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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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/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。通过其控制基因表达、染色质生物化学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 以及特异性识别这些蛋白的其他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”来确定一组与组蛋白和组蛋白结合 DNA 相互作用的蛋白质，从而参与多种细胞流程。因此，识别特定的组蛋白修饰，定量评估相互作用它们介导，修饰组蛋白的酶的表征对于理解染色质至关重要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，因为它的复杂性——五种组蛋白上八十多个不同的氨基酸残基经历了二十多个不同的氨基酸残基已知的翻译后修饰。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未得到充分研究。进一步地，一个绝大多数酶即使经过广泛研究的修饰（例如乙酰化）仍然存在待研究。我们建议解决基本生化工具和可获得的实验方面的关键差距阻碍了对未充分研究的组蛋白修饰和修饰剂的研究的平台。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的平台，进行高通量研究正在研究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。除了未充分研究的组蛋白乙酰转移酶外，我们还将重点关注三类组蛋白修饰：(i) 精氨酸的瓜氨酸化 (ii) 非乙酰基对赖氨酸的酰基修饰 (丙酰化、丁酰化、巴豆酰化)，以及(iii)通过血清素对谷氨酰胺进行单胺修饰和多巴胺。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可以作为活细胞成像的基因编码生物传感器，以及传统的分析，如 ChIP-seq 和 CUT&Tag。在目标 2 中，我们将开发一个高通量识别和定量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询问作家的残余偏好。我们的工作将开发可扩展的“开源”平台，具有成本效益，并且很容易被染色质生物学的其他研究人员采用。此类平台将发挥强大的作用通过对常见和未充分研究的组蛋白进行研究来补充传统的生化测定修改，并解锁染色质生物学中新的高通量测量和研究问题。