Investigating the mechanisms of growth factor independence-1 (Gfi-1)-mediated transcriptional repression of p21cip1 and MBP

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1 The University of Toledo The University of Toledo Digital Repository Theses and Dissertations 2009 Investigating the mechanisms of growth factor independence-1 (Gfi-1)-mediated transcriptional repression of p21cip1 and MBP Qingquan Liu The University of Toledo Follow this and additional works at: Recommended Citation Liu, Qingquan, "Investigating the mechanisms of growth factor independence-1 (Gfi-1)-mediated transcriptional repression of p21cip1 and MBP" (2009). Theses and Dissertations This Dissertation is brought to you for free and open access by The University of Toledo Digital Repository. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of The University of Toledo Digital Repository. For more information, please see the repository's About page.

2 A Dissertation Entitled Investigating the mechanisms of growth factor independence-1 (Gfi-1)-mediated transcriptional repression of p21cip1 and MBP By Qingquan Liu Submitted as partial fulfillment of the requirements for The Doctor of Philosophy in Biology Advisor: Dr. Fan Dong College of Graduate Studies The University of Toledo May 2009

3 Copyright 2009 This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author

4 An Abstract of Investigating the mechanisms of growth factor independence-1 (Gfi-1)-mediated transcriptional repression of p21cip1 and MBP By Qingquan Liu Submitted as partial fulfillment of the requirements for The Doctor of Philosophy in Biology The University of Toledo May 2009 Growth factor independence-1 (Gfi-1) is a zinc-finger transcriptional repressor that plays a critical role in hematopoiesis. Gfi-1 regulates the development of myeloid and lymphoid cells, and controls hematopoietic stem cell self-renewal. Gfi-1 is weakly oncogenic but strongly cooperates with oncoprotein Myc in lymphomagenesis. How Gfi-1 functions in hematopoiesis remains poorly understood. Data presented here demonstrate that Gfi-1 represses p21cip1 and MBP through two distinct mechanisms. Gfi-1 interacts with Myc-interacting zinc-finger protein (Miz-1), a transcriptional activator regulating cell cycle progression and apoptosis, and is recruited by Miz-1 to the promoter of Miz-1 target gene p21cip1, which encodes a potent cell cycle inhibitor, leading to transcriptional repression. Repression of p21cip1 by Gfi-1 is independent of iii

5 direct DNA-binding. Knockdown or deficiency of Gfi-1 results in augmented p21cip1 expression. Interestingly, Gfi-1 forms a ternary Gfi-1/Miz-1/Myc complex on the p21cip1 promoter and collaborates with Myc in the repression of p21cip1. This Miz-1-dependent transcriptional repression by Gfi-1 also applies to other Miz-1 target genes encoding cell cycle inhibitors p15ink4b and p27kip1. Consistent with the mechanism of Miz-1-dependent transcriptional repression, Gfi-1 also represses growth inhibitory cytokine TGF-β-activated p21cip1 independent of DNA-binding. Interestingly, Gfi-1 expression is downregulated by TGF-β, suggesting a role of Gfi-1 in TGF-β-mediated growth inhibition. MBP encodes a cytotoxic granule protein expressed in eosinophils and basophils. Our data identify MBP as a new target of Gfi-1-mediated transcriptional repression. Unlike p21cip1, however, the repression of MBP by Gfi-1 requires Gfi-1 direct DNA-binding as evidenced by the fact that the Gfi-1 dominant negative mutant N382S, which is defective for DNA-binding, relieves the transcriptional repression of MBP by Gfi-1. Indeed, knockdown of Gfi-1 results in enhanced expression of MBP. Expression of the N382S mutant has been shown to cause premature apoptosis of myeloid cells induced to differentiate by G-CSF. Interestingly, overexpression of MBP also results in increased apoptosis during G-CSF-stimulated terminal neutrophilic differentiation, indicating that elevated MBP expression may contribute to the N382S-associated apoptosis of differentiating myeloid cells. These data suggest that the transcriptional repression of MBP by Gfi-1 may contribute to the role of Gfi-1 in regulating granulocyte development. Taken together, our study demonstrates Gfi-1-mediated transcriptional repression of p21cip1 and MBP by two different mechanisms. Gfi-1, via binding to Miz-1, is iv

6 recruited to p21cip1 and other Miz-1 target genes leading to transcriptional repression, and Gfi-1 represses MBP, however, through direct DNA-binding. These findings provide new insights into the transcriptional regulation by Gfi-1 and may have broad implications for better understanding the role of Gfi-1 in normal hematopoiesis and tumorigenesis. v

7 This dissertation is dedicated to my parents Zhenxiang Liu and Pingjiao Tan vi

8 ACKNOWLEGEMENTS I wish to thank many people who have given me support, care, encouragement and help that made the completion of this research and dissertation possible. My deep and sincere gratitude goes to my advisor and mentor, Dr. Fan Dong, for introducing me into this fascinating arena, for his continuous guidance and inspiration throughout my research, and for his faith and confidence in me. The experience of working with him is surely a treasure of my life. I am also truly grateful to the professors on my advisory committee, Dr. Brian Ashburner, Dr. Miles Hacker, Dr. Z. Kevin Pan, Dr. Anthony Quinn, and Dr. William Taylor, for generously investing their time and expertise to better my work. I have been fortunate to have very lovely colleagues and friends all around me during my life at the University of Toledo. I am thankful to my wonderful friend Suchitra, with whom working in the lab had been more fun and efficient. Many thanks must go to Yaling, for teaching me a lot of techniques when I was new, and for making the lab a warmer place like home. My life in Toledo would be lonely without the love and companionship of my dear friends Haying, Shanshan and Ying, with whom I could share all of my emotions. I am indebted to my parents for their tremendous love and support, for inculcating the dedication and discipline in me, and for giving me constant understanding and respect. vii

9 TABLE OF CONTENTS Page ABSTRACT......iii DEDICATION......vi ACKNOWLEDGEMENTS..vii TABLE OF CONTENTS.viii LIST OF FIGURES...xii ABBREVIATIONS..xiv CHAPTER1: INTRODUCTION Hematopoiesis Hematopoietic cytokines Transcription factors in hematopoiesis Leukemia and lymphoma Granulocytes and neutropenia Cell cycle regulation and CDK inhibitors Inhibition of cell proliferation by TGF-β Growth factor independence-1 (Gfi-1) Transcription factor Gfi Gfi-1 in normal hematopoiesis Gfi-1 as a proto-oncoprotein Gfi-1 as a transcriptional repressor Miz-1 in Myc-mediated transcriptional repression.21 viii

10 CHAPTER 2: MATERIALS AND METHODS Cells and materials Construction of plasmids Transfection and generation of stable cell lines Preparation of whole-cell and nuclear extracts Immunoprecipitation and Western blotting analysis GST-pull down assay Oligonucleotide precipitation (oligo-pull down) assay Chromatin immunoprecipitation assay (ChIP) and Re-ChIP Luciferase assay Semi-quantitative reverse transcriptase PCR (RT-PCR) RNA interference Cell proliferation assay Apoptosis Assay 33 CHAPTER 3: RESULTS Miz-1-dependent transcriptional repression of p21cip1 by Gfi Gfi-1 interacts with Miz Gfi-1 and Miz-1 interact with each other via their ZF domains Gfi-1 is recruited to the p21cip1 promoter through association with Miz Gfi-1 represses Miz-1-induced activation of the p21cip1 promoter, and depletion of Gfi-1 leads to enhanced expression of p21cip1.43 ix

11 Gfi-1 represses Miz-1- and TGF-β-activated p21cip1 independent of direct DNA-binding Knockdown of Gfi-1 expression in HL60 and TF-1 cells results in reduced cell proliferation, Gfi-1 functionally collaborates with Myc in the repression of p21cip Gfi-1 forms a ternary complex with Myc through Miz-1 on the p21cip1 promoter The Miz-1-dependent transcriptional repression by Gfi-1 plays a role in regulating other cyclin-dependent kinase inhibitor genes p15ink4b and... p27kip Gfi-1 is downregulated by TGF-β Transcriptional repression of the eosinophil major basic protein (MBP) gene by Gfi Expression of the N382S mutant in myeloid cells leads to upregulation of MBP in response to G-CSF Gfi-1 represses the MBP promoter Expression of the N382S mutant potentiates the induction of the MBP. promoter activity by G-CSF Overexpression of MBP inhibits IL3 and G-CSF-dependent growth and accelerates cell death in response to G-CSF in myeloid cells Overexpression of MBP causes accelerated apoptosis in response to G-CSF Knockdown of Gfi-1 results in MBP upregulation. 67 x

12 CHAPTER 4: DISCUSSION Gfi-1-mediated transcriptional repression of p21cip1 and other Miz-1 target genes Gfi-1 in TGF-β-mediated anti-growth effect Transcriptional repression of MBP by Gfi-1 in granulopoiesis CHAPTER 5: FUTURE DIRECTIONS Define the biological significance of Gfi-1 interaction with Miz Examine the potential role of Gfi-1 in the cellular response to TGF-β Investigate the mechanism by which TGF-β regulates Gfi-1 expression Explore the potential role of Miz-1 in the regulation of Gfi-1 expression and granulopoiesis Further study the mechanism of Gfi-1-mediated repression of MBP and evaluate the role of Gfi-1 in eosinophilic differentiation.. 83 CHAPTER 6: REFERENCES 84 xi

13 LIST OF FIGURES Figure 1 Schematic representation of hematopoiesis and the cytokines regulating hematopoesis.. 4 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Transcription factors that act at the various stages of Hematopoiesis 7 Three types of granulocytes: neutrophil, eosinophil and basophil...9 Schematic representation of cell cycle progression.11 Schematic representation of the Gfi-1 protein structure..14 Schematic view of the role of Gfi-1 in hematopoiesis. 15 Schematic representation of the Miz-1 protein structure.. 23 Gfi-1 interacts with Miz-1 in vitro and in vivo. 36 The C-terminal ZF domains of Gfi-1 are required for Gfi-1. interacting with Miz-1.37 Figure 10 The C-terminal ZF domains of Miz-1 are required for Miz-1 binding to Gfi Figure 11 Gfi-1 is recruited to the p21cip1 core promoter through association with Miz-1 in vitro and in vivo Figure 12 Gfi-1 represses Miz-1-induced activation of the p21cip1 promoter and depletion of Gfi-1 leads to enhanced expression of p21cip1 44 Figure 13 Gfi-1 represses Miz-1- and TGF-β-activated p21cip1 promoter activity independent of direct DNA-binding...46 xii

14 Figure 14 Knockdown of Gfi-1 by shrna in HL60 and TF-1 cells causes reduced cell proliferation...48 Figure 15 Gfi-1 collaborates with Myc in the repression of Miz-1-induced p21cip1 promoter activity...49 Figure 16 Figure 17 Gfi-1 forms a ternary complex with Myc through Miz Gfi-1 forms a ternary complex with Myc through Miz-1 on the p21cip1 core promoter in vivo Figure 18 Gfi-1 represses the transcription of p15ink4b and p27kip1, and Gfi-1 binds to the p15ink4b and p27kip1 core promoters through Miz-1 in vivo.. 56 Figure 19 Figure 20 Gfi-1 is downregulated by TGF-β MBP is upregulated in response to G-CSF in the presence of the N382S mutant...61 Figure 21 Figure 22 Gfi-1 represses the MBP promoter...62 Expression of the N382S mutant potentiates the induction of MBP promoter activity by G-CSF.63 Figure 23 Figure 24 Overexpression of MBP causes reduced cell proliferation and survival.. 65 Overexpression of MBP results in accelerated apoptosis in response to G-CSF...66 Figure 25 MBP expression is increased upon Gfi-1-knockdown in HL60 clone 15 cells.67 Figure 26 Figure 27 Diagrammatic view of the p21cip1 promoter A working model for the regulation of TGF-β response by Gfi-1 78 xiii

15 ABBREVIATIONS ABL AML Bax BCL BCR BM bp BSA PBS CD CDK cdna CDKI C/EBP Abelson tyrosine kinase Acute myeloid leukemia Bcl-2 homologous antagonist/killer B cell lymphoma Breakpoint cluster region Bone Marrow Base pair Bovine serum albumin Phosphate buffered saline Cluster of differentiation Cyclin-dependent kinase Complementary deoxyribonucleic acid Cyclin-dependent kinase inhibitors CAAT/Enhancer Binding Protein Cip1 CDK-interacting protein 1 CLL CLP CMP CML ECM Chronic lymphoid leukemia Common lymphoid progenitor Common myeloid progenitor Chronic myeloid leukemia Extracellular Matrix ELA2 Enzyme elastase 2 EPO Erythropoietin xiv

16 ETO FACS FBS Eight twenty one Fluorescence Activated Cell Sorting Fetal Bovine Serum FLT3 Fms-like tyrosine kinase 3 GC G-CSF Gfi-1 GFP GM-CSF GMP HDAC HEK HLH/LZ HSC IL IP IFN MBP M-CSF MDS MEP Miz-1 NE Germinal center Granulocyte-colony stimulating factor Growth factor independence-1 Green fluorescent protein Granulocyte-macrophage-colony stimulating factor Granulocyte/monocyte progenitor Histone deacetylase Human embryonic kidney Helix loop helix/leucine zipper Hematopoietic stem cell Interleukin Immunoprecipitation Interferon Eosinophil major basic protein Macrophage-colony stimulating factor Myelodysplastic syndrome Megakaryocyte/erythrocyte progenitor Myc-interacting zinc-finger protein-1 Neutrophil elastase xv

17 NHL PBS PIAS3 PML PMSF POZ RAR SCF SCN SNAG STAT Non-Hodgkin s lymphoma Phosphate buffered saline Protein inhibitor of activated STAT3 Acute promyelocytic leukemia Phenylmethylsulfonyl fluoride Poxvirus and zinc-finger All-trans-retinoic acid Stem Cell Factor Severe congenital neutropenia Snail/Slug Signal transducer and activator of transcription Tal1 T-cell acute lymphocytic leukemia 1 TβRE TNF TopBP1 TPO WB WCE ZF TGF-β-response element Tumor necrosis factor Topoisomerase II β-binding protein Thrombopoietin Western blotting Whole-cell extract Zinc-finger xvi

18 CHAPTER 1: INTRODUCTION 1.1. Hematopoiesis Hematopoiesis is the process by which mature and functional blood cells of distinct lineages are produced from hematopoietic stem cells (HSCs). HSCs can undergo self-renewal to sustain the HSC pool and differentiate into multipotential early progenitors, common myeloid progenitors (CMP) and common lymphoid progenitors (CLP) that are committed to lymphoid lineage and myeloid lineage, respectively. Progenitor cells further differentiate and give rise to mature myeloid cells including granulocytes, monocytes/macrophages, erythrocytes and platelets, and lymphoid cells including T cells, B cells and natural killer cells (Fig. 1) [1]. Hematopoietic cells gradually become less capable of proliferation with terminal differentiation and ultimately die by apoptosis [2]. In mouse and human embryogenesis, hematopoiesis takes place in two waves: primitive hematopoiesis and definitive hematopoiesis. Primitive hematopoiesis originates in yolk sack forming blood islands containing embryonic erythroid cells and HSCs. Subsequently, hematopoiesis is shifted to fetal liver, then spleen and as the development progresses, bone marrow eventually becomes the major tissue that manufactures blood cells shortly before birth [3]. Hematopoiesis in fetal liver, spleen and bone marrow is termed definitive hematopoiesis and generally referred to as hematopoiesis. 1

19 Hematopoiesis is a delicate procedure requiring well-orchestrated regulatory mechanisms. The control of hematopoiesis involves cellular interactions between hematopoietic cells and the bone marrow microenviroment (stromal cells and extracellular matrix) as well as a network of cytokines that provides cells with positive or negative proliferation, survival and differentiation signals [1] Hematopoietic cytokines Hematopoietic cytokines are glycoproteins involved in the regulation of hematopoiesis. These cytokines are mainly produced by lymphocytes, monocytes, macrophages, endothelial cells, fibroblasts and stromal cells [1]. Hematopoietic cytokines function through activating cognate receptors, which in turn trigger downstream signaling pathways. Early-acting cytokines, including interleukin-1 (IL-1), IL-3, IL-6, IL-11, Kit ligand, Fms-like tyrosine kinase 3 (FLT3) ligand, and granulocyte macrophage-colony stimulating factor (GM-CSF), act on early progenitors and affect the cell development of multi-lineages whereas more differentiated cells are less responsive to most of these early-acting cytokines. On the contrary, late-acting cytokines act on more committed progenitors to regulate later stages of hematopoietic development of specific cell types [4]. For instance, Macrophage-colony stimulating factor (M-CSF) is required for macrophage maturation. Granulocyte colony-stimulation factor (G-CSF) stimulates terminal granulocytic differentiation towards neutrophils. Erythropoietin (EPO), the first characterized hematopoietic cytokine, promotes the development of erythroid progenitor cells and EPO expression dramatically rises upon anemia or arterial hypoxemia [5]. Thrombopoietin (TPO) stimulates the formation of megakaryocytes, which in turn produce large number of platelets. Some hematopoietic cytokines that act at different 2

20 stages of hematopoiesis are depicted in Fig. 1. Nevertheless several hematopoietic cytokines have been shown to act on both early multipotential progenitor cells and late lineage-specific precursor cells. For instance, GM-CSF not only regulates the formation of early myeloid progenitors but also plays a role in terminal granulocyte and monocyte development. IL-3 supports the proliferation of virtually all of the early progenitor cells and also is indispensable for basophil and megakaryocyte maturation [6]. A few cytokines are known to exert negative effects on hematopoietic cell proliferation. TGF-β is a potent growth inhibitory cytokine that is critical for hematopoietic cell quiescence and differentiation [7]. Tumor necrosis factor (TNF) can exert both inhibitory and stimulatory effects on hematopoietic progenitor cell proliferation through modulating the expression of several hematopoietic cytokines and cytokine receptors [8-10]. Interferons have been shown to suppress hematopoiesis and induce apoptosis, and may play a role in the pathogenesis of bone marrow failures[11-13]. 3

21 GMP GM-CSF, G-CSF Granulocyte IL-6, IL-3,IL-11 CMP IL-3 GM-CSF Kit Ligand GM-CSF, M-CSF EPO Monocyte Erythrocyte Selfrenewal HSC MEP TPO, IL-11 Megakaryocyte IL-3, FLT3 Ligand IL-3, 7 Pre-T IL-2, 4, 7,15 T lymphocyte CLP IL-2, 4, 10, 12 B lymphocyte Pre-B Figure 1: Schematic representation of hematopoiesis and the cytokines regulating hematopoiesis. HSCs either undergo self-renewal or differentiate into early progenitors committed to either myeloid or lymphoid lineage. Progenitor cells further proliferate and differentiate to give rise to mature and functional myeloid cells including granulocytes, macrophages, erythrocytes and megakaryocytes, and lymphoid cells including T lymphocytes and B lymphocytes and natural killer cells. Some of the major hematopoietic cytokines acting at various stages of hematopoiesis are shown. HSC, hematopoietic stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; GMP, granulocyte/monocyte progenitor; MEP, megakaryocyte/erythrocyte progenitor; Pre-T, pre-t cell; Pre-B, pre-b cell. 4

22 1.3. Transcription factors in hematopoiesis Hematopoietic transcription factors, in conjunction with general transcription factors, play a critical role in directing hematopoietic cell specification and lineage-commitment (Fig. 2). A number of in vivo studies, in which the genes encoding hematopoietic transcription factors were selectively inactivated, have provided insights into the essential roles of those factors in normal hematopoiesis. In addition to their roles in normal hematopoiesis, many genes encoding hematopoietic transcription factors are affected by chromosomal rearrangements and translocations that generate abnormal fusion proteins associated with hematological disorders especially leukemias and lymphomas [14]. Similar to the hematopoietic cytokine network described above, the transcription factors involved in hematopoiesis act in a hierarchical manner. Some transcription factors, such as Tal1, GATA2 and AML1, have been shown to affect the development of a broad spectrum of cells. Others such as GATA-1, EKLF and C/EBPε may play their roles in the development of more lineage-committed cells down in the hematopoiesis cascade. Tal1 and AML1, originally identified through translocations in acute T cell leukemia and acute myeloid leukemia, respectively, act at very early stages of hematopoiesis [15]. Tal1-deficiency is embryonic lethal with no recognizable hematopoiesis in the embryo [16]. AML1-null mice embryos lack fetal liver hematopoiesis although primitive hematopoiesis appears to be normal [17]. Notably, the gene encoding AML-1 is the most frequent target of chromosomal translocations in myeloid leukemias [18]. Absence of GATA-2 leads to global hematopoietic deficit in all lineages but the morphology and maturation of individual cells is normal [19]. PU.1 regulates the development of HSC to 5

23 early myeloid and lymphoid progenitors and also directs granulocyte/monocyte progenitors (GMP) in favor of monocytic differentiation. PU.1-null mice show a lack of disparate hematopoietic lineages [14]. CCAAT enhancer binding proteins (C/EBPs) function predominantly in myeloid lineages. C/EBPα is required for common myeloid progenitors (CMPs) to GMP transition whereas C/EBPε is indispensable for terminal neutrophil maturation [20]. GATA-1 and EKLF control the terminal maturation of erythrocytes [14]. Nonetheless, virtually none of the hematopoietic transcription factors are indeed restricted to a single cell type. In general, it is the combinatorial action of transcription factors that establish the gene expression programs leading to ultimate cellular identity. An intimate cross-talk exists between hematopoietic cytokines and transcription factors. Stimulation of cytokines may alter the expression of transcription factors and similarly, changes in transcription factor activities may alter the expression of cytokine signaling components. For instance, C/EBPε expression is induced in response to G-CSF in myeloid cells. PU.1-deficeincy results in decreased expression of the receptors for GM-CSF, G-CSF and M-CSF, and is associated with a lack of mature granulocytes, macrophages and B cells [21-23]. 6

24 GMP C/EBPε RAR Granulocyte C/EBPα CMP PU.1 Monocyte PU.1, C/EBPα GATA-1, EKLF Erythrocyte Selfrenewal HSC GATA-1 MEP GATA-1, 3 Megakaryocyte GATA-2 AML-1 PU.1 c-myb Pre-T GATA-3 T lymphocyte CLP E2A Pax5 B lymphocyte Pre-B Figure 2: Transcription factors that act at the various stages of hematopoiesis Leukemia and lymphoma Leukemia is characterized by clonal expansion of malignant myeloid or lymphoid cells arrested at the various stages of differentiation, which may result from abnormal proliferation and sustained cell survival of hematopoietic cells. According to the origin of leukemic cells and the progression of the diseases, leukemia is classified into acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML) and chronic lymphoid leukemia (CLL). The pathogenesis of leukemia is believed to be the consequence of at least two classes of mutations: Class I mutations cause constitutively activated tyrosine kinases or cytokine receptors, such as BCR/ABL and FLT3 mutants, respectively, which primarily confer cells proliferative or survival advantage whereas to a less extent affect cell differentiation; Class II mutations usually 7

25 lead to abnormal function of transcription factors critical for hematopoietic homeostasis and mainly impair differentiation, such as AML1/ETO and PML/RARα fusions [24, 25]. Lymphoma arises from transformed lymphocytes in lymphatic tissues such as lymph nodes and spleen. Transformed lymphocytes uncontrollably multiply and form a tumor mass in lymphatic tissues and eventually invade other tissues and organs via the lymphatic system. Lymphomas can be generally classified into Hodgkin lymphoma with predictable spreading characteristic and non-hodgkin lymphoma (NHL) that is more likely to spread to other organs. Latest classification of lymphoma tends to base on the cell types, including T, B and natural killer cells, where the malignancy is originated. Abnormalities in oncogenes, tumor suppressor genes and chromosomal translocations have been identified in patients with lymphomas. For instance, proto-oncogene c-myc is constitutively activated upon t(8;14), t(2;8) and t(8;22) chromosomal translocations that are responsible for B cell malignancy in Burkitt s lymphomas [26]. Chromosomal translocations involving genes encoding anti-apoptotic Bcl-2, transcription repressor B cell lymphoma 6 (BCL-6) and tumor suppressor p53 are also associated with various subsets of lymphomas [27, 28] Granulocytes and neutropenia Granulocytes are a group of white blood cells that contain granules in their cytoplasms. The development of granulocytes is termed granulopoiesis. There are three types of granulocytes: neutrophil, basophil, and eosinophil (named according to the staining properties of their cytoplasmic granules) (Figure 3).. 8

26 Neutrophil Eosinophil Basophil Figure 3: Three types of granulocytes: neutrophil, eosinophil, and basophil by Giemsa staining. Neutrophils are the most abundant type of granulocytes accounting for 60-70% of total white blood cells. Neutrophils have 2-5 lobed nuclei and lightly stained granules. They are phagocytic cells functioning as the first line of defense against infections. Eosinophils are less frequent and account for 2-3% of total white blood cells. Eosinophils have 2 lobed nuclei and large granules containing histamine and cationic proteins that are toxic to both parasites and the host tissues. Eosinophils are important effector cells in the defense against parasitic infection and in allergic diseases. Basophils only account for less than 1% of total white blood cells. They usually have 2-lobed nuclei and deeply stained large granules, which store histamine and obscure the underlying nuclei. Basophils function to initiate the inflammatory process at sites of injury and microbe infections [29]. Neutropenia is defined by a low absolute neutrophil count of less than /L in peripheral blood due to impaired production and/or increased destruction of neutrophils. Nutropenias can be categorized into acquired and congenital forms. Acquired neutropenias are caused by infections, immune diseases and side effects of drugs. Congenital neutropenia, frequently referred to as severe congenital neutropenia (SCN), is 9

27 a group of autosomal recessive diseases and the patients are usually born with the condition of lack of neutrophils, and therefore suffer from frequent bacteria infections that could be life-threatening [30]. In addition, patients with SCN are at an increased risk of developing leukemia, myelodysplastic syndrome (MDS) and other bone marrow disorders. G-CSF is a hematopoietic cytokine that plays an essential role in granulopoiesis by supporting the proliferation, differentiation and survival of myeloid progenitor cells. G-CSF particularly induces terminal maturation of granulocytic progenitors towards neutrophils. Therefore administration of recombinant human G-CSF has been successful in the clinical management of some SCN cases. Unfortunately, with prolonged survival due to G-CSF, MDS and AML have emerged in approximately 10% of SCN patients [31, 32] Cell cycle regulation and CDK inhibitors The progression of the cell cycle is tightly regulated by cyclin-dependent kinases (CDKs) that are activated by cyclins and inhibited by CDK inhibitors (CDKIs) (Fig. 4). G1 to S phase progression is controlled by cyclin D, E and A-associated CDKs, whereas cyclin B primarily controls G2 and M phases. CDKIs function through inhibiting cyclin/cdk complexes via physical interaction, thus arresting the cell cycle at different stages depending on the particular cyclin/cdk complex that is inhibited. CDKIs are categorized into two families: a) Ink family proteins including p16ink4a, p15ink4b, p18ink4c and p19ink4d that inhibit cyclin D-CDK4 and cyclin D-CDK6 complexes; b) Cip/Kip family p21cip1, p27kip1 and p57kip2 that inhibit the activities of cyclin D, A and E-associated cyclin-cdk complexes, with the strongest inhibitory activity manifested 10

28 against CDK2 complexes. p21cip1 mainly inhibits cyclin E-CDK2 and cyclin A-CDK2 complexes, thereby blocking G1 to S phase transition and causing G1 cell cycle arrest [33]. Cyclin B/A + CDK2 G0 Cyclin D + CDK4/6 Cyclin E+CDK2 Cyclin A+CDK2 Figure 4: Schematic representation of cell cycle progression Cell cycle arrest is frequently coupled with hematopoietic differentiation and is critical for HSC quiescence and functional integrity. p21cip1 is an important cell cycle regulator involved in maintaining HSC quiescence. p21cip1 -/- HSCs are more prone to cell cycle entry leading to enhanced proliferation and early exhaustion of HSC pool [35]. Furthermore, modulations of p21cip1 and p27kip1 by TGF-β play essential roles in maintaining cell cycle arrest in HSCs and early progenitor cells for proper hematopoietic 11

29 development [36-39]. Although p21cip1 was initially believed to be a specific effector for p53-mediated inhibition of cell proliferation in response to DNA damage [40, 41], subsequent studies have shown that p21cip1 is also activated in response to DNA damage, UV irradiation, and TGF-β independent of p53 [42, 43] Inhibition of cell proliferation by TGF-β TGF-β is an anti-mitogenic cytokine that transmits its signals through a heterodimeric complex of transmembrane serine/threonine kinase receptors: TGF-β receptor type I and type II. The receptor complex then activates the receptor-activated Smads (R-Smads), Smad2 ad Smad3. The activated Smads bind to Smad4, translocate into the nucleus and regulate gene transcription [44]. TGF-β primarily inhibits cell proliferation by inducing G1 cell cycle arrest. Evidence from studies in epithelial cells has shown that TGF-β induces cell cycle arrest through up-modulating the expression or activity of CDKIs, and inhibiting factors essential for CDK activation. For instance, TGF-β induces downregulation of Myc and activation of p15ink4b, p21cip1 and p27kip1 in epithelial cells and keratinocytes [45-47]. TGF-β can also repress the activity of cell cycle progression inhibitor retinoblastoma protein (Rb), downregulate cyclins, and suppress the activation of CDK activating phosphatase Cdc25A [48]. Mutations in the genes involved in TGF-β signaling, including those encoding the TGF-β receptors and TGF-β-signal transducers Smads, cause insensitivity to TGF-β and are associated with the pathogenesis of myeloid and lymphoid malignancies [49]. 12

30 Resistance to TGF-β-mediated cell cycle arrest has been observed in a number types of malignant cells, including cells of hematopoietic origin [50]. TGF-β also controls the quiescence of HSC and primitive hematopoietic progenitor cells and the differentiation of some late progenitor cells [51]. Knockout of the TGF-β gene is embryonic lethal due to the defects in hematopoiesis and vasculogenesis [52]. Nevertheless, the mechanism by which TGF-β acts in hematopoiesis is not clear Growth factor independence-1 (Gfi-1) Transcription factor Gfi-1 The Gfi-1 gene encodes a 423 amino acid nuclear transcription repressor that belongs to a family of proteins including the Gfi-1 homolog Gfi-1B [53], murine proteins Snail and Slug, which are characterized by the C-terminal six C 2 H 2 -type zinc finger (ZF) domains and the N-terminal SNAG domain well conserved among Gfi-1 family proteins [54-56] (Fig. 5). The 3 rd, 4 th and 5 th ZF domains of Gfi-1 are required for its DNA-binding ability, and the SNAG domain carries a nuclear localization signal and the transcriptional repression activity [57, 58]. The less conserved intermediate region between the SNAG domain and ZF domains is still poorly characterized, but it is believed to provide protein-protein interaction interface. In fact all three portions of Gfi-1, the SNAG domain, the middle region and the ZF domains have been shown to be able to mediate protein-protein interactions [59-61]. Gfi-1B is a 330 amino acid protein with C-terminal 6 ZF domains that are 97% identical to the ZF domains in Gfi-1, and a conserved N-terminal SNAG domain. Both Gfi-1 and Gfi-1B bind to the same DNA recognition site containing the AAT/GC core sequence [53, 58, 62-64]. 13

31 SNAG Intermediate Region C 2 H 2 ZFs Nuclear localization Transcriptional repression DNA binding Figure 5: Schematic representation of the Gfi-1 protein structure. Gfi-1 contains an N-terminal SNAG domain, 6 C-terminal ZF domains and a poorly characterized region in between Gfi-1 in normal hematopoiesis Gfi-1 is primarily expressed in hematopoietic system including thymus, bone marrow and spleen [53]. Gfi-1 is highly expressed in HSCs, in early B cells but absent in mature B cells. In T cells, Gfi-1 expression gradually decreases as T cells mature but rises significantly upon antigen-stimulated T cell activation. On the contrary, in granulocyte lineage, Gfi-1 expression increases when granulocyte progenitor cells develop towards mature neutrophils [65, 66]. Activated macrophages also manifest transiently elevated expression of Gfi-1 [67] (Fig. 6). In addition to hematopoietic cells, expression of Gfi-1 is also detectable in lung neuroendocrine cells, intestinal epithelial cells and the developing epithelia of the inner ear hair cells, and Gfi-1 is critical for the differentiation of those cells [68-70]. Several lines of evidence have pointed out the essential role of Gfi-1 in the development of lymphopoiesis and granulopoiesis. Gfi-1-deficient mice manifested 14

32 defective lymphopoiesis with dramatically reduced thymic cellularity, due to impaired development of early uncommitted T cell progenitors, and markedly reduced B cell numbers in bone marrow [71, 72]. Strikingly, Gfi-1-deficient mice were severely Gfi-1 Self-renewal + Gfi-1 CLP + Pre-B Pre-T GMP + + Gfi-1 Gfi-1 B T Neu CMP Gfi-1 Mon + Meg MEP Ery Figure 6: Schematic view of the role of Gfi-1 in hematopoiesis. Cells expressing Gfi-1 are shown with a +. Arrow signifies a required role, and represents an antagonizing role. Ery, erythrocyte; Meg, megakaryocyte; Neu, neutrophil; Mon, monocyte; T, T lymphocyte; B, B lymphocyte. neutropenic with a complete lack of mature neutrophils, but had atypical immature cells sharing both neutrophil and macrophage characteristics with sharing both neutrophil and macrophage characteristics with elevated expression of M-CSF receptor, C/EBPα and PU.1 [74]. The presence of these atypical cells indicates that Gfi-1 acts to promote 15

33 granulocyte development and antagonize the alternative development towards macrophage. This is supported by the observation that Gfi-1 is downregulated upon monocytic differentiation in bipotential HL60 cells [56]. C/EBPε -/- mice also lack mature neutrophils but the atypical cells in C/EBPε -/- mice retain closer resemblance to granulocytes versus monocytes/macrophages, suggesting that Gfi-1 functions upstream of C/EBPε in granulocytes development [75]. Transplantation of Gfi-1-deficient bone marrow cells failed to restore normal granulopoiesis in irradiated recipient mice, indicating that this failure to produce mature granulocytes is intrinsic to the hematopoietic lineage [74]. Gfi-1 mutants that cause loss of Gfi-1 transcriptional repressor activity have been identified in patients with SCN. One such mutant, N382S, carries a substitution of serine (S) for the asparigine (N) at position 382 in the fifth ZF domain. This mutation disrupts the DNA-binding ability of Gfi-1. Patients with N382S manifested extremely low neutrophil count, immature myeloid cells and elevated monocyte production [76, 77]. Another Gfi-1 mutant carries a substitution of arginine for the lysine at position 403 in the sixth ZF domain (K403R) and this mutant is correlated with a less severe phenotype [68, 78]. SCN is most frequently associated with mutations in the gene encoding neutrophil elastase (NE), a primary neutrophil granule protein expressed in neutrophils and monocytes [79]. Mutations in GFI-1 were identified in SCN patients who did not have mutations in NE, suggesting a functional redundancy of mutations in these two genes in the pathogenesis of SCN. Indeed studies have shown that Gfi-1 represses the transcription and enzyme activity of NE [76]. Furthermore a physical interaction between Gfi-1 and NE has been reported [80]. These facts suggest that mutations in GFI-1 may 16

34 contribute to the development of SCN through causing deregulated expression, enzyme activity and trafficking of NE. Significantly, Gfi-1 also plays a critical role in regulating the self-renewal of HSCs by restricting HSC proliferation and maintaining HSC functional integrity [38, 81, 82]. Gfi-1 -/- HSCs are hyperproliferative, causing a higher percentage of the stem cell population entering cell cycle. Although Gfi-1 -/- HSCs are able to produce myeloid and lymphoid progenitors when transplanted alone, they are defective in competitive reconstitution when co-transplanted with wild-type HSCs [82]. The hyperproliferative characteristic of Gfi-1 -/- HSCs strikingly resembles the phenotype of p21cip1 -/- HSCs. Interestingly, p21cip1 expression is absent in Gfi-1 -/- HSC [35]. Gfi-1 knockout studies have further uncovered a role of Gfi-1 in limiting the inflammatory responses. Although Gfi-1 is dispensable for macrophage development, it appears to be essential for macrophage function. Gfi-1 -/- macrophages produce enhanced inflammatory cytokines including TNF, IL-10 and IL-1β upon stimulation of bacteria endotoxin (LPS). Gfi-1 -/- mice show exaggerated inflammatory response to low doses of LPS, that are tolerated by wild-type animals, and suffer high incidence of gram-positive bacteria infections [67, 71, 83, 84]. In line with these notions, Gfi-1 acts upstream of TNF to attenuate endotoxin-induced inflammatory responses in the lung [85]. Expression of Gfi-1B in the hematopoietic system is largely complementary to Gfi-1. Gfi-1B is highly expressed in erythroid cells, megakaryocytes and their progenitor cells, where Gfi-1 is absent. However, Gfi-1B is not detected in granulocytes, activated macrophages or their progenitor cells, or in mature naive and activated lymphocytes, 17

35 where Gfi-1 is present [64]. Several studies have suggested that Gfi-1 and Gfi-1B auto-regulate their gene expression and mutually repress the transcription of each other [62, 63, 86-88]. Gfi-1B is essential for both primitive and definitive erythroid cell development. Gfi-1B deficiency is embryonic lethal due to a lack of primitive red blood cells and Gfi-1B -/- embryonic stem cells failed to contribute to erythrocytes and megakaryocytes formation in adult chimeras [89] Gfi-1 as a proto-oncoprotein Gfi-1 was first identified as a locus of provirus integration in Moloney murine leukemia virus (Mo-MuLV)-induced rat T cell lymphoma lines selected for IL-2-independent growth. The provirus integrations in the Gfi-1 locus were mapped to the Gfi-1 promoter and resulted in long term repeat (LTR)-driven overexpression of Gfi-1 [57], suggesting that deregulated Gfi-1 is oncogenic. Gfi-1 was later found to be a common target of provirus integration in T cell lymphomas induced by Mo-MuLV, mink-cell focus-forming virus, and murine acquired immunodeficiency virus [90, 91]. Expression of a Gfi-1 transgene targeted to T cells is weakly oncogenic in predisposing the mice for T cell lymphoma but strongly cooperates with oncogene c-myc or pim-1 in T-cell lymphomagenesis [92]. Besides rodent lymphomas, human chromosome 1p22, where human GFI-1 is located, is a region hosting chromosomal abnormalities involved in various human neoplasms [93]. Gfi-1 has been implicated in prostate cancer through repressing the gene encoding a vitamin D hydroxylase that has a protective role in the development and/or progression of the disease [94] Gfi-1 also promotes cell proliferation and survival, and protects cells from 18

36 apoptosis. Gfi-1 inhibits IL-2 starvation-induced cell cycle arrest in T cells and suppresses T cell apoptosis stimulated by antigen activation [58, 84]. Gfi-1 also enhances IL-4- and IL-6- dependent T cell proliferation and survival. The ability of Gfi-1 to promote cell survival and inhibit apoptosis can be at least partially attributed to altering the balance between anti-apoptotic proteins and pro-apoptotic proteins of the Bcl-2 family members. Gfi-1 represses pro-apoptotic Bax and Bak in thymocytes, and upregulates anti-apoptotic Bcl-2 and Bcl-X L in CD4 T cells [95, 96] Gfi-1 as a transcriptional repressor Gfi-1 binds to DNA elements containing the consensus DNA sequence AAT/GC through the ZF domains. Gfi-1 potential binding sites have been identified in a number of genes involved in regulating hematopoietic cell proliferation, differentiation and survival. These genes include IL-1, IL-4, CSF-1, G-CSF, TNF-α and -β [56, 82]. Large scale chromatin immunoprecipitation (ChIP) assays have revealed that Gfi-1 occupies the promoters of genes encoding the cyclin-dependent kinase inhibitor p21cip, NE, C/EBPε, C/EBPα, Gfi-1, E2F and other genes that are involved in regulating hematopoietic development [97]. Indeed Gfi-1 has been shown to repress the transcription of Ela2, encoding NE, Gfi-1, Gfi-1B and the gene encoding apoptotic Bax. [53, 76, 95]. Furthermore, both Gfi-1 and Gfi-1B have been shown to repress p21cip1 transcription [53, 97, 98]. In line with this notion, Gfi-1 blocks phorbol ester-induced expression of p21cip1, and Gfi-1-null T-cells express augmented p21cip1 [99]. Most recent studies indicate that Gfi-1 represses the gene encoding monocytic cytokine M-CSF, and elevated M-CSF signaling resulted from the expression of N382S in SCN patients might be 19

37 partially responsible for the impaired neutrophil development [100]. Previous studies have shown that the transcriptional repression activity of Gfi-1 is dependent on the integrity of its N-terminal SNAG domain. However, later evidence indicates that Gfi-1 may repress gene expression via both SNAG domain-dependent and -independent mechanisms. It appears that transcriptional repression by Gfi-1 may involve at least three distinct means: A) SNAG domain-mediated repression: the SNAG domain of Gfi-1 recruits corepressor CoREST, histone demethylase LSD1, and HDAC1 and 2 to Gfi-1 target genes [59]; B) Middle region-mediated repression: the region between SNAG and ZF domains can recruit histone lysine methyltransferase G9a and SUV39H1 and histone deacetylases (HDAC1-3) [60]; C) ZF domains-mediated repression: the C-terminal ZF domains recruit HDACs and co-repressors including ETO, a component of HDAC complexes [61]. In addition, Gfi-1 can regulate transcription through indirect mechanisms. Gfi-1 promotes STAT3-mediated transcriptional activation by sequestering protein inhibitor of activated STAT33 (PIAS3) through association with PIAS3 [101]. Modulation of STAT3 activation by Gfi-1 suggests that Gfi-1 may play a role in a set of cytokine signaling pathways since STAT3 is a downstream molecule in G-CSF, IL-6 and IL-3 signaling. Gfi-1 binds to PU.1 and antagonizes the PU.1-mediated transcriptional activation of the genes encoding M-CSF receptor and monocyte-specific CD64. The antagonistic effect of PU.1-mediated transcriptional activation by Gfi-1 may provide potential explanation for the Gfi-1-associated developmental bias towards granulocytes versus monocytes from GMPs since PU.1 function is essential for monocyte development [102]. Gfi-1 also interacts with tumor suppressor and transcriptional repressor PRDM5, which is 20

38 associated with transcriptional activation, rather than repression, of the genes that are shared targets of both Gfi-1 and PRDM5 [60]. Another recent study has shown that Gfi-1 is a master regulator of micrornas, which are non-protein-coding single-stranded RNA molecules that regulate gene expression. Gfi-1 -/- mice and SCN patients carrying N382S display deregulated micrornas. Overexpression of these micrornas recapitulates a block in G-CSF-induced granulopoiesis that is observed in Gfi-1-deficient mice and N382S-expressing SCN patients [103] Miz-1 in Myc-mediated transcriptional repression Myc family proteins, including evolutionally related c-myc, N-Myc and L-Myc, are helix-loop-helix/leucine zipper (HLH/LZ) transcription factors that contribute to the genesis of a wide range of human cancers. c-myc possesses the most potent oncogenic potential whereas L-Myc is the least among the Myc family members (c-myc will be referred to as Myc henceforth for convenience). Deregulated Myc expression is observed in a large number of hematopoietic malignancies, and transgenic animal models have revealed a critical role of Myc in the generation of leukemias and lymphomas [104]. Cancers with amplified MYC gene are usually associated with poor prognosis. Constitutive expression of Myc reduces growth factor-dependence, prevents cell cycle arrest and impairs differentiation [105]. Downregulation of Myc is a critical event for the growth inhibition induced by TGF-β and is required for TGF-β-mediated activation of p15ink4b, p21cip1 and G1 arrest [106, 107]. Cells overexpressing Myc can overcome cell cycle arrest induced by growth inhibitory signals or differentiation inducers, such as 21

39 TGF-β, p53 activation and phorbol ester, through activating cyclin/cdk complexes and suppressing CDKIs including p27kip1, p15ink4b, p21cip1 and p57kip2 [108, 109]. Recent study shows that Myc stability is markedly prolonged in a number of leukemia cell lines and bone marrow cells from patients with leukemia [110]. Myc can also induces massive apoptosis upon mitogenic signal withdrawal and DNA damage [ ]. Myc can function as either transcriptional activator or repressor. Myc activates transcription when it dimerizes with Max and binds to the Myc consensus DNA recognition sequence termed E-box [114, 115]; Transcriptional repression by Myc is less well characterized. One mechanism involves Myc-interacting zinc-finger protein 1 (Miz-1) [116]. Myc can be recruited to the promoters of Miz-1 target genes including p15ink4b, p21cip1 and Mad4 through association with Miz-1 leading to transcriptional repression [ ]. Repression of p21cip1 and p15ink4b by Myc is critical for Myc-transformed cells to escape cell cycle arrest in response to anti-proliferative signal, differentiation and mitogenic signal withdrawal [118, 120]. A Myc mutant MycV394D, which is defective for Miz-1 interaction but capable of dimerization with Max, does not repress Miz-1-activated transcription and has compromised transformation activity, suggesting that transcriptional repression by Myc through Miz-1 is critical for Myc-mediated transformation [118, 121]. Miz-1 is a poxvirus and zinc finger (POZ) domain containing zinc finger transcription factor with 13 ZF domains at the C-terminus, 12 of which are immediately clustered. The amphipathic helix region located between zinc fingers 12 and 13 is required for Miz-1 binding to Myc (Figure 7). Miz-1 was originally identified to be a Myc-interacting partner [117]. Miz-1 functions as a transcriptional activator and has a 22

40 potent growth arrest effect when ectopically expressed. Nonetheless, the growth inhibitory effect of Miz-1 is alleviated when co-expressed with Myc. Miz-1 activates transcription through binding to the core promoters, which contain the Inr element or the transcription start site, and recruiting p300 acetyltransferase to Miz-1 target genes including p15ink4b, p21cip1 and anti-apoptotic Bcl-2 [115, 118, 122]. Interaction of Myc with Miz-1 displaces p300 from Miz-1 and further recruits corepressors such as DNA methyltransferase, DNMT3a, and histone deacetylases (HDACs) [123, 124]. In addition to Myc, Miz-1-mediated transcriptional activation of p21cip1 is also negatively regulated by transcriptional repressor BCL6 and topoisomerase II β-binding protein (TopBP1), via interacting with Miz-1. BCL6 is essential for germinal center (GC) formation and deregulated BCL6 has been implicated in B-cell lymphomas. Interaction of BCL6 with Miz-1 leads to repression of p21cip1[125]. UV irradiation downregulates TopBP1 expression, and thereby releases Miz-1 from the inhibitory interaction with TopBP1. Free Miz-1 activates p21cip1 in conjunction with activated p53 [118, 126]. POZ ZF 1-12 ZF 13 Transcriptional activation Amphipathic helix Figure 7: Schematic representation of the Miz-1 protein structure. Miz-1 contains a POZ domain at the N-terminus and 13 ZF domains at the C-terminus. Miz-1 has a crucial role in the cellular responses to a number of stimuli including anti-mitogenic, apoptosis and differentiation signals. For instance, TGF-β induces rapid downregulation of Myc through Smad pathway, releasing Miz-1 to activate target gene 23

41 p15ink4b and causing cell cycle arrest in keratinocytes [45]. The induction of p21cip1, and subsequent G1 cell cycle arrest, in response to UV irradiation is Miz-1-depedent and inhibited by Myc through association with Miz-1 [120]. Similarly, the differentiation-induced p21cip1 upregulation is also negatively regulated by Myc through Miz-1 [127]. Moreover, inactivation of Miz-1 by Myc is essential for Myc-mediated apoptosis, which may be partially due to the inhibition of Miz-1 target gene Bcl-2 by Myc [114, 115]. Gene knockout studies indicate a more complex role of Miz-1. Deletion of Miz-1 gene is embryonic lethal from gastrulation deficiency due to massive apoptosis of ectodermal cells. Significantly Miz-1-/- mouse embryos showed similar p21cip1 expression as wild type embryos, but complete loss of p57kip2 suggesting that Miz-1 is required for the expression of p57kip2 but not p21cip1, although induction of p21cip1 upon UV-irradiation is dependent on Miz-1 function [109, 118, 128]. Conditional Miz-1 knockout in mouse keratinocytes resulted in delayed cell cycle exit and aberrant hair follicle development [121, 129]. A most recent study has shown that Miz-1-defeciency in hematopoietic system leads to a block in early B cell development due to impaired cell survival, which can be virtually rescued by Bcl-2 [130] 24