Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.
PLoS One. 2009 Sep 10;4(9):e6995. doi: 10.1371/journal.pone.0006995.
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 5' core with a novel 3' 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.
染色质绝缘子将活跃的转录结构域分隔开，并阻止基因组中异染色质的扩散。对鸡高敏感位点-4（cHS4）元件这一典型绝缘子的研究，确定了近端 250 个碱基对的 cHS4 中的 CTCF 和 USF-1/2 基序，称为“核心”，其具有增强子阻断活性并降低位置效应。然而，核心本身并不能有效地隔离病毒载体。全长 cHS4 具有优异的绝缘性能，但体积过大严重影响载体滴度。我们对 cHS4 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转基因的表达和 cHS4 及转基因启动子的表观遗传变化。我们发现核心仅降低了表达的克隆斑驳性。独特的绝缘子活性位于 cHS4 的远端 400 个碱基对序列中，当与核心结合时，恢复了转基因启动子和绝缘子上的完整绝缘子活性和开放染色质标记。这些数据将已知的规范 5'核心的绝缘活性与具有类似核心性质的新型 3'400 个碱基对元件结合起来。它们共同具有优异的绝缘性能和病毒滴度。我们的数据对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。
