Enhancer-Mediated BDNF Regulation

摘要： 脑源性神经营养因子（BDNF）在神经元的存活、分化和分化中起重要作用。 可塑性，它有助于学习和记忆。BDNF的表达受神经元的高度诱导， 活动，使其能够与感官刺激的接收进行时间协调。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太少或太多BDNF 或在错误的地方被证明会损害大脑的发育，可塑性和功能。神经元活动- BDNF表达的依赖性诱导主要在转录水平介导，通过调节 刺激调节的转录因子。虽然先前的研究寻求证据的机制BDNF 最近的证据表明，在该基因的启动子调控增加的长距离染色质相互作用， BDNF对神经元激活的影响，这表明转录调控在远端的潜在重要性。 活性调节增强子。使用先前发表的ChIP-seq数据集和染色质构象 从这些最近的研究数据，我已经确定了六个假定的远端增强子区域。由于功能 增强子在赋予基因转录调节的特异性，我假设这些增强子 有助于刺激特异性控制BDNF转录。在目标1中，我将测试功能和神经元 活性依赖性调节这些远端增强子在小鼠皮层神经元都使用报告分析 并且在它们的内源性染色质环境中通过用dCas 9融合蛋白激活或抑制增强子。 在目标2中，我将集中在BDNF的基因内增强子上，以检验转录的结合是否与BDNF的转录水平有关的假设。 NPAS 4因子与该增强子的结合介导了海马中的细胞类型特异性调节机制， 恐惧的背景下在体内。这一目标将利用我将用作增强子的转基因dCas 9/CRISPR小鼠 在体内神经回路的背景下调节。这两个目标的完成将阐明 由BDNF调节中的增强子提供。这一建议也将突出转录的意义 调节机制，将上游刺激与其适当的下游反应偶联。

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.