Feng Z M, Wu A Z, Chen C L
Population Council, New York, New York 10021, USA.
Mol Endocrinol. 1998 Mar;12(3):378-90. doi: 10.1210/mend.12.3.0079.
We have previously demonstrated that the basal transcription of rat inhibin alpha-subunit gene in a mouse testicular Leydig tumor cell line, MA-10, depends upon a 67-bp DNA fragment at the position of -163 to -97. Within this promoter region two GATA motifs were observed. In this study, we investigated the possible role of GATA-binding proteins in the regulation of inhibin alpha-subunit gene transcription in testicular cells. Northern blot and RT-PCR analyses showed that mRNAs encoding GATA-binding proteins, GATA-1 and GATA-4, were detected in mouse and rat testis and in MA-10 and rat Sertoli cells. Testis-specific GATA-1 mRNA, which is transcribed from a promoter 8 kb upstream to the erythroid exon I of mouse GATA-1 gene, was also identified in MA-10 cells. Mutations of GATA sequences in alpha-subunit promoter markedly decreased the transcriptional activity of alpha-subunit gene when measured by their ability of transient expression of a bacterial reporter gene, chloramphenicol acetyltransferase (CAT), in MA-10 cells. Cotransfection of alphaCAT chimeric construct with cDNA expression plasmid coding for mouse GATA-1 or GATA-4 protein revealed that GATA-1 but not GATA-4 can transactivate alpha-subunit promoter in a dose-dependent manner. The transactivation by GATA-1 was inhibited if GATA sequences in alpha-subunit promoter were mutated. Furthermore, electrophoretic mobility shift assay demonstrated that GATA-binding proteins present in nuclear extracts of MA-10 cells and rat testis interacted with the GATA motifs in alpha-subunit promoter, and the GATA-1 in these nuclear extracts formed a supershifted immunocomplex with antibody raised against mouse GATA-1 protein. We therefore concluded that the basal transcription of inhibin alpha-subunit gene in testicular MA-10 cells is up-regulated by testicular GATA-1 but not GATA-4 through its interaction with the GATA motifs in alpha-subunit promoter. In summary, we have provided the first evidence of the functional role of a GATA-binding protein in the regulation of testicular gene expression.
我们之前已经证明，大鼠抑制素α亚基基因在小鼠睾丸间质细胞瘤细胞系MA - 10中的基础转录依赖于位于 - 163至 - 97位置的一段67 bp的DNA片段。在该启动子区域内观察到两个GATA基序。在本研究中，我们调查了GATA结合蛋白在睾丸细胞中抑制素α亚基基因转录调控中的可能作用。Northern印迹和RT - PCR分析表明，在小鼠和大鼠睾丸以及MA - 10和大鼠支持细胞中检测到编码GATA结合蛋白GATA - 1和GATA - 4的mRNA。睾丸特异性GATA - 1 mRNA是从小鼠GATA - 1基因红系外显子I上游8 kb的启动子转录而来，在MA - 10细胞中也被鉴定出来。当通过细菌报告基因氯霉素乙酰转移酶（CAT）在MA - 10细胞中的瞬时表达能力来测量时，α亚基启动子中GATA序列的突变显著降低了α亚基基因的转录活性。αCAT嵌合构建体与编码小鼠GATA - 1或GATA - 4蛋白的cDNA表达质粒共转染显示，GATA - 1而非GATA - 4能以剂量依赖的方式反式激活α亚基启动子。如果α亚基启动子中的GATA序列发生突变，GATA - 1的反式激活作用就会受到抑制。此外，电泳迁移率变动分析表明，MA - 10细胞和大鼠睾丸核提取物中的GATA结合蛋白与α亚基启动子中的GATA基序相互作用，这些核提取物中的GATA - 1与针对小鼠GATA - 1蛋白产生的抗体形成了超迁移免疫复合物。因此，我们得出结论，睾丸MA - 10细胞中抑制素α亚基基因的基础转录通过其与α亚基启动子中GATA基序的相互作用，被睾丸GATA - 1而非GATA - 4上调。总之，我们首次提供了GATA结合蛋白在睾丸基因表达调控中功能作用的证据。
