| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1993: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
One of the major unanswered questions in the regulation of erythropoiesis is the mechanism by which hypoxia triggers increased production of erythropoietin (Epo). We have shown that the human hepatoma cell lines Hep3B and HepG_2 produce high levels of Epo mRNA and protein following exposure to hypoxia. Using this culture system, we have shown the evidence that the oxygen sensor is a heme protein and that ligand binding to this heme protein influences Epo production and secretion. Furthermore, we have shown that the presence of promoter and enhancer elements within the 5' flanking region, the first intron and the 3' flanking region of the human Epo gene that are appropriately responsive to hypoxia. However, the effects of these regions were less than expected. This result suggests that these candidate regulatory regions include both positive and negative regulatory elements. In order to investigate positive and negative regulatory elements more in detail, synthetic oligonucleotides are
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used to control Epo transcription by means of an antigene strategy. We demonstrated that CACCC motifs at -60 of the human Epo gene are a positive regulatory element, whereas the GATA sequence at -30 is a negative regulatory element. We also found the presence of transcription factors which specifically bind to the CACCC elements and GATA element, respectively, in the nuclear extract from Hep3B cells. We analyzed the expression of three human GATA (hGATA) factors (GATA-1, -2, and -3) in Hep3B and HepG_2 by RNA blot hybridization analysis. The result clearly showed that both cell lines express hGATA-2 mRNA.The expression of hGATA-2 protein was also identified in an immunohistochemical staining using specific anti-GATA-2 antibody, while the expression of GATA-1 and GATA-3 proteins could not be detected by this analysis. We transfected expression construct of hGATA-2 into quail fibroblast QT6 cells, and found that hGATA-2 in nuclear extract from these cells specifically binds to the GATA element in the human Epo gene promoter. We then transfected the hGATA-2 expression plasmid into Hep3B cells, and examined the expression of Epo mRNA with competitive PCR method. The result showed that the hGATA-2 transfection significantly decreased the expression level of Epo mRNA.These results suggest that GATA-2 binds to the GATA element of the human Epo gene in Hep3B cells and regulate the expression of the Epo gene negatively. Less
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