Title Page

Contents

Abbreviations 12

Summary 15

Chapter Ⅰ. Literature Review The role of protein methyltransferases in immunity 17

Abstract 18

1.1. Introduction 19

1.2. The role of arginine methyltransferases in inflammatory responses 23

1.2.1. PRMT1 24

1.2.2. CARM1 29

1.2.3. PRMT5 30

1.2.4. PRMT6 32

1.3. The role of lysine methyltransferases in inflammatory responses 38

1.3.1. G9a 38

1.3.2. EHMT1 42

1.3.3. SETDB1 42

1.3.4. SMYDs 45

1.3.5. EZH2 46

1.4. Conclusions 50

Chapter Ⅱ. Tumor suppressive functions of METTL21A in gastric cancer 54

Abstract 55

2.1. Introduction 57

2.2. Materials and Methods 60

2.2.1. Materials 60

2.2.2. Preparation of human gastric cancer specimens 62

2.2.3. Cell culture 62

2.2.4. Cloning and site-directed mutagenesis 62

2.2.5. Plasmid transfection and luciferase reporter assay 64

2.2.6. Expression and partial purification of recombinant METTL21A 64

2.2.7. Preparation of protein lysates and immunoblotting 65

2.2.8. Proliferation assay 66

2.2.9. Colony formation assay 66

2.2.10. Invasion assay 66

2.2.11. Wound healing assay 67

2.2.12. shRNA-mediated knockdown of METTL21A and HSPA1 67

2.2.13. siRNA-mediated knockdown of METTL21A 67

2.2.14. Immunoprecipitation 68

2.2.15. In vitro methylation assay 68

2.2.16. mRNA isolation and quantitative reverse transcription PCR 68

2.2.17. RNA sequencing and gene expression analysis 70

2.2.18. Gastritis model 70

2.2.19. Nuclear protein fraction 70

2.2.20. Immunofluorescence assay 71

2.2.21. Statistical analysis 71

2.3. Results 72

2.3.1. Gene landscape of METTL21A 72

2.3.2. Gene expression profiling of METTL21A in gastric cancer and survival analysis 78

2.3.3. METTL21A exhibits tumor-suppressive activity in vitro 84

2.3.4. Pathological role of patient-driven mutation of METTL21A 90

2.3.5. Automethylation of METTL21A at lysine 16 and 215 92

2.3.6. Inverse correlation between METTL21A expression and inflammation 99

2.3.7. METTL21A specifically regulates NF-κB activity 104

2.3.8. Identification of METTL21A substrate 110

2.4. Discussion 118

References 122

논문요약 132

Chapter Ⅰ. Literature Review The role of protein methyltransferases in immunity 9

Table 1. Regulatory mechanisms of PRMTs in immunity 35

Table 2. Regulatory mechanisms of PKMTs in immunity 51

Chapter Ⅱ. Tumor suppressive functions of METTL21A in gastric cancer 9

Table 3. Primary antibodies used in this study. 61

Table 4. Primers sequences used for METTL21A mutagenesis (5'-3') 63

Table 5. Primers sequences used for HSPA1 mutagenesis (5'-3') 65

Table 6. Human primers sequences used for quantitative PCR (5'-3') 69

Table 7. Mouse primers sequences used for quantitative PCR (5'-3') 71

Chapter Ⅰ. Literature Review The role of protein methyltransferases in immunity 10

Figure 1. Mammalian protein arginine methylation encompasses various types. 20

Figure 2. Characteristic domains of arginine protein methyltransferases. 21

Figure 3. The types of lysine methylation. 23

Figure 4. Characteristic domains of lysine protein methyltransferases. 23

Figure 5. Methylation substrates of PRMT1 and the biological functions of this methylation. 28

Figure 6. The role of methyltransferase on T cell differentiation and cytokine secretion. 49

Chapter Ⅱ. Tumor suppressive functions of METTL21A in gastric cancer 10

Figure 7. Identification of Differentially Expressed Genes (DEGs) in MKN1 cells after METTL21A knockdown. 77

Figure 8. Gene expression profiling of METTL21A in gastric cancer. 83

Figure 9. METTL21A exhibits tumor-suppressive activity in vitro. 89

Figure 10. The impact of patient-associated mutations on METTL21A function. 91

Figure 11. Validation of METTL21A automethylation in vivo and in vitro. 98

Figure 12. Negative relationship between METTL21A and inflammatory response. 103

Figure 13. The impact of METTL21A on NF-κB signaling. 109

Figure 14. Identification of substrate of METTL21A. 117

Figure 15. Schematic role of METTL21A in gastric tumorigenesis. 121