REGULATION OF GAMMA CARBOXYLASE GENE TRANSCRIPTION IN LIVER

肝脏中维生素K依赖性羧化酶基因的表达对于维生素K依赖性蛋白中Gla的形成是必需的，因此对于止血是必需的。维生素K依赖性羧化酶基因在肝和肝外组织中的胚胎表达对于胎儿的存活和正常发育也是必不可少的。羧化酶基因的组织特异性表达受发育调控，但其时空调控机制尚不清楚。通过肝细胞分化的细胞系模型中的瞬时基因表达测定，已经鉴定了在羧化酶基因的转录控制中重要的羧化酶基因的5'侧翼区中的DNA序列。这些区域在体内肝细胞发育过程中的功能及其在控制肝外羧化酶基因表达中的重要性尚不清楚。本申请的目的是阐明控制该必需基因在肝脏中发育表达的调控机制。本申请的研究分为两个具体目标。在第一个目标中，负责羧化酶基因表达的转录因子将被分离和分析。 在第二个目标中，将使用转基因动物模型鉴定在体内维生素K依赖性羧化酶基因转录的发育调节中重要的DNA序列。在其体内表达中重要的羧化酶基因的区域将通过在其体内表达中羧化酶基因座的Dnase I超敏性分析来鉴定，将通过在来自未成熟和成熟肝的细胞核中羧化酶基因座的Dnase I超敏性分析来鉴定。 研究DNA-蛋白质相互作用对发育和分化组织中Gla合成的调节。这些研究结果可能为华法林胚胎病的发病机制提供重要的见解。调节肝和肝外Gla合成的机制的知识也可能导致用于治疗和预防几种疾病的替代策略。维生素K依赖性蛋白质合成的选择性组织特异性调节可能导致血栓性疾病临床管理中重要的新抗凝策略。

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.