Dissecting CEBPB Function with Synthetic Biology and Imaging

用合成生物学和影像学剖析 CEBPB 功能

基本信息

批准号：
10553690
负责人：
Timothee Lionnet
金额：
$ 64.54万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-01 至 2026-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10553690
关键词：
Acetylation Address Adverse effects Age Aging Binding Biological Assay Biology Biophysical Process Caloric Restriction Cell Nucleus Cells Chromatin Complex Crowding Custom DNA Binding Domain DNA Library Diet Diffusion Dimensions Dimerization Dominant-Negative Mutation Engineering Exhibits FRAP1 gene Fluorescence Resonance Energy Transfer Genes Genetic Transcription Goals Health Health Benefit Heterodimerization Heterogeneity Image Imaging Device In Vitro Individual Knock-out Libraries Light Longevity Measures Mediating Mediator Messenger RNA Metabolic Modification Molecular Monitor Nuclear PPARG gene Pathway interactions Phase Phenotype Phosphorylation Physical condensation Play Post-Translational Protein Processing Protein Isoforms Proteins Recording of previous events Reporter Reporter Genes Reporting Role Signal Transduction System Technology Testing Tissues Touch sensation Transcription Coactivator Transcription Repressor Transcriptional Activation Transcriptional Regulation Translating Tumorigenicity Visualization age effect anti aging biophysical model design dimer epigenetic memory improved interest lipid biosynthesis mTOR Inhibitor mTOR inhibition pharmacologic programs promoter quantitative imaging recruit restraint scaffold senescence side effect single molecule small molecule synthetic biology therapeutic target tool transcription factor

项目摘要

Project Summary Caloric restriction and reduced mTOR signaling mitigate the adverse effects of aging. They do not translate into effective anti-aging therapies though, because diets are difficult to maintain and mTOR inhibitors exhibit undesirable side effects. The transcription factor CEBPB drives many of the undesirable effects of aging downstream of the TOR pathway. The levels of its short isoform called LIP increase with age; loss of LIP increases lifespan and provides health benefits, while expression of LIP alone increase tumorigenicity. Reprogramming CEBPB and LIP therefore holds great promise as a tool to control aging. Pioneering studies indicated that LIP activates transcription of its target genes, while the canonical long isoform of CEBPB, LAP, is a transcription repressor. Recent evidence draws a more complex picture: - both isoforms are extensively modified by phosphorylation, acetylation (and many others), and those modifications impact their activity; - dimerization of CEBPB with other factors impacts its function; - finally, LIP rescues CEBPB knock-out, suggesting that LIP is not simply a dominant negative inert isoform. Understanding how these regulatory dimensions are integrated by CEBPB isoforms is a pre-requisite to develop technologies able to reprogram aging. Here, we deploy synthetic biology approaches in order to dissect how the CEBPB isoforms, their dimerization partners and post-translational modifications impact the nuclear dynamics of CEBPB and its transcription regulation activity. We also develop synthetic tools to visualize and perturb the LAP:LIP ratio in individual cells. Being able to perturb aging regulators in single cells will enable measuring the contributions of cell-autonomous aging phenotypes and those driven by signals between cells. It will also provide precise tools to dissect one of the common hallmarks of aging: increased heterogeneity of expression.

项目概要热量限制和 mTOR 信号传导减少可以减轻衰老的不利影响。他们不翻译然而，由于饮食难以维持且 mTOR 抑制剂表现出，有效的抗衰老疗法不良副作用。转录因子 CEBPB 会导致许多衰老的不良影响 TOR 通路的下游。其称为 LIP 的短异构体的水平随着年龄的增长而增加。唇部缺失延长寿命并提供健康益处，而 LIP 单独表达会增加致瘤性。因此，重编程 CEBPB 和 LIP 作为控制衰老的工具具有广阔的前景。开创性研究表明，LIP 激活其靶基因的转录，而经典的长 CEBPB 的亚型 LAP 是一种转录抑制因子。最近的证据描绘了一幅更为复杂的图景： - 两种亚型均通过磷酸化、乙酰化（和许多其他）进行广泛修饰，以及修改会影响他们的活动； - CEBPB与其他因素的二聚化影响其功能； - 最后，LIP 拯救了 CEBPB 敲除，表明 LIP 不仅仅是显性负惰性同工型。了解 CEBPB 同工型如何整合这些监管维度是开发能够重新编程衰老的技术。在这里，我们采用合成生物学方法来剖析 CEBPB 亚型及其二聚化伙伴和翻译后修饰影响 CEBPB 及其核动力学转录调控活性。我们还开发了合成工具来可视化和扰乱单个细胞中的 LAP:LIP 比率。能够扰乱单细胞中的衰老调节因子将能够测量细胞自主衰老的贡献表型和由细胞间信号驱动的表型。它还将提供精确的工具来剖析其中之一衰老的共同特征：表达异质性增加。