Title Page
Contents
ABSTRACT 10
Ⅰ. Introduction 13
1.1. Sotos syndrome and human NSD1 13
1.2. Immune-cold and NSD1 deficient tumors 14
1.3. Infertility caused by NSD1 deficiency 16
1.4. Epigenetic regulation by NSD 17
Ⅱ. Materials and Methods 20
2.1. Fly and bacterial strains 20
2.2. Total RNA isolation and Real-time quantitative PCR 21
2.3. Immunohistochemistry 22
2.4. Analysis of polytene chromosome 23
2.5. Cloning of luciferase constructs driven by mutant Attacin A promoter 24
2.6. Transient transfection of S2 cells 25
2.7. Luciferase assay 25
2.8. Western blot analysis 26
2.9. Natural Pe infection of Drosophila larvae and survival rate measurement 27
2.10. Fertility analysis 27
2.11. Statistics 28
Ⅲ. Results 29
3.1. The role of NSD on the innate immunity of Drosophila 29
3.1.1. NSD overexpression in the fat body using the Gal4/UAS system 29
3.1.2. Activation of the innate immunity by overexpressed NSD 30
3.1.3. The ectopic expression of NSD affects the survival of Pe-infected larvae. 40
3.2. The effects of NSD deficiency on oogenesis of Drosophila 43
3.2.1. Generation of the NSD-deficient flies expressing DIAP1 driven by the NSD promoter 43
3.2.2. The effects of NSD deficiency on the fertility of Drosophila 45
3.2.3. The effects of NSD deficiency on ovary development 48
3.2.4. Restoration of normal egg chamber development via inhibition of apoptosis of follicle cells caused by NSD deficiency 53
3.3. The epigenetic regulation of NSD on heterochromatin formation 55
3.3.1. NSD deficiency dramatically decrease the H3K36me2 level on the chromocenter region of polytene chromosomes. 55
3.3.2. NSD deficiency affects heterochromatin formation of chromosome 4 58
3.3.3. H3K9 methyltransferase activity is reduced by NSD deficiency 62
Ⅳ. Conclusion 65
4.1. Effects of Drosophila NSD on immune response 66
4.2. Effects of Drosophila NSD on fertility 67
4.3. Epigenetic regulation of Drosophila NSD on heterochromatin formation of chromosome 4 68
References 71
Abstract (in Korean) 81
Table 1. List of primers for quantitative RT-PCR 21
Table 2. List of primers for cloning mutant AttA-driven reporter constructs 24
Fig. 1. Relative mRNA levels of AMPs in the larvae expressing the ectopic NSD. 33
Fig. 2. Transient transfection of NSD with the AttA promoter containing mutant NF-κB binding sites into S2 cells 37
Fig. 3. NSD activates Relish by the IMD pathway. 39
Fig. 4. Comparison of survival between the larvae overexpressing NSD in the fat body, wild-type larvae, and the NSD-deficient larvae after Pe infection. 42
Fig. 5. DIAP1 was expressed by the NSD promoter using the Gal4/UAS system. 44
Fig. 6. The effects of NSD on fertility 47
Fig. 7. Confocal images of Drosophila ovary. 50
Fig. 8. In NSD-deficient flies, apoptosis is increased during oogenesis. 52
Fig. 9. Immunostaining H3K36 of polytene chromosomes. 57
Fig. 10. Immunostaining HP1 and POF of polytene chromosomes. 61
Fig. 11. Immunostaining H3K9me2 of polytene chromosomes. 64