Investigating the Mechanisms of Growth Factor Independence-1 (Gfi-1)-Mediated Transcriptional Repression of P21cip1 and MBP

A Dissertation

Entitled

Investigating the mechanisms of growth factor independence-1 (Gfi-1)-mediated transcriptional repression of p21Cip1 and MBP

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

This document is copyrighted material. Under copyright law, no parts of this document

may be reproduced without the expressed permission of the author

An Abstract of

Investigating the mechanisms of growth factor independence-1 (Gfi-1)-mediated transcriptional repression of p21Cip1 and MBP

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 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 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.

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

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.

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TABLE OF CONTENTS

Page

ABSTRACT...…………………………………………………………………………....iii

DEDICATION...………………………………………………………………………....vi

ACKNOWLEDGEMENTS……………………………………………………………..vii

TABLE OF CONTENTS……………………………………………………………….viii

LIST OF FIGURES……………………………………………………………………...xii

ABBREVIATIONS……………………………………………………………………..xiv

CHAPTER1: INTRODUCTION…………………………………………………………1

1.1 Hematopoiesis...... 1

1.2 Hematopoietic cytokines………………………………………………2

1.3 Transcription factors in hematopoiesis…………………………………5

1.4 Leukemia and lymphoma...... 7

1.5 Granulocytes and neutropenia……………………………………………8

1.6 Cell cycle regulation and CDK inhibitors………………………………10

1.7 Inhibition of cell proliferation by TGF-β………………………………12

1.8 Growth factor independence-1 (Gfi-1)…………………………………13

1.8.1 Transcription factor Gfi-1……………………………………………13

1.8.2 Gfi-1 in normal hematopoiesis………………………………………14

1.8.3 Gfi-1 as a proto-oncoprotein……………………………………….18

1.8.4 Gfi-1 as a transcriptional repressor…………………………………19

1.9 Miz-1 in Myc-mediated transcriptional repression…………………….21

viii

CHAPTER 2: MATERIALS AND METHODS…………………………………………25

2.1 Cells and materials………………………………………………………25

2.2 Construction of plasmids………………………………………………26

2.3 Transfection and generation of stable cell lines…………………………27

2.4 Preparation of whole-cell and nuclear extracts…………………………28

2.5 Immunoprecipitation and Western blotting analysis……………………29

2.6 GST-pull down assay………………………………………………….29

2.7 Oligonucleotide precipitation (oligo-pull down) assay……………………29

2.8 Chromatin immunoprecipitation assay (ChIP) and Re-ChIP……………30

2.9 Luciferase assay…………………………………………………………31

2.10 Semi-quantitative reverse transcriptase PCR (RT-PCR)…………………32

2.11 RNA interference………………………………………………………32

2.12 Cell proliferation assay…………………………………………………33

2.13 Apoptosis Assay…………………………………………………………33

CHAPTER 3: RESULTS……………………………………………………………34

3.1. Miz-1-dependent transcriptional repression of p21Cip1 by Gfi-1……...... 34

3.1.1. Gfi-1 interacts with Miz-1………………………………………………..34

3.1.2. Gfi-1 and Miz-1 interact with each other via their ZF domains………….37

3.1.3. Gfi-1 is recruited to the p21Cip1 promoter through association with

Miz-1……………………..………………………………………………39

3.1.4. Gfi-1 represses Miz-1-induced activation of the p21Cip1 promoter,

and depletion of Gfi-1 leads to enhanced expression of p21Cip1…….43

3.1.5. Gfi-1 represses Miz-1- and TGF-β-activated p21Cip1 independent………

of direct DNA-binding……………………………………...……………45

3.1.6. Knockdown of Gfi-1 expression in HL60 and TF-1 cells results in………

reduced cell proliferation…………………………………,……..………47

3.1.7. Gfi-1 functionally collaborates with Myc in the repression of p21Cip1 49

3.1.8. Gfi-1 forms a ternary complex with Myc through Miz-1 on the p21Cip1…

promoter……………………………………………………………50

3.1.9. The Miz-1-dependent transcriptional repression by Gfi-1 plays a role in

regulating other cyclin-dependent kinase inhibitor genes p15Ink4b and…...

p27Kip1………………………………………………………………..…54

3.1.10. Gfi-1 is downregulated by TGF-β ……………….……………………58

3.2. Transcriptional repression of the eosinophil major basic protein (MBP) gene

by Gfi-1…………………………………………………………………59

3.2.1. Expression of the N382S mutant in myeloid cells leads to…………

upregulation of MBP in response to G-CSF………………………...... 59

3.2.2. Gfi-1 represses the MBP promoter...... 62

3.2.3. Expression of the N382S mutant potentiates the induction of the MBP.

promoter activity by G-CSF……………………………………………62

3.2.4. Overexpression of MBP inhibits IL3 and G-CSF-dependent growth and

accelerates cell death in response to G-CSF in myeloid cells……………63

3.2.5. Overexpression of MBP causes accelerated apoptosis in response to

G-CSF……………………………………………………………………65

3.2.6. Knockdown of Gfi-1 results in MBP upregulation……………….……67

CHAPTER 4: DISCUSSION…………………………………………………………68

4.1. Gfi-1-mediated transcriptional repression of p21Cip1 and other Miz-1

target genes………………………………………………..…………………70

4.2. Gfi-1 in TGF-β-mediated anti-growth effect…………………………...……75

4.3. Transcriptional repression of MBP by Gfi-1 in granulopoiesis………...……78

CHAPTER 5: FUTURE DIRECTIONS…………………………………………………81

5.1. Define the biological significance of Gfi-1 interaction with Miz-1…………82

5.2. Examine the potential role of Gfi-1 in the cellular response to TGF-β……...82

5.3. Investigate the mechanism by which TGF-β regulates Gfi-1 expression…...82

5.4. Explore the potential role of Miz-1 in the regulation of Gfi-1 expression

and granulopoiesis………….……………………………...………………...83

5.5. 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

LIST OF FIGURES

Figure 1 Schematic representation of hematopoiesis and the cytokines

regulating hematopoesis………………………………….…………….……4

Figure 2 Transcription factors that act at the various stages of Hematopoiesis…7

Figure 3 Three types of granulocytes: neutrophil, eosinophil and basophil………....9

Figure 4 Schematic representation of cell cycle progression……………………….11

Figure 5 Schematic representation of the Gfi-1 protein structure……………….….14

Figure 6 Schematic view of the role of Gfi-1 in hematopoiesis………………….…15

Figure 7 Schematic representation of the Miz-1 protein structure…………..………23

Figure 8 Gfi-1 interacts with Miz-1 in vitro and in vivo…………………….……36

Figure 9 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-1………………………………………………………..………....38

Figure 11 Gfi-1 is recruited to the p21Cip1 core promoter through association with

Miz-1 in vitro and in vivo…………………………………………….…...41

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

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 Gfi-1 forms a ternary complex with Myc through Miz-1……………...... 51

Figure 17 Gfi-1 forms a ternary complex with Myc through Miz-1 on the p21Cip1

core promoter in vivo……………………………………….……………...53

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 Gfi-1 is downregulated by TGF-β …………..…………………….…58

Figure 20 MBP is upregulated in response to G-CSF in the presence of the

N382S mutant………………………………………………………….…..61

Figure 21 Gfi-1 represses the MBP promoter……….…………………………….….62

Figure 22 Expression of the N382S mutant potentiates the induction of MBP promoter

activity by G-CSF………………………………………………………….63

Figure 23 Overexpression of MBP causes reduced cell proliferation and survival..…65

Figure 24 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 Diagrammatic view of the p21Cip1 promoter…………………...………..73

Figure 27 A working model for the regulation of TGF-β response by Gfi-1…………78

xiii

ABBREVIATIONS

ABL Abelson tyrosine kinase

AML Acute myeloid leukemia

Bax Bcl-2 homologous antagonist/killer

BCL B cell lymphoma

BCR Breakpoint cluster region

BM Bone Marrow

bp Base pair

BSA Bovine serum albumin

PBS Phosphate buffered saline

CD Cluster of differentiation

CDK Cyclin-dependent kinase

cDNA Complementary deoxyribonucleic acid

CDKI Cyclin-dependent kinase inhibitors

C/EBP CAAT/Enhancer Binding Protein

Cip1 CDK-interacting protein 1

CLL Chronic lymphoid leukemia

CLP Common lymphoid progenitor

CMP Common myeloid progenitor

CML Chronic myeloid leukemia

ECM Extracellular Matrix

ELA2 Enzyme elastase 2

EPO Erythropoietin

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ETO Eight twenty one

FACS Fluorescence Activated Cell Sorting

FBS Fetal Bovine Serum

FLT3 Fms-like tyrosine kinase 3

GC Germinal center

G-CSF Granulocyte-colony stimulating factor

Gfi-1 Growth factor independence-1

GFP Green fluorescent protein

GM-CSF Granulocyte-macrophage-colony stimulating factor

GMP Granulocyte/monocyte progenitor

HDAC Histone deacetylase

HEK Human embryonic kidney

HLH/LZ Helix loop helix/leucine zipper

HSC Hematopoietic stem cell

IL Interleukin

IP Immunoprecipitation

IFN Interferon

MBP Eosinophil major basic protein

M-CSF Macrophage-colony stimulating factor

MDS Myelodysplastic syndrome

MEP Megakaryocyte/erythrocyte progenitor

Miz-1 Myc-interacting zinc-finger protein-1

NE Neutrophil elastase

NHL Non-Hodgkin’s lymphoma

PBS Phosphate buffered saline

PIAS3 Protein inhibitor of activated STAT3

PML Acute promyelocytic leukemia

PMSF Phenylmethylsulfonyl fluoride

POZ Poxvirus and zinc-finger

RAR All-trans-retinoic acid

SCF Stem Cell Factor

SCN Severe congenital neutropenia

SNAG Snail/Slug

STAT Signal transducer and activator of transcription

Tal1 T-cell acute lymphocytic leukemia 1

TβRE TGF-β-response element

TNF Tumor necrosis factor

TopBP1 Topoisomerase II β-binding protein

TPO Thrombopoietin

WB Western blotting

WCE Whole-cell extract

ZF Zinc-finger

xvi

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.

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].

1.2. 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 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].

GM-CSF, G-CSF Granulocyte GMP

GM-CSF, M-CSF CMP Monocyte IL-3 GM-CSF Kit Ligand EPO IL-6, IL-3,IL-11 Erythrocyte

TPO, IL-11 MEP Megakaryocyte Self- HSC renewal Pre-T IL-2, 4, 7,15 T lymphocyte IL-3, FLT3 Ligand IL-3, 7

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.

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 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].

C/EBPε Granulocyte GMP RAR C/EBPα PU.1 CMP Monocyte

α GATA-1, EKLF PU.1 , C/EBP Erythrocyte GATA-1 GATA-1, 3 MEP Self- Megakaryocyte renewal HSC

GATA-2 AML-1 Pre-T GATA-3 T lymphocyte PU.1 c-Myb

CLP Pax5 E2A B lymphocyte Pre-B

Figure 2: Transcription factors that act at the various stages of hematopoiesis.

1.4. 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 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].

1.5. 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).

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 1.5 × 109/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 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].

1.6. 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 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].

G0 Cyclin B/A + CDK2

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 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].

1.7. 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].

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.

1.8. Growth factor independence-1 (Gfi-1)

1.8.1. 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 C2H2-type zinc finger (ZF)

domains and the N-terminal SNAG domain well conserved among Gfi-1 family proteins

[54-56] (Fig. 5). The 3rd, 4th and 5th 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].

SNAG Intermediate Region C2H2 ZFs

1 2 3 4 5 6

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.

1.8.2. 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 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

B Gfi-1 Pre-B + + CLP Gfi-1 + Pre-T T

+ + Gfi-1 Gfi-1 Neu Self-renewal + + GMP Mon

CMP Gfi-1 +

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 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 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 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].

1.8.3. 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 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-XL in CD4 T cells [95, 96].

1.8.4. 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 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 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].

1.9. 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

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 [111-113].

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 [117-119]. 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 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 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]

CHAPTER 2: MATERIALS AND METHODS

2.1. Cells and materials

Human embryonic kidney (HEK) 293 cells, 293 cells containing SV40 Large

T-antigen (293T) and human epithelial Hela cells derived from cervical carcinoma were

grown in Dulbecco’s Modified Eagle’s medium (DMEM, Mediatech) supplemented with

10% fetal bovine serum (FBS, Mediatech) and 1% penicillin/streptomycin (P/S). Murine

myeloid 32D and L-G cells were cultured in RPMI 1640 medium (Mediatech)

supplemented with 10% FBS, 1% P/S and 10% WEHI-3B cell conditioned medium as a

source of murine IL-3. Human promyelocytic leukemia HL60 and human monocyte-like

lymphoma U937 cells were cultured in 10% FBS supplemented RPMI 1640 medium.

TF-1 cells derived from human erythroid leukemia were maintained in RPMI 1640

medium containing 10% FBS and 2 ng/ml human GM-CSF (PeperoTech). HL60 clone15

cells were cultured in RPMI 1640 supplemented with 10% FBS, 1 mM sodium pyruvate

and 10 mM HEPES. Bone marrow (BM) cells from Gfi-1+/+ and Gfi-1-/- mouse were kindly provided by Dr. Jinfang Zhu (National Institute of Health, NIH). Human TGF-β and G-CSF (PeperoTech) were applied to cells at a concentration of 5 ng/ml and 100 ng/ml, respectively.

Antibodies used are: anti-p21Cip1 (C-19), anti-Gfi-1 (N-20), anti-Miz-1 (N-17),

anti-p27Kip1 (C-19), and anti-Myc (N-262) antibodies from Santa Cruz Biotechnology;

25 anti-Myc-Tag, anti-Flag-Tag and anti-p15 antibodies from Cell Signaling; and anti-β-actin antibody from Sigma.

2.2. Construction of plasmids

Rat Gfi-1 cDNA was kindly provided by Dr. P. N. Tsichlis (Fox Chase Cancer

Center, Philadelphia) and inserted into pBabe-puro vector to generate pBabe-puro-Gfi-1.

The retroviral Gfi-1 expression construct GFP-RV-Gfi-1 containing an internal ribosomal entry sequences (IRES) and humanized green fluorescent protein (GFP) cDNA, was a general gift from Dr. J. Zhu (National Institutes of Health). pBabe-puro-N382S and

GFP-RV-N382S were produced by site-directed mutagenesis. The mutation was confirmed by XhoI digestion as the mutation in Gfi-1 cDNA leading to N382S created an

XhoI site. The rat Gfi-1 cDNA was also inserted into pCDNA3.1B/Myc-His vector

(Invitrogen). The sequence of Myc-tag was replaced by a Flag-tag sequence in frame by inserting an adaptor duplex of Flag-tag sequence between BamH I and Afl II sites. The

Gfi-1-dN mutant lacking the N-terminal SNAG domain, Gfi-1-dZF6 and Gfi-1-dZF3 mutants lacking the C-terminal 6 ZF domains and the ending 3 ZF domains, respectively, were generated using PCR-based strategies.

The GST/Miz-1 construct was generated by cutting out the cDNA encoding the

C-terminal region of Miz-1 from amino acid 329 to 803 from the yeast 2-hybrid

GAL4-AD plasmid and inserting it into pGEX-4T (Invitrogen). Miz-1 full-length cDNA was amplified from 32D cells by RT-PCR and inserted into pCDNA3.1B/Myc-His vector.

The primers used to amplify Miz-1 cDNA areMiz-1 mutants Miz-1-dN lacking the

N-terminal POZ domain and Miz-1-dZF lacking the C-terminal 12 ZF domains were generated using PCR-based strategies.

The 2.4 kb fragment of MBP promoter (-1194/+232) was generated by amplifying two fragments from 32D genomic DNA and then ligated at PasI site. This 2.4 kb MBP promoter sequence was subsequently inserted into pGL3 luciferase reporter vector

(Invitrogen). The cDNA encoding MBP was amplified by RT-PCR from

G-CSF-stimulated 32D cells expressing N382S. MBP cDNA was then inserted into

GFP-RV vector described above.

The luciferase reporter constructs for p21Cip1, p27Kip1 and p15Ink4b have been described previously [131-133].

2.3. Transfection and generation of stable cell lines

32DWT (32D cells transfected with wild-type G-CSF receptor) and L-G cells were transfected with pBabe-puro or pBabe-puro-Gfi-1 by electroporation using Electro

Square Porator ECM 830 (Genentronic), and selected in 1 μg/ml puromycin. Individual clones were obtained by limiting-dilution method. Expression of Gfi-1 was examined by

Western blotting. To generate MBP overexpressing cells, 32DWT cells were co-transfected with GFP-RV-MBP and pBabe-puro at 10:1 ratio and selected in 1 μg/ml puromycin. Cells that were both puromycin-resistant and GFP positive were then subjected to subcloning by limiting-dilution. Expression of MBP was confirmed by

RT-PCR.

To genearate the inducible Gfi-1 and N382S-expressing cells, 32DWT cells were

27 transfected with pUbiq.irtTA [134] by electroporation and selected in 0.8 mg/ml G418.

The plasmid pUbiq.irtTA contained a gene encoding the glucocorticoid/ tetracycline-inducible transactivator and GFP with an IRES. Individual clones were isolated and evaluated for inducible gene expression by luciferase assay. Cells were transfected with pTRE2-Hyg-Luc (Clontech), which contained luciferase gene under the control of a tetracycline-dependent element, followed by dexamethasone and doxycyclin

(Dex/Dox, 100nM and 1 μg/ml, respectively) treatment. Luciferase activity was measured

48 hours after transfection. The cells with strong inducible luciferase activity were chosen and further transfected with pTRE2-Hyg vector, pTRE2-Hyg-Gfi-1 or pTRE2-Hyg-N382S by electroporation. Stable transfectant were selected in 1 mg/ml hygromycin. Expression of Gfi-1 proteins before and after 24 hours treatment with

Dex/Dox was examined by Western blotting.

2.4. Preparation of whole-cell and nuclear extracts

293T cells were transfected with Flag-tagged-Gfi-1, Myc-tagged-Miz-1 or their deletion mutants using the TransIT-LT1 transfection reagent (Mirus Bio Corporation).

Cells were harvested 48 hours later and lysed in lysis buffer (1 M Tris pH 7.5, 150mM

NaCl, 20 mM NaF, 10 mM glycerol phosphate, 1 mM phenylmethylsulfonylfluoride

[PMSF] and 1% Triton X-100). Cell lysates were cleared by centrifugation at 12,000 rpm for 30 min at 4 °C. For the preparation of nuclear extracts, HL-60 cells were washed and resuspended in buffer A (20 mM Hepes pH 7.0, 1 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.1% NP40, 1 mM PMSF, 10% glycerol) and lysed by 15 strokes in a glass dounce homogenizer. After centrifugation at 6,600 rpm for 10 min at 4 °C, pellets were

28 solubilized in buffer A with 300 mM NaCl, sonicated and centrifuged at 12,000 rpm for

30 min. The resulting supernatants were collected as nuclear extracts.

2.5. Immunoprecipitation and Western blotting analysis

Whole-cell and nuclear extracts were subjected to immunoprecipitation using the anti-Flag or anti-Miz1 antibody. Immunocomplexes were recovered with protein A/G beads and washed 5 times with lysis buffer. Samples were boiled in SDS sample buffer and resolved by SDS/PAGE before transfer to immobilon membranes. The membranes were incubated with the appropriate antibodies and the reactive proteins were visualized by enhanced chemiluminescence.

2.6. GST-pull down assay

GST protein and GST/Miz-1 fusion protein were purified from BL21-CodonPlus

Competent Cells (Stratagene) and lysed in phosphate buffered saline (PBS) containing

1% Triton with sonication using Sonic Dismembrator (Model 500, Fisher Scientific).

Purified GST and GST/Miz-1 were immobilized on glutathione sepharose beads and incubated with whole-cell extracts from 293T cells transfected with Flag-tagged-Gfi-1 at

4 °C for overnight. Beads were precipitated and washed 4 times and bound proteins were analyzed for Gfi-1 using the anti-Flag-tag antibody by Western Blotting.

2.7. Oligonucleotide precipitation (oligo-pull down) assay

293T cells were transiently transfected using lipofactamine 2000 (Invitrogen) and

29 collected 48 hours after transfection. Cells were lysed in HKMG buffer (10 mM Hepes pH 7.9, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT and 1% Nonidet p-40)

and whole-cell extracts were incubated at 4 °C overnight with biotinylated

double-stranded oligonucleotide spanning the core promoter of human p21Cip1 (-49/+16)

or p15Ink4b (-12/+18) in the presence of 25-fold excess of poly(dI-dC) (Amersham

Biosciences). The oligonucleotides and bound proteins were then precipitated by

streptavidin beads and washed 4 times with HKMG buffer. Bound proteins were analyzed

by Western Blot analysis.

2.8. Chromatin immunoprecipitation assay (ChIP) and Re-ChIP

293T cells were transiently transfected using lipofactamine 2000 (Invitrogen) and

subjected to chromatin immunoprecipitation (ChIP) assay 48 hours after transfection.

Right before harvesting, the cells were fixed with 1% formaldehyde for 10 min at 37 °C

and terminated with 0.125 M glycine for 5 min at room temperature. Cells were lysed

first in hypotonic buffer (5mM Tris-HCl [pH 7.5] (Fisher), 85mM KCl, and 0.5% NP40).

After centrifugation at 6,000 rpm for 5 min at 4 °C, the pellets of nuclei were lysed in

ChIP lysis buffer (1% SDS, 10 mM EDTA, and 50mM Tris) and sonicated to shear

chromatin DNA to about 500 bp fragments using a Fisher Scientific model 500 sonic

dismembrator. 1/20 of each nuclear lysate was kept for input and the rest was precleared

with protein A/G-agarose beads and rabbit normal IgG (Santa Cruz Biotechnology) for 1

hour and then immunoprecipitated using the anti-Miz-1, anti-Gfi-1 or irrelevant anti-NE

(negative control) antibodies. Precipitated chromatin DNA was eluted with elution buffer

(1% SDS, 1% NaHCO3) and purified using Wizard SV Gel and PCR clean-up System

(Promega). Semi-quantitative PCR was carried out using primer pairs to amplify the promoter regions of human p21Cip1 (-194/+88, -3723/-3400, +3455/+3771, -2991/-2750,

-1548/-1270), p15Ink4b (-100/+45, -3756/-3278, +2995/+3477) and p27Kip1 (+56/+232,

-3190/-2818).

Re-ChIP was described previously [135]. Briefly, single ChIP complexes were eluted with 30 μl 10 mM DTT by incubating for 30 min at 37 °C with agitation. The eluted complexes were then diluted 20 times with Re-ChIP buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl [pH 7.5] ) and subjected to the second immunoprecipitation, and the rest of Re-ChIP thereafter was carried out as the same as

ChIP described above.

2.9. Luciferase assay

32D cells were transfected by electroporation. 12 hours after transfection, cells were treated with or without G-CSF for additional 8 hours and then harvested. Hela cells were transfected using Lipofactamin 2000 (Invitrogen) and harvested 48 hours after transfection with or without TGF-β treatment for 6 hours. Luciferase assays were carried out by lysing the cells in cell culture lysis reagent (CCLR, Invitrogen) and measuring luciferase activities by Lmax luminometer (Molecular Devices). β-galactosidase activities of the co-transfected pCMV-LacZ (A generous gift from Dr. Douglas W. Leaman) was measured by Vmax kinetic microplate reader (Molecular Devices) and used to normalize the luciferase activities. Data represent mean ± SD from triplicated experiments.

2.10. Semi-quantitative reverse transcriptase-PCR (RT-PCR)

2 μg of total cellular RNA isolated using TRIzol reagent (Invitrogen) was reverse transcribed into cDNA by Oligo(dT)-15 primer (Promega) and AMV reverse transcriptase

(AMV-RT, Promega). 2 μl of the reverse transcription reaction was then subjected to PCR amplification with gene-specific primer pairs. The transcripts of genes in different samples were quantitated against the transcript of the house-keeping gene GAPDH. `

`PCR products were resolved on 1% agarose gels, visualized by ethidium bromide staining under UV light and images were captured with a digital camera.

2.11. RNA interference

Short hairpin RNAs (shRNAs) directed against human GFI-1 and cloned into pLKO.1 were purchased from Open Biosystems. The identification of the shRNA constructs are: TRCN0000020464, TRCN0000020465, TRCN0000020466,

TRCN0000020467, and TRCN0000020468. The lentiviruses were produced by co-transfecting 293T cells with the individual shRAN construct along with the packaging plasmids (RRE, RSV and VSVG), using TransIT-LT1 transfection reagent (Mirus Bio).

Transfected cells were cultured in DMEM supplemented with 10% FBS and 0.1% P/S and replaced with DMEM containing 30% FBS and 1% P/S 18 hours after transfection.

The virus-containing medium was harvested at 24 and 48 hours later, pooled and centrifuged to obtain the viral supernatant. HL60, U937 and TF-1 cells were infected with the viral supernatant in the presence of 8 µg/ml of polybrene and selected in 3 µg/ml puromycin 48 hours after infection. Individual clones were isolated by limiting-dilution method in 96 well plates.

2.12. Cell proliferation assay

Cells were seeded at a density of 3 × 105 cells/ml in culture medium. Cell

numbers were monitored by trypan blue exclusion on 7 consecutive days.

2.13. Apoptosis assay

Apoptosis was assessed by Annexin V-PE apoptosis detection kit I (BD

Biosciences). 2 × 105 cells were collected, resuspended in Annexin V binding buffer

and stained with Annexin V-PE and 7 amino-actinomycin (7-AAD) for 15 min at room

temperature. Cells were then analyzed by flow cytometry and data were evaluated using

BD Cellquest software (Becton Dickinson).

CHAPTER 3: RESULTS

3.1. Part I: Miz-1-dependent transcriptional repression of p21Cip1 by

Gfi-1

3.1.1. Gfi-1 interacts with Miz-1

As a transcription factor, Gfi-1 carries out its function by interacting with

co-factors and regulatory proteins. In order to identify new Gfi-1 binding partners to

further investigate the mechanisms by which Gfi-1 functions in myeloid development, a

yeast-two hybrid assay was performed using full-length Gfi-1 as the bait to screen Gfi-1

binding proteins from a human bone marrow cDNA library. One of the positive clones

contained the cDNA encoding the C-terminal portion, starting from the second zinc

finger domain, of Miz-1. To verify the interaction, the Miz-1 cDNA from the clone was

inserted into pGEX-4T vector to generate a construct for the expression of the C-terminal

portion of Miz-1 fused with glutathione S-transferase (GST/Miz-1).

Whole-cell extracts from 293T cells transfected with Flag-tagged Gfi-1 were incubated

with bacterially purified GST or GST/Miz-1 fusion protein immobilized on glutathione

sepharose beads. The precipitated complexes were analyzed for Flag-tagged Gfi-1 by

Western blotting using the anti-Flag-tag antibody. As shown in Fig. 8A, GST/Miz-1

34 fusion protein, but not GST alone, pulled down Gfi-1. Co-immunoprecipitation assays were further carried out to confirm the interaction between Gfi-1 and Miz-1 in vivo. 293T cells were transfected with Myc-tagged Miz-1 alone, or co-transfected with either

Flag-tagged Gfi-1 or Flag-tagged STAT5. Whole-cell extracts were immunoprecipitated with the anti-Flag-tag antibody and analyzed for Myc-tagged-Miz1. As shown in Fig. 8B,

Miz-1 was co-immunoprecipitated with Gfi-1 but not with STAT5. In addition, the interaction between endogenous Gfi-1 and Miz-1 was studied in HL60 promyelocytic leukemic cells (Fig. 1C). HL60 nuclear extract was subjected to immunoprecipitation with the anti-Miz-1 antibody, and then the precipitated proteins were examined for Gfi-1 and Miz-1 by Western blotting. The anti-Miz-1 antibody, but not the species-matched control antibody, co-immunoprecipitated Gfi-1 indicating the interaction between endogenously expressed Gfi-1 and Miz-1 (Fig. 8C). ([136] and the dissertation of Basu,

Suchitra)

A B C

z i Flag-STAT5 - - + IP

/ - Flag-Gfi1 - + 1

S z

Myc-Miz-1 i Marker

G + ++ G

C IP: α-Flag α Flag-Gfi1 α -Flag WB: α-Myc Gfi-1 Kd Kd Miz-1 105 GST/Miz-1 105 75 WB: α-Flag 50 75 35 WCE 50 30 GST WB: α-Myc HL60 293T 293T

Figure 8: Gfi-1 interacts with Miz-1 in vitro and in vivo. (A) Whole-cell extracts from

293T cells transfected with Flag-tagged Gfi-1 were incubated with GST or GST/Miz-1 fusion protein immobilized on glutathione sepharose beads, and then Flag-tagged Gfi-1 was examined by Western blotting using the anti-Flag-tag antibody (upper). Virally purified GST and GST/Miz-1 fusion proteins were stained by GelCode Blue reagent on

SDS-PAGE gel (lower). (B) 293T cells were transfected with Flag-tagged-Gfi-1 or

Flag-tagged-STAT5, along with Myc-tagged-Miz-1. Whole-cell extracts were immunoprecipitated with the anti-Flag-tag antibody and followed by Western blotting using the anti-Myc-tag antibody (top). The expression of the transfected proteins were analyzed by Western blotting using indicated antibodies (middle and bottom). (C)

Nuclear extracts of HL60 cells were subjected to immunoprecipitation with either the anti-Miz-1 antibody or a species-matched control antibody followed by Western blotting using the anti-Gfi-1 and anti-Miz-1 antibodies. (Figure adapted form Basu and Liu et. al.

[136]; done by Qiu, Yaling)

3.1.2. Gfi-1 and Miz-1 interact with each other via their ZF domains

To investigate which region of Gfi-1 is responsible for its interaction with Miz-1, different Gfi-1 deletion mutants (Fig. 9A) were generated and cloned into pCDNA-Flag-tag vector. Gfi-1-dN lacks the N-terminal SNAG domain. Gfi-1-dZF3 and

Gfi-1-dZF6 were devoid of the C-termianl 3 ZF domains and 6 ZF domains, respectively.

Co-immunoprecipitation assays were performed following co-expressing the Flag-tagged

Gfi-1 or Gfi-1 mutants and Myc-tagged Miz-1 in 293T cells. Removal of the SNAG domain did not affect the interaction whereas deletion of the C-terminal 3 ZF domains was enough to abolish the interaction between Gfi-1 and Miz-1 (Fig. 9B). These data indicates that the ZF domains of Gfi-1 were essential for Gfi-1 binding to Miz-1 [136].

ABFlag-Gfi-1-dZF6 - - - - + - SNAG ZF 1-6 Flag-Gfi-1-dZF3 ---+-- Gfi-1 1234 56 Flag-Gfi-1-dN - - + --- Flag-Gfi-1 - + - - -+ Myc/His-Miz-1 + + + + -+ Gfi-1-dN 123456 Kd IP: Flag-Tag 100 WB: Myc-Tag 72 Gfi-1-dZF3 123 50 WB: Flag-Tag Gfi-1-dZF6 35 whole cell extracts

WB: Myc-Tag

Figure 9: The C-terminal ZF domains of Gfi-1 are required for Gfi-1 interacting with Miz-1. (A) Schematic representations of Gfi-1 and Gfi-1 deletion mutants. (B) 293T cells were transfected with Myc-tagged-Miz1 and the Flag-tagged forms of Gfi-1 and

Gfi-1 mutants shown in (A). Whole-cell extracts were examined by Western blotting with the anti-Flag-tag and the anti-Myc-tag antibodies, or immunoprecipitated with the anti-Myc-tag antibody prior to Western blotting with the anti-Flag-tag antibody. (Figure adapted from Basu and Liu et. al. [136] done by Qiu, Yaling)

To map the Miz-1 domain involved in interacting with Gfi-1, Miz-1 mutants with deletion of the N-terminal POZ domain (Miz-1-dN) or the C-terminal 12 ZF domains

(Miz-1-dC) were generated (Fig. 10A). Myc-tagged full-length Miz-1 and the deletion mutants were expressed in 293T cells along with Flag-tagged-Gfi-1 (Fig. 10B). Miz-1-dN interacted with Gfi-1 as effectively as the full-length Miz-1. However Miz-1-dC completely lost the ability to interact with Gfi-1. Therefore the ZF domains of Miz-1 were required for Miz-1 binding to Gfi-1. ([136] and the dissertation of Basu, Suchitra)

Flag-Gfi-1 --++++ Myc-Miz-1 + - + --- Myc-Miz-1- dN - - - + -- A B Myc-Miz-1- dC - - - - + - Kd

POZ ZF 1-12 ZF 13 100 WB: α-Myc Miz-1 IP: α-Flag 72

Miz-1-dN WB: α-Flag

Miz-1-dC 100 WB: α-Myc 72

WB:α-Flag

Figure 10: The C-terminal ZF domains of Miz-1 are required for Miz-1 binding to

Gfi-1. (A) Schematic presentations of Miz-1 and Miz-1 deletion mutants. (B) 293T cells were transfected with Myc-tagged-Miz-1 and Miz-1 mutants along with

Flag-tagged-Gfi-1. Whole-cell extracts were immunoprecepitated with the anti-Flag-tag antibody. Gfi-1 and Miz-1 proteins were examined by Western blottings using the anti-Myc and anti-Flag antibodies before (bottom 2 panels) and after (top 2 panels) immunoprecipitation. (Figure adapted from Basu and Liu et. al. [136] done by Basu,

Suchitra)

3.1.3. Gfi-1 is recruited to the p21Cip1 promoter through association with Miz-1

Myc and BCL6 can be recruited through association with Miz-1 to the promoter regions of Miz-1 target genes p15Ink4b, p21Cip1, and Mad4, leading to repression of their transcription [113, 119, 125, 137]. Gfi-1, as a binding partner of Miz-1, may utilize a similar mechanism to repress Miz-1 target genes. Miz-1 binds to the p21Cip1 core promoter region that contains the transcription start site [125, 127]. To address whether

Gfi-1 is recruited to the p21Cip1 promoter by Miz-1, we performed oligonucleotide precipitation assays. 293T cells were transfected with Myc-tagged-Miz1,

Flag-tagged-Gfi-1 or both. Whole-cell extracts were incubated with biotinylated double-stranded oligonucleotide spanning the p21Cip1 core promoter sequence from –49 bp to +16 bp containing the Miz-1-interacting element [113]. Bound proteins were precipitated with streptavidin-coated beads. As shown in Fig. 11A, Gfi-1 was precipitated with the oligonucleotide only in the presence of Miz-1. Deletion of the six ZF domains, which disrupted Gfi-1 binding to Miz-1 (Fig. 10), abolished the binding of Gfi-1 to the p21Cip1 core promoter oligonucleotide (Fig. 11B). These results suggested that Gfi-1, through interacting with Miz-1, was recruited to the p21Cip1 core promoter.

To assess the binding of Gfi-1 to the p21Cip1 core promoter in vivo, we performed chromatin immunoprecipitation (ChIP) assays. 293T cells were transfected with Gfi-1, Miz-1, or both. Nuclear extracts were prepared and immunoprecipitated with the anti-Miz-1 antibody, anti-Gfi-1 antibody, or the species-matched irrelevant anti-NE antibody as a control. Bound chromatin DNA was then eluted and subjected to semi-quantitative PCR to amplify the p21Cip1 core promoter region (-194/+88) or regions about 3 kb upstream and 3 kb downstream of the transcription start site [113]

[127]. We found that Gfi-1 bound to the p21Cip1 core promoter only when co-expressed with Miz-1. No binding of Gfi-1 to the 3 kb upstream and 3 kb downstream regions was detected (Fig. 11C, left). The expression of Gfi-1 and Miz-1 was examined by Western blotting (Fig. 11C, right).

Duan et al. previously identified two Gfi-1 binding sites located at -2838/-2811 bp and -1383/-1357 bp on p21Cip1 using EMSA assay [60] [97]. We performed ChIP assay to examine whether Gfi-1 bound to the two sites in vivo in HL60 cells, which express endogenous Gfi-1 and Miz-1. Although binding of Gfi-1 to the p21Cip1 core promoter was consistently demonstrated in HL60 cells, we failed to detect binding of Gfi-1 to the two sites identified by Duan et al. using primers to amplify the regions spanning

-2991/-2750 bp and -1548/-1270 bp of the p21Cip1 promoter (Fig. 11D). Therefore, whether Gfi-1 binds to these two sites on the p21Cip1 promoter in vivo is still not clear.

Gfi-1 + - - - - AB Gfi-1-dN - - - ++ - Gfi-1 + + Gfi-1-dZF6 - + + -- Miz-1 -++ Miz-1 + - + - + Gfi-1 Oligonucleotide Gfi-1 precipitation Oligonucleotide Miz-1 precipitation Miz-1

Whole-cell Gfi-1 extract Miz-1 Gfi-1 Whole-cell extract Miz-1

Input -NE -Gf-1 -Miz1 -Gfi-1 α α α α -++ Gfi-1 -+++ -+ + +-+ Miz-1 +- ++ +-+WB

-194 to +88 Gfi-1 Nuclear Extract -3,723 to -3,400 Miz-1

+3,455 to +3,771 β-actin

f E D t

N G

- -

α α

-194 to +88

-2991 to -2750

-1548 to -1270

Fig. 11

Figure 11: Gfi-1 is recruited to the p21Cip1 core promoter through association with

Miz-1 in vitro and in vivo. 293T cells were transfected with Myc-tagged Miz-1,

Flag-tagged Gfi-1 (A) and Gfi-1 deletion mutants (B). Whole-cell extracts were prepared and incubated with the biotinylated double-stranded oligonucleotide spanning -49 bp to +

16 bp of the p21Cip promoter. Bound proteins were precipitated using the streptavidin-coated beads and examined by Western blotting. (C) 293T cells were transiently transfected with Gfi-1 and/or Miz-1 as indicated. Nuclear extracts was sonicated to shear chromatin DNA to about 500 bp fragments and immunoprecipitated with the anti-Miz-1, anti-Gfi-1 and anti-NE (negative control) antibodies. Chromatin

DNA precipitated with antibodies was eluted and subjected to semi-quantitative PCR using primers to amplify the p21Cip1 core promoter region containing the transcription start site (-194/+88). Primer sets aiming to amplify the upstream region (-3723/-3400) and downstream region (+3455/+3771) of the p21Cip1 gene were used as negative control (left). Input shows the PCR amplification from chromatin DNA purified from nuclear extracts prior to immunoprecipitation (middle). Expression of Gfi-1, Miz-1 and

Myc was examined by Western blotting (right). (D) Nuclear extracts of HL60 cells were sonicated and subjected to immunoprecipitation with the anti-Gfi-1 antibody or control antibody. Semi-quantitative PCR was then carried out to amplify indicated regions of the p21Cip1 promoter.

3.1.4. Gfi-1 represses Miz-1-induced activation of the p21Cip1 promoter, and depletion of Gfi-1 leads to enhanced levels of p21Cip1.

Miz-1-induced activation of p21Cip1 and p15Ink4b is repressed by Myc through interaction with Miz-1 [118, 120]. As a transcriptional repressor, the recruitment of Gfi-1 to the p21Cip1 promoter may lead to transcriptional repression of p21Cip1. Luciferase reporter assays were performed in Hela cells to evaluate the effect of Gfi-1 on Miz-1- activated p21Cip1 promoter activity. As shown in Fig. 12A, Miz-1-induced activation of a 2.4 kb fragment of human p21Cip1 promoter [43] was repressed by co-expressed Gfi-1.

To further demonstrate the repression of p21Cip1 by Gfi-1, we addressed whether

Gfi-1 depletion led to increased p21Cip1 expression. HL60 cells were infected with lentivirus carrying pLKO.1 vector (Ctr) or pLKO.1-shRNAs against Gfi-1, and then examined by Western blotting for the expression of Gfi-1 and p21Cip1. We observed that knockdown of Gfi-1 was associated with increased p21Cip1 protein levels (Fig. 12B). We further assessed the expression of p21Cip1 in Gfi-1-/- mice bone marrow cells. As shown in Figure 12C, cells from Gfi-1-/- mice exhibited enhanced levels of p21Cip1 protein and mRNA when compared to bone marrow cells from Gfi-1+/+ mice. These data indicated that Gfi-1 repressed p21Cip1 expression.

expression levelsinGfi-1 of thehumanp21Cip1 transfected withluciferase depletion ofGfi-1leadstoenhancedexpressionp21Cip1.(A) Figure 12:Gfi-1repressesMiz- and p21Cip1expressionlevelswere examined byWestern blotting. lentivirus containingeitherempty pLKO.1 control for normalizing theluciferase activity values. constructs. Western blotting(left)and semi C A

β Ctr -galactosidase activityof co-transfected shRNA Luciferase Activity Miz-1 Gfi-1 p21P (fold activation) 0.5 1.5 2.5 3.5 p21Cip1 β 0 1 2 3 Gfi-1 -actin + - - promoterdoses ofGfi-1expressing alongwithincreasing -/- reporter constructpGL3-p21Pcontaininga2.4-Kbfragment andGfi-1 qatttv TPR(ih) -quantitative RT-PCR(right). D ++ ++ ++ ++ - 1-induced activationofthe

+/+ Gfi-1+/+ mouse bonemarrow cellswereexamined by 44 (Ctr) orpLKO.1-shRNAagainstGfi-1.Gfi-1 Gfi-1-/- p21P (2.4 kb) (2.4 p21P + + β p21Cip1 -actin pCMV-LacZwasused asan internal (B) HL60 cellswere infected with p21Cip1 promoter,and Gfi-1+/+

-/-

Helacells were Gfi-1 (C) GAPDH p21Cip1 p21Cip1

3.1.5. Gfi-1 represses Miz-1- and TGF-β-activated p21Cip1 independent of direct

DNA-binding

Our data demonstrate that Gfi-1 was recruited to the core promoter of p21Cip1 through interacting with Miz-1. We further investigated whether direct DNA-binding of

Gfi-1 is dispensable for Gfi-1-mediated transcriptional repression of p21Cip1. A previously described luciferase reporter construct contains a 111 bp core promoter region devoid of Gfi-1 recognition sites. The activity of the p21P (2.4 kb) and p21P (111 bp) was induced by TGF-β [43]. Our luciferase reporter assays showed that Gfi-1 repressed

Miz-1-induced promoter activity of p21P (2.4 kb) as well as p21P (111 bp), despite of a lack of Gfi-1 recognition site (Fig. 13A). The DN mutant of Gfi-1, N382S, is defective for DNA-binding [66, 76, 77] but capable of interacting with Miz-1 (Figure 11 in the dissertation of Basu, Suchitra). Notably, the N382S mutant was as effective as Gfi-1 in repressing both Miz-1-induced (Fig. 13B and C) and TGF-β-induced (Fig. 13D) activity of p21Cip1 promoter fragments. These results clearly showed that the direct

DNA-binding of Gfi-1 was not required for Gfi-1-mediated repression of p21Cip1.

A B

8 8 7 p21P (111 bp) 6 6 5

3 4

(Arbitrary unit) 2 Luciferase activity (Arbitrary unit) 2

1 activity Luciferase

0 p21P (2.4 kb) + ++- -- 0 p21P (111 bp) - --+ ++ Miz-1 -+++ Miz1 - ++- ++ Gfi-1 --+ - Gfi-1 - --- -+ N382S --- +

C D

3 12 p21P (2.4 kb) p21P (111 bp) 2.5 10

2 8

1.5 6

1 4 (Arbitrary unit) Luciferase activity (arbitrary units)

0.5 Luciferase activity 2

0 0 Miz-1 -+++ TGF-β -+++ Gfi-1 --+- Gfi-1 --+- N382S ---+ N382S ---+

Figure 13: Gfi-1 represses Miz-1- and TGF-β-activated p21Cip1 promoter activity independent of direct DNA-binding. (A) Hela cells were transfected with luciferase reporter constructs containing the 2.4 kb or 111 bp promoter fragment of p21Cip1, along with Miz-1 and Gfi-1 as indicated. Luciferase assay was performed 48 hours after transfection. (B) Hela cells were transfected with p21P (2.4 kb) or (C) p21P (111 bp), along with Gfi-1 or N382S as indicated. Luciferase activity was measured 48 hours after transfection. (D) Hela cells were transfected with p21PSma, Gfi-1 and N382S as indicated. Cells were untreated or treated with 5 ng/ml TGF-β for 6 hours prior to assay for luciferase activity 48 hours after transfection.

3.1.6. Knockdown of Gfi-1 expression in HL60 and TF-1 cells results in reduced cell proliferation

Since Gfi-1 repressed p21Cip1, we reasoned that knockdown of Gfi-1 expression might have a negative effect on cell proliferation. To examine the effect of Gfi-1 knockdown on cell proliferation, human promyelocytic leukemia HL60 cells and erythroid leukemia TF-1 cells were infected with lentivirus containing empty vector (Ctr) or shRNA against Gfi-1. Cells were then selected in puromycin and individual clones were isolated. As shown in Fig. 14A (HL60) and Fig. 14B (TF-1), Gfi-1 expression was effectively reduced in two independent clones of each cell type as compared to cells infected with control lentivirus. The control cells and the clones with Gfi-1-knockdown were maintained in culture and cell numbers were counted on each day by trypan blue exclusion. The result showed that Gfi-1 knockdown moderately reduced the proliferation rate of both HL60 and TF-1 cells.

A HL-60 160 #9 #4 HL-60 Ctr )

5 140 HL-60 shRNA #4 10 RNA RNA 120 HL-60 shRNA #9 Ctr × sh sh 100 Gfi-1 80

mbers ( 60 u

p21Cip1 N 40

actin Cell ß- 0 0123456

Days in culture

B TF-1

5 160 #3 #

) TF-1 Ctr 5 140 TF-1 shRNA #3 10 RNA RNA 120 TF-1 shRNA #5 × sh sh Ctr 100 Gfi-1 80

mbers ( 60 u

p21Cip1 N

ß-actin Cell 0 0123456

Days in culture

Figure 14: Knockdown of Gfi-1 by shRNA in HL60 and TF-1 cells causes reduced cell proliferation. (A) HL60 cells and (B) TF-1 cells were infected with lentivirus containing either empty pLKO.1 vector or pLKO.1-shRNA against Gfi-1 mRNA.

Expression of Gfi-1 and p21Cip1 was examined by Western blotting using the anti-Gfi-1 and anti-p21Cip1 antibodies (A and B, left). Cells infected with empty lentivirus (Ctr) and the clones with Gfi-1 knockdown were maintained in culture and the cell numbers were monitored by trypan blue exclusion for 7 days (A and B, right).

3.1.7. Gfi-1 functionally collaborates with Myc in the repression of p21Cip1

Expression of Gfi-1 significantly accelerates T cell lymphoma development in

Myc transgenic mice suggesting a functional collaboration between Gfi-1 and Myc [92,

138, 139], however, the mechanism by which Gfi-1 collaborates with Myc is not clear.

Since Gfi-1 and Myc both repress p21Cip1 through interacting with Miz-1, we investigated the possibility that Gfi-1 may cooperate with Myc in the repression of p21Cip1. Hela cells were transfected with the p21P luciferase reporter construct along with Miz-1, Gfi-1 and Myc. Expression of either Gfi-1 or Myc alone comparably exerted an about 60% repression on Miz-1-induced activation of p21Cip1 promoter. Notably, co-expression of Gfi-1 and Myc produced an about 85% repression (Fig. 15), indicating a functional collaboration between Gfi-1 and Myc in the repression of p21Cip1.

4 p21P (2.4 kb) 3

1 (arbiturary units) Activity Luciferase 0 Miz-1 -++ + + Gfi-1 --+ -+ Myc --- ++

Figure 15: Gfi-1 collaborates with Myc in the repression of Miz-1-induced p21Cip1

promoter activity. Hela cells were transfected with luciferase reporter construct p21P

along with Miz-1, Gfi-1 and/or Myc as indicated. Luciferase activity was measured 48

hour after transfection.

3.1.8. Gfi-1 forms a ternary complex with Myc through Miz-1 on the p21Cip1 core promoter

We further investigated whether Gfi-1 formed a ternary complex with Myc

through association with Miz-1. 293T cells were transfected with Myc, Flag-tagged-Gfi-1

and Miz-1. Whole-cell extracts were prepared and immunoprecipitated with the

anti-Flag-tag antibody. As shown in Fig. 16A, both Myc and Miz-1 were

co-immunoprecipitated with Gfi-1 in cells expressing all three proteins. However, Gfi-1 failed to interact with Myc in the absence of Miz-1, suggesting that Gfi-1 interacts with

Myc through Miz-1 to form a ternary complex. The same whole-cell extracts were also subjected to oligonucleotide precipitation using oligonucleotide corresponding to –16 to

+49 sequence of the p21Cip1 promoter. As shown in Fig. 16B, both Gfi-1 and Myc can be precipitated by the oligonucleotide when Miz-1 was co-expressed. In the absence of

Miz-1, neither Gfi-1 nor Myc bound to the oligonucleotide. These data indicated that

Miz-1 recruited both Gfi-1 and Myc to the p21Cip1 core promoter.

A B Gfi-1 ++ - Miz-1 -+ + Myc + ++ Myc Myc ++++ Miz-1 --++ Oligo-pull Miz-1 down Flag-Gfi-1 - -++ Gfi-1

Myc Myc IgG heavy chain IP: Flag Whole-cell Miz-1 Miz-1 extract

Gfi-1 Gfi-1

Figure 16: Gfi-1 forms a ternary complex with Myc through Miz-1. (A) 293T cells

were transfected with Flag-tagged Gfi-1, Miz-1 and c-Myc as indicated. 48 hours after

transfection, whole-cell extracts were subjected to immunoprecipitation with the anti-Flag-tag antibody followed by Western blotting analysis using the anti-Flag-tag, anti-Miz-1 and anti-Myc antibodies. (B) 293T cells were transfected the same as described in (A), whole-cell extracts were subjected to oligonucleotide precipitation

using biotinylated oligonucleotide spanning –49 to +16 of the p21Cip1 promoter. Western

blotting was performed using the anti-Flag-tag, anti-Miz1 and anti-Myc antibodies to

examine Gfi-1, Miz-1 and Myc before and after oligonucleotide precipitation.

Re-ChIP assays were performed to address whether the Gfi-1/Miz-1/Myc ternary

complex was formed on the p21Cip1 core promoter in vivo. 293T cells were transfected

with Gfi-1 and Myc along with or without Miz-1. Nuclear extracts were

immunoprecipitated with the anti-Myc antibody first. The protein-chromatin complexes

precipitated with the anti-Myc antibody were eluted and subjected to the second round of

immunoprecipitation with the anti-Gfi-1 antibody. Semi-quantitative PCR was then

51 carried out to amplify the p21Cip1 core promoter region. As shown in Fig. 16A (lanes 1 and 2), Gfi-1 and Myc concurrently bound to the p21Cip1 core promoter only in the presence of Miz-1. A similar result was observed when the anti-Myc antibody and the anti-Gfi-1 antibody were used in a reverse order in the sequential immunoprecipitation procedure (Fig. 17A, lanes 3 and 4). As a negative control, the anti-NE antibody was used instead of the anti-Myc antibody in the sequential immunoprecipitation, and no binding was detected (Fig. 16B). These data indicated that both Gfi-1 and Myc were recruited by Miz-1 to form a Gfi-1/Miz-1/Myc ternary complex on the p21Cip1 core promoter in vivo, leading to their functional cooperation.

1. α -Myc 1 . α -Gfi-1 2. α -Gfi-1 2. α -Myc

Gfi-1 ++ ++ + + + + Myc ++ ++ ++ + + Miz-1 -+ - + - + - + -194 to +88 Gfi-1

-3,723 to -3,400 Miz-1

+3,455 to +3,771 Myc

12 34 Input WB

B -NE -Myc α α . . 2 2 -Gfi-1 -NE α α 1.α-Gfi-1 -194 to +88 Gfi-1

-3,723 to -3,400 Miz-1

+3,455 to +3,771 Myc

1234Input WB

Fig. 17

Figure 17: Gfi-1 forms a ternary complex with Myc through Miz-1 on the p21Cip1 core promoter in vivo. (A) 293T cells were co-transfected with Gfi-1 and Myc along with or without Miz-1. Nuclear extracts were immunoprecipitated with the anti-Myc antibody first. The protein-chromatin complexes precipitated with the anti-Myc antibody were eluted and subjected to the second round of immunoprecipitation with the anti-Gfi-1 antibody (lanes 1 and 2). Re-ChIP was alternatively performed by applying the anti-Gfi-1 antibody first and secondly the anti-Myc antibody in the sequential immunoprecipitation step (lanes 3 and 4). (B) The nuclear extracts from 293T cells expressing Gfi-1, Miz-1 and Myc were subjected to immunoprecipitation with anti-Gfi-1 antibody or with anti-NE for control (lanes 1 and 2); The same nuclear extracts were first immunoprecipitated with anti-Gfi-1 antibody and secondly with either anti-Myc antibody or anti-NE antibody

(lanes 3 and 4). Chromatin DNA after immunoprecipitation was subjected to semi-quantitative PCR using primers to amplify the indicated p21Cip1 promoter regions.

Input shows the PCR amplification from chromatin DNA prior to immunoprecipitation.

Expression of Gfi-1, Miz-1 and Myc in the nuclear extracts was examined by Western blotting using antibodies as indicated.

3.1.9. The Miz-1-dependent transcriptional repression by Gfi-1 plays a role in regulating other cyclin-dependent kinase inhibitor genes p15Ink4b and p27Kip1

Because Gfi-1 repressed p21Cip1 through interaction with Miz-1, it is possible that Gfi-1 may also repress other genes that are activated by Miz-1. p15Ink4b is a Miz-1

target gene and Miz-1-mediated activation of p15Ink4b is negatively regulated by Myc

through interaction with Miz-1 [137, 140]. In addition to p15Ink4b, p27Kip1 contains an

Inr element in the proximal promoter region that is required for its transcriptional repression by Myc [141, 142]. Several groups have reported that the expression of Gfi-1 and p27Kip1 was inversely correlated [96] [47, 84]. Therefore we investigated the role of

Gfi-1 in the regulation of p15Ink4b and p27Kip1. Our data showed that Gfi-1 and the

N382S mutant effectively repressed p15Ink4b (Fig. 18A) and p27Kip1 (Fig. 18D) promoter activities ([136] and the dissertation of Basu, Suchitra), indicating that direct

DNA-binding by Gfi-1 is not essential for the Gfi-1-mediated repression of p15Ink4b and p27Kip1. In ChIP assays, only a weak binding of Gfi-1 to the core promoters of p15Ink4b

(Fig. 18B) or p27Kip1 (Fig. 18E) was detected in the absence of transfected Miz-1, which might result from endogenously expressed Miz-1 by 293T cells. Co-expression of Miz-1 enhanced Gfi-1 binding to the core promoters of p15Ink4b (Fig. 18B) and p27Kip1 (Fig.

18E). Furthermore, Gfi-1 knockdown in HL60 cells by shRNAs was associated with augmented protein levels of p15Ink4b (Fig. 18C) and p27Kip1 (Fig. 18F). These data indicated that Gfi-1 might also play a role in regulating the transcription of p15Ink4b and p27Kip1 through Miz-1.

A B C

9 ChIP Input

A A

8 - - A

N N p15P-113/+70bp Gfi-1 +++ + + N

R R R

) 7

h h h

s s - C s t Miz-1 + ++ - v + +

i v 6

5 p15Ink4b

s r -100/+45

r 4

i b 3

r c Gfi-1

a u -3756/ -3278

(arbitrary univts) ( (arbitrary

LLuciferase activity 2 1 +2995/ +3477 β-actin 0

1 -

i Miz-1 i -+++ z

--Gfi-1

-Gfi-1-

-NE -

Gfi-1 -- + - -Miz1- α α α α α α N382S -- - + α

AB C

ChIP Input 12 p27P-1.6 kb 10 Ctr Cl. 2 Gfi-1 -+--++ ++ Cl. 1 8 - Miz-1 ++- ++ + Gfi-1 6 4 +56/+232 (arbitrary units) p27Kip1 Luciferste Activity 2 0 -3190/-2818 ß-actin Miz-1 - + + + Gfi-1 --+ - --- -Miz-1 -NE -Gfi-1 N382S + -Gfi-1 α α α α

Fig. 18

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. (A) Hela cells were transfected with the promoter reporter construct containing a -113/+75 fragment of p15Ink4b promoter, along with Miz-1, Gfi-1 and N382S as indicated.

Luciferase activity was measured 48 hours after transfection. (B) 293T cells were transfected with Gfi-1, Miz-1 or both. Nuclear extracts were subjected to immunoprecipitation using the antibodies against Miz-1, Gfi-1 or a species-matched irrelevant anti-NE antibody. The indicated regions of p15Ink4b were examined by PCR.

(C) HL60 cells were infected with lentivirus containing either empty pLKO.1 vector (Ctr) or pLKO.1-shRNA against Gfi-1, followed by selection with puromycin. Expression of

Gfi-1 and p15Ink4b was examined by Western blotting. (D) Hela cells were transfected with the promoter reporter construct containing a 1.6 kb fragment of p27Kip1 promoter, along with Miz-1, Gfi-1 and N382S, followed by luciferase assay 48 hours later. (E)

393T cells were transfected with Gfi-1, Miz-1 or both. ChIP assay was carried out by uing the antibodies against Gfi-1, Miz-1 or NE. The indicated regions of p27Kip1 were amplified by PCR. (F) HL60 cells were infected with lentivirus containing either empty pLKO.1 vector (Ctr) or pLKO.1-shRNA against Gfi-1 and then selected with puromycin.

Two independent shRNA transduced clones showing substantially reduced Gfi-1 expression were isolated. Expression of Gfi-1 and p27Kip1 was analyzed by Western blotting. (Figure adapted from Basu and Liu et. al. [136] A and D were done by Basu,

Suchitra)

3.1.10. Gfi-1 is downregulated by TGF-β

The expression of Myc is downregulated by TGF-β in a number of cell types [45,

143, 144]. Like Myc, Gfi-1 also represses TGF-β-induced p21Cip1 and p15Ink4b promoter activities. We therefore examined whether TGF-β affected Gfi-1 expression.

U937 (Fig. 19A), HL60 (Fig. 19B) and TF-1 (Fig. 19C) cells were treated with TGF-β for different days and Gfi-1 expression was examined by Western blotting and semi-quantitative RT-PCR. A marked downregulation of Gfi-1 in response to TGF-β was observed at both protein (Fig. 19 upper panels) and mRNA (Fig. 19 lower panels) levels.

A U937 B HL60 B TF-1

Days in TGF-β TGF-β TGF-β 0 23 1 -+ -+

Gfi-1 Gfi-1 Gfi-1 WB WB WB

β-actin β -actin β -actin

Days in TGF-β TGF-β TGF-β

013 -+ -+

Gfi-1 Gfi-1 Gfi-1 RT-PCR RT-PCR RT-PCR GAPDH GAPDH GAPDH

Figure 19: Gfi-1 is downregulated by TGF-β. U937 (A) treated with 5 ng/ml TGF-β for

days as indicated; HL60 cells (B) and TF-1 cells (C) were untreated or treated with 5

ng/ml TGF-β for 24 hours. Expression of Gfi-1 was examined by Western blotting (upper)

and semi-quantitative RT-PCR (lower).

3.2. Part II: Transcriptional repression of the eosinophil major basic protein (MBP) gene by Gfi-1

3.2.1. Expression of the N382S mutant in myeloid cells leads to upregulation of MBP in response to G-CSF

Expression of N382S, derived from patients with SCN, in murine myeloid 32D cells causes premature cell apoptosis during G-CSF-induced terminal neutrophilic differentiation [66]. We used the IL-3-dependent murine myeloid 32D cells transfected with wild type G-CSF receptor (32DWT) to investigate the role of Gfi-1 in myeloid development in response to G-CSF. 32DWT cells express endogenous Gfi-1 and can undergo terminal neutrophilic differentiation in response to G-CSF. 32DWT cells were stably transfected with either empty pBabe-puro vector (32DWT/Ctr) or with expression construct for N382S pBabe-puro-N382S (32DWT/N382S) [66]. 32DWT/Ctr and

32DWT/N382S cells were treated with G-CSF for 24 hours. Total cellular mRNA was prepared and gene expression profiles were analyzed by Affimetrix Gene Chip analysis.

The expression of MBP gene was observed to be augamented up to 20-fold in response to

G-CSF in 32DWT/N382S cells as compared to 32DWT/Ctr cells.

MBP gene encodes the eosinophil major basic protein (MBP), which is a cytotoxic eosinophil granule protein. Mature eosinophils express four granule cationic proteins and the most abundant is MBP, which is expressed in eosinophils and basophils but not detectable in neutrophils. MBP is essential for the function of eosinophils in defense against parasitic infection and in allergic diseases [145]. Elevated expression of

MBP is a major hallmark of eosinophilic differentiation. MBP is synthesized as non-toxic pre-protein with pre and pro domains that are processed to release the active and cytotoxic form of MBP as cells mature [146].

Semi-quantitative RT-PCR analysis was employed to further examine the effect of

N382S expression on MBP expression. 32DWT/Ctr and 32DWT/N382S cells were stimulated with or without G-CSF for 24 hours and total mRNA was isolated for semi-quantitative RT-PCR. MBP expression was induced by G-CSF in 32DWT/N382S cells but not in 32DWT/Ctr cells (Fig. 20A). We further examined the role of Gfi-1 in

MBP expression in another murine myeloid L-G cell line that express endogenous G-CSF receptor [147]. L-G cells were transfected with either empty pBabe-puro (L-G/Ctr) or pBabe-puro-N382S (L-G/N382S). Expression of N382S alone caused an upregulation of

MBP expression in L-G cells, which was further induced in response to G-CSF (Fig.

20B).

To demonstrate a direct role of Gfi-1 in the regulation of MBP expression, we established 32DWT clones in which the expression of Gfi-1 or N382S was induced by addition of dexamethasone and doxycycline (Dex/Dox). As shown in figure 20C, Gfi-1 proteins were induced upon Dex/Dox treatment for 24 hours in 32DWT/Tet-Gfi-1 cells and 32DWT/Tet-N382S, but not in32DWT/Tet-ctr cells. MBP expression was only detected upon G-CSF treatment in cells that inducibly expressed N382S (Fig. 20C).

Induction of MBP in 32DWT/Tet-N382S could be detected as early as 8 hours after

G-CSF stimulation in 32DWT/N382S cells that had been treated with Dex/Dox for 24 hours (Fig 20D). These findings indicated that Gfi-1 directly repressed MBP and that

DNA-binding activity of Gfi-1 was required in this Gfi-1-mediated repression of MBP.

A B

32DWT/Ctr 32DWT/N382S L-G/Ctr L-G/N382S

G-CSF -+-+ G-CSF - + - +

MBP MBP

GAPDH GAPDH

C D 32DWT/Tet-Ctr 32DWT/Tet-Gfi1 32DWT/Tet-N382S 32DWT/Tet-N382S

Dex/Dox --++ --+ + --++ G-CSF (h) 0 8 24 36

G-CSF -+-+ -+ - + -+-+ MBP

MBP GAPDH

GAPDH

32DWT/ 32DWT/ 32DWT/ Tet-Ctr Tet-Gfi1 Tet-N382S Dex/Dox -+- + -+

Gfi1

β-actin

Figure 20: MBP is upregulated in response to G-CSF in the presence of the N382S mutant. (A) 32DWT/Ctr and 32D/N382S cells. (B) L-G/Ctr and L-G/N382S cells were left untreated or treated with G-CSF for 24 hours, and then MBP expression was examined by semi-quantitative RT-PCR. (C) 32DWT/Tet-ctr, 32DWT/Tet-Gfi-1and 32DWT/

Tet-N382S cells were treated with Dex/Dox for 24 hours followed by G-CSF stimulation for additional 24 hours, MBP expression was examined by semi-quantitative RT-PCR.

Expression of Gfi-1 proteins upon Dex/Dox treatment for 24 hours is shown in the lower panel by Western blotting. (D) 32DWT/Tet-N382S cells were treated with Dex/Dox for 24 hour and followed by G-CSF stimulation for the indicated times. MBP expression was examined by semi-quantitative RT-PCR.

3.2.2. Gfi1 represses the MBP promoter

To demonstrate that Gfi-1 repressed MBP transcription, a 2.4 kb MBP promoter fragment was amplified from the genomic DNA of 32DWT cells and inserted into pGL3 luciferase reporter vector to generate pGL3MBP-P. 32DWT cells were transfected with pGL3MBP-P alone or together with GFP-RV-Gfi1. We observed that Gfi1 repressed both basal and G-CSF-induced MBP promoter activity by about 60% (Fig. 21).

8 140 MBP-P 2.4 Kb Ctr MBP-P 2.4 Kb Ctr+G-CSF 7 120 Gfi-1 Gfi-1+G-CSF 6 100

5 80 4 60 3 (arbitrary univts) Luciferase Activity (arbitrary univts) 40

2 Luciferase Activity 20 1 0 0

Figure 21: Gfi-1 represses the MBP promoter. 32DWT cells were transiently

transfected with the luciferase reporter construct containing a 2.4 kb fragment of mouse

MBP promoter along with or without Gfi-1. Cells were untreated (A) or treated (B) with

G-CSF for 8 hours before harvested for luciferase assays.

3.2.3. Expression of the N382S mutant potentiates the induction of the MBP promoter

activity by G-CSF

We attempted to address the role of the N382S mutant in regulating the MBP

promoter. 32DWT/Tet-N382S cells were left untreated or treated with Dex/Dox for 24

hours followed by transfection of the cells with pGL3MBP-P. 16 hours after transfection,

62 cells were then stimulated with or without G-CSF for additional 8 hours. Induced expression of N382S resulted in a much greater induction of the MBP promoter activity by G-CSF in Dex/Dox treated cells than in cells without Dex/Dox treatment (Fig. 22), indicating that the G-CSF-induced activation of MBP promoter was repressed by Gfi-1, and this repression was relieved by the N382S mutant.

140 Ctr 120 Dex/Dox 100

40 Luciferase activity

(Fold induction by G-CSF) G-CSF) by (Fold induction 20

Figure 22: Expression of the N382S mutant potentiates the induction of the MBP

promoter activity by G-CSF. 32DWT/Tet-N382S cells were left untreated (Ctr) or

treated with Dex/Dox for 24 hours, and then transfected with pGL3-MBP-P. 16 hours

after transfection, half of the Ctr cells and/or Dex/Dox-treated cells were stimulated with

G-CSF for an additional 8 hours and analyzed by luciferase assay. Luciferase activity was

normalized by the co-expressed β-gal activity.

3.2.4. Overexpression of MBP inhibits IL3 and G-CSF-dependent growth and

accelerates cell death in response to G-CSF in myeloid cells

Our previous study has shown that N382S caused premature apoptosis and inhibited

the proliferation of 32DWT cells undergoing terminal neutrophilic differentiation in

63 response to G-CSF [66]. We addressed the effect of MBP expression on myeloid cell growth and survival. MBP cDNA was amplified from G-CSF-stimulated 32DWT/N382S cells by RT-PCR, cloned into GFP-RV vector and stably transfected into 32DWT cells

(32DWT/GFP-MBP). Expression of MBP mRNA was confirmed by RT-PCR in the two independent stable clones transfected with GFP-RV-MBP (32DWT/GFP-MBP#12 and

32DWT/GFP-MBP#14), but not in control (32DWT/GFP-Ctr) cells that were transfected with empty vector (Fig. 23A). 32DWT/GFP-Ctr, 32DWT/GFP-MBP#12 and

32DWT/GFP-MBP#14 cells were maintained in IL-3 or G-CSF medium for 7 days and cell numbers and viabilities were determined by trypan blue exclusion. The results showed that forced expression of MBP inhibited both IL-3 and G-CSF-dependent cell proliferation, and accelerated cell death in response to G-CSF, although did not affect the cell viability in IL-3 (Fig. 23B and C).

A Ctr 12# 14# MBP

B GAPDH

100 1000 Ctr 80 MBP #12 100 MPB #14 60 10 40 Ctr

1 MBP#12 20

Viability (%) (%) Viability MBP#14 Cell Number (log) 0.1 0 01234567 01234567 C days in IL3 days in IL3

100 100 Ctr MBP#12 80 10 MBP#14 60

40 Ctr 1 Viability (%) Viability MBP#12 20

Cell Number (log) MBP#14 0.1 0 01234567 01234567 days in G-CSF days in G-CSF

Figure 23: Overexpression of MBP causes reduced cell proliferation and survival. (A)

Expression of MBP in 32DWT/GFP-Ctr, 32DWT/GFP-MBP#12 and

32DWT/GFP-MBP#14 cells was examined by RT-PCR. Cells as indicated were maintained in IL-3 (B) or G-CSF (C) medium. Cell numbers and viabilities were determined by trypan blue exclusion for 7 days.

3.2.5. Overexpression of MBP causes accelerated apoptosis in response to G-CSF

We performed an annexin V-PE staining assay to address whether the

MBP-associated inhibition of cell proliferation and viability in response to G-CSF was due to enhanced apoptosis. 32DWT/GFP-Ctr cells and the pool of 32DWT/GFP-MBP

65 cells were stained with annexin V-PE/7-AAD after treatment with G-CSF for 4 days and the percentage of apoptotic cells were determined by flow cytometry (Fig. 24).

32DWT/GFP-MBP cells showed a higher percentage of cells that were stained positively by annexin V than 32DWT/GFP-Ctr cells, suggesting that forced expression of MBP resulted in enhanced apoptosis during G-CSF-induced terminal neutrophilic differentiation.

32DWT/GFP- Ctr 32DWT/MBP

26.64% 45.68% AAD - 7

Annexin-V

Figure 24: Overexpression of MBP results in accelerated apoptosis in response to

G-CSF. 32DWT/GFP-Ctr and the pool of 32DWT/GFP-MBP (clone 12 and clone 14) cells were cultured in G-CSF for 4 days followed by Annexin V/7-AAD staining and analyzed by flow cytometry. The percentages of annexin V positive cells (apoptotic) are indicated.

3.2.6. Knockdown of Gfi-1 results in MBP upregulation.

To further demonstrate that Gfi-1 regulated MBP expression, we evaluated the effect of Gfi-1 knockdown on MBP expression level in HL60 clone 15 cells. The HL60 clone 15 cell line is a subline derived from HL60 promyelocytic leukemia cells with the ability to differentiate towards mature eosinophils upon stimulation with differentiation inducers including IL-5 and butyric acid [148, 149]. MBP expression is induced during eosinophilic differentiation of clone 15 HL60 cells [150]. HL60 clone15 cells were infected with lentivirus containing empty vector or shRNA against Gfi-1, and selected with puromycin. Cells survived from selection were examined for Gfi-1 and MBP expression by Western blotting and semi-quantitative RT-PCR, respectively. As shown in

Fig. 25, Gfi-1 expression level was effectively reduced by shRNA. Interestingly, MBP expression was dramatically increased upon knockdown of Gfi-1 in HL60 clone15 cells.

Ctr shRNA

MBP RT-PCR

GAPDH

Gfi-1 WB β-actin

Figure 25: MBP expression is increased upon Gfi-1-knockdown in HL60 clone15

cells. Clone 15 HL60 cells were infected with empty vector or ShRNA against Gfi-1.

Expression of MBP and Gfi-1 is shown by semi-quantitative RT-PCR and Western

blotting, respectively.

CHAPTER 4: DISCUSSION

Gfi-1 is a ZF transcription repressor predominantly expressed in the hematopoietic system and play pivotal roles in hematopoiesis. Gfi-1 regulates the development of granulocytes and lymphocytes, and the self-renewal of HSCs [38, 71]

[82]. Although weakly oncogenic, Gfi-1 strongly cooperates with oncoprotein c-Myc and

Pim-1 in lymphomagenesis [92, 138, 139]. Gfi-1 can regulate gene expression through direct DNA binding and protein-protein interactions [58, 60, 101, 102]. Gfi-1 is capable of regulating the expression of functionally diverse genes encoding cell cycle regulators, cytokines and their receptors, and myeloid specific proteinases, suggesting a master role of Gfi-1 in regulating hematopoietic proliferation and differentiation [151]. How Gfi-1 functions in various hematopoietic cell compartments remains to be elucidated. The mechanism underlying the functional cooperation between Gfi-1 and Myc is also an open question.

Here we show that Gfi-1 interacts with Miz-1, and via Miz-1, is recruited to

Miz-1 target gene p21Cip1 leading to transcriptional repression. This work demonstrates an alternative mechanism by which Gfi-1 regulates gene expression in a manner independent of direct DNA-binding. Furthermore, Gfi-1, through binding to Miz-1, forms a ternary complex with Myc and cooperates with Myc in the transcriptional repression of p21Cip1. Knockdown of Gfi-1 and Gfi-1 deficiency result in elevated p21Cip1

68 expression. Gfi-1 also represses TGF-β-activated p21Cip1 in a

DNA-binding-independent manner. Furthermore, Gfi-1 is downregulated in response to

TGF-β indicating a potential role of Gfi-1 in cellular response to TGF-β. In addition, we have demonstrated that Gfi-1 represses other Miz-1 target genes, including p15Ink4b and p27Cip1, through association with Miz-1.

Our data also identify MBP as a novel target of Gfi-1-mediated transcriptional repression. Expression of the DNA-biding-defective Gfi-1 DN mutant N382S causes dramatic induction of MBP by G-CSF, indicating Gfi-1 represses MBP through direct binding to the MBP promoter. Silencing of Gfi-1 by RNA interference leads to enhanced

MBP expression. Forced overexpression of MBP in myeloid cells inhibits IL-3 and

G-CSF-dependent cell growth, and accelerates apoptosis during G-CSF-stimulated neutrophilic differentiation. The regulation of MBP by Gfi-1 may provide insights into the role of Gfi-1 in granulopoiesis and the pathogenesis of N382S-associated SCN.

Overall, data presented here describe the transcriptional repression by Gfi-1 via two distinct mechanisms: repression of p21Cip1 and other Miz-1 target genes through association with Miz-1; and repression of MBP by Gfi-1 through direct DNA-binding.

These findings provide new insights into the function of Gfi-1 in normal hematopoiesis, cell cycle progression and the mechanism of the collaboration between Gfi-1 and Myc in lymphomagenesis.

4.1. Gfi-1-mediated transcriptional repression of p21Cip1 and other Miz-1 target genes

The oncogenic potential of Gfi-1 has been suggested to involve mainly two mechanisms: 1) promoting cell cycle progression by overriding G1 cell cycle arrest and accelerating S phase entry and 2) protecting cells from apoptosis. Forced expression of

Gfi-1 in T cells disrupts G1 cell cycle arrest and apoptosis induced by IL-2 withdrawal and antigen activation [58]. Therefore it is possible that Gfi-1 regulates genes that are involved in controlling cell survival and cell cycle progression. Early studies showed that increased expression of Gfi-1 was associated with decreased levels of p21Cip1, p27Kip1 and pRb, all of which are negative regulators of cell proliferation [67]. Gfi-1 associates with the promoters of genes encoding other cell cycle regulators such as E2F5, E2F6, p21Cip1 and Myc [98, 151]. Gfi-1 inhibits apoptosis, at least in part, by altering the balance between anti-apoptotic and pro-apoptotic proteins of the Bcl2 family members.

Gfi-1 represses pro-apoptotic Bax and Bak in thymocytes and upregulates anti-apoptotic

Bcl-2 and Bcl-XL in CD4 T cells [95, 96]. Gfi-1 deficiency is associated with

upregulation of the death receptor CD95, the proapoptotic factor Bad and cell cycle

inhibitor p21Cip1, and downregulation of Bcl-2 [99].

Understanding how Gfi-1 regulates gene expression may help in dissecting the role of

Gfi-1 in normal hematopoiesis and tumorigenesis. p21Cip1 mainly functions through

inhibiting cyclin E/Cdk2 activity leading to a late G1 cell cycle arrest. Induction of

p21Cip1 is associated with hematopoietic differentiation, which is frequently

characterized by G1 cell cycle arrest [42]. Several studies have suggested that p21Cip1 is

a target of Gfi-1-mediated transcriptional regulation. For example, overexpression of

Gfi-1 inhibits phorbol ester induced upregulation of p21Cip1 and G1 cell cycle arrest in

Jurkat T cells [83]. In contrast, T cells deficient for Gfi-1 show elevated p21Cip1 expression [99]. Duan et al. showed that Gfi-1 assembles a repressive complex containing histone lysine methyltransferase G9a and HDAC1 on the p21Cip1 promoter leading to transcriptional repression [98, 151]. Notably, Gfi-1 ortholog Gfi-1B, which recognizes the same nucleotide sequences for DNA binding, has been shown to repress p21Cip1 and inhibit IL-6-induced differentiation of myelomonocytic M1 cells [53]. However, the regulation of p21Cip1 by Gfi-1 appears to be cell context-specific. Gfi-1 appears to repress p21Cip1 in T cells and myeloid cells [83, 98] but is required for p21Cip1 expression in HSCs, as p21Cip1 expression is absent in Gfi-1-/- HSCs [38, 82]. Therefore, elucidating the underlying mechanism by which Gfi-1 regulates p21Cip1 has important implication for understanding the role of Gfi-1 in regulating hematopoieitc cell proliferation and differentiation.

The data presented here uncover an alternative mechanism by which Gfi-1

represses p21Cip1. Gfi-1 is recruited to and represses p21Cip1 through association with

Miz-1. Transcriptional repression of p21Cip1 by Gfi-1 is independent of direct DNA

binding of Gfi-1 as demonstrated by 3 lines of evidence: 1) Gfi-1 binds to the

oligo-nucleotide spanning the p21Cip1 core promoter that does not contain potential

Gfi-1 binding site and Gfi-1 represses the activity of the p21Cip1 proximal promoter devoid of Gfi-1 recognition sites; 2) Miz-1 is required for Gfi-1 binding to the p21Cip1 core promoter; and 3) the DNA-binding-defective DN mutant of Gfi-1, N382S, is as effective as wild-type Gfi-1 in repressing p21Cip1 promoter. Consistent with its role in repressing p21Cip1, targeted silencing of Gfi-1 by lentivirus delivery of shRNAs caused

71 increased p21Cip1 expression and reduced cell proliferation in myeloid leukemic HL60 and TF-1 cells. Furthermore, bone marrow cells derived from Gfi-1 knockout mice [152] have elevated levels of p21Cip1.

As stated earlier, Gfi-1 has been previously shown to bind to the p21Cip1 promoter and represses p21Cip1 transcription. Two Gfi-1 binding sites on the p21Cip1 promoter, located at -1.4 kb and -2.8 kb, have been identified by EMSA assay in HL60 cells [151] (Fig. 26). In subsequent ChIP assay, however, the authors showed that Gfi-1 bound to the proximal promoter of p21Cip1 (-391 bp to -265 bp). Therefore it is still unknown whether Gfi-1 binds to the two sites in vivo. Although we consistently demonstrated in vivo binding of Gfi-1 to the p21Cip1 core promoter in HL60 cells, we failed to detect significant binding of Gfi-1 to the two previously identified Gfi-1 binding sites. It remains to be determined whether Gfi-1-mediated repression of p21Cip1 is

Miz-1-dependent or alternatively may involve both Miz-1-dependent and -independent mechanisms.

Miz-1 contacts two sites on the p21Cip1 promoter: site A (-46 to –32) and site B (-105 to –81) [113] (Fig. 26). Our study demonstrates that Gfi-1 associates with the region of the p21Cip1 promoter that contains site A. Whether Miz-1 also recruits Gfi-1 to site B is not clear. Several sp1 sites are present in the proximal region of the p21Cip1 promoter.

Sp1 cooperate with Smads in activating p21Cip1 transcription in response to TGF-β [43,

153]. Whether the recruitment of Gfi-1 by Miz-1 interferes with the transcriptional activation of p21Cip1 by sp1 and Smads needs to be elucidated.

Miz-1 (-105/-81 ) TATA -44 Transcription p53 (-2.3 kb) p53 (-1.4 kb) sp1 (-52 to –84 bp) start site

p21Cip1 Gfi-1 (-2.8 kb) Gfi-1 (-1.4 kb)

? ? TßRE (-86/-70)

Miz-1 (-46/-32 )

Figure 26: Diagrammatic view of the p21Cip1 promoter. The p53, sp1 and Miz-1 binding sites, and the TGF-β-response element (TβRE) are shown. The two Gfi-1 binding sites shown with question markers have not been verified by in vivo experimental approaches.

Interestingly, our data further show that Gfi-1 also represses another Miz-1 target gene p15Ink4b [136]. Similar to Gfi-1-mediated repression of p21Cip1, Gfi-1 is recruited by

Miz-1 to the p15Ink4b core promoter, where it represses transcriptional repression independent of Gfi-1 direct DNA-binding. Moreover, Gfi-1 and Myc show functional cooperation in repressing p15Ink4b promoter activity. Our results reveal p15Ink4b as a novel Gfi-1 target gene, adding to our understanding on how Gfi-1 regulates cell cycle progression.

p27Kip1 is repressed by Myc, but it is not clear whether the repression of p27Kip1 by

Myc is Miz-1-dependent [141]. Notably, p27Kip1 has a TATA-less promoter containing an Inr element [142], suggesting it could be a Miz-1 target gene. Interestingly, there appears to be an inverse correlation between Gfi-1 and p27Kip1 expression. T cells

73 overexpressing retroviral transuded Gfi-1 show substantially lower levels of p27Kip1

[96]. Antigen-induced T-cell activation and IL-4 stimulation in CD4 T cells result in a transient rise of Gfi-1 expression that is accompanied by a decrease in p27Kip1 expression. These results suggest a role of Gfi-1 in the repression of p27Kip1 [83]. We show for the first time that Miz-1 binds to the p27Kip1 core promoter and activities p27Kip1 promoter activity. We further demonstrate that co-expression of Miz-1 significantly enhances binding of Gfi-1 to the p27Kip1 core promoter and Gfi-1 represses

Miz-1-activated p27Kip1 promoter activities. These data indicate that Gfi-1 is also recruited to p27Kip1 and repress Miz-1-mediated transcriptional activation of p27Kip1.

Thus, through binding to Miz-1, Gfi-1 represses a group of Miz-1 target genes that are involved in the negative regulation of cell cycle progression. Interestingly, another Kip family member p57Kip2 has been suggested to be a Miz-1 target gene and its transcription is repressed by Myc through Miz-1 [109, 128, 154]. Whether p57Kip2 is also repressed by Gfi-1 through Miz-1 remains to be investigated.

Gfi-1 functionally cooperates with Myc in lymphomagenesis [92, 138], but the mechanism underlying this cooperation is not clear. Both Gfi-1 and Myc interact with

Miz-1 and inhibit Miz-1-mediated transcriptional activation. Notably, Gfi-1 and Myc associate with different regions of Miz-1. The amphipathic helix located between zinc fingers 12 and 13 is required for Miz-1 binding to Myc [117] whereas Gfi-1 interacts with Miz-1 ZF domains. Our data indicate that Gfi-1 and Myc, through an interaction with Miz-1, form a ternary complex on the p21Cip1 core promoter and synergistically repress p21Cip1. Similar observations were made with the p15Ink4b promoter (data not shown). It is possible that the functional collaboration between Gfi-1 and Myc in

74 repressing Miz-1-mediated activation of p21Cip1 and p15Ink4b may contribute to the cooperation of Gfi-1 with Myc in lymphomagenesis. Both Myc and Gfi-1 can coordinate epigenetic silencing of their target genes by recruiting co-repressors [59, 98, 124, 155]. It is conceivable that the recruitment of both Gfi-1 and Myc to the Miz-1 target genes may bring in a broader spectrum of epigenetic silencing components such as HDACs and histone methyltransferases, leading to a more potent repression of p21Cip1, p15Ink4b and possibly other Miz-1 target genes. It would be interesting to investigate whether the functional cooperation between Gfi-1 and Myc in lymphomagenesis is dependent on their abilities to interact with Miz-1.

Gfi-1 promotes lymphoid cell proliferation [57, 58, 96, 152], however, the role of

Gfi-1 in regulating myeloid cell proliferation is not clear. Our results show that reduced

Gfi-1 expression by RNA interference causes upregulation of p21Cip1, p15Ink4b and p27Kip1, and inhibited cell proliferation in myeloid leukemia cell lines HL60, U937 and

TF-1. These results indicate that Gfi-1 may play a role in promoting myeloid leukemic cell proliferation and could be a potential therapeutic target for the treatment of myeloid leukemias.

4.2. Gfi-1 in TGF-β-mediated anti-growth effect

TGF-β is a potent anti-mitogenic cytokine that inhibits cell cycle progression through G1 phase in many cell types including epithelial, lymphoid, and endothelial cells.

Impaired TGF-β signaling has been implicated in tumorigenesis, for example, loss of expression or inactivation of TGF-β signaling components leading to cell resistance to

TGF-β have been described in a variety of leukemias and lymphomas [43, 51, 156, 157].

The anti-mitogenic response to TGF-β is generally mediated by two classes of rapid gene responses: 1) upregulation of CDK inhibitors p15Ink4b, p21Cip1 and p27Kip1, depending on cell context and 2) downregulation of Myc [107]. Notably, Myc and TGF-β signaling is mutually antagonistic. TGF-β represses Myc transcription whereas Myc inhibits the activation of p15Ink4b and p21Cip1 by TGF-β, which contributes to

Myc-mediated transformation and Myc-associated resistance to TGF-β-induced cell cycle arrest [45, 122, 137, 140, 158-161].

This study demonstrates that Gfi-1 represses TGF-β-induced activation of the p21Cip1 and p15Ink4b promoters in a manner similar to Myc-mediated repression of these two genes. Theses findings suggest that Gfi-1, similar to Myc, may act to antagonize TGF-β-mediated cell cycle arrest, which may contribute to the role of Gfi-1 in tumorigenesis. Indeed, we have observed that overexpression of Gfi-1 in pro-B Ba/F3 cells renders cells less sensitive to the growth-inhibitory effect of TGF-β (data not shown).

Interestingly, the expression of Gfi-1, like c-Myc, is also inhibited by TGF-β in multiple hematopoietic cell lines including HL60, U937, TF-1 and MO7e (data not shown). In agreement with our observation, two recent publications have suggested an inverse correlation between Gfi-1 and TGF-β expression levels. TGF-β mRNA and protein levels are significantly increased in Gfi-1-/- B cells [162]. In CD4 T cells, TGF-β

not only causes down-regulation of basal levels of Gfi-1 but also blocks IL-4-induced

Gfi-1 upregulation. Furthermore Gfi-1 and TGF-β display a reciprocal negative

regulation on T cell fate determination. Downregulation of Gfi-1 was seen at both mRNA

and protein levels, suggesting that TGF-β may inhibit Gfi-1 transcription [163].

Downregulation of Myc by TGF-β is Smad3-dependent and requires the integrity of a

Smad-responsive element (TIE/E2F) in the Myc promoter, which is a complex of TGF-β inhibitory element (TIE) and an E2F binding site responsible for the transcriptional activation of Myc by E2F-4 [106, 164]. However the mechanism by which TGF-β downregulates Gfi-1 remains to be further investigated.

Based on our findings, a working model is proposed, in which Gfi-1 and c-Myc collaborately act to inhibit the biological activity of TGF-β. In the absence of TGF-β or impaired TGF-β signaling, both Gfi-1 and Myc bind to Miz-1, and strongly cooperate in repressing p21Cip1 and other Miz-1 target genes, leading to enhanced cell growth and survival, which may contribute to the pathogenesis of hematological malignancies including lymphoma (Figure 27A). However in the presence of TGF-β, expression of both Gfi-1 and Myc is inhibited. Free Miz-1 activates p21Cip1and other Miz-1 target genes leading to reduced cell growth and survival (Fig. 27B). This working model may help to explain, at least in part, how Gfi-1 cooperates with Myc in lymphomagenesis.

Gfi-1 Myc No Gfi-1 Growth TGF-β Miz-1 Myc Survival p21Cip1 and other Miz-1 target genes

Gfi-1 Growth TGF-β Miz-1 Survival Myc p21Cip1 and other Miz-1 target genes Figure 27: A working model for the regulation of TGF-β response by Gfi-1. (A) In the absence of TGF-β or impaired TGF-β signaling, both Gfi-1 and Myc bind to Miz-1 and cooperate to strongly repress p21Cip1 and other Miz-1 target genes leading to enhanced cell growth and survival, which may contribute to pathogenesis of hematological malignancies including lymphoma. (B) In the presence of TGF-β, expression of Gfi-1 and Myc is inhibited. Free Miz-1 activates p21Cip1and other Miz-1 target genes leading to reduced cell growth and survival.

4.3. Transcriptional repression of MBP by Gfi-1 in granulopoiesis

Targeted disruption of Gfi-1 gene in mice has revealed an essential role of Gfi-1 in granulopoiesis. Gfi-1-/- mice are severely neutropenic with a complete lack of mature neutrophils and the granulocyte progenitor cells are unable to differentiate into neutrophils in response to G-CSF. Moreover the Gfi-1-deficient animals show accumulation of atypical immature myeloid cells sharing characteristics of both the neutrophil and macrophage lineages and this accumulation of atypical cells was increased upon stimulation with G-CSF, suggesting that Gfi-1 may repress some macrophage

78 lineage-specific genes during neutrophil differentiation [71]. These findings indicate that an important role of Gfi-1 is to antagonize the alternative macrophage development from their common granulocyte/monocyte progenitors (GMP).

The critical role of Gfi-1 in granulopoiesis is also supported by the identification of the mutations in GFI1 gene, that lead to dominant negative Gfi-1 mutants in patients with SCN. Although SCN is most frequently associated with mutations in the ELA2 gene, two heterozygous, autosomal dominant mutations in GFI1 leading to production of Gfi-1 mutant proteins N382S and K403R have been reported in SCN patients without ELA2 mutations. N382S abolishes and K403R diminishes the transcriptional repression activity of Gfi-1 both in a dominant negative manner. Interestingly, Gfi-1 represses ELA2 and patients with N382S show increased expression of ELA2, linking the mutations in the two genes to a common phenotype [76].

Our gene Chip analysis revealed that the MBP gene is a target of Gfi-1 transcriptional repression. MBP is one of the four eosinophil cationic proteins that functions as a principle mediator of inflammation and tissue damage in eosinophil-associated allergic responses [165]. MBP is expressed by eosinophils and, and to a lesser extend by basophils, but not by neutrophils. Deposition of MBP from eosinophil granules exerts a toxic effect on cells and plays a role in mediating inflammatory responses [166].

Our study further demonstrates that MBP is activated by G-CSF in 32D cells and

Gfi-1 functions to suppress G-CSF-induced upregulation of MBP. Expression of the

DNA-binding-defective Gfi-1 DN mutant N382S releases the repression of MBP by

Gfi-1, suggesting that Gfi-1 represses MBP through direct DNA binding. In L-G cells, expression of N382S alone is sufficient to cause upregulation of MBP, which is further induced upon G-CSF treatment. Therefore Gfi-1 is capable of repressing both the basal and the G-CSF-induced MBP transcription. This was confirmed by our luciferase assays showing that the activity of a 2.4 kb fragment of the MBP promoter was inhibited by

Gfi-1 in the absence and presence of G-CSF stimulation and expression of N382S significantly potentiates the induction of MBP promoter activity by G-CSF. The 2.4 kb

MBP promoter region contains several Gfi-1 recognition sites. Further experiments are needed to identify the Gfi-1 binding site in the MBP promoter.

Overexpression of MBP in 32D cells marginally inhibited cell proliferation in

IL-3 without affecting the cell viability. However 32D cells over expressing MBP manifested accelerated cell death upon G-CSF-stimulated terminal neutrophilic differentiation. These observations are in agreement with the premature cell death during

G-CSF-induced terminal neutrophilic differentiation in 32D cells expressing N382S mutant. Similarly, N382S did not show apparent impact on cell viability when cells were cultured in IL-3 [66]. It is possible that the upregulated MBP may contribute to the increased cell death induced by N382S upon G-CSF treatment and that cells undergoing neutrophilic differentiation are more sensitive to the toxicity of MBP. Notably, N382S appears to have a more profound effect on G-CSF-dependent survival than MBP overexpression [66], which may result from the fact that apart from MBP, expression of

N382S causes deregulation of other G-CSF-induced genes that are normally repressed by

Gfi-1 such as ELA2.

Gfi-1-/- mice lack neutrophils but have normal eosinophils counts, indicating that

Gfi-1 is not required for eosinophilic differentiation [71]. Whether basophils are affected has not been reported. An interesting question is why Gfi-1-deficiency results in only loss of neutrophils but intact eosinophils. In general, the cellular identity of blood cells is acquired by lineage-specific gene expression. It is believed that the lineage-specific transcription program is coupled with simultaneous suppression of alternative lineage programs [71]. Conceivably, the precursor cells that committed to granulocyte lineage still face the decisions of differentiating into neutrophils, eosinophils or basophile.

Notably, Gfi-1 expression is dramatically increased during G-CSF-stimulated terminal neutrophil differentiation [66]. Our preliminary data show that Gfi-1 expression decreases when HL60 clone 15 cells are induced with butyric acid for eosinophilic differentiation (data not shown). Gfi-1 may play an instructive role in granulocyte fate decision by virtue of suppressing genes involved in eosinophil and basiphil differentiation. It would be interesting to investigate whether suppression of Gfi-1 downregulation influences eosinophilic differentiation.

CHAPTER 5: FUTRUE DIRECTIONS

5.1. Define the biological significance of Gfi-1 interaction with Miz-1

A Gfi-1 mutant defective for Miz-1 interaction but still capable of DNA-binding

and transcriptional repression will be generated and used to examine its effectiveness in repressing Miz-1-activated p21Cip1 and p15Ink4b. This mutant will be expressed under

Gfi-1 knockdown background to evaluate its capacity of rescuing the phenotypes that are

associated with loss of Gfi-1 function. Furthermore, transgenic mice expressing this

mutant can be employed to investigate whether loss of the ability to interact with Miz-1

will impair Gfi-1 functions in regulating granulopoiesis, lymphopoiesis and HSCs

self-renewal.

5.2. Examine the potential role of Gfi-1 in the cellular response to TGF-β

Our findings that Gfi-1 represses TGF-β-induced activation of p21Cip1 and

p15Ink4b, and TGF-β downregulates Gfi-1 expression suggest that Gfi-1 may play a role

in TGF-β-mediated growth inhibition. The effect of Gfi-1 on the cell responsiveness to

TGF-β-mediated growth inhibition and cell cycle arrest will be investigated.

5.3. Investigate the mechanism by which TGF-β regulates Gfi-1 expression

Downregulation of Myc by TGF-β is Smad3-dependent and requires the TIE

element in the Myc promoter [164]. The DNA elements in the Gfi-1 promoter that are 82 responsive to TGF-β and the involvement of Smads in TGF-β-mediated downregulation of Gfi-1 will be studied.

5.4. Explore the potential role of Miz-1 in the regulation of Gfi-1 expression and granulopoiesis

Our preliminary data showed that Miz-1 activated a 2.4 kb GFI-1 promoter, and knockdown of Miz-1 in 32D cells resulted in reduced level of Gfi-1 protein, indicating that Miz-1 may play a role in regulating Gfi-1 expression. ChIP assay will be carried out to examine whether Miz-1 binds to the Gfi-1 core promoter region containing the Inr element. The role of Miz-1 in regulating Gfi-1 in granulopoiesis will be evaluated.

5.5. Further study the mechanism of Gfi-1-mediated repression of MBP and evaluate the role of Gfi-1 in eosinophilic differentiation

Several potential Gfi-1 binding sites are present in the MBP promoter region.

ChIP assay will be performed to determine which site or sites Gfi-1 binds to. The Gfi-1 expression pattern during butyric acid-induced eosinophilic differentiation in HL60 clone

15 cells will be verified, and the effect of Gfi-1 overexpression on eosinophilic differentiation will be examined.

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