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

基本信息

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
项目类别：
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

项目摘要

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剂。此外，组蛋白乙酰化模式的全球异常发生在肿瘤发生过程中，这表明它们可能是转化过程中的相关步骤。然而，对特定组蛋白乙酰化事件的表观遗传作用以及导致其疾病失调失调的机制知之甚少。进一步阐明组蛋白乙酰化的精确作用将需要新的，正交工具使研究人员可以在体内研究单个乙酰化事件。拟议的研究将采用体外进化方法来开发细胞中特异性乙酰化组蛋白的核酶。组蛋白H4赖氨酸16（H4K16）将是由于该残基对染色质的结构和功能的有效作用。源自组蛋白H4的N末端尾巴的肽将在进化过程中用作体外底物，并且将根据其在赖氨酸16处的乙酰化这些肽的能力选择核酶。从最终选择中的单个核酶克隆中，将在自由组蛋白蛋白蛋白中分析乙酰基转移酶活性，从而分析乙酰基转移酶的活性。胜任的核酶将经过设计，以提高针对细胞降解的稳定性，并在细胞中使用高拷贝数U6 snRNA启动子上的盒式底座表达。 H4K16的体内乙酰化将通过Western印迹和从表达组蛋白乙酰转移酶（HAT）核酶的细胞中分离出的内源性组蛋白蛋白的质谱分析来验证。此外，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。这样的研究可能会导致新的诊断和癌症疗法。此外，提出的选择策略最终可用于发展针对其他组蛋白残基甚至非组蛋白蛋白的HAT核酶。公共卫生相关性：组蛋白乙酰化模式的全球异常出现在肿瘤发生过程中，并且是癌细胞的常见标志（1）。这些现象的详细解释（以及对可能的治疗选择的见解）阻碍了研究组蛋白乙酰化的工具的有限可用性（2,3）。拟议的研究旨在开发一种新颖的工具，与目前可实现的方法相比，研究人员可以以更高的分析精度研究组蛋白乙酰化。呢