The Effect of the ref-1 on the Promoter Activity of the GFRα1 and JAG1

Abstract

(I) Ref-1 Induced GFRα1 Promoter Activity in Human fibroblast cell line GM00637. The overall goal of the research in this thesis was to gain the data that will eventually help in elucidating the Human apurinic/apyrimidinic (AP) endonuclease, APE 1/ref-1, induced Glial cell line derived neurotrophic factor family receptor alpha1; GFRα1 expression in Human fibroblast Cell Line GM00637. The mechanisms by which DNA binding of transcription factors NF-κB of GFRα1 promoter regulates the significant induction of functional GFRα1 in APE1 expressing GM00637 cell line. In addition to DNA repair activity, APE 1 is also capable of regulating the DNA binding activity of many transcription factors in vitro by a redox mechanism (ref-1). Here an active NF-κB binding site was identified in the GFRα1 putaive promoter region. By using EMSA super shift assays with NF-κB-p50 was identified as APE1 interacting protein followed by a nuclear translocation of p50 NF-κB subunits binding to NF-κB site of GFRα1 promoter. Further functional reporter assays and by means of in-vivo CHIP indicated that this NF-κB binding DNA elements associated with GFRα1 promoter. Further more, mutation of this site significantly reduced the promoter activity. Therefore APE1 induced human GFRα1 is controlled by NF-κB binding motifs, thereby providing a novel mechanism by which APE1 up-regulates GFRα1 functions via transcription factors of NF-κB binding sites of GFRα1 promoter. Taken together, this thesis demonstrates a novel Promoter analysis of GFRα1 in associated with APE1/ref-1. The studies also indicate that the precise mechanism of APE1/ref-1 mediated GFRα1 induction as well as GFRα1 promoter analysis. (II) Ref-1 Induced JAG1 Promoter Activity in Human fibroblast cell line GM00637. The Jagged 1 protein encoded by JAG1 is the human homolog of the Drosophila jagged protein. Human jagged 1 is the ligand for the receptor notch 1 which is a human homolog of the Drosophila jagged receptor notch. This work reports that, ref-1 induced JAG1 gene expression activity requires EGR-1 DNA binding site activation. The EGR-1 DNA binding sites of JAG1 promoter is capable of regulating the transcription of the ref-1 induced human JAG1 gene promoter. To assess the transcriptional regulation of JAG1 gene, the 5’-flanking region with [-98 to -1473] base pairs of the human JAG1 gene was cloned. By measuring the promoter activity using dozens of serial deleted luciferase constructs, the highest JAG1 transcriptional activity was found at the region from -850 to -382 upstream of ATG from JAG1 gene and the region is associated with three EGR-1 DNA binding sites and one is functional. In particular, it was demonstrated by chromatin immunoprecipitation and EMSA that EGR-1 assembles on the JAG1 promoter and, by site directed mutagenesis experiments, that it significantly reduces its transcriptional effects by acting through the 468 bp minimal promoter. Taken together, this data add to the body of evidence implying that EGR-1 binding site may fulfill a generic role at the promoters of JAG1 in response to ref-1 in GM00637 cells.