re-seeded every second day to provide optimal growth conditions. The exponential growth curve shown in graphs of this work was calculated including the dilution factor which was applied with each step of cell splitting.

2.6. Proliferation65B of Hoxb8 neutrophil progenitors in semi-solid medium Semi-solid medium was used to determine the proliferative capacity of single clones of Hoxb8 neutrophil progenitors. 1x103 progenitor cells were seeded in 3 ml of Methocult medium, supplemented with 10 % FCS, 1 % Pen/Strep, 30 µM β-mercaptoethanol, 1 % SCF supernatant and 1 µM β-estradiol, into 3 cm petri dishes. These small petri dishes were placed into 10 cm petri dishes filled with 10 ml of water to provide humidified culture conditions. Cells were kept in culture for up to 8 days and pictures were taken using the Keyence fluorescence microscope to document colony growth.

2.7. In66B vitro differentiation of Hoxb8 neutrophil progenitor cells For in vitro differentiation of Hoxb8 neutrophil progenitor cells into mature neutrophil granulocytes, progenitor cells were washed twice with PBS + 10 % FCS to remove β-estradiol from the culture media. They were seeded at 1x105 cells/ml in 3 ml

Material and Methods differentiation medium (Optimem Glutamax, 10 % FCS, 1 % Pen/Strep, 30 µM β-mercaptoethanol, 1 % SCF supernatant) into a 6-well plate and cultured for 4 days.

2.8. FACS67B staining of cell surface markers FACS stainings were performed in 96-well V-bottom plates at 4°C. 5x105 cells were washed in PBS with 0.5 % BSA. To prevent unspecific binding of antibodies to Fc- receptors, Fc block was used (1:300 in PBS 0.5 % BSA, 20 min on ice). Fluorescent labelled antibodies were used (1:300) and incubated for 20 min on ice in the dark. Single stain controls for all antibodies were prepared as well as an unstained control sample. Samples were washed with PBS 0.5 %BSA and resuspended in 200 µl PBS for analysis. Analysis was done using BD FACS Canto II (BD Biosciences), results were interpreted using Flow Jo.

2.9. Cell68B death staining To examinate the proportion of dead cells in cell culture, 200 µl of cell suspension were taken out of the culture well and mixed with propidium iodide (1:1000 dilution) prior to measurement. Propidium iodide positive cells were considered as dead cells.

2.10. Giemsa69B staining For morphological analysis of differentiating Hoxb8 neutrophils, Giemsa stainings were performed. 300 µl of cell suspension were diluted in 1 ml PBS, mixed and 300 µl were transferred onto microscope slides using a Cytospin Cytocentrifuge. After centrifugation of the cells onto the microscope slides, they were fixed with methanol for 5 min and afterwards incubated in Giemsa solution (10 % Giemsa solution diluted in Weise buffer) for 40 min. Finally they were washed with water to remove excess dye and analysed by microscopy.

2.11. Lysate70B preparation, SDS-Page and Western Blotting Lysates of Hoxb8 neutrophil progenitor cells as well as differentiated neutrophils were prepared by lysing 3x106 cells in 100 µl clear Laemmli buffer at 95°C for 10 min. Following lysis, the extract was centrifuged at 20,817 g (14,000 rpm) for 5 min. Protein concentration was determined by DC Protein Assay (BioRad) and 50 µg of protein were loaded onto acrylamide gels. Proteins were separated according to their molecular weight by using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Gels were prepared by supplementing EZ-Run Protein Gel Solution

Material and Methods

(12.5 % acrylamide) with APS and TEMED and pouring the mixture into the EZ-Run gel casting system (Thermo Scientific). After 20 min polarization time, gel run was perfomed using 1x EZ running buffer at 80-100 V. To estimate protein size, a prestained protein marker (Fermentas PageRulerTM Prestained Protein Ladder, Thermo Scientific) was run in parallel to the samples. Subsequently, the separated proteins were transferred onto PVDF membranes (Amersham Hybond-P PVDF Membrane, GE Healthcare) using a wet blotting system (Bio-Rad). The PVDF membrane was activated my soaking in methanol. Transfer was performed in Transfer Buffer at 150 mA over night at 4°C.Protein loading was checked by Ponceau staining. To prevent unspecific binding of antibodies, membranes were blocked by incubating in 5 % milk powder in TBS-T for 1 hour with gentle agitation. Membranes were then incubated with primary antibody in 5 % milk overnight at 4°C and washed at least three times in TBS-T. Afterwards, the membranes were incubated with secondary antibody coupled to horse radish peroxidase (HRP) for 1 hour at RT. The membrane was washed again for at least three times with TBS-T and then incubated with chemiluminescence solution for 5 min. Chemiluminescent signals were detected either on film or using Intas Lab Image (Kapelan Bio-Imaging, Germany) and quantified using the Intas Lab Image 1D software.

2.12. Inhibition71B of translation using cycloheximide To assess proteasomal turnover of proteins, Hoxb8 neutrophil progenitor or differentiated neutrophils were seeded at 1x106 cells/ml in 3 ml of progenitor or differentiation medium. Cycloheximide, a substance blocking de novo protein synthesis, was added at 10 µg/ml for 0 h, 1 h, 2 h, 4 h, 8 h, 16 h and 24 h. As Hoxb8 neutrophils are sensitive to treatment with cycloheximide, they were transduced with a retroviral vector expressing the anti-apoptotic Bcl-2 family protein Bcl-XL. At the indicated time points, cells were harvested and lysed in 100 µl clear Laemmli buffer at 95°C for 10 min.

2.13. RNA72B extraction and cDNA synthesis Total RNA was isolated of 4x106 Hoxb8 neutrophil progenitor or differentiated cells using Trizol-Chloroform extraction followed by purification using the High Puro RNA isolation kit (Roche). Cells were washed with PBS and resuspended in 500 µl Trizol. 100 µl chloroform were added and the mixture was shaken for 15 seconds by hand.

Material and Methods

After 2-3 min incubation at RT, cell lysates were centrifuged at 12,000 g for 15 min at 4°C. The aquaeous phase on top of the Trizol was transferred to a clean Eppendorff tube and mixed with 400 µl binding buffer of the RNA isolation kit. Further steps were conducted according to manufacturer’s instructions. Subsequently, quality and quantity of isolated RNA were determined using Nanodrop (ND-2000 UV-vis Spectrophotometer, PeqLab). 1 µg of RNA was used for cDNA synthesis by reverse transcription using Transcript First strand cDNA synthesis kit (Roche).

2.14. Quantitative73B real-time PCR (qRT-PCR) Relative mRNA expression was determined by quantitative real-time PCR using the Light Cycler Taqman Master kit together with the Universal Probe Library system (Roche). A master mix containing all the components listed below except for the DNA was prepared and pipetted into light cycler capillaries (Roche) kept in pre-cooled centrifuge adapters (Roche). Afterwards, cDNA (diluted 1:50) was added to the capillaries which were centrifuged at 425 g (2000 rpm) for 15 seconds before they were mounted onto the Light Cycler Carousel.

Final concentration Primer forward (stock 20 µM) 0.4 µM Primer reverse (stock 20 µM) 0.4 µM UPL probe 0.2 µM 5x Taq Man Master water cDNA Total volume 20 µl

Relative expression of the target gene was normalized to HPRT reference gene expression.

The PCR program used is shown below.

UNG Incubation 40°C 2 min 1 cycle Pre-Incubation 95°C 10 min 1 cycle Amplification 95°C 10 s 45 cycles

Material and Methods

60°C 30 s 72°C 10 s Cooling 40°C 30 s 1 cycle

2.15. Luciferase74B reporter assay To perform reporter assays to examine C/EBPα activity during differentiation, Hoxb8 neutrophil progenitor cells were transduced with the lentiviral construct pf3xC/EBPα- Luciferase-Reporter carrying a puromycin resistance gene. Cells were selected for two weeks using 3 µg/ml puromycin before any experiments and keep in progenitor outgrowth medium supplemented with puromycin all the time. For the reporter assay, cells were differentiated on 4 consecutive days to obtain Hoxb8 neutrophils of all differentiation stages on one day. On the day of analysis, cells were counted, washed and seeded into 96-well plates. 200,000 cells were seeded in 150 µl medium (progenitor outgrowth or differentiation medium, respectively) into 96-wells in duplicates and then 30 µl D-Luciferin (100 µg/ml) were added. After incubation for

10 min (37°C, 5 %CO2), luminescence was measured for 10 s using the Tecan infinite M200 reader.

2.16. Determination75B of IL-6 and TNF levels by ELISA Hoxb8 neutrophil progenitor and d4 differentiated cells were seeded at 1x106 cells/ml in 1 ml of progenitor or differentiation medium into 12-well plates. Cells were stimulated with 1 mg/ml LPS for 8 h. Supernatants were harvested and used for enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. ELISA plates were coated with specific capture antibody at 4°C for 16 h. Plates were washed with PBS 0.5 % BSA and blocked with 1x ELISA diluent. Standard as well as samples (1:5 diluted) were added into appropriate wells and incubated for 2 h at RT. Plates were washed with PBS 0.5 % BSA and biotinylated detection antibody was added for 1 h. After washing with PBS 0.5 % BSA, HRP- Streptavidin solution was added and incubated for 30 min. Plates were washed and ELISA colorimetric TMB reagent was added for 15 min. The reaction was stopped using 1 M phosphoric acid. Plates were analysed using Tecan infinite M200 reader. Measurements were taken at 450 nm and 570 nm as reference wavelength.

Material and Methods

2.17. Adoptive76B transfer of Hoxb8 neutrophil progenitor and BM cells into mice For adoptive transfer experiment, 6-8 week old female C57Bl/6 mice were used. 5x106 Hoxb8 neutrophil progenitor cells (pMIG-R1, pMIG-FLAG-C/EBPα wt or C/EBPα K313) along with 0.5x106 unfractionated bone marrow cells (C57Bl/6) were resuspended in 200 µl PBS 1 % FCS and transferred via tail-vein injection into C57Bl/6 mice which had been lethally irradiated (2 x 5.5 Gy) the day before transfer. Mice were bled or sacrificed at the indicated time points and blood, bone marrow and spleen were harvested. Hoxb8- and BM-derived cells were discriminated based on GFP expression. Cells were then stained for the myeloid markers CD11b, Gr-1 and c-kit.

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IX. Appendix8B

Index36B of figures Figure 1: Hematopoietic progenitors ...... 12 Figure 2: Granulopoiesis...... 14 Figure 3: Scheme illustrating C/EBPα functional domains ...... 15 Figure 4 Schematic representation of the Hoxb8 system ...... 28 Figure 5: Hoxb8 neutrophils differentiate into mature granulocytes in vitro ...... 30 Figure 6: C/EBPαfl/fl LysMcretg Hoxb8 neutrophils change over cultivation time ...... 32 Figure 7: Lef1-/- Hoxb8 neutrophils are unable to mature and have lower C/EBPα levels ...... 34 Figure 8: Lef1-/- Hoxb8 neutrophils are able to differentiate and express C/EBPα .... 35 Figure 9: GFP intensity of Hoxb8 neutrophils transduced with pMIG-C/EBPα wt/K313/BRM2 is comparable even upon differentiation ...... 36 Figure 10: Proliferation of Hoxb8 neutrophil progenitors changes upon C/EBPα expression ...... 38 Figure 11: Proliferation of Hoxb8 neutrophil progenitors changes upon p30/p42 C/EBPα expression ...... 39 Figure 12: Proliferation of Hoxb8 neutrophil progenitors changes upon expression of wt/K313/BRM2 C/EBPα in parallel to p30 C/EBPα ...... 40 Figure 13: Proliferation in semi-solid media ...... 41 Figure 14: Cell numbers of C/EBPα wt/K313/BRM2 expressing Hoxb8 neutrophils during differentiation ...... 42 Figure 15: Differentiation of Hoxb8 neutrophils upon C/EBPα expression ...... 44 Figure 16: Differentiation of Hoxb8 neutrophils upon expression of different C/EBPα isoforms ...... 46 Figure 17 Differentiation of Hoxb8 neutrophils upon expression of different C/EBPα C-terminal mutations in combination with the N-terminal truncation mutation p30 ... 47 Figure 18: Expression of Neutrophil Elastase is reduced in C/EBPα K313 expressing cells ...... 49 Figure 19: Cytokine secretion of C/EBPα wt/K313 expressing Hoxb8 neutrophils ... 50 Figure 20: C/EBPα Luciferase Reporter Activity in living cells ...... 51 Figure 21: Survival of Hoxb8 neutrophils and expression of Bcl-2 family proteins .... 55

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Figure 22: C/EBPα levels in wt Hoxb8 neutrophils as well as upon expression of FLAG-wt/K313 C/EBPα ...... 56 Figure 23: C/EBPα protein levels differ between C/EBPα wt/K313/BRM2 expressing ER-Hoxb8 cells ...... 57 Figure 24: C/EBPα protein levels of p30/wt/p42 C/EBPα expressing ER-Hoxb8 cells ...... 58 Figure 25: C/EBPα-transduced ER-Hoxb8 cells show much higher protein levels than wt Hoxb8 neutrophils ...... 59 Figure 26: Protein stability is not significantly changed in C/EBPα K313 expressing Hoxb8 neutrophils ...... 60 Figure 27: Hek293FT cells show lower C/EBPα levels of C/EBPα K313 compared to C/EBPα wt protein ...... 61 Figure 28: Leukocyte cell counts show equal engraftment of C57Bl/6 mice by BM cells and ER-Hoxb8 neutrophils with minor differences in the spleen ...... 63 Figure 29: The proportion of GFP-positive cells varies strongly upon expression of C/EBPα wt/K313 ...... 64 Figure 30: More CD11b/Gr-1 double-positive Hoxb8 neutrophils expressing C/EBPα K313 are detectable on day 8 and 10 after transfer ...... 66 Figure 31: C/EBPα protein levels are drastically increased in human AML samples with C/EBPα K313 mutations...... 67

Index37B of tables

Table 1: Retroviral expression vectors used for transduction of Hoxb8 progenitor cell lines ...... 37

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Abbreviations38B

°C Degrees centigrade -/- Knockout µg Microgram µl Microliter µM Micromolar APS Ammonium persulfate AML Acute myeloid leukaemia AML1 Acute myeloid leukemia 1 protein, also called RUNX1 Bak Bcl-2 homologous antagonist/killer BaP Basophil progenitor Bax Bcl-2-associated X protein Bcl-2 B-cell lymphoma 2

Bcl-XL B-cell lymphoma gene x (long form) BH3 Bcl-2 homology domain Bim Bcl-2 interacting mediator of cell death BM Bone marrow BMCP Basophil-mast cell progenitor BR Basic region BR-LZ Basic region leucine zipper BRM Basic region mutation BSA Bovine serum albumin bzip Basic leucine zipper proteins CD Cluster of differentiation Cdk Cyclin-dependent kinase cDNA Complementary DNA C/EBP CCAAT/enhancer binding protein cFLIP Caspase-8-inhibitory protein CGD Chronic granulomatous disease CHX Cycloheximide CLP Common lymphoid progenitor CML Chronic myelogenous leukaemia CMP Common myeloid progenitor

CO2 Carbon dioxide

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CSF Colony-stimulating factor C-terminal Carboxyterminal CV1 Coefficient of variance DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide DNA Desoxyribonucleic acid DTT Dithiothreitol E Embryonic stage E2F E2 factor E2F-DP E2 factor dimerization partner ECL Enhanced chemiluminescence ECM Extracellular matrix EIF2 Eukaryotic Initiation Factor 2 EIF4E Eukaryotic translation initiation factor 4E EoP Eosinophil progenitor ER Estrogen receptor ER-Hoxb8 Estrogen receptor regulated Homeobox B8 protein Erk Extracellular signal-regulated kinase Et al. Et alii (and others) ETO Eight-Twenty One oncoprotein FACS Fluorescence-activated cell sorting Fc Fragment, crystallisable FCS Fetal calf serum fMLP Formylmethionyl-leucyl-phenylalanine FL Fetal liver Flip Flice-inhibitory protein fl, flox “flanked by LoxP”; DNA sequence between two lox P sites Flt-3 Fms like tyrosine kinase 3 G-CSF Granulocyte-colony stimulating factor Gfi-1 Growth factor independent-1 GFP Green fluorescent protein GM-CSF Granulocyte macrophage colony-stimulating factor GMP Granulocyte macrophage precursor GSF3R Granulocyte colony stimulating factor 3 receptor GW Gateway

H2O Water

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Appendix h Hour(s) HA Human influenza hemagglutinin HCl Hydrogen chloride HDAC Histone deacetylase HEK Human embryonic kidney cell line HI Heat inactivated Hoxb8 Homeobox B8 protein HSC Hematopoietic stem cell IAP inhibitor of apoptosis IFN Interferon IL Interleukin IRES Inner ribosome entry site i.v. Intravenous kb Kilo base pairs kDa Kilodalton l Liter ITD Internal tandem duplication Lef-1 Lymphoid-enhancer binding factor 1 LMPP Lymphoid-primed multipotent progenitor LPS Lipopolysaccharide LSK Lin- Sca-1+ c-kit+ LysMCre Cre recombinase expressed under the LysM (Lysin Motif) promoter M Molar Mcl-1 Myeloid cell leukemia-1 MCP Mast cell progenitor MDP Monocyte-dendritic cell progenitor MEF Mouse embryonic fibroblast MEP Megakaryocyte-erythrocyte progenitor mg Milligram min Minute(s) ml Milliliter mM Millimolar MOMP Mitochondrial outer membrane permeabilisation MPO Myeloperoxidase MPP Multipotent progenitor mRNA messenger RNA

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Appendix

N-terminal Aminoterminal NaCl Sodium chloride NADPH Nicotinamide adenine dinucleotide phosphate NE Neutrophil elastase NET Neutrophil extracellular trap NF-κB Nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells p21/cdki p21, also cyclin-dependent kinase inihibitor p30 30 kDa C/EBPα p42 42 kDa C/EBPα PAMP Pathogen associated molecular pattern PBS Phosphate buffered saline PCR Polymerase chain reaction Pen/Strep Penicillin/Streptomycin pH potentia hydrogenii (power of hydrogen) PI Propidium iodide PMN Polymorphonuclear leukocyte PVDF Polyvinylidene fluoride qRT-PCR Quantitative real time PCR RNA Ribonucleic acid ROS Reactive oxygen species RPMI Roswell Park Memorial Institute medium RT Room temperature RUNX1 Runt-related transcription factor 1, also called AML1 s Second(s) SCF Stem cell factor SCN Severe congenital neutropenia SDS Sodium dodecyl sulfate SDS-PAGE SDS polyacrylamide gel electrophoresis SEM Standard error of the mean SLE Systemic lupus erythematosis TBS Tris buffer saline TBS-T TBS with 0.05 % Tween TE Transactivation element TEMED Tetramethylethylenediamine tg Transgenic TNF Tumor necrosis factor

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Appendix

TOR Target of rapamycin TRAIL Tumor necrosis factor related apoptosis inducing ligand Trib2 Tribbles homolog 2 Ubc9 Ubiquitin-conjugating enzyme 9 WB Western blot Wnt Wingless-related integration site (portmanteau of int and Wg) wt Wild type

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Appendix

X. Acknowledgements9B Foremost I would like to thank Prof. Dr. Georg Häcker for providing me the opportunity to perform this study in his laboratory. I am thankful for the constructive discussions, your motivation and never ending ideas that opened up new perspectives for this project and made it interesting and challenging. Your trust and support throughout my PhD encouraged me a lot.

Special thanks to Dr. Ian Gentle, who was always available to give experimental and technical advice. I am thankful for all the input and insightful discussions which always helped me to think clearly. Thank you for all your patience and knowledge and your encouragement when things did not go as we expected. I could not wish for, nor have gotten a better mentor.

I additionally would like to thank Juliane Vier, without whom lab life would be impossible. You were always there to answer every single question and to help when technical problems occurred. Special thanks also go to Sophie Krüger, the heart and soul of the western blot lab. You are also always there in the lab when help is needed and I will miss the conversations we had when I was working next to you.

I want to give special thanks to Michaela Ohmer for sharing the office and innumerable lunch breaks with me. Thank you for all the useful software tips and your willingness to help- not only in regards of work. And thank you for the enjoyable nights we spent at festivities.

I also want to thank Dr. Susanne Kirschnek and Dr. Arnim Weber for helpful discussions and input. Of course many thanks go to all the other present and former lab members for providing such a pleasant atmosphere. The scientific diversity in our group makes lab life interesting and broadened my knowledge substantially.

Special thanks go to my friends who always encouraged me during my studies.

Most importantly, I would like to thank my family for always supporting me in everything I do and for providing me with all the opportunities which brought me here. Without your constant belief in me I would not be where and who I am. Thank you!

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