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 characterized. The transcriptional start point (tsp) was identified by primer 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 the 1.5 kb 5' flanking region was analyzed by deletion constructs using a firefly luciferase-encoding gene (luc) expression vector transiently transfected into primary rat Sertoli cells and other SCF positive and negative cell lines. For all the cells and cell lines examined, a -119 bp to +43 bp fragment including the tsp was sufficient for SCF promoter activity, and the core promoter activity was not significantly changed by inclusion of upstream sequences as far as -1461 bp. These results indicate that additional sites outside of this promoter region are needed to define the cell-specific regulatory elements of SCF expression. The transcriptional activities of all SCF deletion constructs treated with cyclic adenosine 3',5'-monophosphate (cAMP) and forskolin were increased two- to threefold, indicating that SCF transcription in Sertoli cells is regulated by a cAMP-dependent pathway in the proximal promoter region.
Jiang
C
C
Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA.
Hall
S J
SJ
Boekelheide
K
K
D016475:3T3 Cells / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D012708:Sertoli Cells / Q000187:drug effects / Q000502:physiology*; D019089:Stem Cell Factor / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D005576:Colforsin; D000242:Cyclic AMP; D008156:Luciferases; D013755:Tetradecanoylphorbol Acetate