Structural and functional determinants of biomolecular condensates in transcription organization

转录组织中生物分子凝聚体的结构和功能决定因素

• 批准号: 10714536
• 负责人: Jan-Hendrik Spille
• 金额: $ 33.92万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-22 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714536
• 关键词: Binding Sites; Biological Process; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromatin Structure; Data; Development; Disease; Elements; Enhancers; Epigenetic Process; Genetic Transcription; Genomics; Health; Informal Social Control; Knowledge; Link; Membrane; Microscopy; Modeling; Modification; Molecular; Mus; Nature; Nuclear; Organelles; Phase; Physical condensation; Proteins; Regulatory Element; Research; Resolution; Structure; Surface; Testing; Time; Transcriptional Regulation; Vision; biophysical model; embryonic stem cell; genome-wide analysis; programs; scaffold; self organization; superresolution microscopy; transcription factor

Many cellular processes use phase separation to sequesters biomolecules into membraneless organelles or condensates. This concept is of particular importance in the cell nucleus. Most nuclear condensates are associated with specific chromatin loci. Thus, chromatin organization and biomolecular condensate formation are closely related. Topology, epigenetic modification, or enzymatic activity are possible links between the chromatin scaffold and protein condensation. We have previously described condensates that concentrate transcription machinery at super-enhancers but understanding of cell-to-cell variability and locus specific features that nucleate condensates is missing. Preliminary data indicates that active chromatin decorates the surface of transcription condensates below the resolution limit of conventional microscopy. Making use of recent developments in multiplexed super-resolution microscopy of both chromatin elements and protein factors, we will characterize the nature of the chromatin enrichment layer and determine if it reflects the condensate surface as an active interface that regulates transcription. We will for the first time directly observe the interplay of local chromatin topology, epigenetic signatures, and condensate formation in a specific model locus in mouse embryonic stem cells. By scrutinizing with genomic resolution how each of the regulatory elements in the locus interacts with the condensate we will determine if chromatin structures such as enhancer hubs nucleate condensates, or if condensates induce specific chromatin topologies by connecting active elements. Finally, we will investigate at the molecular level how condensate constituents interact with the microenvironment and associated chromatin elements, and test the hypothesis that retention of transcription factors boosts binding site occupancy and stabilizes condensates at regulatory chromatin. Targeted perturbation and locus-specific observation will allow us to answer this question and begin to identify genome-wide rules that predict where precisely chromatin-supported condensates form. The overall vision for this research program is to develop an integrated view of chromatin organization and condensate formation. Experimental data will inspire new conceptual frameworks and help us integrate the full complexity of condensates into paradigms of biological function such as transcription regulation. We expect that new biophysical models of self-organization and self-regulation will emerge from our studies of intracellular condensates.

许多细胞过程使用相分离将生物分子隔离到无膜细胞器中 或凝析油。这一概念在细胞核中特别重要。大多数核凝析油 与特定的染色质基因座相关。因此，染色质组织和生物分子缩合物 编队是密切相关的。拓扑、表观遗传修饰或酶活性是可能的联系 在染色质支架和蛋白质凝聚之间。我们之前曾描述过凝析油 将转录机制集中在超级增强子上，但了解细胞间的可变性 以及核凝析油缺失的轨迹特定特征。初步数据显示，活跃 染色质修饰转录缩合物的表面，低于常规的分辨率极限 显微镜。利用多路超分辨率显微技术的最新进展 染色质的成分和蛋白质的因素，我们将表征染色质的丰富性 层，并确定它是否将凝析油表面反射为调节 抄写。我们将首次直接观察到局部染色质拓扑的相互作用， 小鼠胚胎干细胞特定模型基因座的表观遗传特征和凝集物的形成 细胞。通过用基因组分辨率研究基因座中的每个调控元件是如何相互作用的 利用凝集物，我们将确定染色质结构，如增强子中心是否成核 冷凝物，或如果冷凝物通过连接活性元素而诱导特定的染色质拓扑结构。 最后，我们将在分子水平上研究凝析油的成分如何与 微环境和相关的染色质元素，并检验假设 转录因子增加结合位点的占有率，并稳定调节染色质的凝集物。 有针对性的扰动和特定地点的观察将使我们能够回答这个问题，并开始 确定全基因组规则，准确预测染色质支持的缩合物形成的位置。 这项研究计划的总体愿景是发展染色质组织的综合观点 和凝析油的形成。实验数据将启发新的概念框架，并帮助我们 将凝聚体的全部复杂性整合到生物功能的范例中，如转录 监管。我们预计新的自组织和自我调节的生物物理模型将会出现。 来自我们对细胞内冷凝物的研究。