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.

项目总结/摘要 真核生物基因组DNA与蛋白质和RNA广泛结合形成染色质。通过其控制 在基因表达过程中，染色质生物化学和结构的变化是几乎所有细胞过程的基础。 与基因组DNA结合的组蛋白的翻译后修饰在基因组的表达中起着特别关键的作用。 调节染色质结构和功能。某些组蛋白残基的修饰会影响 组蛋白与DNA的相互作用以及其他特异性识别这些蛋白质的蛋白质的相互作用 修改.组蛋白修饰的特定模式充当“组蛋白代码”以确定组蛋白修饰的集合。 与组蛋白和组蛋白结合的DNA相互作用的蛋白质，因此参与多种细胞 流程.因此，识别特定的组蛋白修饰，定量评估相互作用， 它们介导，修饰组蛋白的酶的特性对于理解染色质是必不可少的 调节复杂的细胞行为。然而，解开组蛋白密码是一个艰巨的挑战，因为它 复杂性-超过八十个不同的氨基酸残基上的五个组蛋白的蛋白质经历超过二十个不同的 已知的翻译后修饰。尽管一些组蛋白修饰的研究取得了重大进展， 例如乙酰化和甲基化，绝大多数组蛋白修饰仍然研究不足。此外，A 绝大多数酶，即使是经过广泛研究的修饰，如乙酰化， 替补演员我们建议解决关键的差距，在基本的生化工具和可访问的实验 这些平台阻碍了对未充分研究的组蛋白修饰和修饰剂的研究。具体来说，我们将 利用下一代酵母表面展示系统作为高通量研究的可扩展平台， 未充分研究的组蛋白修饰和修饰剂，以及工程生化试剂的平台， 染色质研究除了研究不足的组蛋白乙酰转移酶，我们将重点放在三类 组蛋白修饰：（i）精氨酸的瓜氨酸（ii）非乙酰基对赖氨酸的酰基修饰 （丙酰化、丁酰化、巴豆酰化），和（iii）谷氨酰胺被5-羟色胺和 多巴胺在目标1中，我们将开发一个平台，用于高效生成具有高特异性的亲和试剂 它可以作为基因编码的生物传感器，用于活细胞成像，以及传统的分析， ChIP-seq和CUT&Tag。在目标2中，我们将开发一个用于高通量鉴定和定量的平台 由组蛋白修饰介导的结合相互作用。在目标3中，我们将开发一个高吞吐量平台 对作者的剩余偏好的询问。我们的工作将开发可扩展的“开源”平台， 具有成本效益，并且易于被染色质生物学中的其他研究人员采用。这些平台将作为强大的 通过对常见的和未充分研究的组蛋白进行研究，补充了传统的生物化学测定 修饰，并解锁新的高通量测量和染色质生物学研究问题。