Probing the Formation and Function of Transcription Hubs-Equipment Supplement

探究转录中枢的形成和功能-设备补充

基本信息

批准号：
10581220
负责人：
Danfeng Cai
金额：
$ 24.92万
依托单位：
JOHNS HOPKINS UNIVERSITY
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：
2021-08-01 至 2026-05-31
项目状态：
未结题

项目摘要

PROJECT SUMMARY 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.

项目概要转录中心是基因组中转录活性升高的热点。转录中心集中蛋白质和核酸，对于细胞转录至关重要对细胞身份和细胞类型特异性功能很重要的基因，而它们的失调会导致许多疾病，例如神经退行性疾病和癌症。尽管人们越来越感兴趣对于转录中心的理解，还有很多未知的地方。我们研究的目标是解决使用先进成像和蛋白质组学，关于转录中心的两个基本问题技术。第一个问题是：转录中心是如何形成的？转录中心在细胞核中表现为不同的焦点，并且假设液-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，用于了解如何转录中心形成。 YAP 是一种转录共激活因子，可激活细胞中重要的基因增殖和生存。高渗应激后，诱导信号激活 YAP 靶标基因转录，我们发现YAP随着时间的推移形成具有不同成分的枢纽，与它们的不同功能一致（首先对可访问的染色质进行聚类，然后激活转录第二）。我们将使用候选和公正的方法来识别蛋白质具有活细胞成像的 YAP 中枢组件，然后测试调节这些组件的效果可及染色质组织和转录激活中的蛋白质。我们也将使用单粒子追踪探测 YAP 转录中心的生物物理性质，了解它们是否通过相分离或替代机制形成。第二个问题关于转录 hubs 是：转录中枢如何激活转录？我们假设转录中心可以介导增强子-启动子相互作用以激活转录。我们将使用多色现场- 细胞成像以可视化 Myc 增强子和启动子（YAP 的靶基因）的定位，以及了解它们与 YAP 转录中心的相对位置如何导致 Myc 转录激活。这些研究将共同阐明转录中心形成的分子机制和功能。更好地理解这些机制将对形成和其他转录中心的功能，并揭示受损疾病的机制转录中心的形成。