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Tamiko Nakajima,
Rie Sano,
Yoichiro Takahashi, Rieko Kubo,
Keiko Takahashi,
Yoshihiko Kominato,
Junichi Tsukada,
Haruo Takeshita,
Toshihiro Yasuda,
Makoto Uchikawa,
Kazumi Isa,
Kenichi Ogasawara
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ABSTRACT: BACKGROUND: The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes. STUDY DESIGN AND METHODS: In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional Bm individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element. RESULTS: Sequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element. CONCLUSION: These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual.
Transfusion 04/2013; · 3.22 Impact Factor
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Rie Sano,
Tamiko Nakajima,
Keiko Takahashi, Rieko Kubo,
Yoshihiko Kominato,
Junichi Tsukada,
Haruo Takeshita,
Toshihiro Yasuda,
Kazuto Ito,
Takayuki Maruhashi,
Akihiko Yokohama,
Kazumi Isa,
Kenichi Ogasawara,
Makoto Uchikawa
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ABSTRACT: The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell-specific manner. Electrophoretic mobility-shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with B(m) phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the B(m) allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells.
Blood 03/2012; 119(22):5301-10. · 9.90 Impact Factor
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ABSTRACT: Gene expression is driven by promoters, enhancers, silencers, and other cis-regulatory elements upstream and downstream of the gene. Previous studies of the regulation of human ABO gene transcription have focused mainly on the 5' region, including the core promoter and the region proximal to it. However, as the involvement of the 3' flanking region in transcriptional regulation has not yet been examined, we focused on this issue. The 3' region approximately 2.2kb downstream of the ABO gene was PCR-amplified and inserted into a cloning vector, followed by sequence determination and preparation of luciferase reporter vectors. Transient transfections into KATOIII and K562 cells were performed using various reporter plasmids containing the 3' region. The 3' region of the ABO gene, which was characterized by a high degree of sequence repetition, was effectively cloned by a single-copy cloning method. Transfections in KATOIII and K562 cells showed that negative elements were demonstrable within the 3' region. These observations suggest that negative regulatory elements seem to be present in the 3' region of ABO in both epithelial and erythroid lineages. As we had observed a negative region just upstream of the ABO promoter, transcription from ABO could be negatively regulated by repressive regions just upstream of the promoter and downstream of the gene. Further studies of the enhancer will be required for elucidating the molecular basis of ABO gene expression.
Legal Medicine 01/2011; 13(1):22-9.
