Fenstermacher D A, Joseph D R
The Laboratories for Reproductive Biology, The University of North Carolina, Chapel Hill 27599, USA.
Mol Endocrinol. 1997 Aug;11(9):1387-400. doi: 10.1210/mend.11.9.9981.
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.
大鼠雄激素结合蛋白（ABP）基因受两个启动子转录调控：P1启动子调控从外显子1起始的转录本表达，而P(A)调控包含外显子A的转录本。P1启动子指导支持细胞分泌的ABP的细胞特异性基因调控。在本研究中，使用荧光素酶报告系统和三种细胞系，包括表达ABP基因的支持细胞系（MSC-1），进一步检测了P1的支持细胞调控序列。缺失定位实验确定，MSC-1细胞中充分活性所需的序列包含在起始位点的619 bp内，并鉴定了几个显示荧光素酶活性增加的区域：-583 bp至-564 bp、-503 bp至-484 bp和-114 bp至-65区域。每个区域在MSC-1细胞中的活性比在MA10睾丸间质细胞或NIH3T3成纤维细胞中高得多（高达120倍）。使用几种分子生物学技术鉴定了核结合蛋白及其结合序列。使用凝胶阻滞试验鉴定了由MSC-1、MA10和NIH3T3细胞的核蛋白形成的、特异性结合-114至-65 bp区域的复合物。此外，通过脱氧核糖核酸酶（DNase）I足迹法鉴定了该区域的反向重复序列5'-AGGGTCAGTGTCCCT-3'。通过扫描诱变鉴定了-503至-484 bp区域内的调控元件，但通过凝胶阻滞或DNase I保护试验未发现与该序列结合的蛋白质。该元件的特征性核心序列为5'-GGAGGC-3'。第三个调控区域（-583至-564位残基）在凝胶迁移试验中结合了一种使游离DNA探针迁移率降低的蛋白质复合物。使用几种技术，将结合序列鉴定为5'-TTCATAGTATCCATTAAAC-3'。总之，这些数据鉴定了几个转录调控序列及其结合蛋白，它们似乎在ABP基因的支持细胞特异性表达中发挥作用。
