Institut Pasteur, Université Paris Cité, CNRS UMR 3691, Imaging and Modeling Unit, F-75015 Paris, France.
IGH, University of Montpellier, CNRS UMR 9002, Montpellier, France.
Nucleic Acids Res. 2023 Apr 11;51(6):2614-2632. doi: 10.1093/nar/gkad034.
Genome-wide chromosome conformation capture (Hi-C) has revealed the organization of chromatin into topologically associating domains (TADs) and loops, which are thought to help regulate genome functions. TADs and loops are understood as the result of DNA extrusion mediated by the cohesin complex. However, despite recent efforts, direct visualization and quantification of this process in single cells remains an open challenge. Here, we use polymer simulations and dedicated analysis methods to explore if, and under which conditions, DNA loop extrusion can be detected and quantitatively characterized by imaging pairs of fluorescently labeled loci located near loop or TAD anchors in fixed or living cells. We find that under realistic conditions, extrusion can be detected and the frequency of loop formation can be quantified from fixed cell images alone, while the lifetime of loops and the speed of extrusion can be estimated from dynamic live-cell data. Our delineation of appropriate imaging conditions and the proposed analytical methods lay the groundwork for a systematic quantitative characterization of loop extrusion in fixed or living cells.
全基因组染色体构象捕获（Hi-C）揭示了染色质组织成拓扑关联域（TAD）和环，这些结构被认为有助于调节基因组功能。TAD 和环被理解为黏合蛋白复合物介导的 DNA 挤压的结果。然而，尽管最近进行了努力，在单细胞中直接可视化和定量此过程仍然是一个未解决的挑战。在这里，我们使用聚合物模拟和专用的分析方法来探索是否以及在何种条件下可以通过对固定或活细胞中位于环或 TAD 锚点附近的荧光标记基因座对进行成像来检测和定量表征 DNA 环挤压。我们发现，在现实条件下，可以从固定细胞图像中检测到挤出，并可以定量确定环的形成频率，而环的寿命和挤出速度可以从动态活细胞数据中估计。我们对适当的成像条件的描述和提出的分析方法为在固定或活细胞中对环挤出进行系统的定量表征奠定了基础。
