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Analysis of transcriptional regulation of the human ABO genes during differentiation of erythroid lineage

红系分化过程中人类 ABO 基因的转录调控分析

基本信息

批准号：
13670418
负责人：
KOMINATO Yoshihiko
金额：
$ 2.37万
依托单位：
TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670418/
关键词：
the human ABO blood group genes transcription promoter assay alternative promoter usage DNA methylation LINE ABO antigens Alu ABO式血液型遺伝子発現転写制御 CpGアイランドプロモーター細胞分化

项目摘要

We have studied the expression of the human histo-blood group ABO genes during erythroid differentiation using the ex vivo culture of AC133^-CD34^+ cells obtained from peripheral blood. The AC133^-CD34^+ cells are rich in erythroid-committed progenitors, and more than 90 % of the colonies produced from those cells are pure erythroid colonies. The AC133^-CD34^+ cells were primarily cultured in serum-free medium supplemented with TPO, FL and SCF for 7 days, followed by the secondary culture with the addition of EPO for the next 7 days. The ABO genes were expressed in the cells at day 7. The 5'-RACE analysis of RNA from those cells has revealed a novel transcription start site, which appeared to mark an alternative starting exon (1a) comprising 27 base pairs at the 5' end of a CpG island which contains a constitutive promoter of exon 1 in the ABO genes. The results from the RT-PCR specific to exon 1a indicated that the cells of both erythroid and epithelial lineages utilize this exon as the transcription starting exon. The transient transfection experiments showed that the region just upstream of the transcription start site possesses the promoter activity in a cell type-specific manner, when placed 5' adjacent to the reporter luciferase gene. The results from bisulfite genomic sequencing and RT-PCR analysis indicated that hypermethylation of the distal promoter region correlated with the absence of the transcripts containing exon 1a, whereas hypermethylation in the interspersed repeats 5' adjacent to the distal promoter was commonly observed in all the cell lines examined. These results suggest that a functional alternative cell-type specific promoter is located between the hypermethylated region of repetitive elements such as Alu and LINE and the CpG island containing a constitutive promoter of exon 1 in the ABO genes.

我们使用从外周血中获得的AC 133 ^-CD 34 ^+细胞的离体培养物研究了红系分化过程中人类组织血型ABO基因的表达。AC 133 ^-CD 34 ^+细胞富含红系定向祖细胞，这些细胞产生的集落中超过90%是纯红系集落。将AC 133 ^-CD 34 ^+细胞在添加TPO、FL和SCF的无血清培养基中原代培养7天，然后在添加EPO的培养基中继代培养7天。ABO基因在第7天在细胞中表达。对来自这些细胞的RNA的5 '-RACE分析揭示了一个新的转录起始位点，该位点似乎标记了在CpG岛的5'端包含27个碱基对的替代起始外显子（1a），该CpG岛含有ABO基因中外显子1的组成型启动子。对外显子1a特异性的RT-PCR结果表明，红系和上皮细胞系的细胞都利用该外显子作为转录起始外显子。瞬时转染实验表明，转录起始位点上游的区域具有细胞类型特异性的启动子活性，当放置在报告荧光素酶基因的5'端附近时。亚硫酸氢盐基因组测序和RT-PCR分析的结果表明，远端启动子区域的高甲基化与含有外显子1a的转录本的缺失相关，而在所有检测的细胞系中通常观察到远端启动子5'附近的散布重复序列的高甲基化。这些结果表明，一个功能性的替代细胞类型特异性启动子位于之间的高甲基化区域的重复元件，如Alu和LINE和CpG岛含有组成型启动子的外显子1的ABO基因。