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Abstract

Testicular GATA-1 factor upregulates the promoter activity of rat inhibin a-subunit gene in MA-10 Leydig tumor cells 
Feng,Zong-Ming; Wu,Ai Zhen; Chen,Ching-Ling C.
Molecular Endocrinology 12(3): 378-390
Publication date: 1998


We have previously demonstrated that the basal transcriptionof rat inhibin -subunit gene in a mouse testicular Leydig tumorcell line, MA-10, depends upon a 67-bp DNA fragment at the positionof -163 to -97. Within this promoter region two GATA motifs wereobserved. In this study, we investigated the possible role of GATA-bindingproteins in the regulation of inhibin -subunit gene transcriptionin testicular cells. Northern blot and RT-PCR analyses showedthat mRNAs encoding GATA-binding proteins, GATA-1 and GATA-4, weredetected in mouse and rat testis and in MA-10 and rat Sertoli cells.Testis-specific GATA-1 mRNA, which is transcribed from a promoter8 kb upstream to the erythroid exon I of mouse GATA-1 gene, wasalso identified in MA-10 cells. Mutations of GATA sequencesin -subunit promoter markedly decreased the transcriptionalactivity of -subunit gene when measured by their ability oftransient expression of a bacterial reporter gene, chloramphenicolacetyltransferase (CAT), in MA-10 cells. Cotransfection of CATchimeric construct with cDNA expression plasmid coding for mouseGATA-1 or GATA-4 protein revealed that GATA-1 but not GATA-4can transactivate -subunit promoter in a dose-dependent manner.The transactivation by GATA-1 was inhibited if GATA sequencesin -subunit promoter were mutated. Furthermore, electrophoreticmobility shift assay demonstrated that GATA-binding proteinspresent in nuclear extracts of MA-10 cells and rat testis interactedwith the GATA motifs in -subunit promoter, and the GATA-1 inthese nuclear extracts formed a supershifted immunocomplex with antibodyraised against mouse GATA-1 protein. We therefore concluded thatthe basal transcription of inhibin -subunit gene in testicular MA-10cells is up-regulated by testicular GATA-1 but not GATA-4 through itsinteraction with the GATA motifs in -subunit promoter. In summary,we have provided the first evidence of the functional role of aGATA-binding protein in the regulation of testicular gene expression.