The expression of the vitamin K-dependent ␥-glutamyl carboxylase gene in liver is developmentally regulated. Since the gene product catalyzes an essential posttranslational modification of the vitamin K-dependent blood coagulation proteins, the regulation of carboxylase expression is critical for hemostasis. We analyzed the activity of the rat carboxylase gene 5-regulatory DNA sequences in rat hepatoma cell lines at different states of differentiation. These studies demonstrated that the 2.6-kb 5-flanking sequence has differentiation-dependent transcriptional activity. Transient gene expression assays, examining the effects of nested deletions and site-directed mutagenesis of putative regulatory sequences, together with electrophoretic mobility shift assays (EMSAs) were used to identify sequences critical for the developmentally regulated transcription of the rat carboxylase gene. We identified a DNA sequence (؊76 to ؊65; GTTCCGGCCTTC) not known to bind to transcription factors, yet which functions as an upstream promoter element. In vivo genomic DNA footprinting confirms the presence of
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