Structure and function of the zeta-globin upstream regulatory element.
Structure and function of the zeta-globin upstream regulatory element.
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zeta-珠蛋白上游调控元件的结构和功能。
DOI：
10.1093/nar/24.24.4978
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发表时间：
1996
影响因子：
14.9
通讯作者：
Yang,WQ
中科院分区：
文献类型：
--
作者：
Sabath,DE;Koehler,KM;Yang,WQ
人β-珠蛋白启动子在5′侧翼区含有一个强的正调控元件，称为β-珠蛋白上游调控元件（URE）。在这项研究中，我们定义了URE功能所需的最小序列，并表征了相关的蛋白质-DNA相互作用。缺失实验表明，URE跨越位于转录起始位点5′端220 - 279 bp之间的60 bp区域。该区域的进一步细分表明存在多重作用序列。电泳迁移率变动分析表明，红系转录因子加塔-1结合的位点在-230，和Sp1和一个未知的因子结合的CCACC位点在-240。未鉴定的CCACC因子与另外两种CCACC因子EKLF和BKLF/TEF-2不同。第三种复合物含有一种新的DNA结合活性，与−269至−255区域的一个位点相互作用，称为URE结合因子（URE-BF）。该因子存在于表达β-珠蛋白的K562细胞中，但不存在于不表达β-珠蛋白的人红细胞系OCIM 1细胞系中。URE-BF似乎与加塔因子相互作用，因为含有加塔位点的未标记寡核苷酸的存在可以阻止URE-BF复合物的形成。最后，增加从-230加塔位点到两个上游位点的距离导致β-珠蛋白启动子活性的逐渐降低。没有迹象表明要求加塔-1与其他上游因子位于DNA螺旋的同一侧。这些结果表明β-珠蛋白启动子功能高度依赖于至少三种不同因子结合的60 bp区域。这些因素中的两个可能代表DNA结合蛋白，以前没有确定为重要的珠蛋白基因表达的调节。很可能这些因素在物理上相互作用，形成一个功能性调节单位。
The human ζ-globin promoter contains a strong positive regulatory element in the 5′ flanking region, designated the ζ-globin upstream regulatory element (URE). In this study, we define the minimal sequences required for URE function and characterize the associated protein-DNA interactions. Deletion experiments show that the URE spans a 60 bp region located between 220 and 279 bp 5′ to the transcription start site. Further subdivision of this region shows that multiplecisacting sequences are present. Electrophoretic mobility shift assays demonstrate that the erythroid transcription factor GATA-1 binds a site at −230, and Sp1 and an unidentified factor bind a CCACC site at −240. The unidentified CCACC factor is distinct from two other CCACC factors, EKLF and BKLF/TEF-2. A third complex contains a novel DNA-binding activity that interacts with a site in the −269 to −255 region, designated URE binding factor (URE-BF). This factor is present in K562 cells that express ζ-globin, but is absent in the OCIM1 cell line, a human erythroid cell line that does not express ζ-globin. URE-BF appears to interact with a GATA factor, since formation of the URE-BF complex can be prevented by the presence of unlabeled oligonucleotides containing GATA sites. Finally, increasing the distance from the −230 GATA site to the two upstream sites causes a progressive decrease in ζ-globin promoter activity. There is no indication of a requirement for GATA-1 to be on the same side of the DNA helix as the other upstream factors. These results show that ζ-globin promoter function is highly dependent on a 60 bp region to which at least three different factors bind. Two of these factors may represent DNA-binding proteins not previously identified as important for regulation of globin gene expression. It is likely that these factors interact physically to create a functional regulatory unit.