Scalable platforms for understudied histone modifications and modifiers

用于未充分研究的组蛋白修饰和修饰剂的可扩展平台

• 批准号: 10567849
• 负责人: Albert Keung
• 金额: $ 32.58万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-06 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10567849
• 关键词: Acetylation; Address; Adopted; Affinity; Amino Acids; Antibodies; Arginine; Binding; Binding Proteins; Biochemical; Biochemistry; Biological; Biological Assay; Biological Process; Biology; Biosensor; Bypass; Cell physiology; ChIP-seq; Chemicals; Chromatin; Chromatin Structure; Complement; Complex; Cost Effectiveness Analysis; DNA; DNA Binding; DNA Modification Process; Disease; Dopamine; Engineering; Enzymes; Epigenetic Process; Eukaryota; Gene Expression; Gene Expression Regulation; Gene Silencing; Generations; Genetic Transcription; Genome; Genomic DNA; Glutamine; Goals; Health; Heart; Heritability; Histone Code; Histones; Human; Lysine; Measurement; Mediating; Methylation; Modification; Neurotransmitters; Pattern; Play; Post-Translational Protein Processing; Protein Engineering; Proteins; Quality Control; RNA; Reader; Reagent; Recombinant Proteins; Regulation; Research; Research Personnel; Role; Serotonin; Set protein; Site; Specificity; Structure; Surface; System; Transcriptional Regulation; Work; Writing; Yeasts; acyl group; biochemical tools; cell behavior; cell growth regulation; combinatorial; cost efficient; epigenetic silencing; gene repression; genome-wide; histone acetyltransferase; histone modification; histone-binding proteins; live cell imaging; monoamine; next generation; open source; preference; tool; virtual

PROJECT SUMMARY/ABSTRACT Eukaryotic genomic DNA is extensively associated with proteins and RNAs to form chromatin. Through its control of gene expression, changes in chromatin biochemistry and structure underlie nearly all cellular processes. Post-translational modifications of histone proteins that bind genomic DNA play an especially critical role in regulating chromatin structure and function. Modifications of certain histone residues influence the binding of histone proteins to DNA as well as the interactions of other proteins that specifically recognize these modifications. The specific pattern of histone modifications acts as a “histone code” to determine the set of proteins that interact with histones and histone-bound DNA, and consequently participate in diverse cellular processes. Therefore, identification of specific histone modifications, quantitative assessment of the interactions they mediate, and characterization of enzymes that modify histones is essential for understanding chromatin regulation of complex cellular behavior. However, unraveling the histone code is a daunting challenge due to its complexity – over eighty different amino acid residues on five histone proteins undergo over twenty distinct known post-translational modifications. Despite significant advances in research on some histone modifications such as acetylation and methylation, the vast majority of histone modifications remains understudied. Further, a vast majority of enzymes that write even extensively researched modifications like acetylation remain understudied. We propose to address the critical gap in essential biochemical tools and accessible experimental platforms that has hindered research on understudied histone modifications and modifiers. Specifically, we will harness next generation yeast surface display systems as scalable platforms for high throughput studies on understudied histone modifications and modifiers, as well as platforms for engineering biochemical reagents for chromatin research. In addition to understudied histone acetyltransferases, we will focus on three classes of histone modifications: (i) citrullination of arginine (ii) acyl modification of lysine by non-acetyl groups (propionylation, butyrylation, crotonylation), and (iii) monoamine modification of glutamine by serotonin and dopamine. In Aim 1, we will develop a platform for efficient generation of affinity reagents with high specificity that can serve as genetically encoded biosensors for live cell imaging, as well as in conventional analyses like ChIP-seq and CUT&Tag. In Aim 2, we will develop a platform for high throughput identification and quantification of binding interactions mediated by histone modifications. In Aim 3, we will develop a platform for high throughput interrogation of residue preferences of writers. Our work will develop “open source” platforms that are scalable, cost-efficient, and easily adopted by other investigators in chromatin biology. Such platforms will serve as strong complements to traditional biochemical assays by enabling research on both common and understudied histone modifications, and unlocking new high throughput measurements and research questions in chromatin biology.

项目摘要/摘要 真核基因组DNA与蛋白质和RNA广泛相关，形成染色质。通过其控制 基因表达，几乎所有细胞过程的染色质生物化学和结构的变化。 结合基因组DNA的组蛋白的翻译后修饰在 调节染色质结构和功能。某些组蛋白残留物的修饰会影响结合 Hisstone蛋白与DNA以及其他明确识别这些蛋白的相互作用 修改。组蛋白修饰的特定模式充当“组蛋白代码”，以确定一组 与组蛋白和结合组蛋白结合的DNA相互作用的蛋白质，因此参与了大型细胞 过程。因此，鉴定特定组蛋白修饰，相互作用的定量评估 它们介导并表征修饰组蛋白的酶对于理解染色质至关重要 调节复杂的细胞行为。但是，由于其揭开组蛋白代码是一个艰巨的挑战 复杂性 - 超过八十种不同的氨基酸保留在五种Hisstone蛋白上，经历了二十多个不同的氨基酸 已知的翻译后修改。尽管研究某些组蛋白修饰方面有重大进展 例如，乙酰化和甲基化，绝大多数组蛋白修饰仍然可以理解。此外， 绝大多数编写甚至进行了广泛研究的修饰（例如乙酰化）的酶仍然存在 研究了。我们建议解决基本生化工具中的关键差距和可访问的实验 阻碍了对理解的组蛋白修饰和修饰符的研究的平台。具体来说，我们会的 利用下一代酵母表面显示系统作为可扩展平台，用于高通量研究 正在研究的组蛋白修饰和修饰符，以及用于工程生化试剂的平台 染色质研究。除了理解的组蛋白乙酰转移酶外，我们还将重点放在三类 组蛋白的修饰：（i）精氨酸（II）非乙酰基基团对赖氨酸的修饰 （丙酰化，丁酰化，共旋to）和（iii）通过5-羟色胺和5-羟胺的单胺修饰 多巴胺。在AIM 1中，我们将开发一个平台，以有效地生成具有高特异性的亲和力试剂 可以用作实用细胞成像的一般编码生物传感器，以及在常规分析中 chip-seq和cut＆tag。在AIM 2中，我们将开发一个用于高通量识别和量化的平台 由组蛋白修饰介导的结合相互作用。在AIM 3中，我们将为高通量开发一个平台 审问作家的居住偏好。我们的工作将开发可扩展的“开源”平台， 具有成本效益，并在染色质生物学领域的其他研究者轻松采用。这样的平台将是强大的 通过对共同和可理解的组蛋白进行研究来完成传统的生化测定 修改并解锁染色质生物学中的新的高通量测量和研究问题。