REGULATION OF GAMMA CARBOXYLASE GENE TRANSCRIPTION IN LIVER

维生素K依赖的羧基酶基因在肝脏中的表达对维生素K依赖的蛋白质中GLA的形成是必不可少的，因此对止血是必不可少的。维生素K依赖的羧基酶基因在肝脏和肝外组织中的胚胎表达对胎儿的存活和正常发育也是必不可少的。羧基酶基因的组织特异性表达是发育调节的，但其温度空间调节的潜在机制尚不清楚。在肝细胞分化的细胞系模型中，通过瞬时基因表达分析，已经确定了在羧基酶基因转录调控中起重要作用的5‘侧翼区的DNA序列。这些区域在体内肝细胞发育过程中的功能以及它们在控制肝外羧基酶基因表达中的重要性尚不清楚。这项应用的目的是阐明控制这一必需基因在肝脏中发育表达的调控机制。在这一应用中的研究分为两个具体目的。在第一个目标中，将分离和分析负责羧基酶基因表达的转录因子。在第二个目标中，将使用转基因动物模型来鉴定在体内维生素K依赖的羧基酶基因转录的发育调节中重要的DNA序列。在体内表达的羧基酶基因的重要区域将通过DNase I超敏分析来确定其在体内表达的羧基酶基因位置将通过DNase I超敏分析在未成熟和成熟的肝脏细胞核中的羧基酶基因位置来确定。DNA-蛋白质相互作用在发育和分化组织中调节GLA合成的研究。这些研究结果可能会为华法林胚胎病的发病机制提供重要的见解。对调节肝脏和肝外GLA合成的机制的了解也可能导致替代治疗和预防几种疾病的策略。选择性的组织特异性调节维生素K依赖的蛋白质合成可能导致新的抗凝策略，这在血栓性疾病的临床治疗中非常重要。

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.