Introduction 9

Fig. 1.1. Stages of gene expression in cell. 17

Fig. 1.2. A typical promoter structure showing modular organization of TFBSs. Taken from Werner 2003. 19

Fig. 1.3. Detail diagrammatic structure of the genomic configuration of exon-intron of the GFRα1 gene. 20

Fig. 1.4. Multifunctional activities of the human AP endonuclease. Ape1/ref-1 is a multifunctional protein involved in BER, transcription factor regulation, and oxidative signaling. In DNA BER, it functions as an AP endonuclease. It is also involved in the... 29

Fig. 1.5. Schematic diagram of important residues and putative phosphorylation sites in Ape1/ref-1. CKI, CKII, and PKC have been shown to phosphorylate Ape1/ref-1 in vitro, however, the other phosphorylation sites are putative. There were no phosphorylation sites... 30

I. Ref-1 Induced GFRα1 Promoter Activity in Human fibroblast cell line GM00637. 10

Fig. I-1. Relative transcript levels of the GFRα1 receptor in GM00637 cells. A. Messenger RNA levels were determined by a RT-PCR method and normalized to GAPDH transcripts. B. Relative GFRα1 protein content in ref-1 expressed cell lines were identified by western blot... 52

Fig. I-2. Nucleotide sequence of the 5'flanking region of the GFRα1 gene. Nucleotides are numbered corresponding to the translational initiation site at + 1 at the start. On the based of Ensemble data, first and second exon as shown highlighted, Binding sites for the transcription... 53

Fig. I-3. Primer extension analysis for GFRα1 transcription start sites. Reverse oligonucleotide as indicated above was end-labeled with [γ-32P] ATP and used in a primer extension experiment with 20 ㎍ of total cellular RNA isolated from ref-1 expressing... 56

Fig. I-4.A. Schematic representation of GFRα1 promoter-reporter chimeras with putative NF-κB binding sites. Scheme of the 5'-upstream region of the human GFRα1 promoter showing major luciferase constructs which were used to identify the promoter activity and... 57

Fig. I-4.B. Relative luciferase activities of GFRα1 promoter-deletion constructs. GFRα1 promoter-deletion constructs were transiently transfected into ref-1 expressing GM00637 cells. Forty eight hour after transfection, cells were harvested for assaying luciferase activities. The... 58

Fig. I-5.A. General overview of 5'deleted luciferase reporter constructs in human GRFα1 gene focusing to purposed NF-κB regulatory region. 59

Fig. I-5.B. Relative luciferase activities of NF-κB oriented GFRα1 promoter-reporter constructs. The GFRα1 promoter-reporter constructs each as shown in Fig.I-5.A and the PGL3 luciferase reporter vector, an internal control, were transiently co-transfected... 59

Fig. I-5.C. General overview of 3' deleted luciferase reporter constructs in human GRFα1 gene focusing to purposed NF-κB regulatory region. 60

Fig. I-5.D. Relative luciferase activities of 3' deleted NF-κB oriented GFRα1 promoter-reporter constructs. The GFRα1 promoter-reporter constructs each as shown in Fig.I-5.A and the PGL3 luciferase reporter vector, an internal control, were transiently co-transfected... 60

Fig. I-6. Identification of the NF-κB binding site in the sub-region -575/-66 of the GFRα1 promoter. A. NF-κB binding to the sub-region -348/-332 of the GFRα1 promoter determined by EMSA assays. 5'-Biotin labeled synthetic GFRα1 promoter... 62

Fig. I-7. Effects of NF-κB-BS-WT and its mutants on the SV40 promoter-directed reporter gene expression. The NF-κB-BS-WT and various mutants as shown in schematic drawing in the left were inserted upstream of the SV40 promoter in the pGL-3 basic reporter... 64

Fig. I-8. Determination of functional NF-κB-binding site in the GFRα1 promoter region-575/-66. [32P]-labeled synthetic GFRα1 promoter fragments containing putative NF-κB BS#I binding sites as shown in fig. I-8 and 5 ㎍ nuclear extracts (NE) prepared from ref-1 expressed... 66

Fig. I-9. Determination of cellular NF-κB binding to the GFRα1 gene promoter by the ChIP assay. GM00637,GM00637-pcDNA3.1 and GM00637-ref-1 cells were incubated from 1 x 106 cells for each group, and immunoprecipitated with an antibody all NF-κB-p50... 67

Fig. I-10. Ref-1 regulation to the functionalities of the GFRα1 promoter. Ref-1 regulated GFRα1 transcriptional activity was determined by using various ref-1 expression constructs with site directed mutations and NF-κB GFRα1 Promoter-reporter constructs (-575/-66) were... 69

II. Ref-1 Induced JAGl Promoter Activity in Human fibroblast cell line GM00637. 12

Fig. II-1. Relative transcript levels of the JAG1 receptor in GM00637 cells. A. Messenger RNA levels were determined by a RT-PCR method and normalized to GAPDH transcripts. B. Relative JAG1 protein content in ref-1 expressed cell lines were identified by western blot... 70

Fig. II-2. Nucleotide sequence of the 5'flanking region of the JAG1 gene. Nucleotides are numbered corresponding to the translational initiation site at + 1 at start. Binding sites for the transcription factors EGR-1 and others are boxed. Primers which were used for primer... 72

Fig. II-3. Primer extension analysis for JAG1 transcription start sites. 74

Fig. II-4.A. Schematic representation of JAG1 promoter-deletion constructs. 76

Fig. II-4.B. Luciferase activities in ref-1 expressing GM00637 cells transfected with JAG1 promoter-deletion constructs. JAG1 promoter-deletion constructs were transiently transfected into ref-1 expressed GM00637 cells. 48 h after transfection, cells were... 76

Fig. II-5.A. General overview of 5'deleted luciferase reporter constructs in human JAG1 gene focusing to purposed EGR-1 regulatory region. 77

Fig. II-5.B. Relative luciferase activities of EGR-1 oriented JAG1 promoter-reporter constructs. The JAG1 promoter-reporter constructs each as shown in Fig.I-5.A and the PGL3 luciferase reporter vector, an internal control, were transiently co-transfected... 78

Fig. II-6.A. Identification of the EGR-1 binding site in the sub-region -850/-382 of the JAG1 promoter. A. EGR-1 binding to the subregion -638/-622 of the JAG1 promoter determined by EMSA assays. 5'-Biotin labeled synthetic JAG1 promoter... 79

Fig. II-7. Effects of EGR-1-BS-WT and its mutants on the SV40 promoter-directed reporter gene expression. The EGR-1-BS-WT and EGR-1 BS #II mutant as shown in schematic drawing in the left were inserted upstream of the SV40 promoter in the pGL-3... 80

Fig. I-8. Determination of functional EGR-1-binding sites in the JAG1 promoter region -850/382. 5'-biotin labeled synthetic JAG1 promoter fragments containing putative EGR-1 binding sites as shown in Fig. II-8 and 5 ㎍ nuclear extracts (NE) prepared from ref-1... 82

Fig. II-9. Determination of cellular EGR-1 binding to the JAG1 promoter by the ChIP assay. GM00637, GM00637-pcDNA3.1 and GM00637-APE1 cells were incubated from 2 x 106 cells for each group, and immunoprecipitated with an antibody against all EGR-1 Anti... 83

Fig. II-10. Ref-1 regulation to the functionalities of the JAC1 promoter. Ref-1 regulated JAG1 transcriptional activity was determined by using various ref-1 expression constructs with site directed mutations and EGR-1 JAG1 Promoter-reporter constructs (-850/-382) were... 85