Loyd Melanie R, Okamoto Yasuhiro, Randall Mindy S, Ney Paul A
Department of Biochemistry, Rm 4064, Thomas Tower, St Jude Children's Research Hospital, 332 N Lauderdale St, Memphis, TN 38105-2794, USA.
Blood. 2003 Dec 1;102(12):4223-8. doi: 10.1182/blood-2003-02-0574. Epub 2003 Aug 14.
High-level alpha-globin expression depends on cis-acting regulatory sequences located far upstream of the alpha-globin cluster. Sequences that contain the alpha-globin positive regulatory element (PRE) activate alpha-globin expression in transgenic mice. The alpha-globin PRE contains a pair of composite binding sites for the transcription factors activating protein 1 and nuclear factor erythroid 2 (AP1/NFE2). To determine the role of these binding sites in alpha-globin gene transcription, we mutated the AP1/NFE2 sites in the alpha-globin PRE in mice. We replaced the AP1/NFE2 sites with a neomycin resistance gene (neo) that is flanked by LoxP sites (floxed). Mice with this mutation exhibited increased embryonic death and alpha-thalassemia intermedia. Next, we removed the neo gene by Cre-mediated recombination, leaving a single LoxP site in place of the AP1/NFE2 sites. These mice were phenotypically normal. However, alpha-globin expression, measured by allele-specific RNA polymerase chain reaction (PCR), was decreased 25%. We examined the role of the hematopoietic-restricted transcription factor p45Nfe2 in activating expression through these sites and found that it is not required. Thus, we have demonstrated that AP1/NFE2 binding sites in the murine alpha-globin PRE contribute to long-range alpha-globin gene activation. The proteins that mediate this effect remain to be determined.
高水平的α-珠蛋白表达依赖于位于α-珠蛋白基因簇上游远处的顺式作用调控序列。含有α-珠蛋白正调控元件（PRE）的序列可在转基因小鼠中激活α-珠蛋白表达。α-珠蛋白PRE包含一对转录因子激活蛋白1和核因子红系2（AP1/NFE2）的复合结合位点。为了确定这些结合位点在α-珠蛋白基因转录中的作用，我们对小鼠α-珠蛋白PRE中的AP1/NFE2位点进行了突变。我们用一个两侧带有LoxP位点（floxed）的新霉素抗性基因（neo）取代了AP1/NFE2位点。具有这种突变的小鼠表现出胚胎死亡率增加和中间型α地中海贫血。接下来，我们通过Cre介导的重组去除了neo基因，在AP1/NFE2位点的位置留下了一个单一的LoxP位点。这些小鼠的表型正常。然而，通过等位基因特异性RNA聚合酶链反应（PCR）测量，α-珠蛋白表达下降了25%。我们研究了造血限制转录因子p45Nfe2通过这些位点激活表达的作用，发现它并非必需。因此，我们证明了小鼠α-珠蛋白PRE中的AP1/NFE2结合位点有助于α-珠蛋白基因的远程激活。介导这种效应的蛋白质仍有待确定。
