Analysis of promoter and androgen regulatory sequences required for optimal transcription of the rat androgen-binding protein gene.
The androgen-binding protein (ABP) gene P1 promoter directs cell-specific gene regulation of ABP secreted by Sertoli cells. A recent study using the mouse Sertoli cell line (MSC-1) with a luciferase reporter system demonstrated Sertoli cell-specific gene expression with 619 bp of P1 DNA. Furthermore, based on studies of the rat and human genes, several controversies developed over the promoter characteristics, including the promoter type, the transcription start site, and whether the gene is regulated directly by androgens. In this study, the answers to several of these controversies were deciphered using the MSC-1 cell model. The results of mutagenesis experiments were consistent with the presence of the major transcription start site at 36 bp upstream of the initiating Met residue. Modification of the initiator sequence at the start site reduced activity in MSC-1 and NIH3T3 fibroblast cells. Mutation of a putative modified TATA sequence or conversion to the consensus TATA sequence had no effect on activity. Modification of a consensus RNA splice sequence at the start site also had no effect on activity. Furthermore, a minor start site was localized 179 bp upstream of the major site using reverse transcriptase-polymerase chain reaction with various P1 primers (primer walking), primer extension, and cDNA cloning. RNA transcripts from the minor site contain an untranslated 5' exon but apparently encode the same protein as the major transcript. The effect of androgens on P1 expression was also investigated. Cotransfection experiments with pCMVAR, which encodes the androgen receptor, demonstrated that dihydrotestosterone had no effect on the activity in MSC-1 cells. Taken together, these experiments and previous studies indicate that the rat ABP promoter P1 is regulated at the major start site by an initiator element without a TATA sequence, and the gene appears not to be directly regulated by follicle-stimulating hormone or androgens.
Fenstermacher
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Curriculum in Genetics and Molecular Biology, Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, USA.
Joseph
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D016475:3T3 Cells; D000727:Androgen-Binding Protein / Q000235:genetics*; D000728:Androgens / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic / Q000502:physiology*
D000727:Androgen-Binding Protein; D000728:Androgens; D017931:DNA Primers; D018076:DNA, Complementary