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

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，这表明它们可能是转化过程中的相关步骤。然而，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，使研究人员能够在体内研究单一乙酰化事件。这项研究将采用体外进化的方法来开发核酶，它可以特异性地乙酰化细胞中的组蛋白。组蛋白H4赖氨酸16（H4 K16）将是初始靶标，这是由于： 这种残基对染色质结构和功能的潜在影响。来自组蛋白H4的N-末端尾部的肽将在进化过程中充当体外底物，并且核酶将基于它们在赖氨酸16处乙酰化这些肽的能力来选择。将在游离组蛋白、核小体核心颗粒和核小体阵列的背景下测定来自最后一轮选择的单个核酶克隆的乙酰转移酶活性。将对感受态核酶进行工程改造以提高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。H4 K16的体内乙酰化将通过从表达组蛋白乙酰转移酶（HAT）核酶的细胞分离的内源性组蛋白的蛋白质印迹和质谱分析来验证。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H4 K16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，具有更高的分析精度。这些研究可能会导致新的癌症诊断和治疗方法。此外，所提出的选择策略可能最终用于进化靶向其他组蛋白残基甚至非组蛋白蛋白的HAT核酶。

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.