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首页> 外文期刊>Molecular and Cellular Biology >The Chicken β-Globin 5′HS4 Boundary Element Blocks Enhancer-Mediated Suppression of Silencing
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The Chicken β-Globin 5′HS4 Boundary Element Blocks Enhancer-Mediated Suppression of Silencing

机译：鸡β-球蛋白5'HS4边界元件阻滞增强子介导的沉默抑制

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A constitutive DNase I-hypersensitive site 5′ of the chicken β-globin locus, termed 5′HS4 or cHS4, has been shown to insulate a promoter from the effect of an upstream enhancer and to reduce position effects on mini-white expression in Drosophilacells; on the basis of these findings, it has been designated a chromatin insulator. We have examined the effect of the cHS4 insulator in a system that assays both the level of gene expression and the rate of transcriptional silencing. Because transgenes flanked by insulator elements are shielded from position effects in Drosophilacells, we tested the ability of cHS4 to protect transgenes from position effects in mammalian cells. Flanking of an expression vector with the cHS4 insulator in a colony assay did not increase the number of G418-resistant colonies. Using lox/cre-based recombinase-mediated cassette exchange to control integration position, we studied the effect of cHS4 on the silencing of an integrated β-geo reporter at three genomic sites in K562 erythroleukemia cells. In this assay, enhancers act to suppress silencing but do not increase expression levels. While cHS4 blocked enhancement at each integration site, the strength of the effect varied from site to site. Furthermore, at some sites, cHS4 inhibited the enhancer effect either when placed between the enhancer and the promoter or when placed upstream of the enhancer. These results suggest that the activity of cHS4 is not dominant in all contexts and is unlikely to prevent silencing at all genomic integration sites.
机译：鸡β-珠蛋白基因座的DNase I组成型超敏位点5'（称为5'HS4或cHS4）已显示可隔离启动子与上游增强子的作用，并减少对迷你白蛋白的位置影响在果蝇细胞中的表达;基于这些发现，它被称为染色质绝缘子。我们已经检查了cHS4绝缘子在分析基因表达水平和转录沉默率的系统中的作用。由于侧翼带有绝缘子元件的转基因在果蝇细胞中不受位置效应的影响，因此我们测试了cHS4保护转基因免受哺乳动物细胞中位置效应影响的能力。在集落分析中将表达载体与cHS4绝缘子连接在一起并没有增加G418抗性集落的数量。使用基于lox / cre的重组酶介导的盒交换来控制整合位置，我们研究了cHS4对K562红白血病细胞中三个基因组位点处整合的β-geo报告基因沉默的影响。在该测定中，增强子起抑制沉默的作用，但不增加表达水平。尽管cHS4阻止了每个整合位点的增强，但效应的强度因地点而异。此外，在某些位点，当置于增强子和启动子之间或置于增强子的上游时，cHS4抑制了增强子的作用。这些结果表明，cHS4的活性并非在所有情况下都占主导地位，并且不太可能在所有基因组整合位点均阻止沉默。

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