Context-dependent EKLF responsiveness defines the developmental specificity of the human epsilon-globin gene in erythroid cells of YAC transgenic mice.
Context-dependent EKLF responsiveness defines the developmental specificity of the human epsilon-globin gene in erythroid cells of YAC transgenic mice.
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背景依赖性 EKLF 反应性定义了 YAC 转基因小鼠红细胞中人ε珠蛋白基因的发育特异性。
DOI：
10.1101/gad.822500
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发表时间：
2000
影响因子：
10.5
通讯作者：
Engel,JD
中科院分区：
文献类型：
--
作者：
Tanimoto,K;Liu,Q;Grosveld,F;Bungert,J;Engel,JD
我们探索了转基因小鼠中表达的人β-珠蛋白YAC中确定性阶段β-珠蛋白转录失活的机制。我们专注于珠蛋白CAC和CAAT启动子基序，因为先前的实验室和临床研究表明这些元件在珠蛋白基因激活中起关键作用。将红系克吕佩尔样因子（EKLF）的高亲和力CAC结合位点置于β-珠蛋白启动子中与成人β-珠蛋白启动子中相应的位置，从而同时切除直接重复（DR）元件。这种突变导致EKLF-非依赖性β-珠蛋白转录在确定性红细胞生成。β-珠蛋白CAAT序列中的第二个4-bp取代，同时破坏了第二个DR元件，进一步增强了β-珠蛋白转录的异位定形红细胞激活，这令人惊讶地成为EKLF依赖性的.最后，我们研究了从胚胎或成人红系细胞制备的核提取物中结合这些元素在体外的因素，我们确定了一种新的DR结合蛋白（DRED），其性质与预期的最终阶段β-珠蛋白阻遏物一致。我们的结论是，在确定性红细胞生成过程中β-珠蛋白转录的抑制是通过抑制因子的结合介导的，该抑制因子阻止EKLF激活β-珠蛋白基因。
We explored the mechanism of definitive-stage ɛ-globin transcriptional inactivity within a human β-globin YAC expressed in transgenic mice. We focused on the globin CAC and CAAT promoter motifs, as previous laboratory and clinical studies indicated a pivotal role for these elements in globin gene activation. A high-affinity CAC-binding site for the erythroid krüppel-like factor (EKLF) was placed in the ɛ-globin promoter at a position corresponding to that in the adult β-globin promoter, thereby simultaneously ablating a direct repeat (DR) element. This mutation led to EKLF-independent ɛ-globin transcription during definitive erythropoiesis. A second 4-bp substitution in the ɛ-globin CAAT sequence, which simultaneously disrupts a second DR element, further enhanced ectopic definitive erythroid activation of ɛ-globin transcription, which surprisingly became EKLF dependent. We finally examined factors in nuclear extracts prepared from embryonic or adult erythroid cells that bound these elements in vitro, and we identified a novel DR-binding protein (DRED) whose properties are consistent with those expected for a definitive-stage ɛ-globin repressor. We conclude that the suppression of ɛ-globin transcription during definitive erythropoiesis is mediated by the binding of a repressor that prevents EKLF from activating the ɛ-globin gene.