Characterization of the c-kit promoter: Participation of the Sp -family of transcription factors in modulating hematopoietic c-kit expression

The c-kit proto-oncogene encodes a tyrosine kinase receptor that plays an important role in mammalian hematopoiesis. Expression of the human gene in hematopoietic cell types is regulated at the level of transcription initiation. Previous experiments have implicated a region 183 base pairs upstream from the translational initiation site to be critical for promoter activity. This proximal region is GC-rich, contains no TATA-element, and harbors multiple transcription initiation sites. The focus of this dissertation was to define promoter element(s) in the proximal promoter and identify trans-acting factors that potentially modulate c-kit expression in hematopoietic cell types.;The approach taken was first to identify protein-DNA interaction(s) in c-kit expressing human hematopoietic cell lines. A protected region between base pairs -112 and -158 was defined by in vitro DNase I footprinting analysis of nuclear extracts from HEL and MEG-01 cell lines. This region contains consensus sites for the Sp-family of transcription factors, and purified Sp1 protects a similar region. Electrophoretic mobility shift assays reveal that Sp1 as well as isoforms of Sp3, independently interacted with this protected region in vitro. These interactions are dependent on two Sp-specific consensus sites located in this region. Additionally, mutation or deletion of these sites reduces c-kit proximal promoter activity in transient expression analysis. Cotransfection of c- kit proximal promoter-luciferase constructs with either Sp1 or Sp3 into Sp-deficient Drosophila SL2 cells demonstrated that both Sp1 and Sp3 affect promoter activity. However, cotransfection of a small isoform of Sp3 inhibited both Sp1 and Sp3-mediated activation.;To substantiate the in vitro findings, nuclear protein interactions on the c-kit proximal promoter were investigated in human hematopoietic cell lines. In vivo footprinting by LM-PCR identified occupancy of the c-kit proximal promoter by nuclear protein(s) in both c-kit expressing and non-expressing hematopoiefic cell lines. No interactions were observed in the c-kit non-expressing HeLa cell line. These studies imply that proximal promoter occupancy, presumably by the Sp-family of transcription factors, modulate c-kit expression in hematopoietic cell types.