Multiple core homeodomain binding motifs differentially contribute to transcriptional activity of the murine gonadotropin-releasing hormone receptor gene promoter

Multiple homeodomain (Hbox) proteins have been shown to organize expression of key markers of gonadotropes. Nine putative Hbox-binding sites, characterized by the homeospecific TAAT motif, are located within the proximal 600 bp of the murine GnRHR promoter. Homeoproteins bind separate Hbox sites within this promoter, supporting basal- and endocrine-directed transcription. The function of the most proximal sites (Hbox1 and Hbox2) in the murine GnRHR is unknown; thus, understanding of the global contribution of homeospecific TAAT sites to promoter function is incomplete. Site-directed mutagenesis revealed that loss of Hbox2 reduced promoter activity in a cell-specific manner, having no effect in alphaT3-1 cells but reducing promoter function in LbetaT2 cells, another gonadotrope-derived cell line representing a later developmental stage. In contrast, eliminating Hbox1 reduced basal activity in both lines. This region displayed specific binding to homeoprotein Oct-1. Mutagenesis of a previously identified Oct-1-binding site in concert with Hbox1 led to further reduction in activity. We suggest that the two most proximal homeodomain-binding sites in the murine GnRHR promoter may regulate the promoter in a developmentally dependent fashion and that Oct-1 acts at multiple but distinct TAAT sites to support basal transcription.