Mechanisms regulating the formation and repair of neuronal activity-induced DNA breaks and their effects on learning behavior

项目摘要 经历对动物的行为有显著的影响。在分子水平上，新基因的启动 神经元中的转录对于经验驱动的适应行为的发展至关重要。此外， 神经元活性依赖转录程序的缺陷表现为认知障碍和神经功能障碍 精神错乱。因此，了解神经元活动依赖的转录是如何协调的具有重要意义。 在这方面的一个令人惊讶的新发现是，神经元活动的各种范例，包括暴露在 学习行为，诱导拓扑异构酶，拓扑异构酶IIb(Top2B)，以产生DNA双链 神经元基因组中特定位置的断裂(DSB)。这些活性诱导的DSB在体内富含 突出的早期反应基因(ERGs)的启动子，如Fos、Npas4和Egr1，以及DSB促进 这些ERG的快速转录。这些观察描述了一个耐人寻味的机制，它管理着 神经元活性依赖的转录。然而，究竟是如何形成和修复活动诱导的 DSB是如何被控制的，DSB如何刺激快速ERG诱导，以及DSB修复缺陷如何影响活动- 依赖的转录和学习行为仍然不清楚。这些主题将是本项目的重点。 该项目的初步数据表明，神经元刺激触发了Top2B的快速去磷酸化和 修饰其DNA切割活性。利用高分辨率成像和生化方法，拟议的 实验将揭示调节Top2B产生DSB的依赖于活动的信号机制 特定的基因组基因座。全基因组活动诱导的双链断裂的一个重要特征是它们形成于 由CTCF占用。CTCF染色质环路对基因增强子-启动子的拓扑障碍 接触，在初步研究中，即使在没有神经元的情况下，敲除CTCF也会提高ERG水平 刺激。这些结果表明，CTCF抑制ERG的表达，而活性诱导的DSB可以 是一种在ERG上快速覆盖CTCF强制的拓扑约束的机制。为了检验这一假设， 染色体构象捕捉(3C)将用于揭示活性诱导部位的染色质相互作用 并阐明DSB如何影响这些相互作用。此外，CTCF在调节启动子- 在基于CRISPR的特定CTCF位点突变之后，将探索ERGs上的增强子偶联。神经元 活性诱导的DSB通过非同源末端连接(NHEJ)修复。评估DSB的作用 在体内修复，应用先前利用的芯片序列策略来定位响应于 小鼠海马区的生理性学习行为。使用此信息并通过使用类似的 方法在NHEJ缺陷小鼠模型中，拟议的实验将识别全基因组位置 易受DSB影响的海马区，并研究DSB修复缺陷如何影响活动依赖 抄写。最后，NHEJ缺陷小鼠将受到适当的行为任务，以测试如何修复 活动诱导的DSB会影响学习和记忆。

Project Summary Experiences have a remarkable influence on animal behavior. At the molecular level, the initiation of new gene transcription in neurons is crucial for the development of experience-driven adaptive behaviors. Moreover, defects in neuronal activity-dependent transcription programs manifest in cognitive deficits and neurological disorders. Understanding how neuronal activity-dependent transcription is orchestrated is therefore significant. A surprising new finding in this regard is that various paradigms of neuronal activity, including exposure to learning behaviors, induce the topoisomerase, topoisomerase IIb (Top2B), to generate DNA double strand breaks (DSBs) at specific loci within the genome of neurons. These activity-induced DSBs are enriched within the promoters of prominent early response genes (ERGs), such as Fos, Npas4, and Egr1, and DSBs facilitate the rapid transcription of these ERGs. These observations describe an intriguing mechanism that governs neuronal activity-dependent transcription. However, precisely how the formation and repair of activity-induced DSBs is controlled, how DSBs stimulate rapid ERG induction, and how defective DSB repair affects activity- dependent transcription and learning behaviors remain obscure. These topics will be the focus of this project. Preliminary data for this project indicate that neuronal stimulation triggers rapid Top2B dephosphorylation and modifies its DNA cleavage activity. Employing high-resolution imaging and biochemical methods, the proposed experiments will unveil the activity-dependent signaling mechanisms that modulate Top2B to generate DSBs at specific genomic loci. A defining feature of genome-wide activity-induced DSBs is that they form at sites co- occupied by CTCF. Chromatin looping by CTCF creates topological barriers to gene enhancer-promoter contacts, and in preliminary studies, knockdown of CTCF elevated ERG levels even in the absence of neuronal stimulation. These results suggest that CTCF constrains ERG expression, and that activity-induced DSBs could be a mechanism to rapidly override CTCF-enforced topological constraints at ERGs. To test this hypothesis, chromosome conformation capture (3C) will be utilized to reveal chromatin interactions at sites of activity-induced DSBs and clarify how DSBs affect these interactions. Additionally, the roles of CTCF in regulating promoter- enhancer coupling at ERGs will be explored following CRISPR-based mutation of specific CTCF sites. Neuronal activity-induced DSBs are repaired through nonhomologous end joining (NHEJ). To assess the role of DSB repair in vivo, previously utilized ChIP-seq strategies were applied to map DSBs formed in response to physiological learning behaviors in the mouse hippocampus. Using this information and by employing similar methods in an NHEJ-deficient mouse model, the proposed experiments will identify genome-wide sites that are vulnerable to DSB accrual in the hippocampus, and study how defective DSB repair affects activity-dependent transcription. Finally, NHEJ-deficient mice will be subjected to appropriate behavioral tasks to test how the repair of activity-induced DSBs impacts learning and memory.