Enhancer-Mediated BDNF Regulation

增强子介导的 BDNF 调节

• 批准号: 10660955
• 负责人: Arthy Narayanan
• 金额: $ 4.02万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10660955
• 关键词: Address; BDNF gene; Binding; Binding Sites; Biochemical; Biological Assay; Brain-Derived Neurotrophic Factor; CRISPR/Cas technology; ChIP-seq; Characteristics; Chimeric Proteins; Chromatin; Chromatin Loop; Clustered Regularly Interspaced Short Palindromic Repeats; Coupling; Data; Data Set; Development; Distal; Enhancers; Fright; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Growth Factor; Guide RNA; Hippocampus; Impairment; In Situ Hybridization; Learning; Link; Logic; Luciferases; Measures; Mediating; Memory; Messenger RNA; Methods; Molecular Conformation; Mus; NPAS4 gene; Neurons; Nucleic Acid Regulatory Sequences; Play; Publishing; RNA; Regulation; Regulatory Element; Reporter; Repression; Role; Signal Transduction; Specificity; Stimulus; Techniques; Testing; Transcriptional Regulation; Transfection; Transgenic Organisms; Untranslated RNA; Up-Regulation; cell type; conditioned fear; epigenome editing; experience; fear memory; impaired brain development; in vivo; neural circuit; neuronal survival; promoter; protein expression; recruit; response; sensory stimulus; transcription factor

ABSTRACT: Brain Derived Neurotrophic Factor (BDNF) plays essential roles in neuronal survival, differentiation, and plasticity, and it contributes to learning and memory. The expression of BDNF is highly induced by neuronal activity, allowing it to function in temporal coordination with the reception of sensory stimuli. Proper control of Bdnf expression is essential to its function, because expressing too little or too much BDNF at the wrong times or in the wrong places has been shown to impair brain development, plasticity, and function. Neuronal activity- dependent induction of BDNF expression is mediated primarily at the transcriptional level, via the regulation of stimulus-modulated transcription factors. Although prior studies sought evidence for mechanisms of Bdnf regulation at this gene’s promoters, recent evidence shows increased long-range chromatin interactions with Bdnf upon neuronal activation, which suggests the potential importance of transcriptional regulation at distal activity-regulated enhancers. Using previously published ChIP-seq datasets and the chromatin conformation data from these recent studies, I have identified six putative distal enhancer regions. Given the function of enhancers in conferring specificity upon the regulation of gene transcription, I hypothesize that these enhancers contribute to the stimulus-specific control of Bdnf transcription. In Aim 1, I will test the function and neuronal activity-dependent regulation of these distal enhancers in mouse cortical neurons both using reporter assays and in their endogenous chromatin context by activating or repressing the enhancers with dCas9-fusion proteins. In Aim 2, I will focus on an intragenic enhancer of BDNF to test the hypothesis that the binding of the transcription factor NPAS4 to this enhancer mediates a cell-type specific mechanism of regulation in the hippocampus in the context of fear in vivo. This aim will take advantage of transgenic dCas9/CRISPR mice that I will use for enhancer regulation in the context of neural circuits in vivo. Completion of these two aims will elucidate the specificity provided by enhancers in Bdnf regulation. This proposal will also highlight the significance of transcriptional regulatory mechanisms in coupling upstream stimuli to its appropriate downstream response.

抽象的： 脑源性神经营养因子 (BDNF) 在神经元存活、分化和发育过程中发挥重要作用 BDNF 的表达高度受神经诱导。 活动，使其能够与感觉刺激的接收进行时间协调的适当控制。 Bdnf 表达对其功能至关重要，因为在错误的时间表达过少或过多的 BDNF 或在错误的地方已被证明会损害大脑发育、可塑性和神经元活动。 BDNF 表达的依赖性诱导主要在转录水平上介导，通过调节 尽管先前的研究寻求 Bdnf 机制的证据。 该基因启动子的调控，最近的证据表明，与 神经激活时的 Bdnf，这表明远端转录调控的潜在重要性 使用先前发布的 ChIP-seq 数据集和染色质构象。 根据这些最近研究的数据，我确定了六个假定的远端增强子区域的功能。 增强子赋予基因转录调控特异性，我发现这些增强子 有助于 Bdnf 转录的刺激特异性控制。在目标 1 中，我将测试功能和神经。 小鼠皮质神经元中这些远端增强子的活性依赖性调节均使用报告基因检测 并在内源染色质环境中通过 dCas9 融合蛋白激活或抑制增强子。 在目标 2 中，我将重点关注 BDNF 的基因内增强子，以测试转录结合的假设 该增强子的因子 NPAS4 介导海马体中细胞类型特异性的调节机制 这个目标将利用我将用作增强剂的转基因 dCas9/CRISPR 小鼠。 体内神经回路的调节将阐明这两个目标的特异性。 该提案还将强调转录的重要性。 将上游刺激与其适当的下游响应耦合的调节机制。