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

基本信息

批准号：
10596091
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
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10596091
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 Darkness 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 Mappings Genome Stability Genomics Hippocampus Knowledge Learning Ligation Light Location Maps Mass Spectrum Analysis Mediating Memory Methods Molecular Mus Mutation NPAS4 gene Nervous System Neurons Nonhomologous DNA End Joining Nuclear Pathway interactions Pattern Performance Phosphoric Monoester Hydrolases Phosphorylation Physiological Play Process Protein Dephosphorylation Proteins Role Shapes Signal Transduction Site Stimulus Synapses Testing Topoisomerase chromosome conformation capture cognitive performance conditioned fear experience experimental study gene induction genome-wide genome-wide analysis 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

项目摘要

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行为产生了显着影响。在分子水平上，新基因的启动神经元中的转录对于发展经验驱动的自适应行为至关重要。而且，神经元活动依赖性转录程序的缺陷表现出在认知缺陷和神经功能中疾病。因此，了解神经元活性依赖性转录的序列是重要的。在这方面，一个令人惊讶的新发现是神经元活动的各种范例，包括暴露于学习行为，诱导拓扑异构酶，拓扑异构酶IIB（top2b）生成DNA双链神经元基因组内的特定基因座的断裂（DSB）。这些活动引起的DSB在突出的早期反应基因（ERG）的启动子，例如FOS，NPAS4和EGR1和DSB，促进这些ERG的快速转录。这些观察结果描述了一种引人入胜的机制神经元活动依赖性转录。但是，精确地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DSB维修有缺陷的影响 - 依赖的转录和学习行为仍然晦涩难懂。这些主题将是该项目的重点。该项目的初步数据表明，神经元刺激会触发快速的TOP2B去磷酸化和修饰其DNA裂解活性。采用高分辨率成像和生化方法，提出了实验将揭示活动依赖性信号传导机制，该机制调节TOP2B以生成DSB 特定的基因组基因座。全基因组活性诱导的DSB的一个定义特征是它们在位点形成由CTCF占领。 CTCF染色质循环为基因增强子促进剂创建拓扑障碍接触和初步研究中，即使没有神经元，CTCF的敲低也会升高ERG水平刺激。这些结果表明CTCF限制了ERG表达，并且活动诱导的DSB可以成为迅速覆盖ERGS的CTCF强化拓扑约束的机制。为了检验这一假设，染色体构象捕获（3C）将用于揭示活动诱导的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基于CRISPR的特定CTCF位点突变之后，将探索ERGS的增强耦合。神经元通过非同源末端连接（NHEJ）修复活性引起的DSB。评估DSB的作用在体内维修，以前利用的芯片序列策略用于响应于小鼠海马中的生理学习行为。使用此信息并采用类似的 NHEJ缺陷小鼠模型中的方法，所提出的实验将确定全基因组位点在海马中容易受到DSB的影响，并研究有缺陷的DSB维修如何影响活动依赖性转录。最后，NHEJ缺陷小鼠将受到适当的行为任务，以测试如何维修活动引起的DSB会影响学习和记忆。