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(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重要调节因子。此外，组蛋白乙酰化模式的全球异常发生在肿瘤发生过程的早期，这表明它们可能是转化过程中的相关步骤。然而，特殊的组蛋白乙酰化事件的表观遗传学作用及其在疾病中导致其失调的机制尚不清楚。进一步阐明组蛋白乙酰化的确切作用将需要新的， 使研究人员能够研究体内单个乙酰化事件的正交工具。这项拟议的研究将使用体外进化方法来开发核酶，这种核酶可以特异性地使细胞中的组蛋白乙酰化。组蛋白H4赖氨酸16(H4K16)将是最初的靶标，因为 这种残基对染色质的结构和功能有很大的影响。在进化过程中，来自组蛋白H4 N端的多肽将作为体外底物，核酶将根据它们在赖氨酸16位乙酰化这些多肽的能力进行选择。最后一轮选择的单个核酶克隆将在游离组蛋白、核小体核心颗粒和核小体阵列的背景下被检测乙酰基转移酶的活性。合格的核酶将被设计成增加对细胞降解的稳定性，并使用基于高拷贝数U6 SnRNA启动子的卡盒在细胞中表达。H4K16在体内的乙酰化将通过Western印迹和从表达组蛋白乙酰基转移酶(HAT)核酶的细胞中分离出的内源性组蛋白的质谱分析来验证。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，与目前可以实现的方法相比，具有更高的分析精度。这样的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被用于进化针对其他组蛋白残基甚至非组蛋白蛋白的HAT核酶。 与公共卫生相关：组蛋白乙酰化模式的全球异常在肿瘤形成过程中很早就出现并积累，是癌细胞的共同标志(1)。对这些现象的详细解释(以及对可能的治疗选择的洞察)由于研究组蛋白乙酰化的工具有限而受阻(2，3)。这项拟议的研究旨在开发一种新的工具，使研究人员能够与目前可实现的方法相比，以更高的分析精度研究组蛋白乙酰化。好了！