Wenger R H, Kvietikova I, Rolfs A, Camenisch G, Gassmann M
Institute of Physiology, University of Zürich-Irchel, Zürich, Switzerland.
Eur J Biochem. 1998 May 1;253(3):771-7. doi: 10.1046/j.1432-1327.1998.2530771.x.
The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3' to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3' enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3' HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3' HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.
缺氧诱导因子-1（HIF-1）是一种转录激活因子，参与诸如促红细胞生成素等氧调节基因的表达。暴露于低氧分压（缺氧）后，HIF-1与位于促红细胞生成素基因3'端的缺氧反应元件结合，并赋予促红细胞生成素表达的激活作用。促红细胞生成素3'增强子（CGTG）的保守核心HIF-1结合位点（HBS）包含一个已知为胞嘧啶甲基化潜在靶点的CpG二核苷酸。我们发现HBS的甲基化消除了HIF-1与DNA的结合以及缺氧报告基因的激活，这表明无甲基化的HBS对于HIF-1功能是必需的。通过使用甲基化敏感限制酶的基因组Southern印迹法评估了各种人类细胞系和小鼠器官中促红细胞生成素3'HBS的体内甲基化模式。在产生促红细胞生成素的细胞系Hep3B中，该位点基本上无甲基化，而在不同的HepG2亚系中发现促红细胞生成素蛋白表达与促红细胞生成素3'HBS甲基化程度之间存在直接相关性。然而，在不表达促红细胞生成素的人类细胞系和小鼠组织中该位点部分无甲基化这一发现表明，可能存在一种普遍的选择压力来保持该位点无甲基化，而与促红细胞生成素的表达无关。
