DNA topology and alternative DNA structures are implicated in regulating diverse biological processes. Although biomechanical properties of these structures have been studied extensively in vitro, characterization in vivo, particularly in multicellular organisms, is limited. We devised new methods to map DNA supercoiling and single-stranded DNA in Caenorhabditis elegans embryos and diapause larvae. To map supercoiling, we quantified the incorporation of biotinylated psoralen into DNA using high-throughput sequencing. To map single-stranded DNA, we combined permanganate treatment with genome-wide sequencing of induced double-stranded breaks. We found high levels of negative supercoiling at transcription start sites (TSSs) in embryos. GC-rich regions flanked by a sharp GC-to-AT transition delineate boundaries of supercoil propagation. In contrast to TSSs in embryos, TSSs in diapause larvae showed dramatic reductions in negative supercoiling without concomitant attenuation of transcription, suggesting developmental-stage-specific regulation. To assess whether alternative DNA structures control chromosome architecture and gene expression, we examined DNA supercoiling in the context of X-Chromosome dosage compensation. We showed that the condensin dosage compensation complex creates negative supercoils locally at its highest-occupancy binding sites but found no evidence for large-scale supercoiling domains along X Chromosomes. In contrast to transcription-coupled negative supercoiling, single-strandedness, which is most pronounced at transcript end sites, is dependent on high AT content and symmetrically positioned nucleosomes. We propose that sharp transitions in sequence composition at functional genomic elements constitute a common regulatory code and that DNA structure and propagation of torsional stress at regulatory elements are critical parameters in shaping important developmental events.

中文翻译：

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全基因组分析揭示了 DNA 序列组成、转录活性和核小体定位在驱动线虫 DNA 超螺旋和螺旋不稳定中的功能相互作用

DNA 拓扑和替代 DNA 结构涉及多种生物过程的调节。尽管这些结构的生物力学特性已在体外进行了广泛的研究，但体内的表征，特别是在多细胞生物中，是有限的。我们设计了新方法来绘制秀丽隐杆线虫胚胎和滞育幼虫中 DNA 超螺旋和单链 DNA 的图谱。为了绘制超螺旋图，我们使用高通量测序对生物素化补骨脂素与 DNA 的掺入进行了定量。为了绘制单链 DNA 图谱，我们将高锰酸盐处理与诱导双链断裂的全基因组测序相结合。我们发现胚胎转录起始位点（TSS）处存在高水平的负超螺旋。GC 丰富的区域两侧是 GC 到 AT 的急剧转变，描绘了超螺旋传播的边界。与胚胎中的 TSS 相比，滞育幼虫中的 TSS 显示负超螺旋显着减少，但没有伴随转录减弱，这表明发育阶段特异性调节。为了评估替代 DNA 结构是否控制染色体结构和基因表达，我们在 X 染色体剂量补偿的背景下检查了 DNA 超螺旋。我们表明，凝缩蛋白剂量补偿复合物在其最高占有率结合位点局部产生负超螺旋，但没有发现沿 X 染色体存在大规模超螺旋结构域的证据。与转录偶联的负超螺旋相反，单链在转录末端位点最为明显，它依赖于高 AT 含量和对称定位的核小体。我们提出，功能基因组元件的序列组成的急剧转变构成了共同的调控密码，并且调控元件的DNA结构和扭转应力的传播是塑造重要发育事件的关键参数。