Control of protein degradation and transcriptional dynamics in the auxin response

生长素反应中蛋白质降解和转录动力学的控制

• 批准号: 10549582
• 负责人: JENNIFER L NEMHAUSER
• 金额: $ 37.42万
• 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: 2023-06-10 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10549582
• 关键词: Auxins; Biochemical; Biology; Cells; Complex; Development; Engineering; Eukaryota; Excision; Future; Genetic Transcription; Genome; Heart; Kinetics; Link; Logic; Malignant Neoplasms; Maps; Mediating; Mediator; Modeling; Molecular; Molecular Medicine; Morphogenesis; Organogenesis; Outcome; Pathway interactions; Plants; Property; Proteins; RNA Polymerase II; Reaction Time; Repression; Research Project Grants; Resolution; Structure; System; Testing; Therapeutic; Transcription Initiation; Transcription Repressor; Transcriptional Activation; Transcriptional Regulation; Transgenic Plants; Translating; Ubiquitin; Variant; Work; Yeasts; cell behavior; chromatin modification; design; gene repression; genetic corepressor; human disease; in vivo; insight; novel; protein complex; protein degradation; response; small molecule; spatiotemporal

Coordinating the timing of responses both within and between cells is critical for multicellular behaviors like development. One way to evolve (or engineer) a molecular pacer is via induced turnover of transcriptional repressors acting at multiple loci—a solution observed in diverse eukaryotes. Yet we know remarkably little about how protein degradation kinetics translate into downstream transcriptional responses that lead to outcomes like changes in cell fate. One possible reason is the lack of available models for high-resolution structure-function analysis of degradation-linked transcriptional activation. We have leveraged the auxin response, at the heart of nearly every aspect of plant biology, as a model to investigate general principles underlying ubiquitin-mediated degradation and its connection to transcriptional activation and morphogenesis. The small-molecule triggered degradation in the auxin pathway offers a unique advantage for these studies, and has facilitated our engineering of auxin-induced degradation and transcriptional activation in yeast. Our extensive work with our ‘AuxInYeast’ system has led to a central hypothesis: the auxin system functions as a developmental timer in plants, and similar logic circuits act in many eukaryotes. One recent insight into the molecular mechanism underlying the auxin timer was our discovery that transcriptional repression in the auxin circuit, conferred by a Groucho/Tup1/TLE-type corepressor called TPL, requires interaction with the Mediator complex. Our results suggest a new model of transcriptional regulation where corepressors can stabilize the Pre-Initiation Complex in the absence of RNA Polymerase II, priming loci for rapid activation. In addition, we have shown in transgenic plants that the rate of degradation-triggered removal of the TPL corepressor sets the pace of de novo organogenesis in the root. Here, we will rigorously test the emergent model that corepressor- based priming facilitates coordination of rapid transcriptional bursts at multiple loci across the genome, and facilitates cell-cell synchrony during morphogenesis. Specific research projects will: (1) Deliver a high spatiotemporal resolution, integrated, functional map of a single synthetic yeast locus transitioning from repressed to active state, including composition/placement of protein complexes and chromatin modifications; (2) Dissect the mechanism of a novel autonomous TPL repression domain we have identified that is found in thousands of proteins, and quantify the impacts on repressive function of cancer-associated variants in select proteins; (3) Build an in vivo cell fate tracker to probe the connection between synchrony in transcription and morphogenesis. Together, the proposed work will provide a mechanistic framework for degradation-initiated transcriptional activation in the auxin response, and potentially provide insights into fundamental properties in common with many corepressor-primed systems. These insights can inform our understanding of degradation- and transcription-associated human disease, as well as guiding future design of synthetic circuits using auxin components for therapeutic applications.

协调细胞内和细胞之间的反应时间对于多细胞行为至关重要，如 发展。进化(或设计)分子步行者的一种方式是通过诱导转录的翻转 作用于多个位点的抑制物--在不同的真核生物中观察到的一种溶液。然而，我们对此知之甚少 关于蛋白质降解动力学如何转化为下游转录反应从而导致 结果就像细胞命运的变化。一个可能的原因是缺乏可用于高分辨率的模型 降解相关转录激活的结构-功能分析。我们已经利用了生长素 反应，几乎是植物生物学各个方面的核心，作为研究一般原理的模型 泛素介导的降解及其与转录激活和形态发生的关系。 生长素途径中的小分子引发的降解为这些研究提供了独特的优势， 并促进了生长素在酵母中诱导降解和转录激活的工程实现。我们的 对我们的‘AuxInYEast’系统的广泛研究导致了一个中心假说：生长素系统的功能是 植物中的发育计时器，以及许多真核生物中类似的逻辑电路。一项最新洞察 生长素计时器背后的分子机制是我们发现生长素的转录抑制 由称为TPL的Groucho/Tup1/TLE类型的辅抑制子授予的电路需要与调解器交互 很复杂。我们的结果提示了一种新的转录调控模式，在这种模式下，辅抑制子可以稳定 预引发复合体在没有RNA聚合酶II的情况下，启动基因座进行快速激活。此外，我们 已经在转基因植物中表明，由降解引发的TPL辅阻遏子的移除速度设置了 根中新器官发生的速度。在这里，我们将严格测试核心抑制器- 基于启动的有助于协调基因组上多个基因座的快速转录爆发，以及 在形态发生过程中促进细胞间的同步化。具体的研究项目将：(1)提供高 单个合成酵母基因座的时空分辨率、集成功能图 抑制到活性状态，包括蛋白质复合体的组成/放置和染色质修饰； (2)剖析了我们发现的一个新的自主TPL抑制结构域的机制。 数以千计的蛋白质，并量化选择的癌症相关变体对抑制功能的影响 蛋白质；(3)建立体内细胞命运追踪器，以探索转录同步性与 形态发生。总之，拟议的工作将为退化启动提供一个机制框架 在生长素反应中的转录激活，并潜在地为深入了解 在许多辅酶阻滞剂启动的系统中常见。这些洞察力可以帮助我们理解退化-- 和转录相关的人类疾病，以及指导未来使用生长素的合成电路设计 用于治疗应用的组件。