DNA sequences and their binding proteins required for Sertoli cell-specific transcription of the rat androgen-binding protein gene.
The rat androgen-binding 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.
Fenstermacher
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The Laboratories for Reproductive Biology, The University of North Carolina, Chapel Hill 27599, USA.
Joseph
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D000727:Androgen-Binding Protein / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics; D004586:Electrophoresis / Q000379:methods; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D012708:Sertoli Cells / Q000502:physiology*; D014158:Transcription, Genetic
D000727:Androgen-Binding Protein; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D008156:Luciferases; D003850:Deoxyribonuclease I