Probing the Formation and Function of Transcription Hubs

探究转录中心的形成和功能

• 批准号: 10275593
• 负责人: Danfeng Cai
• 金额: $ 40.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10275593
• 关键词: Address; Biophysics; Cell Nucleus; Cell Proliferation; Cell Survival; Cells; Chromatin; DNA; Disease; Enhancers; Genes; Genetic Transcription; Genome; Goals; Image; Impairment; Lead; Light; Liquid substance; Malignant Neoplasms; Mediating; Molecular; Nature; Nerve Degeneration; Nucleic Acids; Phase; Positioning Attribute; Proteins; Proteomics; Research; Signal Transduction; Stress; System; Techniques; Testing; Time; Transcription Coactivator; Transcriptional Activation; Work; cell type; interest; live cell imaging; particle; promoter

ABSTRACT Transcription hubs are hotspots in the genome with elevated transcription activities. Concentrating proteins and nucleic acids, transcription hubs are essential for cells to transcribe genes important for cell identity and cell type-specific functions, while their dysregulation leads to many diseases such as neurodegeneration and cancer. Although there is growing interest in understanding transcription hubs, a lot remains unknown. The goal of our research is to address two fundamental questions about transcription hubs, using advanced imaging and proteomics techniques. The first of these questions is: how are transcription hubs formed? Transcription hubs appear as distinct foci in the nucleus, and it is hypothesized that liquid-liquid phase separation is responsible for transcription hubs formation. We will use Yes-associated Protein (YAP) transcription hub, an ideal system established in my postdoctoral work to understand how transcription hubs form. YAP is a transcription coactivator activating genes important in cell proliferation and survival. After hyperosmotic stress, an inducible signal to activate YAP target gene transcription, we have found that YAP forms hubs with different components over time, coincident with their different functions (clustering accessible chromatins first, and activating transcription second). We will use candidate and unbiased approaches to identify protein components of YAP hubs with live-cell imaging, and then test the effect of modulating these proteins in accessible chromatin organization and transcription activation. We will also use single particle tracking to probe the biophysical nature of YAP transcription hubs, to understand if they form by phase separation or alternative mechanisms. The second question regarding transcription hubs is: how do transcription hubs activate transcription? We hypothesize that transcription hub can mediate enhancer-promoter interaction to activate transcription. We will use multicolor live- cell imaging to visualize localization of enhancer and promoter of Myc (a target gene of YAP), and see how their relative position to YAP transcription hub lead to Myc transcription activation. Together, these studies will elucidate molecular mechanisms of transcription hub formation and function. A better understanding of these mechanisms will have implications for the formation and function of other transcription hubs, and shed light on mechanisms of diseases of impaired transcription hub formation.

抽象的 转录轮毂是基因组中具有升高转录活性的热点。 浓缩蛋白质和核酸，转录轮毂对于细胞转录至关重要 对于细胞身份和细胞类型特异性功能重要的基因，而其失调导致 许多疾病，例如神经变性和癌症。尽管对 了解转录中心，很多仍然未知。我们研究的目的是解决 使用高级成像和蛋白质组学，有关转录中心的两个基本问题 技术。这些问题中的第一个是：如何形成转录轮毂？转录 集线器在细胞核中显示为不同的灶，并假设液态液相 分离负责转录中心的形成。我们将使用与是相关的蛋白 （yap）转录中心，这是我的博士后工作中建立的理想系统，以了解如何 转录轮毂形式。 YAP是一种在细胞中重要的转录共激活因子激活基因 增殖和生存。过度渗透应力后，激活YAP目标的诱导信号 基因转录，我们发现YAP随着时间的推移形成具有不同组件的集线器， 与它们的不同功能一致（首先将染色蛋白聚类并激活 转录第二）。我们将使用候选和公正的方法来识别蛋白质 带有活细胞成像的YAP集线器的组件，然后测试调节这些的效果 可访问的染色质组织和转录激活中的蛋白质。我们还将使用单个 粒子跟踪以探测YAP转录中心的生物物理性质，以了解它们是否是否 通过相分离或替代机制形成。关于转录的第二个问题 集线器是：转录轮毂如何激活转录？我们假设该转录集线器 可以介导增强剂促进剂相互作用以激活转录。我们将使用多色现场 细胞成像可视化MYC（YAP的靶基因）的增强子和启动子的定位，并且 查看它们与YAP转录集线器的相对位置如何导致MYC转录激活。 总之，这些研究将阐明转录中心形成的分子机制和 功能。更好地了解这些机制将对形成和 其他转录中心的功能，并阐明了受损疾病的机制 转录集线器形成。