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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：
10797570
负责人：
Danfeng Cai
金额：
$ 14.43万
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.

项目摘要转录中心是基因组中具有升高的转录活性的热点。集中蛋白质和核酸，转录枢纽是细胞转录所必需的对细胞特性和细胞类型特异性功能重要的基因，而它们的失调导致许多疾病，如神经变性和癌症。尽管人们越来越关注理解转录枢纽，还有很多未知的东西。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，技术.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不同的焦点，并且假设液-液相分离负责转录枢纽的形成。我们将使用Yes相关蛋白 (YAP)转录中心，一个理想的系统，建立在我的博士后工作，以了解如何转录枢纽形成。雅普是一种转录辅激活因子，激活细胞内重要基因增殖和生存。高渗胁迫后，激活雅普靶点的诱导信号基因转录，我们发现雅普随着时间的推移形成具有不同组分的枢纽，与它们的不同功能一致（首先聚集可接近的染色质，转录第二）。我们将使用候选和无偏的方法来识别蛋白质用活细胞成像检测雅普枢纽的组成部分，然后测试调节这些组成部分的效果。蛋白质在可接近的染色质组织和转录激活。我们还将使用单粒子追踪来探测雅普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。第二个关于抄写的问题转录枢纽是如何激活转录的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。我们将使用实时- 细胞成像以可视化Myc（雅普的靶基因）的增强子和启动子的定位，和看看它们与雅普转录中心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枢纽形成的分子机制，功能更好地理解这些机制将对形成和其他转录枢纽的功能，并阐明受损的疾病的机制，转录中心的形成。