2002 Fiscal Year Final Research Report Summary
Analysis of transcriptional regulation of the human ABO genes during differentiation of erythroid lineage
| Project/Area Number |
13670418
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Legal medicine
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| Research Institution | TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY |
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Principal Investigator |
KOMINATO Yoshihiko Toyama Medeical and Pharmaceutical University, Faculty of Medicine, Assistant Professor, 医学部, 講師 (30205512)
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| Co-Investigator(Kenkyū-buntansha) |
HATA Yukiko Toyama Medeical and Pharmaceutical University, Faculty of Medicine, Assistant, 医学部, 教務職員(研究職) (30311674)
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| Project Period (FY) |
2001 – 2002
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| Keywords | the human ABO blood group genes / transcription / promoter assay / alternative promoter usage / DNA methylation / LINE / ABO antigens / Alu |
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| Research Abstract |
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.
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