Multiple Regulatory Elements in the 5′-Flanking Sequence of the Human ε-Globin Gene*
Multiple Regulatory Elements in the 5′-Flanking Sequence of the Human ε-Globin Gene*
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人类 ε-珠蛋白基因 5′-侧翼序列中的多个调控元件*
DOI：
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发表时间：
1998
影响因子：
4.8
通讯作者：
A. Schechter
中科院分区：
文献类型：
--
作者：
Jin Li;C. Noguchi;W. Miller;R. Hardison;A. Schechter
我们之前报道过，在转染实验的基础上，人胚胎（ε）珠蛋白基因的5′侧翼区域存在一个沉默元件，位于帽位点5′-270个碱基对处，该元件对该基因提供负调控。转基因小鼠的实验表明了这种ε-珠蛋白沉默子的生理重要性，但也表明ε-珠蛋白基因表达的下调可能涉及ε-珠蛋白基因侧翼的其他负面元件。现在，我们使用具有缺失突变的报告基因构建体和瞬时转染测定，将 ε-珠蛋白基因调控的分析扩展到包括跨度高达 6 KB 对 5' 的侧翼区域至基因座控制区域。我们已经鉴定并表征了其他强负调控区域，以及几个影响转录激活的正区域。 -3 kilobase对的负调控区（εNRA-I和εNRA-II），两侧是正控制元件，对红系K562和非红系HeLa细胞中的ε-球蛋白启动子有很强的影响，并且包含几个转录因子GATA-1的结合位点，如DNA-蛋白质结合测定所证明的。 εNRA-II 内的 GATA-1 位点是阴性对照所直接需要的。 εNRA-I 和 εNRA-II 在异源启动子上均具有活性，因此似乎充当转录沉默子。位于-1.7 KB 对的另一个阴性控制区 (εNRB) 不表现出一般的沉默子活性，因为当与 ε-珠蛋白最小启动子结合使用时，εNRB 不会影响转录活性。 εNRB 的负面影响是红系特异性的，但不是阶段特异性的，因为它可以抑制 K562 红系细胞以及成人红系细胞原代培养物中的转录活性。与其他灵长类和其他哺乳动物物种的系统发育 DNA 序列比较显示，ε-珠蛋白基因的侧翼序列同源性程度不同寻常，包括在这些功能和 DNA-蛋白质结合分析中确定的几个区域，这为它们的潜在重要性提供了替代证据。我们认为，随着发育的进展，ε-珠蛋白基因表达的下调涉及这些负调控元件、εNRA-I/εNRA-II、εNRB、ε-珠蛋白沉默子以及ε-珠蛋白基因5'-侧翼区域中可能的其他负向和正向元件的复杂、协作的相互作用。
We have previously reported, on the basis of transfection experiments, the existence of a silencer element in the 5′-flanking region of the human embryonic (ε) globin gene, located at −270 base pairs 5′ to the cap site, which provides negative regulation for this gene. Experiments in transgenic mice suggest the physiological importance of this ε-globin silencer, but also suggest that down-regulation of ε-globin gene expression may involve other negative elements flanking the ε-globin gene. We have now extended the analysis of ε-globin gene regulation to include the flanking region spanning up to 6 kilobase pairs 5′ to the locus control region using reporter gene constructs with deletion mutations and transient transfection assays. We have identified and characterized other strong negative regulatory regions, as well as several positive regions that affect transcription activation. The negative regulatory regions at −3 kilobase pairs (εNRA-I and εNRA-II), flanked by a positive control element, has a strong effect on the ε-globin promoter both in erythroid K562 and nonerythroid HeLa cells and contains several binding sites for transcription factor GATA-1, as evidenced from DNA-protein binding assays. The GATA-1 sites within εNRA-II are directly needed for negative control. Both εNRA-I and εNRA-II are active on a heterologous promoter and hence appear to act as transcription silencers. Another negative control region located at −1.7 kilobase pairs (εNRB) does not exhibit general silencer activity as εNRB does not affect transcription activity when used in conjunction with an ε-globin minimal promoter. The negative effect of εNRB is erythroid specific, but not stage-specific as it can repress transcription activity in both K562 erythroid cells as well as in primary cultures of adult erythroid cells. Phylogenetic DNA sequence comparisons with other primate and other mammalian species show unusual degree of flanking sequence homology for the ε-globin gene, including in several of the regions identified in these functional and DNA-protein binding analyses, providing alternate evidence for their potential importance. We suggest that the down-regulation of ε-globin gene expression as development progresses involves complex, cooperative interactions of these negative regulatory elements, εNRA-I/εNRA-II, εNRB, the ε-globin silencer and probably other negative and positive elements in the 5′-flanking region of the ε-globin gene.
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DOI：
10.1006/geno.1996.4458
发表时间：
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期刊：
Genomics.
影响因子：
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作者：
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DOI：
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期刊：
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影响因子：
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DOI：
10.3109/10425179409010191
发表时间：
1994
期刊：
DNA sequence : the journal of DNA sequencing and mapping
影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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DOI：
10.1073/pnas.86.18.7033
发表时间：
1989
影响因子：
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