Dissecting CEBPB Function with Synthetic Biology and Imaging

项目摘要 热量限制和mTOR信号传导减少减轻了衰老的不利影响。他们不翻译 然而，由于饮食难以维持，mTOR抑制剂表现出 不良副作用。转录因子CEBPB驱动了许多老化的不良影响 TOR通路的下游。其短亚型LIP的水平随着年龄的增长而增加; LIP的丧失 增加寿命并提供健康益处，而单独表达LIP增加致瘤性。 因此，重编程CEBPB和LIP作为控制衰老的工具具有很大的希望。 开创性的研究表明，LIP激活其靶基因的转录，而经典的长 CEBPB的同种型CEBPB是一种转录阻遏物。最近的证据描绘了一幅更为复杂的画面： - 这两种异构体都被磷酸化、乙酰化（和许多其他）广泛修饰， 修改影响其活动; - CEBPB与其他因素的二聚化影响其功能; - 最后，LIP挽救了CEBPB敲除，表明LIP不仅仅是一种显性负惰性基因 同种型 了解CEBPB亚型如何整合这些调控维度是 开发能够重新编程老化的技术。 在这里，我们部署合成生物学方法，以剖析如何CEBPB亚型，其 二聚化伴侣和翻译后修饰影响CEBPB的核动力学及其 转录调节活性。 我们还开发了合成工具来可视化和扰动单个细胞中的LIP：LIP比率。能够 在单个细胞中扰动老化调节器将能够测量细胞自主老化的贡献 表型和由细胞之间的信号驱动的表型。它还将提供精确的工具， 衰老的共同标志：表达的异质性增加。

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.