Comprehensive identification of E3 ubiquitin ligases that degrade heart, lung, and blood-relevant transcription factors

全面鉴定可降解心脏、肺和血液相关转录因子的 E3 泛素连接酶

• 批准号: 10677457
• 负责人: Chase Cameron Suiter
• 金额: $ 4.25万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677457
• 关键词: Ablation; Address; Affinity Chromatography; Amino Acid Motifs; Bar Codes; Binding; Biological; Biological Assay; Biology; Blood; Bone Marrow; Cell physiology; Cells; ChIP-seq; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Computing Methodologies; Coupled; DNA; DNA Binding; Data; Databases; Development; Disease; Engineering; Ensure; Equilibrium; Erythropoietin; Failure; Fellowship; Flow Cytometry; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Goals; Guide RNA; Heart; Heart Diseases; Hematological Disease; Homeostasis; Human; Human Genome; Hypoxia; Individual; Investigation; Knowledge; Libraries; Ligase; Link; Lung; Lung diseases; Maintenance; Maps; Mass Spectrum Analysis; Measures; Mediating; Methodology; Methods; Molecular; Mutate; Onset of illness; Organism; Output; Oxygen; Pathologic; Patients; Peptides; Phenotype; Play; Post-Translational Regulation; Production; Proteins; Proteolysis; Records; Red Blood Cell Count; Regulation; Reporter; Research; Role; Series; Specificity; Substrate Interaction; System; Testing; Therapeutic Intervention; Tissues; Training; Transcript; Transcriptional Regulation; Translating; Ubiquitin; Ubiquitin-mediated Proteolysis Pathway; Ubiquitination; Universities; Washington; analytical tool; body system; cell type; combinatorial; gene discovery; human disease; improved; insight; interest; lymph nodes; multicatalytic endopeptidase complex; new therapeutic target; next generation; next generation sequencing; novel; novel strategies; overexpression; preempt; prevent; programs; protein aminoacid sequence; protein degradation; protein protein interaction; proteostasis; red fluorescent protein; response; statistics; stem; therapeutic target; transcription factor; ubiquitin ligase; ubiquitin-protein ligase

Project Summary Faithful development and maintenance of individual cell types in the heart, lungs, and blood (HLB) are dependent upon cell type-specific decoding of the genome. These cell type-specific transcriptional programs are the result of the coordinated actions of many transcription factors (TFs), which regulate transcriptional output in a temporal and locus specific fashion. Due to their central role in HLB biology, a comprehensive molecular understanding of TF function would reveal the contribution of TFs to maintaining homeostasis, as well as inform how disruption of this balance can lead to the progression of HLB disorders. While methods to understand how TFs regulate gene expression exist, we lack methods and analytical tools to assay how TFs themselves are regulated post-translationally. By narrowly focusing on well-known phenotypes (DNA-binding) with well-developed methods (CHIP-seq, CUT&Tag), we fail to capture biological information at other regulatory levels, such as the post-translational control of TF degradation by E3 ubiquitin ligases. Importantly, this failure is not one of interest, but rather stems from the dearth of facile, scalable methods to study post-translation regulation of TFs. To address this critical shortcoming, I will develop a novel assay capable of measuring the effect of genetic perturbations on TF protein abundance at a scale that is not possible with current methods (Aim 1). I will initially apply this method to a library of 127 TFs previously demonstrated to have tissue specific expression in heart, lung, blood, bone marrow, or lymph node 1, and then subsequently scale the method to study the effect of genetic perturbation on 2000 TFs to comprehensively discover E3 ubiquitin ligases that regulate TF abundance. (Aim 2). Finally, I will map the minimal peptides necessary for observed changes in protein levels (Aim 3). This study will provide a target-linked map of E3-TF pairs, and lead to a broader understanding of the factors controlling transcription factor protein levels. Further, we will gain insights into the role of post-translational TF regulation in homeostasis as well as the mechanisms by which this homeostasis becomes dysregulated. Importantly, the E3-TF pairs discovered in this project will be of interest as therapeutic targets, with each pair representing a novel protein-protein interaction to target for stabilization or ablation. As a part of the fellowship, I will also undergo a training plan aimed at expanding my abilities as an experimental biologist as well as improving my knowledge of computational methods and statistics. The research and training plan will be performed in collaboration with my sponsor and co-sponsor, Dr. Jay Shendure and Dr. Ning Zheng, both at the University of Washington. My advisors were specifically chosen because they each have outstanding records of performing cutting edge research and training the next generation of scientific thought leaders.

项目摘要 心脏、肺和血液（HLB）中单个细胞类型的忠实发育和维持是 这取决于基因组的细胞类型特异性解码。这些细胞类型特异性转录程序 是许多转录因子（TF）协同作用的结果，这些转录因子调节转录水平， 以时间和位置特定的方式输出。由于它们在HLB生物学中的核心作用， 对TF功能的分子理解将揭示TF对维持体内平衡的贡献， 并告知这种平衡的破坏如何导致HLB障碍的进展。虽然方法， 了解TF如何调节基因表达的存在，我们缺乏方法和分析工具来分析TF如何调节基因表达。 它们本身是事后监管的。通过狭隘地关注众所周知的表型（DNA结合） 使用成熟的方法（CHIP-seq，CUT和Tag），我们无法在其他地方捕获生物信息。 调节水平，如通过E3泛素连接酶对TF降解的翻译后控制。重要的是， 这种失败并不是人们感兴趣的，而是由于缺乏简便的、可扩展的研究方法 转录因子的翻译后调节。 为了解决这个关键的缺点，我将开发一种新的检测方法，能够测量基因的影响， 在当前方法不可能的规模上对TF蛋白丰度进行扰动（目标1）。我会 首先将该方法应用于127个TF文库，所述TF先前被证明在 心脏、肺、血液、骨髓或淋巴结1，然后随后缩放该方法以研究 对2000个TF进行遗传扰动，以全面发现调节TF的E3泛素连接酶 丰饶。(Aim 2）的情况。最后，我将绘制观察到的蛋白质水平变化所需的最小肽 (Aim 3）。这项研究将提供一个E3-TF对的靶点连锁图，并导致对E3-TF对的更广泛的理解。 控制转录因子蛋白水平的因子。此外，我们将深入了解 翻译后TF调节稳态以及这种稳态的机制， 变得失调。重要的是，在这个项目中发现的E3-TF对将作为治疗性的感兴趣。 靶点，每一对代表一种新的蛋白质-蛋白质相互作用，以靶点稳定或消融。 作为奖学金的一部分，我还将接受一项旨在扩大我作为一名 实验生物学家以及提高我的计算方法和统计学知识。的 我的研究和培训计划将与我的赞助商和共同赞助商Jay博士合作进行 Shendure和Ning Zheng博士，都在华盛顿大学。我的顾问是专门挑选的 因为他们每个人都有出色的记录进行尖端研究和培训下一个 一代科学思想领袖。