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

自1994年克隆出人类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。该酶催化组氨酸产生组胺，其表达仅限于造血细胞系中的肥大细胞和嗜碱性细胞。我们在这里阐明了细胞特异性表达在细胞系中，这种特异性是在转录水平上控制的。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活跃，而且在非表达细胞中也活跃。对HDC启动子区域的详细分析表明，GC盒对于transactivation是必不可少的。此外，HDC基因的启动子区域在表达HDC的细胞中被证明对dna酶I和限制性内切酶敏感，这表明这些细胞中的反式作用因子很容易进入启动子区域。此外，用HpaII和MspI核酸内核酸酶和亚硫酸氢钠分析基因组DNA的Southern blot分析显示，HDC表达细胞系的启动子区域被选择性地非甲基化。此外，在瞬时表达分析中，HDC启动子的甲基化降低了荧光素酶报告活性，这表明启动子区域的甲基化对HDC基因的表达具有重要的功能。这些结果表明，DNA甲基化的改变是调控HDC基因细胞特异性表达的机制之一。我们证明了HDC启动子的染色体构型和甲基化对肥大细胞和嗜碱性粒细胞特异性表达很重要。阐明细胞分化和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去甲基化机制本身。我们目前的努力集中在这些方面。

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.