| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
DNA methylation is also an important factor in regulatory gene expression during growth and development (1-6). In general, DNA methylation is associated with the inhibition of gene expression. We, already reported that both the binding of the transcription factor GABP (GA binding protein) to and the activation of the thyrotropin receptor (TSHR) gene promoter were prevented by methylation of CpG sites at -93bp (base pairs) and -85bp in 5'-flanking region of the gene. These CpG sites were highly methylated in FRT cells and completely demethylated in FRTL-5 cells, consistent with TSHR gene expression in the latter, but not in the former (7). These results suggested that methylation of specific CpG sites and the presence of methylation-sensitive transcription factors contribute to gene regulation. In the present study, we examined the mechanism underlying insulin gene at the point of epigenetics, the level of DNA methylation.
At first, to determine the methylation status of CpG sites both i
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n RIN cells (βcell) and HepG2 cells (non βcell), the insulin gene promoter was amplified and sequenced according to the method of Frommer et al.(8). We found that CpG sites were highly demethylated in RIN cells. In contrast, CpG sites were highly methylated in HepG2 cells. Next, we examined the effect of histonedeacetylase inhibitor (Depsipeptide) or 5-Azacytidine. Treatment with the HDAC inhibitor or 5-Azacytidine could not lead to an increase in expression of insulin gene in AR42J cells, BRL cells and HepG2 cells using RT-PCR and immunohistochemistry. Then, PDX-1 gene was stable-transfected into HepG2 cells or BRL cells. Treatment with both the HDAC inhibitor and 5-Azacytidine could lead to a little increase in expression of insulin gene in these stable transfected HepG2 cells and BRL using RT-PCR. However the expression of insulin gene was not detected using Northern blot analysis and immunohistochemistry. These findings suggest that epigenetics may contribute to the expression of insulin gene, but it looks difficult to express insulin gene in non-βcells using PDX-1, the HDAC inhibitor and 5-Azacytidine.
On the other hand, we already found that GABP bound to the human insulin gene promoter. In this study, we also found that GABP was located only in Langerhans island using immunohistochemistry. This localization may suggest the importance of GABP to the expression of insulin gene. In future study, we think it will be interesting to examine the epigenetics of islet specific gene like insulin gene during the differentiation. Less
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