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.

项目概要 经验对动物行为有显着影响。在分子水平上，新基因的启动 神经元的转录对于经验驱动的适应性行为的发展至关重要。而且， 神经元活动依赖性转录程序的缺陷表现为认知缺陷和神经功能障碍 失调。因此，了解神经元活动依赖性转录是如何协调的具有重要意义。 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（Top2B），产生DNA双链 神经元基因组内特定位点的断裂（DSB）。这些活动诱导的 DSB 富集于 重要早期反应基因 (ERG) 的启动子，例如 Fos、Npas4 和 Egr1，以及 DSB 促进 这些 ERG 的快速转录。这些观察描述了一种有趣的机制 神经元活动依赖性转录。然而，究竟如何形成和修复活动诱导的 DSB 受到控制，DSB 如何刺激快速 ERG 诱导，以及有缺陷的 DSB 修复如何影响活动 - 依赖转录和学习行为仍然不清楚。这些主题将是该项目的重点。 该项目的初步数据表明，神经元刺激会触发 Top2B 快速去磷酸化， 改变其 DNA 切割活性。采用高分辨率成像和生化方法，提出的 实验将揭示调节 Top2B 生成 DSB 的活动依赖性信号传导机制 特定的基因组位点。全基因组活性诱导的 DSB 的一个决定性特征是它们在共同位点形成 被CTCF占用。 CTCF 形成的染色质环对基因增强子-启动子造成拓扑障碍 在初步研究中，即使在神经元缺失的情况下，CTCF 的敲除也会升高 ERG 水平。 刺激。这些结果表明 CTCF 限制 ERG 表达，并且活动诱导的 DSB 可以 是一种快速克服 ERG 上 CTCF 强制拓扑约束的机制。为了检验这个假设， 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 DSB 并阐明 DSB 如何影响这些相互作用。此外，CTCF 在调节启动子中的作用 在特定 CTCF 位点的基于 CRISPR 的突变之后，将探索 ERG 的增强子耦合。神经元 活性诱导的 DSB 通过非同源末端连接 (NHEJ) 进行修复。评估 DSB 的作用 体内修复，之前使用的 ChIP-seq 策略被应用于绘制响应形成的 DSB 小鼠海马体的生理学习行为。使用此信息并采用类似的 方法在 NHEJ 缺陷小鼠模型中，所提出的实验将识别全基因组位点 易受海马 DSB 累积的影响，并研究缺陷 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，以测试修复情况如何 活动诱发的 DSB 会影响学习和记忆。