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

基本信息

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
项目类别：
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

项目摘要

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膜细胞器中或冷凝。这个概念在细胞核中尤其重要。大多数核冷凝物与特定的染色质基因座有关。因此，染色质组织和生物分子冷凝物形成密切相关。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冷凝之间。我们先前已经描述了冷凝水将转录机械集中在超级增强剂上，但对细胞间变异性的理解和基因座的特定特征，而成核凝结物缺失。初步数据表明活动染色质在传统的分辨率限制以下的转录表面装饰表面显微镜。利用两者的多路复用超分辨率显微镜的最新发展染色质元素和蛋白质因子，我们将表征染色质富集的性质层并确定它是否反映冷凝水面作为调节的活动界面转录。我们将首次直接观察局部染色质拓扑的相互作用，在小鼠胚胎茎中的特定模型基因座中的表观遗传签名和凝结物形成细胞。通过用基因组分辨率仔细检查基因座中的每个调节元素如何相互作用使用冷凝物，我们将确定染色质结构（例如增强子中心）是否成核凝结物，或者如果凝结物通过连接活性元件诱导特定的染色质拓扑。最后，我们将在分子水平上研究冷凝水成分如何与微环境和相关的染色质元素，并检验了保留的假设转录因子增强了结合位点的占用率并稳定在调节染色质处的冷凝物。有针对性的扰动和特定于基因座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，这些规则可以预测精确染色质的冷凝物形成的位置。该研究计划的总体愿景是开发染色质组织的综合视图和凝结物的形成。实验数据将激发新的概念框架并帮助我们将冷凝物的全部复杂性整合到生物学功能的范式中，例如转录规定。我们预计，新的自组织和自我调节的生物物理模型将出现根据我们对细胞内冷凝物的研究。