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DNA Sequences and Their Binding Proteins Required for Sertoli Cell-Specific Transcription of the Rat Androgen-Binding Protein Gene

The Curriculum in Genetics and Molecular Biology (D.A.F.) Department of Pediatrics (D.R.J.) The Laboratories for Reproductive Biology The University of North Carolina Chapel Hill, North Carolina 27599

The rat androgen-bindng protein (ABP) gene is transcriptionally regulated from two promoters: the P₁ promoter regulates expression of transcripts starting at exon 1, whereas P_A regulates transcripts containing exon A. The P₁ promoter directs cell-specific gene regulation of ABP secreted by Sertoli cells. In this study, the Sertoli cell-regulatory sequences of P₁ were further examined using a luciferase reporter system with three cell lines, including a Sertoli cell line (MSC-1) that expresses the ABP gene. Deletion mapping experiments determined that the sequences required for full activity in MSC-1 cells were included within 619 bp of the start site and identified several regions that demonstrated increased luciferase activity: the -583 bp to -564 bp, -503 bp to -484 bp, and -114 bp to -65 regions. The activities contributed by each region were much higher (up to 120-fold) in MSC-1 cells than in MA10 Leydig or NIH3T3 fibroblast cells. Nuclear-binding proteins and their binding sequences were identified using several molecular biology techniques. Complexes formed by nuclear proteins of MSC-1, MA10, and NIH3T3 cells, which bind specifically to the -114 to -65-bp region, were identified using gel retardation assays. Furthermore, the inverted repeat sequence in this region, 5'-AGGGTCAGTGTCCCT-3' was identified by deoxyribonuclease (DNase) I footprinting. The regulatory element contained within the -503 to -484-bp region was identified by scanning mutagenesis, but no protein was found that bound to this sequence by gel retardation or DNase I protection assays. This element is characterized by the core sequence, 5'-GGAGGC- 3'. The third regulatory region (residues -583 to -564) bound a protein complex that retarded mobility of the free DNA probe in a gel shift assay. Using several techniques, the binding sequence was identified as 5'-TTCATAGTATCCATTAAAC-3'. In summary, these data have identified several transcriptional regulatory sequences and their binding proteins, which appear to play a role in the Sertoli cell-specific expression of the ABP gene.

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