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（FOS）是许多主要驱动因素抗药性癌症特别折磨儿童。 FO的子集包含本质上无序的转录因子和其他转录共同调节器的区域（IDR）。最近的工作证明了许多转录因子和共同调节器通过由IDR在其序列中介导的相互作用并形成转录冷凝物。基因座的基因与这些冷凝物相关的关联是显着更新的，在基因调控和细胞命运中的含义PS。我们假设一部分富含IDR的FO的子集形成了通过PS的异常转录缩合，这会导致异常的基因表达和肿瘤发生。此外，我假设这些形成的FO 异常的转录冷凝物以IDR序列富集，以前尚未证明相分开，并且这些序列特征决定了构象和材料特性 FOS和由此产生的凝结物。了解FO衍生的IDR的生物物理特性是重要的是，其他人可以设计针对所得冷凝物的理论。识别形式的fos 在生理条件下冷凝水，我根据患者衍生的转录本选择了100个FOS来转染作为EGFP标记的蛋白进入哺乳动物细胞。我将筛选这100个fos的形成点使用半自动荧光共聚焦显微镜测定法表示PS。确定IDR 负责PS的序列，我将从形成点形的FOS衍生成大肠杆菌的IDR下表达向量的过表达和纯化。我将使用浊度测定和共聚焦显微镜进行评估这些IDR是否经历PS并在什么条件下进行。探测特定的分子相互作用并识别可能驱动PS的潜在新型基序或“贴纸”，我将使用核磁共振（NMR）测量PS时IDR的动力学和分子间接触的变化的光谱。此外，我将将序列/贴纸基序组成与构象和材料特性相关联 fo衍生的IDR。我将使用NMR和荧光相关光谱来得出扩散系数报告冷凝水内外IDR的流体动力半径。我将进一步使用萨克斯测量冷凝水和冷凝物内分子间间距外的回旋半径。最后，我将在光漂白后使用荧光回收来评估流体动力学和材料状态冷凝水。这些完整的多学科研究将使我能够测试讨论的假设上方并提高我们对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。