Abstract
An imperfect estrogen receptor half-site response element direct-repeat, located within the TATA-less promoter of the human luteinizing hormone receptor (hLHR), was identified as an inhibitory site for Sp1/Sp3-driven basal transcription. Isolation of proteins recognizing this site by yeast one-hybrid screening of a human placenta cDNA library revealed three nuclear orphan receptors, EAR2, EAR3/COUP-TFI, and TR4. Electrophoresis mobility shift assays demonstrated that the in vitro translated nuclear orphan receptors specifically bound the direct-repeat motif of the hLHR promoter. Also, endogenous EAR2 and EAR3/COUP-TFI from JAR cell and human testis and TR4 from testes bound this motif in electrophoresis mobility shift assays. Functional analyses in CV-1 cells showed that EAR2 and EAR3/COUP-TFI repressed the hLHR promoter 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 through binding to the same cis-element. The stimulation was reversed by coexpression of EAR2 or EAR3/COUP-TFI, indicating their competitive binding for this site. Such recognition of a common cognate site by the proteins with antagonistic functions implies that a net regulation of the hLHR gene may result from the relative availability of repressors and activator in a physiological state. This also may contribute to the differential expression of the hLHR gene in gonadal and non-gonadal tissues.
Highlights
Characterization of the human LHR gene has demonstrated genetic polymorphism with at least two forms of sequence heterogeneity by the presence or absence of a 6-bp insertion that encodes a leucine-glutamine dipeptide within the exon I coding region [3]
We demonstrated that the inhibition of the human luteinizing hormone receptor (hLHR) gene in JAR cells was caused by endogenous EAR2 and EAR3/COUPTFI proteins binding to the direct-repeat motif
An imperfect EREhs direct-repeat motif, compatible with the recognition site bound by members of RAR/TR/orphan nuclear receptor subfamily, was identified in this study as an inhibitory element for hLHR gene transcription in JAR cells
Summary
Characterization of the human LHR gene has demonstrated genetic polymorphism with at least two forms of sequence heterogeneity by the presence or absence of a 6-bp insertion that encodes a leucine-glutamine dipeptide within the exon I coding region [3]. Electrophoresis mobility shift assays (EMSAs) and mutagenesis revealed that multiple specific DNA-protein complexes were formed between the hLHR EREhs and JAR cell nuclear extracts [5]. This indicated that more than one protein could converge upon this common cognate site, it does not exclude the possibility that the bands represent several truncated forms of a single protein. Three human nuclear orphan receptors, EAR2, EAR3/COUP-TFI, and TR4, were cloned and shown to bind the EREhs region in which an imperfect direct-repeat motif was identified as a cognate binding site. We demonstrated that the inhibition of the hLHR gene in JAR cells was caused by endogenous EAR2 and EAR3/COUPTFI proteins binding to the direct-repeat motif
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