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Elucidation of the control mechanism of transcription of human L-histidine decarboxylase and trial of the control of histamine production

阐明人L-组氨酸脱羧酶转录控制机制并尝试控制组胺产生

基本信息

批准号：
08670101
负责人：
OHTSU Hiroshi
金额：
$ 1.34万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Since we cloned human L-histidine decarboxylase (HDC) gene in 1994, we have been investigating the control mechanism of cell specific transcription of this gene. This enzyme catalyze to produce histamine from histidine and its expressions are restricted to mast cells and basophils in hematopoietic cell lineages. We clarified here the cell specific expression in cell lines and this specificity were controlled at transcriptional level. However, in transient transfection analysis, the reporter constructs with HDC promoter were active not only in cells expressing the endogenous gene but also in non-expressing cells. Detailed analyzes of the HDC promoter region revealed that a GC box is essential for transactivation. Also, the promoter region of HDC gene proved to be sensitive to DNase I and restriction endonucleases exclusively in HDC expressing cells, suggesting that the promoter region is readily accessible to trans-acting factor (s) in those cells. Furthermore, Southern blot analysis with HpaII and MspI endonucleases and sodium bisulfite analysis of genomic DNA revealed the promoter region in HDC expressing cell lines to be selectively unmethylated. In addition, methylation of the HDC promoter in vitro reduced the luciferase reporter activity in transient expression analysis, suggesting that methylation of the promoter region is functionally important for HDC gene expression. These results imply that alteration of DNA methylation is one of the mechanisms regulating cell-specific expression of the HDC gene. We provided proof that chromosomal configuration and methylation of the HDC promoter are important for mast-cell and basophil specific expression. It will be exciting to elucidate the mechanism how the chromatin structure or the mehtylation state changes during cell differentiation and/or induction. These experiments also may lead to clarify the demethylation mechanism itself. Our current efforts are focused on these points.

自1994年克隆人L-组氨酸脱羧酶（HDC）基因以来，我们一直在研究该基因的细胞特异性转录调控机制。该酶催化组氨酸生成组胺，其表达仅限于造血细胞系中的肥大细胞和嗜碱性粒细胞。我们在这里阐明了细胞系中的细胞特异性表达，并且这种特异性在转录水平上受到控制。然而，在瞬时转染分析中，具有HDC启动子的报告构建体不仅在表达内源基因的细胞中有活性，而且在非表达细胞中也有活性。对HDC启动子区的详细分析表明，GC盒是转录激活所必需的。此外，HDC基因的启动子区被证明仅在表达HDC的细胞中对DNA酶I和限制性内切酶敏感，这表明在这些细胞中启动子区容易接近反式作用因子。此外，Southern印迹分析与HpaII和MspI核酸内切酶和亚硫酸氢钠分析的基因组DNA显示的启动子区域中的HDC表达细胞系被选择性非甲基化。此外，体外HDC启动子的甲基化降低了瞬时表达分析中的荧光素酶报告基因活性，表明启动子区域的甲基化对HDC基因表达具有重要的功能。这些结果表明，DNA甲基化的改变是调节HDC基因的细胞特异性表达的机制之一。我们提供的证据表明，染色体构型和甲基化的HDC启动子是重要的肥大细胞和嗜碱性粒细胞的特异性表达。阐明细胞分化和/或诱导过程中染色质结构或甲基化状态变化的机制将是令人兴奋的。这些实验也可能导致澄清脱甲基化机制本身。我们目前的努力集中在这些方面。