Regulation of the murine NMDA-receptor-subunit NR2C promoter by Sp1 and fushi tarazu factor1 (FTZ-F1) homologues

We have cloned the 5'-region of the murine N-methyl-D-aspartate (NMDA) rece ptor channel subunit NR2C (GluR epsilon 3) gene and characterized the cis- and trans-activating regulatory elements responsible for its tissue specifi c activity. By using a native epsilon 3-promoter/lacZ-construct and various 5'-deletion constructs, we compared beta-galactosidase expression in non-n euronal NIH3T3 cells and in neuronal epsilon 3-gene-expressing HT-4 cells a nd show that large parts of the epsilon 3 promoter are responsible for the repression of the epsilon 3 gene in non-neuronal cells. Deletion of exon 1 sequences led to an enhancement of epsilon 3 transcription, suggesting a ro le of the 5'-untranslated region in epsilon 3 gene regulation. Sequence ana lysis of the promoter region revealed potential binding sites for the trans cription factor Spl, the murine fushi tarazu factor1 (FTZ-F1) homologues, e mbryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF-1), a s well as for the chicken ovalbumin upstream promoter transcription-factor (COUP-TF). Electrophoretic mobility shift assays confirmed specific binding of Spl, SF-I and COUP-TFI. Whereas point mutation studies indicate that, i n neuronal HT-4 cells, Spl is apparently not critically involved in basal e psilon 3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of Q-promoter-driven reporter gene expression upon cotransfection of an SF1-expression vector, which was reverted by dele tion and point mutation of the SF1 binding site.