Programmable control over histone acetylation at human regulatory elements using precision epigenome editing

使用精确表观基因组编辑对人类调控元件的组蛋白乙酰化进行可编程控制

• 批准号: 10669331
• 负责人: Isaac Hilton
• 金额: $ 56.88万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10669331
• 关键词: Acetylation; Architecture; Automobile Driving; Biological Phenomena; CRISPR screen; CRISPR/Cas technology; Cell Nucleus; Cells; Chimeric Proteins; Chromatin; Chromatin Loop; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA; Data; Deacetylase; Deacetylation; Disease; Dissection; Engineering; Enhancers; Epigenetic Process; Future; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genetic Transcription; Genome; Genomics; Global Change; Goals; Health; Histone Acetylation; Histone Deacetylase; Histone Deacetylation; Histones; Human; Human Activities; Human Genome; In Vitro; Knock-out; Light; Link; Location; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Mutation; Nucleosomes; Outcome; Pathologic; Pathology; Patients; Pattern; Pharmaceutical Preparations; Play; Post Translational Modification Analysis; Post-Translational Protein Processing; Process; Production; Proteins; RNA; Regulator Genes; Regulatory Element; Role; Signal Transduction; Source; System; Technology; Testing; Therapeutic; Transcription Coactivator; Untranslated RNA; Variant; Vorinostat; Work; Writing; base; cell type; combinatorial; cytotoxic; developmental disease; epigenetic therapy; epigenome editing; epigenomics; experience; experimental study; genome-wide; histone acetyltransferase; human disease; improved; inhibitor; innovation; insight; nervous system disorder; new technology; nuclease; prevent; programs; promoter; protein complex; scaffold; screening; selective expression; side effect; small molecule; stem; tool; virtual

PROJECT SUMMARY/ABSTRACT Dysregulated gene expression contributes to nearly every human disease. The level at which we understand the inherent complexity of gene regulation ultimately dictates our ability to link and/or manipulate biomolecular changes and pathology. Beyond their function as a structural substrate, histone proteins play key roles in gene expression by selectively gating access to DNA and thereby guiding when and how transcriptional machinery engages the human genome. Histone-based gene regulatory control largely stems from post-translational modifications (PTMs) to histone proteins themselves. One type of PTM, histone lysine acetylation, is particularly critical for gene regulation and human health, because overall acetylation levels tightly correlate with genomic activity and gene expression, and inappropriate histone acetylation patterns are linked to diverse human diseases. In fact, small molecules that globally change histone acetylation across the human genome have emerged as important therapeutics. However, in many patients these drugs can be ineffective and/or can result in toxic side effects. Furthermore, small molecules that globally alter histone acetylation patterns cannot be used to dissect how changes at specific locations in the human genome drive disease pathology. Robust tools that enable precise and targeted control over endogenous histone acetylation are urgently needed, because these technologies could illuminate the function(s) that this complex epigenomic signature plays within the human genome and open the door to new sophisticated epigenetic therapies. In this project, we will fulfill this urgent need by building synthetic and precisely targetable biomolecules that mimic the spectrum of activities displayed by natural human histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Aim 1). Specifically, we will combine the programmability of the nuclease null CRISPR/Cas9 (dCas9) scaffold with different classes of human HATs/HDACs and we will use these technologies to probe the selectivity of different HATs/HDACs in vitro and within native human chromatin. We will integrate our results with epigenomic profiling data to define how epigenetic marks, nucleosome occupancy, and cis regulatory element proximity influence the effects of histone acetylation at human enhancers and promoters. We will also use mass spectrometry, dCas9-based transcriptional activators and HATs, and genome-scale knockout screening to define the proteins/protein complexes that support histone acetylation-based gene activation (Aim 2). Finally, we will establish the impact of precisely targeted histone acetylation/deacetylation on enhancer activity and enhancer-promoter interactions (Aim 3). Experiments will be conducted at testbed human loci that have broad significance to human health and mechanistic epigenetics, and that will serve as proof-of-principle for interrogating virtually any regulatory element or locus in the human genome in future efforts. To accomplish the aims of this project, we have assembled an experienced team with synergistic expertise in CRISPR/Cas- based epigenome editing, histone PTM analysis, and functional dissection of enhancer activity.

项目概要/摘要 基因表达失调会导致几乎所有人类疾病。我们的理解水平 基因调控固有的复杂性最终决定了我们连接和/或操纵生物分子的能力 变化和病理。除了作为结构底物的功能外，组蛋白在基因中发挥着关键作用 通过选择性地门控 DNA 的获取来表达，从而指导转录机制的时间和方式 参与人类基因组。基于组蛋白的基因调控很大程度上源于翻译后 组蛋白本身的修饰（PTM）。一种类型的 PTM（组蛋白赖氨酸乙酰化）尤其重要 对于基因调控和人类健康至关重要，因为总体乙酰化水平与基因组密切相关 活性和基因表达以及不适当的组蛋白乙酰化模式与不同的人类有关 疾病。事实上，在整个人类基因组中全局改变组蛋白乙酰化的小分子已经 成为重要的治疗方法。然而，对许多患者来说，这些药物可能无效和/或可能导致 在毒副作用方面。此外，不能使用全局改变组蛋白乙酰化模式的小分子 剖析人类基因组特定位置的变化如何驱动疾病病理学。强大的工具 迫切需要对内源组蛋白乙酰化进行精确和有针对性的控制，因为这些 技术可以阐明这种复杂的表观基因组特征在人类体内发挥的功能 基因组并为新的复杂表观遗传疗法打开了大门。 在这个项目中，我们将通过构建合成且可精确靶向的生物分子来满足这一迫切需求 模拟天然人类组蛋白乙酰转移酶 (HAT) 和组蛋白的活性谱 脱乙酰酶 (HDAC)（目标 1）。具体来说，我们将结合核酸酶无效的可编程性 具有不同类别的人类 HAT/HDAC 的 CRISPR/Cas9 (dCas9) 支架，我们将使用这些技术 探索体外和天然人类染色质内不同 HAT/HDAC 的选择性。我们将整合 我们的结果与表观基因组分析数据来定义表观遗传标记、核小体占据和顺式 调节元件的邻近性影响组蛋白乙酰化对人类增强子和启动子的影响。我们 还将使用质谱、基于 dCas9 的转录激活剂和 HAT 以及基因组规模敲除 筛选以定义支持基于组蛋白乙酰化的基因激活的蛋白质/蛋白质复合物（目标 2）。最后，我们将确定精确靶向组蛋白乙酰化/脱乙酰化对增强子的影响 活性和增强子-启动子相互作用（目标 3）。实验将在人类基因座测试平台上进行 对人类健康和机械表观遗传学具有广泛的意义，这将作为原理验证 用于在未来的工作中询问人类基因组中几乎任何调控元件或位点。为了完成 为了实现这个项目的目标，我们组建了一支经验丰富的团队，在 CRISPR/Cas 领域具有协同专业知识- 基于表观基因组编辑、组蛋白 PTM 分析和增强子活性的功能剖析。