Probing the prevalence and molecular mechanisms of phase separation by fusion oncoproteins

摘要 由染色体易位形成的融合癌蛋白(FOS)是许多 尤其是对儿童具有抗药性的癌症。FOS的一个子集包含内在的无序 转录因子和其他转录共调节因子的区域(IDR)。最近的研究表明 一系列转录因子和共调控因子通过 在其序列中由IDR介导的相互作用形成转录缩合物。基因座的基因 与这些凝集物相关的是显著上调，暗示PS参与基因调控和细胞命运。 我们假设富含IDR的FOS子集通过PS形成异常的转录缩合物， 这会导致基因表达异常和肿瘤发生。此外，我假设这些FOS形成了 异常转录缩合物富含IDR序列，这是以前没有证明的 相分离，且这些序列特征决定了构象和材料属性 FOS和由此产生的冷凝物。了解FO来源的IDRs的生物物理性质是 重要的是，这样其他人就可以设计针对所产生的冷凝物的疗法。要识别该表单的FOS 在生理条件下，我根据患者的转录本选择了100个FOS进行转染 作为绿色荧光蛋白标记的蛋白质进入哺乳动物细胞。我将对这100个FOS进行筛选，以确定它们是否形成了斑点 使用半自动荧光共聚焦显微镜分析显示PS。确定IDR的步骤 负责PS的序列，I将从形成点状FOS的IDR亚克隆到大肠杆菌中 高效表达和纯化的表达载体。我会用浊度分析和共聚焦显微镜 评估这些IDR是否接受PS，以及在什么情况下。探索特定的分子间 相互作用和识别潜在的新基序，或驱动PS的“贴纸”，我将使用核磁共振 (核磁共振)光谱，以测量动力学和分子间接触的变化，在PS上的IDR。 此外，我将把序列/粘贴基序的组成与构象和材料属性联系起来 FO派生的IDR。我将使用核磁共振和荧光相关光谱来推导扩散系数， 关于凝析油内部和外部IDR的水动力半径的报告。我将进一步使用SAXS来 测量凝析油外的回转半径和凝析油内的分子间距。 最后，我将使用光漂白后的荧光恢复来评估流体动力学和材料状态 凝析油。这些互补的、多学科的研究将使我能够检验所讨论的假设。 以上内容促进了我们对PS在体外和细胞致癌行为中的作用的理解 融合癌蛋白。

ABSTRACT Fusion oncoproteins (FOs) that are formed by chromosomal translocations are the primary drivers of many treatment-resistant cancers that afflict children in particular. A subset of FOs contain intrinsically disordered regions (IDRs) from transcription factors and other transcriptional co-regulators. Recent work has demonstrated that a number of transcription factors and co-regulators undergo liquid-liquid phase separation (PS) through interactions mediated by IDRs within their sequences and form transcriptional condensates. Genes at loci associated with these condensates are significantly upregulated, implicating PS in gene regulation and cell fate. We hypothesize that a subset of FOs enriched in IDRs form aberrant transcriptional condensates through PS, which results in aberrant gene expression and oncogenesis. Furthermore, I hypothesize that these FOs that form aberrant transcriptional condensates are enriched in IDR sequences that were not previously demonstrated to phase separate, and that these sequence features determine both the conformational and material properties of the FOs and the resulting condensates. Understanding the biophysical properties of FO-derived IDRs is important so that others can design therapeutics targeting the resulting condensates. To identify FOs that form condensates under physiological conditions, I selected 100 FOs based on patient-derived transcripts to transfect into mammalian cells as eGFP-tagged proteins. I will screen these 100 FOs for the formation of puncta that are indicative of PS using a semi-automated fluorescence confocal microscopy assay. To determine the IDR sequences responsible for PS, I will subclone IDRs derived from puncta-forming FOs into Escherichia coli expression vectors for overexpression and purification. I will use turbidity assays and confocal microscopy to assess whether these IDRs undergo PS and under what conditions. To probe the specific intermolecular interactions and identify potentially novel motifs, or “stickers”, that drive PS, I will use nuclear magnetic resonance (NMR) spectroscopy to measure changes in dynamics and intermolecular contacts for the IDRs upon PS. Additionally, I will relate sequence/sticker motif composition with the conformational and material properties of FO-derived IDRs. I will use NMR and fluorescence correlation spectroscopy to derive diffusion coefficients that report on the hydrodynamic radius of the IDRs inside and outside of condensates. I will further use SAXS to measure the radius of gyration outside of condensates and the intermolecular spacing within condensates. Finally, I will use fluorescence recovery after photobleaching to assess the fluid dynamics and material state of condensates. These complementary, multi-disciplinary studies will enable me to test the hypotheses discussed above and advance our understanding of the role of PS in the in vitro and cellular behavior of cancer-driving fusion oncoproteins.