Elucidation of glucose responsive transcription factor complexes of L-type pyruvate kinase gene

1. 我们发现转录辅激活因子PGC1α与碳水化合物应答转录因子ChREBP在体外和细胞中相互作用，这两种蛋白都过表达。GST下拉分析显示，ChREBP的7-150和659-760氨基酸与PGC1α的650-797氨基酸之间存在相互作用。这种相互作用导致ChREBP与l型丙酮酸激酶基因启动子的结合受到抑制，导致该基因的转录活性降低2。另一个转录因子Hex在体外被观察到与ChREBP结合。报告基因试验表明，海克斯对海克斯活性没有影响。此外，在两个蛋白都过表达的细胞中，Hex不与ChREBP相互作用。利用GST ChREBP与酵母双杂交系统对ChREBP结合蛋白进行搜索。然而，到目前为止，还没有获得候选蛋白。我们克隆了一个大鼠ChREBP基因的启动子片段，确定了其序列和多个转录起始位点。基因报告分析显示-163和-32之间有几个转录调控区域。它们的区域分别是Sp结合位点、固醇调节位点和NF-Y结合位点，在肝细胞中与这些蛋白结合。Sp1与NF-Y、Sp1与SREBP1之间存在功能协同作用。

1. We found that transcriptional coactivator PGC1α interacted with carbohydrate responsive transcription factor ChREBP in vitro and also in cells that both proteins were overexpressed. GST pull-down assay revealed that this interaction occurred between amino acids 7-150 and 659-760 of ChREBP, and amino acids 650-797 of PGC1α. This interaction caused inhibition of ChREBP binding to L-type pyruvate kinase gene promoter that resulted in decrease in transcriptional activity of its gene.2. Another transcription factor Hex was observed to bind to ChREBP in vitro. However, Hex did not affect activity of Hex by reporter gene assay. Moreover, Hex did not interact with ChREBP in cells that both proteins were overexpressed.3. ChREBP binding proteins were searched using GST ChREBP and yeast two hybrid system. However, so far no candidate proteins were obtained.4. We cloned a fragment of rat ChREBP gene containing promoter region, determined its sequence and multiple transcription start sites. Gene reporter assays revealed several transcriptional regulatory regions between -163 and -32. Their regions were Sp binding site, sterol regulatory site and NF-Y binding site and were bound by these proteins in hepatocytes. Functional synergisms were found between Sp1 and NF-Y, and Sp1 and SREBP1.