DNA-protein interactions in the proximal zeta-globin promoter: identification of novel CCACCC- and CCAAT-binding proteins.
DNA-protein interactions in the proximal zeta-globin promoter: identification of novel CCACCC- and CCAAT-binding proteins.
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DOI：
10.1006/bcmd.1998.0185
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发表时间：
1998-06
期刊：
影响因子：
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通讯作者：
D. Sabath;Karen M. Koehler;Wei Qing Yang;Van N. Phan;J. Wilson
中科院分区：
文献类型：
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作者：
D. Sabath;Karen M. Koehler;Wei Qing Yang;Van N. Phan;J. Wilson
人类zeta-珠蛋白基因以组织和发育特异性模式表达，其表达仅限于胚胎卵黄囊血岛的原始红系细胞。转基因小鼠的研究表明，近端zeta-珠蛋白启动子包含的序列，有助于表达的阶段特异性，但没有系统的功能研究近端zeta-珠蛋白启动子中的顺式元件已被报道。在本文中，我们表明，在zeta珠蛋白启动子的保守序列元件的数量是重要的瞬时转染K562红白血病细胞，组成型表达zeta珠蛋白的启动子活性。这些包括位于-105的加塔站点、位于-93的CCACC站点、位于-65的CCAAT盒和位于-29的TATA盒。在该系统中，-78处高度保守的CCTCC序列对于ζ-珠蛋白启动子活性并不重要。这些位点的突变不会导致OCIM 1细胞（一种不表达ζ-珠蛋白的红系细胞）启动子活性增加，这表明这些位点都不是发育沉默子。电泳迁移率变动分析表明，K562和OCIM 1核提取物含有与-105加塔，-65 CCAAT和-29 TATA位点相互作用的DNA结合活性。此外，K562细胞而不是OCIM 1细胞具有结合-93 CCACC位点的活性。加塔-1与加塔位点相互作用。K562 CCACC结合蛋白不同于Sp1、Sp2、Sp3、Sp 4、EKLF和BKLF。K562和OCIM 1核提取物中存在特异性-65 CCAAT结合活性，其不同于其他CCAAT结合蛋白，包括CBF/NF-Y、C/EBP、NF-1和CP 2。因此，我们已经确定了两个新的因素，可能有助于组织或发育阶段特异性表达zeta珠蛋白。
The human zeta-globin gene is expressed in a tissue- and developmental-specific pattern, with expression confined to primitive erythroid cells of the embryonic yolk sac blood islands. Transgenic mouse studies have shown that the proximal zeta-globin promoter contains sequences that contribute to the stage-specificity of expression, but no systematic functional studies of the cis elements in the proximal zeta-globin promoter have been reported. In this paper, we show that a number of conserved sequence elements in the zeta-globin promoter are important for promoter activity in transiently transfected K562 erythroleukemia cells, which constitutively express zeta-globin. These include a GATA site at -105, a CCACC site at -93, a CCAAT box at -65, and a TATA box at -29. A highly conserved CCTCC sequence at -78 is not important for zeta-globin promoter activity in this system. Mutations at these sites do not result in increased promoter activity in OCIM1 cells, an erythroid line that does not express zeta-globin, suggesting none of these sites is a developmental silencer. Electrophoretic mobility shift assays show that K562 and OCIM1 nuclear extracts contain DNA-binding activities that interact with the -105 GATA, -65 CCAAT, and -29 TATA sites. In addition K562 cells, but not OCIM1 cells, have an activity that binds the -93 CCACC site. GATA-1 interacts with the GATA site. The K562 CCACC-binding protein is distinct from Sp1, Sp2, Sp3, Sp4, EKLF, and BKLF. A specific -65 CCAAT-binding activity is present in K562 and OCIM1 nuclear extracts that is distinct from other CCAAT-binding proteins including CBF/NF-Y, C/EBP, NF-1, and CP2. Thus, we have identified two novel factors that may contribute to the tissue or developmental stage-specific expression of zeta-globin.