To determine whether the chorionic gonadotropin beta gene is regulated by E box-binding transcription factors. RESULTS Developmentally appropriate and cell-specific gene transcription is regulated in many organs by transcription factors binding to the E box motif. Examination of the human chorionic gonadotropin beta subunit 5'-flanking DNA revealed three E box-like motifs at -274 (A box), -135 (B box), and -126 (C box) relative to the start site of transcription. To examine whether these elements play a role in gene transcription, the A box, the B/C boxes or all three sites were mutated and activity was compared to wild-type activity. Transfection of JEG-3 choriocarcinoma cells demonstrated that individual E box mutations reduced basal transcription by approximately 50%, but fold-induction by cAMP (a hallmark of trophoblast function) was not dramatically changed. Mutation of all three E boxes led to a 75% reduction in basal transcription, but not the fold-induction by cAMP. Gel mobility-shift assays with the A box revealed two bands which were not specific for trophoblast cells, and supershift analysis with specific antisera to a variety of E box-binding proteins demonstrated that while none of the specific complexes contained the bHLH dimer partners E12/E47, at least one band contained the widely expressed transcription factor USF-1. FUTURE DIRECTIONS USF-1 has recently been demonstrated to bind an E box-like element in the common glycoprotein hormone alpha subunit promoter and contribute to transcriptional activation of this gene in mouse pituitary gonadotropin tumor cells. This suggests a role in placental CG expression as well, and we will examine whether USF-1 is part of a mechanism promoting coordinate expression of the CG alpha and beta subunits during trophoblast differentiation. KEY WORDS placenta, gene transcription, helix-loop-helix, chorionic gonadotropin, USF-1
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