Endocrinology, Vol 137, 5407-5414, Copyright © 1996 by Endocrine Society

ARTICLES

Human stem cell factor promoter deoxyribonucleic acid sequence and regulation by cyclic 3',5'-adenosine monophosphate in a Sertoli cell line

WE Taylor, H Najmabadi, M Strathearn, NT Jou, M Liebling, T Rajavashisth, N Chanani, L Phung and S Bhasin
Division of Endocrinology, Metabolism, and Molecular Medicine, Charles R. Drew University of Medicine and Science, Los Angeles, California 90059, USA.

Stem cell factor (SCF) gene expression is regulated by FSH in testicular Sertoli cells. Many functions of FSH are mediated through the second messenger cAMP. We show that cAMP activates transcription of the human SCF promoter in a Sertoli cell line. The human SCF promoter was cloned in cosmid vector pWE15, and its DNA sequence was determined for the promoter region extending 2.3 kilobase pairs upstream from the translation start site at +184 bp. The in vivo messenger RNA (mRNA) start site, by primer-extension studies, was located in exon 1 at +109 bp in human testis mRNA, and at +99 bp in mouse SF7 Sertoli cell line or GC1 germ cell line mRNA. To test which regions of the SCF promoter are necessary for regulation by cAMP, a series of 5'-end deletions of this region were cloned onto the luciferase reporter gene in plasmid pXP1. The SCF promoter region was fused to luciferase downstream (at +120) from its +109 mRNA start site, extending upstream a variable distance to BstXI (-162), BamHI (-313), Bgl2 (-853), or XbaI (-2185). The shortest of these fragments extending only to -162 bp, contains possible SP1 and AP-2 elements. When mouse Sertoli SF7 or human JEG.3 cell lines were transfected with these plasmids, all of the mutants were regulated by 8Br-cAMP or forskolin, as expected for the SCF gene, whereas FSH and TPA had no effect. In the shortest promoter deletion - 162, luciferase expression from SF7 cells in serum-free media was at a moderate basal level, but it was induced in six h about 2-fold by 8Br- cAMP, and over 7-fold by forskolin (an adenylate cyclase activator) to high levels, similar to the SV40 positive control promoter. In SCF-luc plasmids extending to -853 or -2185, luciferase expression was still inducible by 8Br-cAMP and forskolin to high levels, but basal promoter activity was repressed to levels over 15-fold lower, in both the absence or presence of testosterone in the media for SF7 cells. The distal portion of the human SCF promoter (between -313 and -853, and also -853 and -2185) inhibits the basal level of transcription, while the proximal region (5' of -162) can mediate activation by cAMP.

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