Genetically encoded designer inhibitors for functional epigenomics

描述(申请人提供)：该项目的长期目标是建立一套强大的技术，使特殊和时间控制表观基因组。我们假设，通过选择性地和生化地干扰单个蛋白质(甚至蛋白质中的单个结构域)，我们可以高精度地控制表观基因组。因此，该项目旨在利用最先进的蛋白质工程技术来产生高性能的结合到表观遗传调控蛋白的蛋白，这些蛋白可以遗传编码用于细胞内。我们将利用我们在过去十年中首创和改进的设计师结合蛋白平台，称为“单体”。单体是使用纤维连接蛋白III型(FN3)支架构建的设计结合蛋白。与传统抗体及其片段不同，FN3缺乏二硫键，因此单体在还原条件下折叠成其功能形式，如细胞内的细胞核和细胞质。因此，单体体特别适合作为基因编码的细胞内抑制物来对抗表观遗传调节蛋白。通过利用我们在过去十年中开发的尖端技术，我们将产生与许多表观遗传调控蛋白具有高亲和力和精致特异性的单体体。我们将开发配套技术，使单体的新应用能够理解和控制表观遗传调控过程。具体地说，我们提出了以下目标：目的1.为染色质修饰的读者和作者开发高特异性、高亲和力的单体。目的2.开发利用单体作为针对表观遗传调节蛋白的遗传编码抑制物的技术，并验证来自目标1的单体是否可在细胞内使用。目的3.发展“化学表观遗传学”技术，利用单体抑制剂，特别是CHD7，在神经脊分化和电荷综合征的背景下，对发育细胞中的染色质调节器进行时间控制。我们将向社区提供这些强大的工具。我们组建了一支技能互补的专家团队，该项目将利用通过合作者网络提供的资源和专业知识。我们在创新和推动研究界方面有着良好的记录。在这个项目中获得的技术和知识将为表观基因组学研究界提供新的和强大的工具，并导致控制表观基因组的新方法，从而对人类健康产生积极影响。总而言之，这个项目与RFA的愿景完全一致。

DESCRIPTION (provided by applicant): The long-term goal of this project is to establish a set of powerful technologies that enable special and temporal control of the epigenome. We hypothesize that, by selectively and biochemically perturbing a single protein (or even single domain within a protein), we can control the epigenome with high precision. Thus, this project aims to utilize state-of-the-art protein engineering technologies to generate high-performance binding proteins to epigenetic regulatory proteins that can be genetically encoded for intracellular use. We will utilize the designer binding protein platform, termed "monobody" that we have pioneered and refined over the last decade. Monobodies are designer binding proteins built using the fibronectin type III (FN3) scaffold. Unlike conventional antibodies and their fragments, FN3 lacks disulfide bonds and thus monobodies fold into their functional form under reducing conditions, such as the nucleus and cytoplasm within cells. Therefore, the monobodies are particularly suited as genetically encoded, intracellular inhibitors against epigenetic regulatory proteins. By utilizing sophisticated technologies that we have developed over the last decade, we will generate monobodies to many epigenetic regulatory proteins that have high affinity and exquisite specificity. We will develop companion technologies that enable novel applications of monobodies toward understanding and controlling epigenetic regulatory processes. Specifically, we propose the following aims: Aim 1. To develop high-specificity, high-affinity monobodies to readers and writers of chromatin modifications. Aim 2. To develop technologies for using monobodies as genetically encoded inhibitors directed to epigenetic regulatory proteins and validate monobodies from Aim 1 for intracellular use. Aim 3. To develop "chemoepigenetic" technologies for temporal control of the chromatin regulators in developing cells using monobody inhibitors, in particular CHD7 in the context of neural crest differentiation and the CHARGE syndrome. We will make these powerful tools available to the community. We have assembled a team of experts with complementary skills and this project will leverage resources and expertise available through a network of collaborators. We have a strong track record of innovations and enabling the research community. Technologies and knowledge gained in this project will provide the epigenomics research community with novel and powerful tools and lead to new approaches to controlling the epigenome for positively impacting human health. Together, this project is perfectly aligned with the vision of the RFA.