Abruzzo L V, Reitman M
Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1770.
J Biol Chem. 1994 Dec 23;269(51):32565-71.
Upstream of the chicken beta-globin gene cluster are four DNase I-hypersensitive sites (HS1-4). Hypersensitive sites located upstream of the mammalian beta-globin clusters have enhancer activity and mediate position-independent gene expression. In contrast, a region inside the chicken cluster has enhancer activity and mediates position-independent expression. Here we investigate the function of the chicken upstream sites, which are different from the mammalian ones in sequence, number, and distance from the genes. Each was tested for its effect on reporter gene expression in transfected primary erythroid cells. HS2 and HS3 (4.4 and 6.4 kilobases upstream of rho-globin) showed significant enhancer activity while HS1 and HS4 (1.6 and 11 kilobases upstream of rho-globin) did not. A 237-base pair region of HS2 contained the sequences necessary for enhancer activity. Proteins from erythroid extracts bound HS2 in seven different regions; six of these sites were characterized. GATA-1 bound to four of the sites. Each site contributed to the enhancer activity of HS2. Two other sequences bound proteins that may be related to Sp1 and erythroid krüppel-like factor. Surprisingly, mutations in these elements, which disrupted protein binding, did not affect enhancer activity. Thus, the observed enhancer activity of HS2 is due to the four GATA sites. The existence of multiple GATA sites in both chicken HS2 and the mammalian upstream sites may be due to evolution from a common element with preservation of only very short sequences or to convergent evolution. These observations highlight the crucial role for GATA proteins in globin regulation.
鸡β-珠蛋白基因簇的上游有四个脱氧核糖核酸酶I高敏位点（HS1 - 4）。位于哺乳动物β-珠蛋白簇上游的高敏位点具有增强子活性，并介导位置独立的基因表达。相比之下，鸡基因簇内部的一个区域具有增强子活性并介导位置独立的表达。在此，我们研究鸡上游位点的功能，这些位点在序列、数量以及与基因的距离方面与哺乳动物的不同。对每个位点在转染的原代红细胞中对报告基因表达的影响进行了测试。HS2和HS3（位于ρ-珠蛋白上游4.4和6.4千碱基处）显示出显著的增强子活性，而HS1和HS4（位于ρ-珠蛋白上游1.6和11千碱基处）则没有。HS2的一个237碱基对区域包含增强子活性所需的序列。来自红细胞提取物的蛋白质在七个不同区域与HS2结合；其中六个位点得到了表征。GATA - 1与其中四个位点结合。每个位点都对HS2的增强子活性有贡献。另外两个序列结合了可能与Sp1和红细胞类krüppel样因子相关的蛋白质。令人惊讶的是，这些元件中的突变破坏了蛋白质结合，但并未影响增强子活性。因此，观察到的HS2的增强子活性归因于四个GATA位点。鸡HS2和哺乳动物上游位点中多个GATA位点的存在可能是由于从一个共同元件进化而来，仅保留了非常短的序列，或者是由于趋同进化。这些观察结果突出了GATA蛋白在珠蛋白调控中的关键作用。
