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The Role of Evi3 in B-cell Differentiation and Leukaemia

Salma AlDallal

[Thesis].The University of Manchester;2014.

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Abstract

Although, many types of human leukaemia in now understood, the causes of human B-cell lymphocytic leukaemias are not clearly understood. Research showed that many genes that are required for B-cell development and maturation are mis-expressed in leukaemia, suggesting that alteration during B-cell development leads to transformation. Studies into the genetic causes of B-cell leukaemia on both humans and animal models are required to understand normal cellular development and improve treatments. Evi3 (ZFP521) is recently identified transcription factor with 30 zinc-finger domains which is over-expressed in recombinant inbred mouse strains with B-cell leukaemia. However, during normal B-cell development, Evi3 is expressed at all stages. In this study we aim to understand the role of Evi3 in B-cell development and leukaemia. In this study we have demonstrated that transcription factors HoxC13 and PU.1 are synergistically regulate Evi3 expression and they are expressed in immature and mature B-cells indicating that they may play a key role in regulating Evi3 expression. We have also confirmed that these transcription factors bind to their recognition sequences in Evi3 promoter. Furthermore, we have shown that knocking down Evi3 resulted in a reduction in cell viability and increased in the number of dead cells. Moreover, we have shown that genes regulating B-cell proliferation were down-regulated in cells with Evi3 knockdown. Pax5 is a transcription factor that is essential for the later development of B-cell. Furthermore, Pax5 is a potential Evi3 target gene and its expression was shown to be upregulated in tumour cells with viral insertion at Evi3 locus. Additionally, we have demonstrated that co-transfection of Evi3 and Ebf1 may upregulate Pax5 expression. Given together, these new experiments will provide a key insight on the role of Evi3 in B-cell development and B-cell proliferation. Hence, Evi3 could be a new possible target for treatment in B-cell leukaemia.

Keyword(s)

Evi3

Bibliographic metadata

Type of resource:

text

Content type:

Thesis

Type of thesis:

Traditional format

Thesis title:

The Role of Evi3 in B-cell Differentiation and Leukaemia

Author(s) list:

Salma AlDallal

Degree type:

PhD

Publication date:

2014-06-25

Institution:

The University of Manchester

Total pages:

306

Table of contents:

Table of ContentsList of Figures 8List of Tables 11List of Abbreviation 12Declaration 18Copy Right Statement 18Acknowledgement 19Abstract 20CHAPTER 1 21Introduction 211. General Introduction 221.1 The Immune System 221.2 Cells of the Immune System 251.2.1 Lymphoid Cells 251.2.2 Mononuclear Cells 261.2.3 Granulocytic Cells 271.3 Haematopoiesis 281.4 B-cell Development 311.5 B-cell Genes 351.5.1 PU.1 351.5.2 IKAROS 361.5.3 E2A 361.5.5 Sox4 391.5.6 Sox11 421.5.7 Pax5 431.5.8 mb-1 (cd79a) 441.5.9 Cyclin D 441.6 Cancer 461.6.1 Genetic Defects in Cancer 471.6.2 Genomic Instability 471.7 Hallmarks of Cancer 511.7.1 Sustaining Proliferative Signalling 531.7.2 Evading Growth Suppressors 531.7.3 Resisting Cell Death 531.7.4 Enabling Replicative Immortality 541.7.5 Inducing Angiogenesis 541.7.6 Activating Invasion and Metastasis 541.8 Cancer and the Immune System 551.9 Leukaemia 561.9.1 Acute Myeloid Leukaemia (AML) 571.9.2 Acute Lymphoblastic Leukaemia (ALL) 581.9.3 Chronic Myeloid Leukaemia (CML) 591.9.4 Chronic Lymphoblastic Leukaemia (CLL) 601.10 Mouse Models for Leukaemia 641.10.1 The Need for an In-vivo Model System 641.10.2 The Perfect Leukaemia Model 641.10.3 The Mouse is an Outstanding Model 641.11 AKXD Recombinant Inbred (RI) Mouse Strains 651.12 Evi3 661.13 Hypothesis 72CHAPTER 2 73Materials & Methods 732. General Protocols 742.1.1 Site Directed Mutagenesis 742.1.1.1 Site-Directed Mutagenesis PCR Primer Sequences 742.1.1.2 Site-Directed Mutagenesis-PCR 742.1.1.3 Site-Directed Mutagenesis Restriction Digest (Kinase, Ligase & DpnI (KLD) Treatment) 752.1.2 Site-Directed Mutagenesis Transformation 752.1.2.1 Site directed Mutagenesis 752.1.2.2 Bacterial Colony Culture 752.1.2.3 Culture of Bacterial Colonies 752.1.3 Plasmid Extraction and Purification 752.1.4 Gel Electrophoresis 762.1.5 DNA Band Purification 762.1.6 DNA Sequencing and Sequence Purification 772.1.6.1 DNA Sequencing 772.1.6.2 Sequencing Purification 772.1.7 cDNA Production 782.1.7.1 RNA Isolation from Tissues & Cells 782.1.8 Measuring RNA/DNA concentrations 782.1.9 cDNA Production 782.1.10 DNAse Treatment of RNA and Reverse Transcription 782.1.11 Preparation of Heat-Shock Competent Cells 792.1.11.1 Evi3 restriction Enzymes 792.1.11.2 HoxC13 Restriction Enzymes 792.1.11.3 PU.1 Restriction Enzymes 792.1.12 Preparation of Heat-Shock Competent Cells 792.1.13 Preparation of Dual Luciferase Reporter Assay Reagents 792.1.14 Sequence Alignment and Database Searches 802.1.15 Designing Expression Vector 802.1.16 Evi3 Binding Site in Pax5 and Cyclin D1 812.2 Biochemical Analysis 822.2.1 Protein Isolation from Cells 822.2.2 SDS-PAGE 822.2.3 Western Immunoblotting Protocol 822.3 Non-Radioactive Electrophoretic Mobility Shift Assay (EMSA) 832.3.1 Reticulocyte Lysate Preparation 852.4 EMSA with SYBR Green Stain 872.4.1 Antibodies Used in EMSA with SYBR Green Assay 882.5 Cell Culture and Transfection 882.5.1 Cell lines 882.5.2 Cell Culture 882.6 Transient Transfection 892.7 Luciferase Reporter Assay 892.8 Real-Time Quantitative PCR 902.9 Evi3 shRNA Transfection 912.9.1 Evi3 shRNA Plasmids 912.9.2 Plasmid Explanation 912.10 Cell Based Assays 922.10.1 Viability Assay 922.10.2 Caspase-3/7 Assay 932.10.3 Trypan Blue Stain 932.10.4 Rescue Assay 932.10.5 Calculations 942.11 Northern Blotting 952.11.1 Samples 952.11.2 Transfection 952.113 RNA Extraction and Quantification 952.11.4 DIG-RNA Labeling 962.11.5 Formaldehyde Gel preparation 962.11.6 Sample Preparation 962.11.7 Running the Formaldehyde Gel 962.11.8 Viewing the Formaldehyde Gel 962.11.9 RNA Transformation and Fixation 972.11.10 Hybridization 972.11.11 Stringency Washes 972.11.12 Immunological Detection 982.12 Statistical Difference 982.13 Vector maps 982.14 qPCR melting Curves 982.15 Chemicals & Suppliers 982.16 Plasmid Gifts 99CHAPTER 3 100PU.1 and HoxC13 Regulation of Evi3 1003. Introduction 1013.1 Haematopoiesis 1013.2 PU.1 1013.3 Hox Genes 1053.4 Evi3 1093.5 Previous Unpublished Work 1103.6 Aims 1143.7 Results 1153.7.1 HoxC13 Binding site and Sequence Analysis of Human Evi3 Promoter 1153.7.2 HoxC13 Expression 1173.7.3 Evi3 Activation 1193.7.4 Introducing Mutation in the HoxC13 Binding Site 1243.7.5 Binding of PU.1 and HoxC13 on Evi3 Promoter 1243.8 Discussion 136CHAPTER 4 140A Role for Evi3 in B-cell Proliferation 1404. Introduction 1414.1 Evi3 Function 1414.2 Aims 1434.3 Results 1444.3.1 Validation of shRNA plasmids in BCL1 Cells 1444.3.2 Cell Viability Assay 1464.3.3 Trypan Blue Stain 1464.3.4 Apoptosis 1474.3.5 ZFP423 Expression in Knockdown Cells 1554.3.6 Full Length Evi3 Rescue 1574.3.7 Rescue with PU.1 and HoxC13 1644.4 Discussion 172CHAPTER 5 176Identification of B-cell Gene Expression Defects 176Following Evi3 Knockdown 1765.1 Introduction 1775.2 Genes Involved in B-cell Proliferation 1785.2.1 LRF 1785.2.2 Runx1 1805.2.3 E2F2 1825.2.4 Ikaros 1845.3 Genes Involved in B-cell Apoptosis 1845.3.1 BTG2 1845.4 Genes Known to be Downstream of Evi3 1855.4.1 B29 1855.4.2 Cyclin D1 1865.4.3 Pax5 1895.5 Other B-cell Genes 1915.5.1 Rag1 1915.5.2 Emb 1925.6 Aims 1935.7 Results 1945.7.1 Expression Analysis of Candidate Evi3 Target Genes 1945.7.2 Rescue Assays 2065.7.3 Pax5 Activation 2205.7.4 Evi3 Mode of Gene Regulation 2225.8 Discussion 226CHAPTER 6 235Overall Discussion, Limitations & Future 235Directions 2356.1 Overall Discussion 2366.2 Limitations 2476.3 Future Directions 249Reference List 250Appendices 267

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