Center for Quantitative Biology, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University.
Center for Quantitative Biology, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University;
J Vis Exp. 2021 Sep 8(175). doi: 10.3791/62974.
The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.
染色体易位产生的融合基因已在许多实体瘤或白血病中被发现。EWS-FLI1 属于 FET 家族融合癌蛋白，是尤文肉瘤中最常涉及的融合基因之一。这些 FET 家族融合蛋白通常在其 N 端具有 FET 蛋白的低复杂度结构域 (LCD)，在 C 端具有 DNA 结合结构域 (DBD)。由于 LCD-LCD 和 LCD-DBD 相互作用，EWS-FLI1 已被证实可在其靶结合部位形成生物分子凝聚物，这些凝聚物可募集 RNA 聚合酶 II 以增强基因转录。然而，这些凝聚物如何在其结合部位组装仍然不清楚。最近，一种单分子生物物理学方法——DNA Curtains 被应用于可视化 EWS-FLI1 凝聚物的组装过程。在这里，讨论了 DNA Curtains 在研究靶 DNA 上生物分子凝聚物组装的应用中的详细实验方案和数据分析方法。
