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

项目摘要 心脏、肺和血液中单个细胞类型的忠实发育和维持(HLB)是 依赖于基因组的特定细胞类型的解码。这些特定于细胞类型的转录程序 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 以特定于时间和地点的方式输出。由于它们在HLB生物学中的核心作用，一个全面的 对转铁蛋白功能的分子理解将揭示转铁蛋白对维持动态平衡的贡献，如 以及这种平衡的破坏如何导致HLB紊乱的进展。而方法是 了解转录因子是如何调控基因表达的，我们缺乏方法和分析工具来分析转录因子是如何存在的 他们自己受到翻译后的监管。通过狭隘地关注众所周知的表型(DNA结合) 使用成熟的方法(芯片序列、切割和标签)，我们无法在其他地方捕获生物信息 调控水平，例如E3泛素连接酶对Tf降解的翻译后控制。重要的是 这种失败并不是一个令人感兴趣的问题，而是因为缺乏方便、可扩展的研究方法 翻译功能的翻译后调控。 为了解决这一关键缺陷，我将开发一种新的测试方法，能够测量基因的影响 对转铁蛋白丰度的扰动达到了目前方法所不可能达到的程度(目标1)。这就做 最初将该方法应用于先前证明具有组织特异性表达的127个TF的文库 心、肺、血、骨髓或淋巴结1，然后再用标尺的方法研究其作用效果。 综合发现调节转铁蛋白的E3泛素连接酶对2000个转铁蛋白的遗传扰动 富足。(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多肽 (目标3)。这项研究将提供E3-Tf对的靶点连锁图谱，并导致对 控制转录因子蛋白水平的因素。此外，我们还将深入了解 翻译后转铁蛋白在动态平衡中的调节以及这种动态平衡的机制 变得不受监管。重要的是，在这个项目中发现的E3-Tf对将具有治疗作用 靶点，每一对代表一种新的蛋白质-蛋白质相互作用，以稳定或消融靶点。 作为奖学金的一部分，我还将接受一项旨在扩大我作为 实验生物学家，以及提高我的计算方法和统计知识。这个 研究和培训计划将与我的赞助人和共同赞助人Jay博士合作执行 Shendure和郑宁博士，两人都在华盛顿大学。我的顾问是被特别挑选出来的 因为他们每个人都有出色的尖端研究和培训记录 一代科学思想领袖。

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.