Functional characterization of the basal promoter of the murine LH receptor gene in immortalized mouse Leydig tumor cells.

Abstract

The nuclear proteins of the LH receptor (LHR) expressing murine Leydig tumor cells (mLTC-1), binding to the LHR primary promoter, were studied by gel retardation assays. Nuclear extracts of HeLa cells, not expressing LHR, were used as control. Protein binding was characterized to the first 173 base pairs (bp) of the LHR 5'-untranslated region, comprising the basal transcriptional promoter activity in mLTC-1 cells, and accounting for the Leydig cell-specific LHR expression. The promoter fragment is GC-rich and contains several Sp1 sites, one activating protein 2 (AP-2) site, and a putative SF-1 binding site. Three subfragments of the 173 bp promoter, I (bases -1 to -55), II (-56 to -102) and III (-103 to -173), were separately analyzed. Fragments II and III formed several complexes with mLTC-1 and HeLa cell nuclear extracts. One complex with fragments II and III, using mLTC-1 and HeLa cell extracts, was similar to that formed with purified Sp1, and it could be removed by an Sp1 oligo and supershifted by an Sp1 antibody. Both fragments formed additional complexes with mLTC-1 cell extracts with no specificity for Sp1. Partly similar, though weaker, complexes were seen with HeLa cell extracts. The most clearcut differences between the protein/DNA complexes formed with LHR expressing mLTC-1 cells and non-expressing (HeLa, COS, HEK 293 and MSC-1) cells were found with fragment I. Extracts of the non-expressing cells formed one prominent protein/DNA complex which was missing in mLTC-1 cells. Purified Sp1 also bound to this fragment. The fragment containing the putative SF-1 binding site did not form any protein/DNA complexes with mLTC-1 cell proteins. In conclusion, the murine LHR primary promoter binds, in addition to the Sp1 and AP-2 transcription factors, several other proteins. The Sp1 protein can bind into at least three different sites in the basal promoter. The other binding proteins differ most clearly between LHR expressing and non-expressing cells in the promoter fragment closest to the translation start site, suggesting a key role for this part of the promoter in cell-specific LHR expression.