Interrogating the Structural Basis of Nuclear Receptor Activation on Chromatin

项目总结 核受体(Nr)是配体门控的转录因子，控制着重要的生物过程。 包括生殖、发育、新陈代谢和免疫。NRs是~16%使用的药物的目标 临床应用包括节育、炎症性疾病和代谢紊乱。NR是 认为通过改变染色质的动态来改变基因的表达水平来发挥作用 共调节子、染色质重构体的招募，并最终通过激活转录 机械设备。然而，天然橡胶和天然橡胶复合体的动态性质使其研究变得具有挑战性 全长蛋白质和蛋白质组件调控基因表达的分子机制 传统的结构生物学技术。尽管NRs在基因表达激活中起着核心作用，但我们的 对它们如何在染色质分子水平上调节靶基因程序的机械理解是 很大程度上是未知的。这部分归因于缺乏全长受体的结构生物学数据和 在其功能相关的NRS上几乎完全没有生化、生物物理或结构数据 染色质上下文。建立在赞助商国内实验室已有的基础上，结合 申请人之前在生化和生物物理方法方面的专业知识，以及对自然保护领域的深入了解； 该项目试图确定名为RoRγT的模型NR如何与共调节蛋白SRC3组装 和p300来改变染色质的动力学和翻译后修饰状态，染色质是NR的标志 靶基因激活。拟议的研究计划有两个主要目标：(1)确定RoRγT和 共调节蛋白复合体与核小体结合，以及(2)确定RoRγT和辅助调节因子如何 蛋白质复合体修饰组蛋白，改变核小体动力学。这将通过使用 将传统生物化学和包括低温电子在内的最新技术相结合 显微镜(冷冻-EM)和结构蛋白质组学。通过研究这些蛋白质复合体是如何组装和作用的 在核小体方面，我们将获得新的见解，了解RoRγT，更广泛地说，是如何调节NR超级家族的 核小体动力学、修饰状态，并最终在基因调控中发挥作用。这一点的影响 这项工作将帮助我们了解一个重要的药物靶点是如何发挥作用的，并将为未来的研究铺平道路 调查一类重要的毒品目标。

PROJECT SUMMARY Nuclear receptors (NRs) are ligand-gated transcription factors that control important biological processes including reproduction, development, metabolism, and immunity. NRs are the target of ~16% of drugs used in the clinic for applications that include birth control, inflammatory disease, and metabolic disorders. NRs are thought to function by altering the dynamics of chromatin to alter expression levels of genes through the recruitment of coregulators, chromatin remodelers, and ultimately through the activation of transcriptional machinery. However, the dynamic nature of NRs and NR complexes has made it challenging to study the molecular mechanisms by which full length proteins and protein assemblies regulate gene expression using traditional structural biology techniques. Despite the central role that NRs play in gene expression activation, our mechanistic understanding of how they regulate target gene programs at the molecular level of chromatin is largely unknown. This is attributed, in part, to the dearth of structural biology data for full-length receptors and the near complete absence of biochemical, biophysical, or structural data on NRs in their functionally relevant chromatin context. Building on established foundations in sponsor’s home laboratory, combined with the applicant’s prior expertise in biochemical and biophysical methods and an intimate understanding of the NR field, this project seeks to determine how a model NR named RORγT assembles with coregulatory proteins SRC3 and p300 to alter chromatin dynamics and post-translational modification status of chromatin, a hallmark of NR target gene activation. The proposed research program has two major goals: (1) to determine how RORγT and coregulatory protein complexes engage with nucleosomes, and (2) to determine how RORγT and coregulator protein complexes modify histones and alter nucleosome dynamics. This will be accomplished using a combination of traditional biochemistry, and state-of-the-art technologies including cryogenic electron microscopy (cryo-EM) and structural proteomics. By studying how these protein complexes assemble and act on nucleosomes, we will gain new insight into how RORγT, and more broadly the NR superfamilty, modulates nucleosome dynamics, modification status, and, ultimately, function in gene regulation. The implications of this work will help us understand how an important drug target functions and will pave the way for future research investigating an important class of drug targets.