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 真核生物基因组DNA广泛地与蛋白质和RNA结合形成染色质。通过它的控制 在基因表达中，染色质生化和结构的变化几乎是所有细胞过程的基础。 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。某些组蛋白残基的修饰影响结合 组蛋白与DNA的相互作用以及其他识别这些组蛋白的蛋白质之间的相互作用 修改。组蛋白修饰的特定模式起到“组蛋白密码”的作用，以确定 与组蛋白和组蛋白结合的DNA相互作用，从而参与不同细胞的蛋白质 流程。因此，鉴定特定的组蛋白修饰，定量评估相互作用 它们起中介作用，而修饰组蛋白的酶的特性对于理解染色质是必不可少的 对复杂细胞行为的调节。然而，解开组蛋白密码是一项艰巨的挑战，因为它 复杂性--五种组蛋白上80多种不同的氨基酸残基经历了20多种不同的 已知的翻译后修饰。尽管在一些组蛋白修饰的研究中取得了重大进展 例如乙酰化和甲基化，绝大多数组蛋白修饰仍未得到充分研究。此外，一个 绝大多数酶，即使是经过广泛研究的修饰，如乙酰化，仍然存在 未得到充分研究。我们建议解决必要的生化工具和可获得的实验的关键差距 阻碍了对未充分研究的组蛋白修饰和修饰物的研究的平台。具体来说，我们将 利用下一代酵母表面展示系统作为可扩展平台，用于高通量研究 研究不足的组蛋白修饰和修饰剂，以及用于工程生化试剂的平台 染色质研究。除了未被充分研究的组蛋白乙酰转移酶外，我们还将重点介绍三类 组蛋白修饰：(I)精氨酸的瓜氨酸化(Ii)非乙酰基对赖氨酸的酰化修饰 (3)5-羟色胺和5-羟色胺对谷氨酰胺的单胺修饰 多巴胺。在目标1中，我们将开发一个高效生成高特异性亲和试剂的平台 它可以作为遗传编码的生物传感器用于活细胞成像，以及在常规分析中，如 芯片序列和裁剪标签。在目标2中，我们将开发一个高通量识别和量化的平台 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的平台 询问作家的残留物偏好。我们的工作将开发可扩展的“开源”平台， 性价比高，易于被染色质生物学的其他研究人员采用。这样的平台将发挥强大的作用 通过对常见的和未被研究的组蛋白的研究来补充传统的生化分析 改进和解锁染色质生物学中新的高通量测量和研究问题。