Probing the Formation and Function of Transcription Hubs

探究转录中心的形成和功能

• 批准号: 10456213
• 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/10456213
• 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.

摘要 转录中心是基因组中具有较高转录活性的热点。 转录中心集中了蛋白质和核酸，是细胞转录所必需的。 对细胞特性和细胞类型特定功能很重要的基因，而它们的失调导致 许多疾病，如神经退行性变和癌症。尽管人们对……越来越感兴趣 对于转录中心的理解，仍有许多未知之处。我们研究的目标是解决 关于转录中心的两个基本问题，使用先进的成像和蛋白质组学 技巧。第一个问题是：转录中心是如何形成的？转录 中心在核中表现为明显的焦点，并假设液-液相 分离负责转录中心的形成。我们将使用YES相关蛋白 (YAP)转录中心，这是我在博士后工作中建立的一个理想系统，可以了解如何 转录中心的形成。YAP是一种转录共激活因子，激活细胞中重要的基因 扩散和生存。高渗应激后，激活YAP靶标的可诱导信号 基因转录，我们发现随着时间的推移，YAP形成了具有不同成分的枢纽， 符合它们不同的功能(先将可访问的染色质聚类，然后激活 转录秒)。我们将使用候选和无偏见的方法来识别蛋白质 用活细胞成像检测YAP集线器的组件，然后测试调制这些组件的效果 可及染色质组织和转录激活中的蛋白质。我们还将使用Single 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 通过相分离或替代机制形成。关于抄写的第二个问题 Hubs是：转录中心如何激活转录？我们假设转录中心 可以介导增强子-启动子相互作用来激活转录。我们将使用多彩现场直播- 细胞成像显示YAP靶基因Myc增强子和启动子的定位 看看它们相对于YAP转录中心的位置如何导致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。对这些机制的更好理解将对形成和 其他转录中心的功能，并阐明受损疾病的机制 转录中心的形成。