Inhibin and activin gene expression in the ovary and in cultured granulosa cells is modulated by the pituitary gonadotropins FSH and LH. In granulosa cells, two messenger RNAs (mRNAs) of 4.3 and 3.3 kilobases (kb) encoding the common βB-chain of inhibin and activin are observed. We demonstrate here that FSH or pharmacological agents that elevate intracellular levels of cAMP induce expression of the 4.3-kb mRNA to a greater extent than that of the 3.3-kb mRNA. To elucidate the mechanism(s) by which the two βB transcripts are generated, we characterized the rat βB gene and used probes from various regions of the gene to examine the structures of the βB mRNAs. RNA blot analysis using probes from the 5’-nontranslated and flanking regions of the βB gene revealed that the 4.3- and 3.3-kb transcripts differ in the length of their 5’-nontranslated regions. S1 nuclease protection and primer extension assays were used to map two adjacent transcriptional start sites 1051 and 1052 basepairs (bp) up-stream of the most 3’-start site previously reported for the βB gene. These novel start sites are used to generate the 4.3-kb mRNA, whereas transcription of the 3.3-kb mRNA initiates at down-stream start sites. DNA sequence analysis did not reveal any consensus CCAAT or TATA box elements up-stream of the novel start sites. Multiple potential binding sites for the transcription factors SP1 and activator protein-2 are present throughout a region of the βB gene from −1461 to 82 bp. Fusion genes were constructed that contain βB sequences from −1460 to 14 bp, −1460 to −914 bp, and -913 to 14 bp controlling luciferase reporter gene expression. Analysis of βB promoter activity in transiently transfected primary granulosa cells demonstrated that sequences flanking both the up- and down-stream transcriptional start sites have substantial basal promoter activity; however, forskolin did not affect expression of the βB/luciferase fusion genes.
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