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

探讨融合癌蛋白相分离的普遍性和分子机制

基本信息

批准号：
10313054
负责人：
Scott Daniel Gorman
金额：
$ 4.8万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-13 至 2022-05-12
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10313054
关键词：
Address Amino Acid Sequence Automobile Driving Behavior Binding Biological Assay Cell Culture Techniques Cells Child Chromatin Chromosomal translocation Collaborations Communities Confocal Microscopy Databases Development Diffusion Disease Drug Targeting Escherichia coli Fluorescence Fluorescence Recovery After Photobleaching Fluorescence Resonance Energy Transfer Fusion Oncogene Proteins Gene Expression Gene Expression Regulation Genes Genetic Transcription Hela Cells In Vitro Link Liquid substance Malignant Neoplasms Mammalian Cell Measures Mediating Molecular Molecular Biology Molecular Conformation NMR Spectroscopy Nuclear Nuclear Magnetic Resonance Oils Parents Patients Phase Physiological Positioning Attribute Prevalence Process Prognosis Property Protein Sequence Analysis Proteins RNA Polymerase II Radial Reporting Roentgen Rays Role Saint Jude Children&apos s Research Hospital Sequence Analysis Source Spectrum Analysis Structure Testing Training Transcript Water Work analysis pipeline base behavior influence biophysical properties cancer cell cell behavior cell transformation chromatin remodeling design expression vector gene translocation high throughput screening improved interest intermolecular interaction multidisciplinary novel overexpression recruit refractory cancer screening structural biology targeted treatment therapeutic development therapeutic target transcription factor tumorigenesis

项目摘要

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.

抽象的由染色体易位形成的融合癌蛋白（FO）是许多疾病的主要驱动因素难治性癌症尤其困扰儿童，FO 的一部分本质上是紊乱的。最近的工作已经证明了转录因子和其他转录共调节因子的区域（IDR）。许多转录因子和共调节因子通过液-液相分离（PS）进行由 IDR 在其序列内介导的相互作用并在位点处形成转录缩合物。与这些缩合物相关的蛋白显着上调，表明 PS 参与基因调控和细胞命运。我们发现富含 IDR 的 FO 子集通过 PS 形成异常凝聚物，这会导致异常的基因表达和肿瘤发生。此外，我发现这些 FO 的形成。异常的转录凝聚物富含 IDR 序列，而这些序列之前并未被证实相分离，并且这些序列特征决定了构象和材料特性了解 FO 及其产生的冷凝物的生物物理特性是至关重要的。重要的是，其他人可以设计针对由此产生的冷凝物的治疗方法，以识别形成的 FO。生理条件下的凝集，我根据患者来源的转录本选择了 100 个 FO 进行转染作为 eGFP 标记蛋白进入哺乳动物细胞，我将筛选这 100 个 FO 的斑点形成情况。使用半自动荧光共聚焦显微镜测定来指示 PS 以确定 IDR。 PS 的负责序列，我会将来自点状形成 FO 的 IDR 亚克隆到大肠杆菌中我将使用浊度测定和共聚焦显微镜来进行表达载体的过度表达和纯化。评估这些 IDR 是否经历 PS 以及在什么条件下探测特定的分子间。相互作用并识别驱动 PS 的潜在新颖图案或“贴纸”，我将使用核磁共振 (NMR) 光谱法测量 PS 上 IDR 的动力学和分子间接触变化。此外，我会将序列/贴纸图案的组成与构象和材料特性联系起来我将使用 NMR 和荧光相关光谱来导出 FO 衍生的 IDR。关于凝结水内部和外部 IDR 的流体动力学半径的报告我将进一步使用 SAXS 来进行。测量冷凝物外部的回转半径和冷凝物内的分子间距。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。以上并促进我们对 PS 在癌症驱动的体外和细胞行为中的作用的理解融合癌蛋白。