Mechanisms of embryonic stem cell division and differentiation

Transcription

1 Mechanisms of embryonic stem cell division and differentiation Josephine Wbite.'.... Department of Molecular Biosciences (Biochemistry) The University of Adelaide Adelaide, South Australia Submitted for the degree of Doctor of Philosophy March,2004

2 Thesis Summary The regulatory mechanisms governing dramatic proliferative changes during early mouse development are not well understood. This thesis aims to address this question using in vitro model systems of mouse embryogenesis. In particular, this thesis aimed to assess the function of the elevated, constitutive levels of cyclin dependent kinase 2 (CDK2) activity in embryonic stern (ES) and early primitive ectoderm-like (EPL) cells and the changes associated with differentiation into EPL embryoid bodies, in vitro equivalent of differentiation primarily to a mesodermal fate. It was determined that active CDK2 complexes associate with an increased proportion of substrates in pluripotent ES and EPL cells compared to EPL embryoid bodies. In addition, this thesis assessed the presence of other G 1 CDK activity, determining that ES cells have high levels of constitutive CDK6 activity, which is refractory to inhibition by p 16. Lineage specific decreases in CDK6 activity highlighted the complexities regulating cell proliferation during differentiation. Due to the reported constitutive E2F target gene expression in ES cells, this thesis also -aimed to further analyse the regulation and activity of E2F transcription factors and pocket proteins in ES cells. It was demonstrated that constitutive phosphorylation of pi 07 and increased E2F-4 stability in ES cells contributes to increased levels of free E2F-4, that binds E2F target gene promoters in vivo. The importance of CDK regulation of pi 07 in ES cells was demonstrated by analysis of ectopic expression of phosphorylation-resistant mutant pi 07. The increased sensitivity of EPL cells to ectepic pi 07 highlighted differences in pluripotent cell populations. In addition, it was determined that differential regulation of pl07 during differentiation was associated with increased pi 07 binding to E2F target gene promoters ' d decreased E2F target gene expression. Differentiation associated changes in regulation and activity of cell cycle regulators demonstrated in this thesis are important for understanding the pre-gastrulating mouse embryo and to enable regulation of pluripotent cell differentiation for therapeutic use.

3 TABLE OF CONTENTS CHAPTER 1: Introduction 1.1 The mammalian cell cycle Regulation of cyclin dependent kinase activity Cyclin Binding Phosphorylation of cyclin dependent kinases Cyclin dependent kinases inhibitors Functions of cyclin dependent kinase activity Cyclin D-CDK4/ Cyclin E-CDK CyclinA-CDK Regulation ofgl/s phase progression E2F Transcrption Factors The pocket protein family Deregulation of the cell cycle in cancer The cell cycle and embryogenesis Early mouse embryogenesis Cell cycle regulation and early mouse embryogenesis In vitro model systems of early mouse embryogenesis Cell cycle regulation of embryonic stem cells Cell cycle regulation during differentiation Aims CHAPTER 2: Materials and Methods 2.1 Abbreviations Bacteral Manipulations Materials Buffers and Solutions Bacterial Strains and Media Bacterial Transformations..." Mini Plasmid Preparation from Bacteria Maxi Plasmid Preparation from Bacteria Preparation of bacterially expressed GST fusion proteins Tissue,Culture Methods M terials Buffers and Solutions Cell Lines Derivation ofmefs Media Gelatinised Tissue Culture plates Maintenance of Cell Cultures Preparation of Embryoid Bodies Harvesting cells and embryoid bodies Thawing cell lines Stable ES Cell Line Establishment Via Electroporation Static Cell Synchronisation Block/Release ES Cell Synchronisation Proteasome Inhibitor treatment of ES cell and embryoid bodies Molecular Methods Materials... 43

4 Chemicals and Reagents Buffers and Solutions Miscellaneous Materials Plasmids Antibodies Oligonucleotides Radiochemicals cdna Fragments Used For Probe Synthesis Preparation of whole cell protein extracts Preparation of nuclear and cytoplasmic fractionated protein extracts Determination of Protein Concentration Fractionation of whole-cell protein extracts by gel filtration SDS-P AGE analysis Tris-Tricine PAGE analysis Coomassie Gel Analysis Western Analysis Immunoprecipitations Kinase Assays Luciferase Assays :. : Chromatin Immunoprecipitations (ChIPs) Sonication optimisation PCR optimisation Preparation of blocked Protein A Sepharose ChIP washing optimisation Equalising chromatin input ChIP assays Flow Cytometry RNA extraction Norther Transfer Preparation of e -P]-Labelled DNA Probes Hybridisation and washing...57 CHAPTER 3: Reassortment of active cyclin-cdk2 complexes during differentiation 3.1 Introduction Analysis of G lis regulatory proteins in fractionated extracts Analysis of kinase activity in fractionated extracts Analysis of cell cycle independent cyclin E-associated kinase activity in pluripotent cells... : Analysis of cyclin E-associated kinase activity during differentiation Summary CHAPTER 4: Analysis of CDK4 and CDK6 activity in embryonic stem cells and differentiation 4.1 Introduction Analysis of CDK4/6 activity in ES cells Analysis of CDK6 activity in ES cells Analysis of p 16 inhibition of CDK6 activity in ES cells Analysis of CDK6 activity as ES cells differentiate Summary... 90

5 CHAPTER 5: Analysis of the pl071e2f-4 pathway in embryonic stem cells 5.1 Introduction p107 and E2F4 regulation in ES cells E2F4 activity in ES cells In vitro functional analysis ofpl07 in ES cells In vivo functional analysis ofp107 in ES cells Discussion CHAPTER 6: Analysis of the pl07-e2f pathway in differentiation 6.1 Introduction Characterisation ofp107/e2f-4 pathway as ES cells differentiate Changes in E2F activity as ES cells differentiate Association with E2F target gene promoters as ES cells differentiate Function ofpl07-e2f pathway as ES cells differentiate Discussion CHAPTER 7: Final discussion 7.1 Roles ofcdk2 activity in ES cells and differentiation Putative roles ofcdk6 activity in ES cells and differentiation pl07 and E2F-4 ES cells and differentiation...: Similarties between ES cells and tumour cells APPENDIX.. REFERENCES ,