Dissecting CEBPB Function with Synthetic Biology and Imaging

项目摘要 卡路里限制和mTOR信号的减少减轻了衰老的不利影响。他们不会翻译 有效的抗衰老疗法，因为饮食很难维持，而且mTOR抑制剂表现出 不良副作用。转录因子CEBPB驱动了衰老的许多不良影响 在TOR路径的下游。其称为LIP的短亚型的水平随着年龄的增长而增加； 延长寿命和提供健康益处，而表达LIP本身就增加了致瘤性。 因此，重新编程CEBPB和LIP作为控制衰老的工具大有可为。 开创性的研究表明，LIP激活了其目标基因的转录，而规范的Long CEBPB的异构体LAP是一种转录抑制因子。最近的证据描绘了一幅更复杂的图景： -这两种异构体都通过磷酸化、乙酰化(和许多其他)和那些 修改会影响他们的活动； -CEBPB的二聚化以及影响其功能的其他因素； -最后，LIP拯救了CEBPB基因敲除，表明LIP不仅仅是一种显性的负惰性 异构体。 了解CEBPB异构体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，我们使用合成生物学的方法来剖析CEBPB的异构体、它们的 二聚化伙伴和翻译后修饰对CEBPB及其核动力学的影响 转录调节活性。 我们还开发了合成工具来可视化和干扰单个细胞中的LAP：LIP比率。能够 在单个细胞中扰动衰老调节剂将使测量细胞自主衰老的贡献成为可能 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解剖其中一个 衰老的共同特征：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.