Nabirochkin S, Ossokina M, Heidmann T
Unité de Physicochimie et Pharmacologie des Macromolécules Biologiques, CNRS URA147, Institut Gustave Roussy, Villejuif, France.
J Biol Chem. 1998 Jan 23;273(4):2473-9. doi: 10.1074/jbc.273.4.2473.
The 5'-untranslated region of the Drosophila gypsy retrotransposon contains an "insulator," which disrupts the interactions between enhancer and promoter elements located apart. The insulator effect is dependent on the suppressor of Hairy-wing (su(Hw)) protein, which binds to reiterated sites within the 350 base pairs of the gypsy insulator, whereby it additionally acts as a transcriptional activator of gypsy. Here, we show that the 350-base pair su(Hw) binding site-containing gypsy insulator behaves in addition as a matrix/scaffold attachment region (MAR/SAR), involved in interactions with the nuclear matrix. In vitro experiments using nuclear matrices from Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, we show that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Finally, experiments within cells in culture demonstrate differential effects of the gypsy MAR sequence on reporter genes, namely no effect under conditions of transient transfection and a repressing effect in stable transformants, as expected for a sequence involved in chromatin structure and organization. A model for the gypsy insulator, which combines within a short "compacted" retroviral sequence three functional domains (insulator, enhancer, and the presently unraveled MAR/SAR) dispersed within more extended regions in other "boundary" domains, is discussed in relation to previously proposed models for insulation.
果蝇gypsy逆转录转座子的5'非翻译区包含一个“绝缘子”，它会破坏相距较远的增强子和启动子元件之间的相互作用。绝缘子效应依赖于毛翅抑制因子（su(Hw)）蛋白，该蛋白与gypsy绝缘子350个碱基对中的重复位点结合，此外它还作为gypsy的转录激活因子发挥作用。在此，我们表明包含350个碱基对的su(Hw)结合位点的gypsy绝缘子还具有基质/支架附着区域（MAR/SAR）的功能，参与与核基质的相互作用。使用果蝇、小鼠和人类细胞的核基质进行的体外实验表明，gypsy绝缘子具有特异性结合，而在逆转录转座子内的任何其他序列中均未观察到这种结合。此外，我们表明gypsy绝缘子与先前表征的MAR/SAR一样，与拓扑异构酶II和组蛋白H1特异性相互作用，即与核基质的两个重要组成部分相互作用。最后，细胞培养实验证明了gypsy MAR序列对报告基因的不同影响，即在瞬时转染条件下无影响，而在稳定转化体中具有抑制作用，这与参与染色质结构和组织的序列预期相符。我们结合先前提出的绝缘模型，讨论了一个关于gypsy绝缘子的模型，该模型在一个短的“紧凑”逆转录病毒序列中整合了三个功能域（绝缘子、增强子和目前已揭示的MAR/SAR），这些功能域分散在其他“边界”域的更广泛区域内。
