Nuclear orphan receptors regulate transcription of the gene for the human luteinizing hormone receptor

imperfect estrogen receptor half-site response element direct-repeat, locat ed within the TATA-less promoter of the human luteinizing hormone receptor (hLHR), was identified as an inhibitory site for Sp1/Sp3-driven basal trans cription. Isolation of proteins recognizing this site by yeast one-hybrid s creening of a human placenta cDNA library revealed three nuclear orphan rec eptors, EARS, EAR3/COUP-TFI, and TR4. Electrophoresis mobility shift assays demonstrated that the in vitro translated nuclear orphan receptors specifi cally bound the direct-repeat motif of the hLHR promoter. Also, endogenous EARS and EAR3/COUP-TFI from JAR cell and human testis and TR4 from testes b ound this motif in electrophoresis mobility shift assays. Functional analys es in CV-1 cells showed that EARS and EAR3/COUP-TFI repressed the hLHR prom oter activity by up to 70% in a dose dependent and sequence-specific manner . Conversely, TR4 activated the hLHR promoter activity up to 2.5-fold throu gh binding to the same cis-element. The stimulation was reversed by coexpre ssion of EARS or EARS/COUP-TFI, indicating their competitive binding for th is site. Such recognition of a common cognate site by the proteins with ant agonistic functions implies that a net regulation of the hLHR gene may resu lt from the relative availability of repressors and activator in a physiolo gical state. This also may contribute to the differential expression of the hLHR gene in gonadal and non-gonadal tissues.