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The control of human histidine decarboxylase gene expression and elucidation of the regulatory mechanism of histamine synthesis

人组氨酸脱羧酶基因表达的控制及组胺合成调控机制的阐明

基本信息

批准号：
06670097
负责人：
OHTSU Hiroshi
金额：
$ 1.34万
依托单位：
TOHOKU UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Histamine is synthesized from histidine catalyzed with histidine decarboxylase (HDC). The expression of this HDC gene is restricted in mast cells and basophils in the hematopoietic cells. By RNA blot analysis it was shown that the HMC-1 which is a unique human mast cell line express HDC mRNA but K562 which is one of human erythroid cell lines does not. HMC-1 cells were shown to transcribe higher HDC mRNA than K562 cells by nuclear run-on assay. These results suggest the existence of cell lineage specific cis-acting elements for the regulation of the transcription of HDC gene. By transient transfection of the plasmids containing various deletion mutants of 5' flanking region inserted into just upstream of luciferase reporter gene into HMC-1 and K562 cells, it is suggested that the flagment between-153 bp and-52 bp is essential for the transcription. In this fragment there are several consensus binding elements for transcription factors. After preparing finer deletion mutants and transfection to descriminate the activity of those element, it was clarifiedthat GC box between-64 bp and-52 bp is important for basal transcription. We further assessed the binding activity of the nuclear protein of HMC 1 and K562 to synthetic oligonucleotide including this GC box sequence. It was proved that there are Sp1 protein in both HMC-1 and K562 which bind to this fragment but not to the GC box-mutated fragment. We further searched for tissue/cell specific enhancer and/or suppressor between-7 Kb from transcription initiation site to 3 Kb down stream of the last exon, but there are no candidates yet. It is inevitable to do further experiment to elucidate the tissue/cell specific cis-elements.

组胺是由组氨酸在组氨酸脱羧酶（HDC）的催化下合成的。该HDC基因的表达仅限于造血细胞中的肥大细胞和嗜碱性粒细胞。RNA印迹分析表明，人肥大细胞系HMC-1表达HDC mRNA，而人红系细胞系K562不表达HDC mRNA。细胞核连续实验显示HMC-1细胞比K562细胞转录更高的HDC mRNA。这些结果提示HDC基因转录调控存在细胞系特异性顺式作用元件。将荧光素酶报告基因上游5'侧翼区缺失突变体质粒瞬时转染HMC-1和K562细胞，结果表明-153 ~-52 bp的标记区是荧光素酶报告基因转录所必需的。在该片段中有几个转录因子的共有结合元件。通过制备更精细的缺失突变体和转染来描述这些元件的活性，明确了-64 bp和-52 bp之间的GC盒对于基础转录是重要的。我们进一步评估了HMC 1和K562的核蛋白与包括该GC盒序列的合成寡核苷酸的结合活性。证明HMC-1和K562细胞中均存在Sp1蛋白，Sp1蛋白与该片段结合，但不与GC盒突变片段结合。我们进一步寻找了转录起始位点-7Kb到最后一个外显子下游3 Kb之间的组织/细胞特异性增强子和/或抑制子，但还没有候选者。因此，进一步研究组织/细胞特异性顺式元件是不可避免的。