Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, United States of America.
PLoS One. 2011;6(8):e23234. doi: 10.1371/journal.pone.0023234. Epub 2011 Aug 12.
To study transcriptional regulation by distant enhancers, we devised a system of easily modified reporter plasmids for integration into single-copy targeting cassettes in clones of HuH7, a human hepatocellular carcinoma. The plasmid constructs tested transcriptional function of a 35-kb region that contained the rat albumin gene and its upstream flanking region. Expression of integrants was analyzed in two orientations, and compared to transient expression of non-integrated plasmids. Enhancers were studied in their natural positions relative to the promoter and localized by deletion. All constructs were also analyzed by transient transfection assays. In addition to the known albumin gene enhancer (E1 at -10 kb), we demonstrated two new enhancers, E2 at -13, and E4 at +1.2 kb. All three enhancers functioned in both transient assays and integrated constructs. However, chromosomal integration demonstrated several differences from transient expression. For example, analysis of E2 showed that enhancer function within the chromosome required a larger gene region than in transient assays. Another conserved region, E3 at -0.7 kb, functioned as an enhancer in transient assays but inhibited the function of E1 and E2 when chromosomally integrated. The enhancers did not show additive or synergistic behavior,an effect consistent with competition for the promoter or inhibitory interactions among enhancers. Growth arrest by serum starvation strongly stimulated the function of some integrated enhancers, consistent with the expected disruption of enhancer-promoter looping during the cell cycle.
为了研究远距离增强子的转录调控，我们设计了一种易于修饰的报告质粒系统，用于整合到 HuH7 克隆的单拷贝靶向盒中，HuH7 是一种人肝癌细胞系。这些质粒构建体测试了包含大鼠白蛋白基因及其上游侧翼区域的 35kb 区域的转录功能。整合子的表达以两种方向进行分析，并与非整合质粒的瞬时表达进行比较。通过删除来研究增强子在相对于启动子的天然位置及其定位。所有构建体也通过瞬时转染测定进行了分析。除了已知的白蛋白基因增强子（E1 在 -10kb）之外，我们还证明了两个新的增强子，E2 在 -13 和 E4 在 +1.2kb。这三个增强子在瞬时测定和整合构建体中均具有功能。然而，染色体整合与瞬时表达显示出几个差异。例如，E2 的分析表明，增强子在染色体中的功能需要比瞬时测定更大的基因区域。另一个保守区域 E3 在 -0.7kb，在瞬时测定中作为增强子发挥作用，但在染色体整合时抑制 E1 和 E2 的功能。增强子没有表现出加性或协同作用，这种效应与启动子的竞争或增强子之间的抑制相互作用一致。血清饥饿引起的生长停滞强烈刺激了一些整合增强子的功能，这与细胞周期中增强子-启动子环的预期中断一致。
