Pandya K, Donze D, Townes T M
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Biol Chem. 2001 Mar 16;276(11):8239-43. doi: 10.1074/jbc.M008457200. Epub 2000 Nov 22.
Erythroid Kruppel-like Factor (EKLF) is an erythroid-specific transcription factor that plays a critical role in gamma- to beta-globin gene switching during development. To identify essential domains required for EKLF transactivation function, we cotransfected a human erythroleukemia cell line (K562) with a locus control region gamma/Luc-beta/Cat reporter and an EKLF expression vector. In this assay EKLF mediates a 500-fold induction of beta/CAT expression compared with controls. To map essential transactivation domains, progressive NH(2)-terminal and internal deletion mutants of EKLF were constructed. All EKLF mutants were expressed at wild-type levels, localized to the nucleus, and bound DNA. When mutant EKLF proteins were tested for beta/CAT activation, a novel transactivation domain was identified. This novel domain, encompassing amino acids (aa) 140-358, is sufficient for maximal beta/CAT activation. An 85-amino acid subdomain within this region (aa 140-225) is essential for its activity. Interestingly, this central transactivation subdomain is functionally redundant with the amino-terminal domain (aa 1-139). Thus, EKLF possesses at least two potent transactivation domains that appear to function in a redundant manner.
红系 Kruppel 样因子（EKLF）是一种红系特异性转录因子，在发育过程中的γ珠蛋白基因向β珠蛋白基因转换中起关键作用。为了确定 EKLF 反式激活功能所需的必需结构域，我们将一个基因座控制区γ/Luc-β/Cat 报告基因和一个 EKLF 表达载体共转染到人红白血病细胞系（K562）中。在该检测中，与对照相比，EKLF 介导β/CAT 表达诱导了 500 倍。为了绘制必需的反式激活结构域图谱，构建了 EKLF 的渐进性 NH(2) -末端和内部缺失突变体。所有 EKLF 突变体均以野生型水平表达，定位于细胞核，并与 DNA 结合。当测试突变型 EKLF 蛋白对β/CAT 的激活作用时，鉴定出一个新的反式激活结构域。这个新结构域，包含氨基酸（aa）140 - 358，足以实现最大程度的β/CAT 激活。该区域内一个 85 个氨基酸的亚结构域（aa 140 - 225）对其活性至关重要。有趣的是，这个中央反式激活亚结构域在功能上与氨基末端结构域（aa 1 - 139）冗余。因此，EKLF 拥有至少两个有效的反式激活结构域，它们似乎以冗余方式发挥作用。
