Although much is known about the secretory dynamics and post translational regulation of GnRH, little information is available concerning the mechanisms which control its tissue specific expression. With the use of two GnRH secreting neuronal cell lines derived from transgenic mice, and human placental cell lines immortalized using a temperature sensitive SV40 T antigen mutant, the applicant proposes to delineate tissue specific and species specific mechanisms of transcriptional regulation of GnRH production.
Specific Aim 1 will elucidate the specific cis acting DNA sequences that mediate hypothalamic and placental expression of GnRH. Gene transfer studies with deletion analysis of the rat and human promoter will characterize those DNA regions necessary for tissue specific expression. DNase footprinting and gel retardation assays will further define promoter regions that interact with tissue specific nuclear proteins. Targeted site directed mutagenesis will examine the functional significance of these regions in transient transfection assays. Methylation interference assays of promoter sequences will be used to confirm contacts with proteins.
Specific Aim 2 will identify and characterize the trans acting nuclear protein DNA or protein protein interactions that confer hypothalamic and placental expression of the GnRH promoter. Once the cis acting DNA sequences have been rigorously defined, four strategies will be employed to identify and isolate DNA binding proteins: ligand screening, protein purification, PCR amplification of putative transcription factor family members, and differential display. Molecular cloning by differential display will be used to isolate developmentally regulated transcription factors by comparing expressed mRNAs from hypothalamic cells derived at early (Gn 10 cells) and late (GT1 7 cells) stages of GnRH neuronal migration and placental cells derived from early (SPA 2 9 cells) and late (HPA 1) gestation.
