The 3′ Region of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to Its Core and Is Required for Full Insulator Activity
The 3′ Region of the Chicken Hypersensitive Site-4 Insulator Has Properties Similar to Its Core and Is Required for Full Insulator Activity
复制标题
DOI：
10.1371/journal.pone.0006995
复制
发表时间：
2009-09-10
期刊：
影响因子：
3.7
通讯作者：
Malik, Punam
中科院分区：
文献类型：
--
作者：
Arumugam, Paritha I.;Urbinati, Fabrizia;Malik, Punam
染色质绝缘子分离活性转录结构域并阻断异染色质在基因组中的传播。对鸡超敏位点4（cHS 4）元件（原型绝缘子）的研究已经在cHS 4的近端250 bp处鉴定出CTCF和USF-1/2基序，称为“核心”，其提供增强子阻断活性并减少位置效应。然而，单独的核心不能有效地隔离病毒载体。全长cHS 4具有出色的绝缘性能，但其大尺寸严重影响了载体滴度。我们对cHS 4侧翼慢病毒载体进行了结构-功能分析，并分析了造血干细胞克隆后代中的转基因表达以及cHS 4和转基因启动子中的表观遗传变化。我们发现，核心只减少表达的克隆杂色。独特的绝缘子活性存在于远端400 bp cHS 4序列中，当与核心结合时，恢复了完整的绝缘子活性，并在转基因启动子和绝缘子上打开染色质标记。这些数据巩固了已知的绝缘活性的典型的59核心与一个新的39 400 bp的元素与性能类似的核心。总之，它们具有优异的绝缘性能和病毒滴度。我们的研究结果对于理解绝缘子功能的分子基础和基因治疗载体的设计具有重要意义。
Chromatin insulators separate active transcriptional domains and block the spread of heterochromatin in the genome. Studies on the chicken hypersensitive site-4 (cHS4) element, a prototypic insulator, have identified CTCF and USF-1/2 motifs in the proximal 250 bp of cHS4, termed the "core'', which provide enhancer blocking activity and reduce position effects. However, the core alone does not insulate viral vectors effectively. The full-length cHS4 has excellent insulating properties, but its large size severely compromises vector titers. We performed a structure-function analysis of cHS4 flanking lentivirus-vectors and analyzed transgene expression in the clonal progeny of hematopoietic stem cells and epigenetic changes in cHS4 and the transgene promoter. We found that the core only reduced the clonal variegation in expression. Unique insulator activity resided in the distal 400 bp cHS4 sequences, which when combined with the core, restored full insulator activity and open chromatin marks over the transgene promoter and the insulator. These data consolidate the known insulating activity of the canonical 59 core with a novel 39 400 bp element with properties similar to the core. Together, they have excellent insulating properties and viral titers. Our data have important implications in understanding the molecular basis of insulator function and design of gene therapy vectors.