Noncanonical E2F Regulation in the Neuronal DNA Damage Response

项目摘要/摘要 E2F转录程序，它控制细胞周期承诺和增殖过程 细胞，在神经元DNA损伤后上调。神经元DNA损伤与细胞周期失调 是神经退行性疾病的特征，也与精神疾病有关。尽管 E2F在增殖中的作用已被广泛研究，E2F在非增殖细胞中的作用，如 就像在神经元中一样，受到的关注较少。新的证据表明E2F在DNA损伤中发挥作用 修复，独立于其在细胞周期进入中的作用。这种作用在神经元中可能特别重要，它是 特别容易受到DNA损伤。尽管E2F诱导与DNA损伤的解决有关 神经元的大量细胞制备，这种整体分析不能解释潜在的混杂异质性 在样本中。单细胞分析是检验E2F活性和DNA之间关系的必要手段 并将E2F动态与同一细胞中的功能结果联系起来。 迈耶实验室专门从事高通量显微镜数据的单细胞分析，该实验室已经 最近开发了一种用于检测E2F活性的荧光生物传感器。使用活体显微技术的定量显微镜 E2F活性和DNA损伤的固定细胞读数将允许将信号历史映射到细胞命运 以数千个细胞为单位的单个细胞分辨率的结果。我的假设是，在有丝分裂后神经元中，E2F是 在亚致死性DNA损伤后，可逆地激活以驱动DNA修复，而不复制DNA；然而，如果 DNA损伤激活E2F超过S期进入门槛，E2F诱导DNA复制和 细胞凋亡。通过表征E2F程序在神经元中是如何调节的，以及它如何对DNA做出贡献 损伤修复，这项研究将支持确定潜在的治疗目标，以保存基因 以基因毒性应激为特征的脑部疾病的完整性。 这项研究项目是我独立职业生涯培训的重要组成部分 研究人员，使用高通量单细胞方法研究复杂疾病的生物学。我的龙- 学期目标是成为一名内科科学家，以精神病学家的身份执业，同时管理一个基础科学实验室 在一家学术医院。这份提案中概述的计划以及我的赞助商论文的指导 委员会和Tri-I医学博士项目的领导，将帮助我实现这些职业抱负。

PROJECT SUMMARY/ABSTRACT The E2F transcriptional program, which controls cell cycle commitment and progression in proliferating cells, is upregulated following DNA damage in neurons. Neuronal DNA damage and cell cycle dysregulation are features of neurodegeneration that have also been associated with psychiatric diseases. Although the function of E2F during proliferation has been extensively studied, the role of E2F in nonproliferating cells, such as in neurons, has received less attention. Emerging evidence indicates that E2F plays a role in DNA damage repair, independent of its role in cell cycle entry. This role may be especially important in neurons, which are uniquely vulnerable to DNA damage. Although E2F induction has been linked to resolution of DNA damage in bulk-cell preparations of neurons, such bulk analyses fail to account for potentially confounding heterogeneity within samples. Single cell analysis is necessary to examine the relationship between E2F activity and DNA damage and to link E2F dynamics to functional outcomes in the same cells. The Meyer Lab specializes in single-cell analysis of high-throughput microscopy data, and the lab has recently developed a fluorescent biosensor of E2F activity for this purpose. Quantitative microscopy using live- and fixed-cell readouts of E2F activity and DNA damage will allow signaling history to be mapped to cell fate outcomes at single-cell resolution in thousands of cells. My hypothesis is that in postmitotic neurons, E2F is reversibly activated to drive DNA repair without DNA replication following sublethal DNA damage; however, if DNA damage activates E2F beyond the threshold for S-phase entry, E2F induces DNA replication and apoptosis. By characterizing how the E2F program is regulated in neurons and how it contributes to DNA damage repair, this study will support the identification of potential treatment targets for preserving genetic integrity in brain disorders characterized by genotoxic stress. This research project represents an important component of my training for a career as an independent investigator, employing high-throughput single-cell methods to study the biology of complex diseases. My long- term goal is to become a physician-scientist, practicing as a psychiatrist while also running a basic science lab in an academic hospital. The plan outlined in this proposal along with the mentorship of my sponsor, thesis committee, and the leadership of the Tri-I MD- PhD program, will help me achieve these career aspirations.