The expression of the vitamin K-dependent carboxylase gene in liver is essential for Gla formation in the vitamin K-dependent proteins, and is therefore essential for hemostasis. The embryonic expression of the vitamin K-dependent carboxylase gene, in hepatic and extrahepatic tissues, is also essential for survival and normal development of the fetus. The tissue-specific expression of the carboxylase gene is developmentally regulated, yet the mechanism underlying its temperospatial regulation is unknown. DNA sequences in the 5' flanking region of the carboxylase gene that are important in the transcriptional control of the carboxylase have been identified by transient gene expression assays in cell line models of hepatocytes differentiation. The functions of these regions during hepatocyte development in vivo and their importance in the control of extrahepatic carboxylase gene expression is unknown. The objectives of this application are to elucidate the regulatory mechanisms that control the developmental expression of this essential gene in liver. Studies in this application are divided into two specific aims. In the first aim, transcription factors responsible for carboxylase gene expression will be isolated and analyzed. In the second aim, DNA sequences important in the developmental regulation of vitamin K-dependent carboxylase gene transcription in vivo will be identified using transgenic animal models. Regions of the carboxylase gene important in its in vivo expression will be identified by Dnase I hypersensitivity analysis of the carboxylase gene locus in its in vivo expression will be identified by Dnase I hypersensitivity analysis of the carboxylase gene locus in nuclei from immature and mature liver. Studies of DNA-protein interactions at regulation of Gla synthesis in developing and differentiated tissues. The findings of the proposed studies may provide important insights into pathogenesis of the warfarin embryopathy. Knowledge of the mechanisms that regulate hepatic and extrahepatic Gla-synthesis may also lead to alternative strategies for treatment and prevention of several diseases. Selective tissue-specific modulation of vitamin K-dependent protein synthesis may lead to novel anti-coagulation strategies important in the clinical management of thrombotic disorders.
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