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3T3-L1分化过程中，GLUT4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)参与了GLUT4基因的调控。类似的机制也可能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质量的主要激素调节基因，在3T3-L1前脂肪细胞向脂肪细胞分化的过程中表达。为了确定DNA甲基化是否参与调节瘦素基因的表达，我们检测了3T3-L1分化过程中甲基化和甲基化敏感转录因子的水平。用瘦素启动子瞬时转染报告构建体显示，不转录瘦素基因的前脂肪细胞表现出足够的反式激活，这表明存在额外的调节机制。我们确定了启动子上的8个cpG位点，它们都是高度甲基化的(…超过92%)的前脂肪细胞。其中7个位点在分化过程中表现出不同程度的去甲基化，而位于.nt-54的位点仍保持甲基化。在凝胶迁移率改变分析中，当CpG位点甲基化时，从NT-115到NT-70的DNA片段与前脂肪细胞的核提取物产生了一条甲基化敏感带。西南分析确定了一个52 kDa的蛋白与甲基化的探针强烈结合，还鉴定了这个52 kDa的蛋白与一个55 kDa的蛋白相同，它与甲基化的GLUT4基因结合。启动子活性因CpG位点甲基化而降低，直到nT-115，但不到nT-70。这些结果提示，在3T3-L1细胞脂肪细胞分化过程中，特定CpG位点的甲基化和相同的甲基化敏感蛋白可能参与了GLUT4和Leptin基因的表达。我们现在正在尝试克隆这个甲基化敏感蛋白，并对获得的阳性克隆进行测序。我们将在未来的研究中对这一克隆进行分析。较少

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