목차

I. INTRODUCTION 1
1-1. Transcriptional mechanism of pluripotent embryonic stem cell 2
1-2. The master regulator of pluripotency : Oct4 3
1-3. The cell cycle of embryonic stem cells 5
1-4. Connection between pluripotency regulators and the cell cycle of embryonic stem cells 7
1-5. Aurora kinase b-protein phosphatase 1 axis during the cell cycle 8
1-6. Purpose of this study 9
II. MATERIALS AND METHODS 13
2-1. Cells and culture conditions 14
2-2. Mitotic Arrest-Release and Cell Cycle Analysis 14
2-3. Immunofluorescence and Confocal Microscopy 15
2-4. DNA Constructs 15
2-5. Generation of stably expressing Flag-Oct4 ZHBTc4 ESCs 16
2-6. Reporter gene assay 16
2-7. In vitro kinase assay 17
2-8. In vitro phosphatase assay 18
2-9. Immunoprecipitation and Western blot 18
2-10. Nascent RNA Analysis 19
2-11. Lentiviral shRNA-mediated knockdown 19
2-12. Real-Time qPCR, Chromatin Immunoprecipitation (ChIP) Assay 20
2-13. ChIP-Seqeuncing 20
2-14. Self renewal assay 21
2-15. Reprogramming 21
2-16. Nano-LC-ESI-MS/MS Analysis of Phosphorylation Sites in Oct4 21
III. RESULTS 26
3-1. Identification of Oct4 phosphorylation at serine 229 residue 27
3-2. Phosphorylated Oct4 at serine 229 is highly enriched in G2/M phase and dissociated from chromatin 34
3-3. Aurora kinase b binds Oct4 and phosphorylates Oct4 at serine 229 in a cell cycle dependent manner 44
3-4. Protein phosphatase 1 binds Oct4 and dephosphorylates serine 229 in Oct4 in G1 phase 52
3-5. PP1-mediated dephosphorylation of Oct4(S229) correlates with the resetting of pluripotency in the next G1 phase 60
3-6. Oct4 targets and resets cell cycle related genes in the next G1 phase 68
3-7. Oct4 ChIP-seq reveals that resetting regions enriched in pluripotency and cell cycle related genes 74
3-8. Oct4 mutants?Oct4(S229D) and Oct4(F271A)?effect the loss of pluripotency and alter the cell cycle by impeding gene expression 77
IV. DISCUSSION 87
V. REFERENCES 93
VI. ABSTRACT IN KOREAN 99