NEGATIVE CONTROL OF THE RAT INHIBIN ALPHA-SUBUNIT PROMOTER IN MA-10 LEYDIG TUMOR-CELLS

The promoter/regulatory sequences responsible for the transcription of the rat inhibin alpha subunit gene in the testis were identified by t he transient expression in an MA-10 Leydig tumour cell line of a bacte rial reporter gene, chloramphenicol acetyltransferase (CAT), which was driven by different regions of the 5' flanking sequence of the inhibi n alpha subunit gene. The CAT activity was elevated when the 2.0 kb 5' flanking alpha subunit gene fragment was progressively shortened from its 5' end, and a maximal increase was reached when the CAT gene was driven by an alpha subunit gene promoter extending to - 163 bp. This c onstruct was termed A alpha BstCAT. Furthermore, when either the - 2.0 to - 1.6 kb or the - 2.0 to - 1.0 kb alpha subunit DNA fragment was f used to A alpha BstCAT, the CAT activity was markedly suppressed, indi cating the presence of negative regulatory DNA elements (NREs) in the upstream region of the gene. The cyclic AMP (cAMP) responsiveness of t he alpha subunit gene, which was dependent upon the putative cAMP resp onse element within the 67 bp alpha subunit promoter, was not affected by the upstream NREs. The inhibitory effect was also demonstrated whe n the - 2.0 to - 1.0 kb fragment was placed in either orientation with respect to the alpha subunit promoter or to a thymidine kinase promot er, suggesting that the NRE(s) can act as a silencer. Based on our obs ervations we conclude that the basal expression of the rat inhibin alp ha subunit gene in testicular MA-10 cells may, at least in part, be co ntrolled by the upstream silencer(s) and NRE(s).