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

神经元活动诱导的 DNA 断裂形成和修复的调节机制及其对学习行为的影响

• 批准号: 10376801
• 负责人: Ram Madabhushi
• 金额: $ 40.12万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376801
• 关键词: Ablation; Adaptive Behaviors; Affect; Animal Behavior; Architecture; Behavior; Behavioral; Binding Sites; Biochemical; C-terminal; CRISPR/Cas technology; Calcineurin; Calcium; ChIP-seq; Chromatin; Chromatin Loop; Clustered Regularly Interspaced Short Palindromic Repeats; Cognitive deficits; Coupling; Cues; DNA; DNA Double Strand Break; DNA-PKcs; Data; Defect; Development; Distant; Double Strand Break Repair; ERG gene; Early Promoters; Enhancers; Event; Exposure to; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic Transcription; Genome; Genome Stability; Genomics; Hippocampus (Brain); Knowledge; Lead; Learning; Ligation; Light; Location; Maps; Mass Spectrum Analysis; Mediating; Memory; Methods; Molecular; Mus; Mutation; NPAS4 gene; Nervous system structure; Neurons; Nonhomologous DNA End Joining; Nuclear; Pathway interactions; Pattern; Performance; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Play; Process; Protein Dephosphorylation; Proteins; Role; Signal Transduction; Site; Stimulus; Synapses; Testing; Topoisomerase; base; chromosome conformation capture; cognitive performance; conditioned fear; experience; experimental study; gene induction; genome-wide; genomic locus; high resolution imaging; in vivo; knock-down; learned behavior; long term memory; morris water maze; mouse model; mutant; nervous system disorder; programs; promoter; repaired; response; transcription factor; transcriptome sequencing

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.

项目摘要 经验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性行为的发展至关重要。此外，委员会认为， 神经元活性依赖性转录程序的缺陷表现为认知缺陷和神经功能障碍。 紊乱因此，了解神经元活动依赖性转录是如何编排的是很重要的。 在这方面，一个令人惊讶的新发现是，神经元活动的各种范式，包括暴露于 学习行为，诱导拓扑异构酶，拓扑异构酶IIb（Top 2B），产生DNA双链 在神经元基因组内的特定位点处的断裂（DSB）。这些活性诱导的DSB富集在 主要早期反应基因（ERG）的启动子，如Fos、Npas 4和Egr 1，以及DSB， 这些ERG的快速转录。这些观察描述了一种有趣的机制， 神经元活性依赖性转录。然而，究竟是如何形成和修复活动诱导的， DSB是受控的，DSB如何刺激快速ERG诱导，以及缺陷DSB修复如何影响活动- 依赖性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，神经元刺激触发快速Top 2B去磷酸化， 改变其DNA切割活性。采用高分辨率成像和生物化学方法， 实验将揭示调节Top 2B产生DSB的活性依赖性信号机制， 特定的基因位点全基因组活性诱导的DSB的一个定义特征是它们形成于与DNA结合的位点， 被CTCF占领。CTCF染色质成环对基因增强子-启动子的拓扑屏障作用 接触，并在初步研究中，敲低CTCF升高ERG水平，即使在没有神经元的情况下， 刺激.这些结果表明，CTCF抑制ERG表达，并且活动诱导的DSB可以 成为一种机制，以快速覆盖CTCF强制的拓扑约束在ERG。为了验证这个假设， 染色体构象捕获（3C）将用于揭示活性诱导位点的染色质相互作用 争端解决机构，并澄清争端解决机构如何影响这些相互作用。此外，CTCF在调节启动子- 将在特定CTCF位点的基于CRISPR的突变之后探索ERG处的增强子偶联。神经元 活性诱导的DSB通过非同源末端连接（NHEJ）修复。评估争端解决机构的作用 在体内修复中，应用先前利用的ChIP-seq策略来映射响应于体内修复而形成的DSB。 小鼠海马的生理学习行为。利用这些信息，并通过使用类似的 方法在NHEJ缺陷小鼠模型中，所提出的实验将确定基因组范围内的位点， 易受海马DSB累积的影响，并研究缺陷的DSB修复如何影响活动依赖性 转录。最后，NHEJ缺陷小鼠将接受适当的行为任务，以测试修复如何进行 活动诱导的DSB影响学习和记忆。