In vitro evolution of ribozymes capable of site-specific histone acetylation

能够进行位点特异性组蛋白乙酰化的核酶的体外进化

• 批准号: 8308862
• 负责人: Jonathan Thomas Sczepanski
• 金额: $ 4.92万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8308862
• 关键词: Acetyl Coenzyme A; Acetylation; Acetyltransferase; Biological Assay; Catalytic RNA; Cells; Chromatin; Chromatin Modeling; Chromatin Structure; DNA Repair; DNA biosynthesis; Development; Disease; Engineering; Enzymes; Epigenetic Process; Event; Evolution; Gene Expression Profile; Gene Silencing; Genetic Transcription; Histone Acetylation; Histone H4; Histones; In Vitro; Individual; Lead; Link; Lysine; Mammalian Cell; Mass Spectrum Analysis; Methods; Modification; N-terminal; Nuclear; Nucleosome Core Particle; Nucleosomes; Pattern; Peptides; Physical condensation; Process; Proteins; RNA; Regulation; Research; Research Personnel; Role; Site; Structure; Tail; Technology; Transcript; Transcriptional Regulation; U6 small nuclear RNA; Western Blotting; base; cancer cell; cancer therapy; histone acetyltransferase; histone modification; in vivo; insight; novel; novel diagnostics; promoter; thioester; tool; tumorigenesis

DESCRIPTION (provided by applicant): The posttranslational acetylation of histone proteins is a crucial regulator of chromatin structure and function. Furthermore, global abnormalities in histone acetylation patterns occur early during the course of tumorigenesis, suggesting they may be relevant steps in the transformation process. However, the epigenetic role of particular histone acetylation events, and the mechanisms leading to their dysregulation in diseases, is poorly understood. Further elucidation of the precise role of histone acetylation will require new, orthogonal tools that allow researchers to study single acetylation events in vivo. The proposed research will employ in vitro evolution methods to develop ribozymes that site-specifically acetylate histone proteins in cells. Histone H4 lysine 16 (H4K16) will be the initial target due to the potent effect of this residue on the structure and function of chromatin. Peptides derived from the N-terminal tail of histone H4 will serve as in vitro substrates during evolution, and ribozymes will be selected based on their ability to acetylate these peptides at lysine 16. Individual ribozyme clones from the final round of selection will be assayed for acetyltransferase activity in the context of free histone proteins, nucleosome core particles, and nucleosomal arrays. Competent ribozymes will be engineered for increased stability against cellular degradation and expressed in cells using a cassette base on the high copy number U6 snRNA promoter. In vivo acetylation of H4K16 will be verified by Western blot and mass spectrometry analysis of endogenous histone proteins isolated from cells expressing histone acetyltransferase (HAT) ribozymes. In addition, a comprehensive transcriptome analysis will be conducted on cells expressing HAT ribozymes. Ribozymes developed using these methods will allow researchers to study H4K16 acetylation, as well as the enzymes associated with this modification, with greater analytical precision compared to approaches that are currently achievable. Such studies may lead to new diagnostics and therapies for cancer. In addition, the proposed selection strategy may eventually be used to evolve HAT ribozymes that target other histone residues or even non- histone proteins. PUBLIC HEALTH RELEVANCE: Global abnormalities in histone acetylation patterns appear early and accumulate during the course of tumorigenesis and are a common hallmark of cancer cells (1). A detailed explanation for these phenomena (and insight into possible treatment options) is impeded by the limited availability of tools for studying histone acetylation (2,3). Th proposed research aims to develop a novel tool that will allow researchers to study histone acetylation with greater analytical precision compared to approaches that are currently achievable. !

描述（由申请人提供）：组蛋白的翻译后乙酰化是染色质结构和功能的关键调节因子。此外，组蛋白乙酰化模式的整体异常发生在肿瘤发生过程的早期，表明它们可能是转化过程中的相关步骤。然而，人们对特定组蛋白乙酰化事件的表观遗传作用以及导致其在疾病中失调的机制知之甚少。进一步阐明组蛋白乙酰化的确切作用将需要新的、 正交工具使研究人员能够研究体内的单个乙酰化事件。 拟议的研究将采用体外进化方法来开发可在细胞中对组蛋白进行位点特异性乙酰化的核酶。组蛋白 H4 赖氨酸 16 (H4K16) 将成为最初的目标，因为 该残基对染色质的结构和功能的强大影响。源自组蛋白 H4 N 末端尾部的肽将在进化过程中充当体外底物，并且将根据核酶在赖氨酸 16 处乙酰化这些肽的能力来选择核酶。将在游离组蛋白、核小体核心颗粒和核小体阵列的背景下分析来自最后一轮选择的各个核酶克隆的乙酰转移酶活性。感受态核​​酶将被设计以提高对抗细胞降解的稳定性，并使用基于高拷贝数 U6 snRNA 启动子的盒在细胞中表达。 H4K16 的体内乙酰化将通过对从表达组蛋白乙酰转移酶 (HAT) 核酶的细胞中分离的内源组蛋白进行蛋白质印迹和质谱分析来验证。此外，还将对表达 HAT 核酶的细胞进行全面的转录组分析。 使用这些方法开发的核酶将使研究人员能够研究 H4K16 乙酰化以及与这种修饰相关的酶，与目前可实现的方法相比，分析精度更高。此类研究可能会带来新的癌症诊断和治疗方法。此外，所提出的选择策略最终可能用于进化针对其他组蛋白残基甚至非组蛋白的HAT核酶。 公共健康相关性：组蛋白乙酰化模式的整体异常出现较早，并在肿瘤发生过程中累积，是癌细胞的常见标志 (1)。研究组蛋白乙酰化的工具有限，阻碍了对这些现象的详细解释（以及对可能的治疗方案的深入了解）(2,3)。拟议的研究旨在开发一种新颖的工具，使研究人员能够以比目前可实现的方法更高的分析精度来研究组蛋白乙酰化。 ！