Transcription forms and remodels supercoiling domains unfolding large scale chromatin structures [Agilent ChIP-chip]

This study was designed to investigate DNA supercoiling across the human genome and to understand how supercoiling domains impact on higher levels of genome organisation. DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes. The binding of bTMP, as a reporter for DNA supercoiling, was investigated in RPE1 cells. Experiments were biological replicates

本研究旨在探究整个人类基因组范围内的DNA超螺旋（DNA supercoiling）现象，并解析超螺旋结构域如何影响基因组的高级组织形式。DNA超螺旋是DNA双链扭转的固有结果，对基因表达调控与DNA复制至关重要。然而，此前尚未明确人类细胞中基因组尺度下的DNA超螺旋状态。为绘制超螺旋图谱，本研究以生物素化三甲基补骨脂素（biotinylated-trimethylpsoralen，bTMP）作为DNA结构探针，证实人类基因组可被组织为超螺旋结构域。超螺旋结构域由RNA聚合酶与拓扑异构酶的活性参与形成并重塑，其侧翼区域存在GC-AT边界与CCCTC结合因子（CTCF）结合位点。欠旋结构域具有转录活性，富集拓扑异构酶I、‘开放’染色质纤维与脱氧核糖核酸酶I（DNase I）位点，但拓扑异构酶II的分布则较为匮乏。此外，DNA超螺旋还会影响染色质压缩的其他层级：欠旋结构域在细胞学层面呈现解凝状态，且存在拓扑约束，同时可通过短RNA的转录过程实现解压缩。本研究提出，超螺旋结构域可构建利于基因激活的拓扑微环境，这也为基因沿染色体成簇分布提供了进化层面的解释。本研究以bTMP作为DNA超螺旋的报告探针，在视网膜色素上皮细胞（RPE1）中探究了其结合情况，所有实验均设置了生物学重复。