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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要心脏、肺和血液 (HLB) 中各个细胞类型的忠实发育和维持是依赖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）协调作用的结果，这些转录因子调节转录以时间和位点特定方式输出。由于它们在 HLB 生物学中的核心作用，综合对 TF 功能的分子理解将揭示 TF 对维持稳态的贡献，如并告知这种平衡的破坏如何导致 HLB 疾病的进展。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 如何存在它们本身受到翻译后调节。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&Tag），我们无法在其他方法中捕获生物信息监管水平，例如 E3 泛素连接酶对 TF 降解的翻译后控制。重要的是，这种失败并不是出于兴趣，而是源于缺乏简便、可扩展的研究方法 TF 的翻译后调控。为了解决这个关键缺点，我将开发一种能够测量遗传影响的新方法对 TF 蛋白丰度的扰动达到了当前方法无法实现的程度（目标 1）。我会最初将此方法应用于先前已证明具有组织特异性表达的 127 个 TF 文库心脏、肺、血液、骨髓或淋巴结 1，然后按比例缩放方法来研究效果对2000个TF进行遗传扰动，全面发现调节TF的E3泛素连接酶丰富。（目标 2）。最后，我将绘制观察到的蛋白质水平变化所需的最小肽图（目标 3）。这项研究将提供 E3-TF 对的靶标关联图谱，并导致对 E3-TF 对的更广泛理解。控制转录因子蛋白水平的因素。此外，我们还将深入了解稳态中翻译后转录因子的调节以及这种稳态的机制变得失调。重要的是，该项目中发现的 E3-TF 对作为治疗药物将引起人们的兴趣。目标，每一对代表一种新的蛋白质-蛋白质相互作用，以实现稳定或消融。作为奖学金的一部分，我还将接受一项培训计划，旨在扩展我作为一名研究人员的能力实验生物学家以及提高我的计算方法和统计学知识。这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 Cheng 博士，均来自华盛顿大学。我的顾问是专门挑选的因为他们每个人都有进行前沿研究和培训下一代的杰出记录一代科学思想领袖。