Two Inr elements are important for mediating the activity of the proximal promoter of the human gonadotropin-releasing hormone receptor gene

Abstract

Differential usage of several transcription start sites in the human GnRH receptor gene was evident in human brain and pituitary. To locate the promoter responsible for a cluster of the 3′ CAP sites from -635 to -578 (relative to ATG) found in the pituitary, a proximal promoter element was identified at -677/-558 by 5′ and 3′ deletion mutant analysis. The promoter element drove a 13.1 ± 0.6-fold increase in reporter gene activity in an orientation-dependent manner in the mouse gonadotrope-derived αT3-1 cells. Within the core promoter element, two functional AT-rich Inr motifs, interacting with the same protein factor with different affinities, were identified. By Southwestern blot analysis and competitive gel mobility shift assays, multiple nuclear factors (36-150 kDa) were found to interact specifically with the core promoter element. Interestingly, these nuclear proteins also interacted with a previously identified distal promoter of the human GnRH receptor gene. Taken together, our studies suggested that these two promoters share common protein factors to regulate transcription initiations at two different regions. Additional mechanisms are needed to modulate the efficiencies of individual promoters for developmental and/or tissue-specific regulations.