The regulation of the GLUT4 gene expression during the differentiation of 3T3-L1 cells by the transcriptional represser

我们最近发现，在3 T3-L1分化过程中，GLUT 4基因特定CpG位点的甲基化以及甲基化敏感的转录因子（55 kDa）有助于GLUT 4基因的调控。类似的机制也可以调节脂肪细胞分化过程中诱导的其他基因的表达。小鼠瘦素基因是食欲和脂肪细胞量的主要激素调节因子，在3 T3-L1前脂肪细胞向脂肪细胞分化期间表达。为了确定DNA甲基化是否参与调节瘦素基因表达，我们检测了3 T3-L1分化过程中甲基化和甲基化敏感性转录因子的水平。瞬时转染的报告结构与瘦素启动子显示，前脂肪细胞不转录的瘦素基因显示足够的转录，这表明存在一个额外的监管机制。我们在启动子中鉴定了8个CpG位点，最多到nt-161，它们都是高度甲基化的（ ...更多信息 > 92%）。这些位点中的7个在分化过程中表现出不同程度的去甲基化，而nt-54处的位点保持甲基化。在电泳迁移率变动分析中，当CpG位点被甲基化时，从nt-115到nt-70的DNA片段与来自前脂肪细胞的核提取物产生甲基化敏感条带。Southwestern分析鉴定了与甲基化探针强烈结合的52 kDa蛋白，并且还鉴定了该52 kDa蛋白与结合甲基化GLUT 4基因的55 kDa蛋白相同。启动子活性被CpG位点的甲基化降低，直至nt-115，但不直至nt-70。这些结果表明，特定的CpG位点的甲基化和相同的甲基化敏感蛋白可能有助于GLUT 4和瘦素基因表达在脂肪细胞分化过程中在3 T3-L1细胞。我们正在尝试克隆这种甲基化敏感蛋白，并对获得的阳性克隆进行测序。我们将在未来的研究中分析这个克隆。少

We recently showed that methylation of specific CpG sites of the GLUT4 gene, as well as a inethylation sensitive transcription factor (55kDa), contribute to GLUT4 gene regulation during 3T3-L1 differentiation. Similar mechanisms may also regulate the expression of other genes induced during adipocyte differentiation. The mouse leptin gene, a major hormonal regulator of appetite and fat cell mass, expresses during the differentiation of 3T3-L1 preadipocytes to adipocytes. To determine if DNA methylation is involved in regulating leptin gene expression, we examined the level of methylation and methylation-sensitive transcription factors during 3T3-L1 differentiation. Transient transfection of reporter constructs with the leptin promoter showed that preadipocytes that do not transcribe the leptin gene show enough transactivation, suggesting the presence of an additional regulatory mechanism. We identified eight CpG sites in the promoter up to nt -161, all of which were highly methylated ( … More >92 %) in preadipocytes. Seven of these sites showed varying degree of demethylation during differentiation, while the site at.nt -54 remained methylated. In electrophoretic mobility-shift assays, DNA fragments from nt -115 to nt -70 generated a methylation-sensitive band with nuclear extracts from preadipocytes when the CpG sites were methylated. Southwestern analysis identified a 52 kDa protein that binds strongly to the methylated probes, and also identified this 52 kDa protein is same as a 55 kDa protein that binds to the methylated GLUT4 gene. Promoter activity was reduced by methylation of the CpG sites up to nt -115, but not up to nt -70. These results suggest that methylation of specific CpG sites and the same methylation-sensitive protein may contribute to GLUT4 and leptin gene expression during adipocyte differentiation in 3T3-L1 cells. We are now trying the cloning this methylation-sensitive protein, and sequencing the obtained positive clone. We will analyze this clone in future study. Less