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.

协调细胞内和细胞间的反应时间对于多细胞行为至关重要， 发展进化（或工程）分子起搏器的一种方法是通过诱导转录周转 阻遏物作用于多个位点-在不同的真核生物中观察到的解决方案。然而我们对地球上 关于蛋白质降解动力学如何转化为下游转录反应， 比如细胞命运的改变。一个可能的原因是缺乏高分辨率的可用模型 降解相关转录激活的结构-功能分析。我们利用生长素 反应，在植物生物学的几乎每一个方面的核心，作为一个模型，调查一般原则 潜在的泛素介导的降解及其与转录激活和形态发生的联系。 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， 并且促进了我们在酵母中生长素诱导的降解和转录激活的工程化。我们 对我们的“酵母生长素”系统的广泛研究导致了一个中心假设：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。最近一个关于 生长素计时器的分子机制是我们发现生长素中的转录抑制 由称为TPL的Groucho/Tup 1/TLE型辅阻遏物赋予的回路需要与中介体相互作用 复杂.我们的结果提示了一种新的转录调控模型，其中辅阻遏物可以稳定转录。 在不存在RNA聚合酶II的情况下的预起始复合物，引发位点快速活化。另外我们 已经在转基因植物中表明，降解触发的TPL辅阻遏物的去除速率设定了 根中从头器官发生的速度。在这里，我们将严格测试共阻遏蛋白的涌现模型- 基础启动促进跨基因组多个基因座的快速转录爆发的协调， 在形态发生期间促进细胞-细胞同步。具体的研究项目将：（1）提供一个高 一个单一的合成酵母基因座的时空分辨率，整合，功能图从 抑制到活性状态，包括蛋白质复合物的组成/放置和染色质修饰; (2)剖析一个新的自主TPL抑制结构域的机制，我们已经确定，发现在 数以千计的蛋白质，并量化对癌症相关变异体的抑制功能的影响， （3）构建体内细胞命运追踪器，以探测转录中的同步性与 形态发生总之，拟议的工作将提供一个机制框架， 转录激活生长素反应，并可能提供洞察的基本性质， 与许多辅阻遏物引发的系统相同。这些见解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合成电路的设计 用于治疗应用的组分。