CLONING AND CHARACTERIZATION OF THE 5'-FLANKING REGION OF THE STEM-CELL FACTOR GENE IN RAT SERTOLI CELLS

In order to elucidate the molecular basis of stem cell factor (SCF, or steel factor/kit ligand) expression in Sertoli cells of rat testis, 1 .5 kb of the 5' flanking region of the SCF gene was isolated and chara cterized. The transcriptional start point (tsp) was identified by prim er extension assay and a rapid amplification of cDNA ends (RACE) assay . A TATA box was found 29 base pairs (bp) upstream from the tsp, and a number of transcription factor consensus sequences, including several AP2 and Spl sites, were identified. The transcriptional activity of t he 1.5 kb 5' flanking region was analyzed by deletion constructs using a firefly luciferase-encoding gene (luc) expression vector transientl y transfected into primary rat Sertoli cells and other SCF positive an d negative cell lines. For all the cells and cell lines examined, a -1 19 bp to +43 bp fragment including the tsp was sufficient for SCF prom oter activity, and the core promoter activity was not significantly ch anged by inclusion of upstream sequences as far as -1461 bp. These res ults indicate that additional sites outside of this promoter region ar e needed to define the cell-specific regulatory elements of SCF expres sion. The transcriptional activities of all SCF deletion constructs tr eated with cyclic adenosine 3',5'-monophosphate (cAMP) and forskolin w ere increased two- to threefold, indicating that SCF transcription in Sertoli cells is regulated by a cAMP-dependent pathway in the proximal promoter region.