Functional Genomic Analysis of Neural Activity-Regulated Enhancer Deactivation

 描述（由申请人提供）：成年人大脑最显著的特征之一是其响应感官体验以学习新信息并适应性地响应环境提出的新要求而经历结构和功能可塑性的能力。这种神经可塑性的细胞机制需要活性依赖的基因转录。强调这种经验依赖性基因表达对人类大脑的生物学意义是诱导基因表达的分子组分与人类认知发育障碍（如自闭症）之间的明确遗传关联。活性调节基因表达的一个基本方面是选择性失活的立即早期基因（IEG），最初和迅速诱导的神经元活动。这与延迟应答基因（DRG）的延迟、延长的转录形成对比。IEG的选择性失活被认为对塑造细胞可塑性至关重要，因为IEG蛋白产物（如ARC）的精确水平决定了神经网络内突触连接的强度。我们打算通过利用最近对神经活性依赖性增强子的全面鉴定来揭示这种IEG特异性失活的机制。关于IEGs和DRG之间的动力学转录差异是否由增强子、启动子或两者顺式编码，存在相互矛盾的模型。因此，我们有充分的条件来测试这些预测中的每一个。我们将通过三种关键方法实现这一目标：在目标1中，我们将使用ChIP-seq和eRNA-sequencing来监测神经活动时间过程中全基因组增强子的活性，以确定在持续存在的活动中失活的增强子子集。在目标2中，我们将分离每个感兴趣的增强子序列，并在全局和无偏的报告基因测定中研究其内在活性，以鉴定那些在持续活性下稳定的特异性增强子序列，从而可能协调IEG负调控。在目标3中，我们将在代表性的基因组位点评估内源性增强子景观是否足以编码独立于核心启动子序列的IEG特异性失活。我们将通过使用基因组工程技术来交换两个IEG和两个DRG的核心启动子以确定IEG增强子是否具有内在的负调节活性，其在延长的去极化条件下下调靶启动子的转录，从而实现目标3。这项工作将确定协调IEG负调控的顺式调控序列，并将提供基本的洞察活动调节增强子功能和编码的诱导调控逻辑，区分启动子和增强子的机制。它将推进我们对IEG失活分子机制的追求，并有可能确定新的分子靶点，以帮助开发治疗认知或记忆衰退疾病的疗法。

 DESCRIPTION (provided by applicant): One of the most remarkable features of the adult brain is its capacity to undergo structural and functional plasticity in response to sensory experience to learn new information and to adaptively respond to new demands posed by the environment. The cellular mechanisms that underlie this neural plasticity require activity-dependent gene transcription. Underscoring the biological significance of this experience-dependent gene expression to the human brain are the clear genetic associations between the molecular components of inducible gene expression and human developmental disorders of cognition, such as autism. A fundamental aspect of activity-regulated gene expression is the selective deactivation of the immediate early genes (IEGs) that are initially and rapidly induced by neuronal activity. This is in contrast to the delayed, prolonged transcription of delayed response genes (DRGs). The selective deactivation of IEGs is thought to be critically important for shaping cellular plasticity, as the precise levels of IEG protein products, such as ARC, determine the strength of synaptic connections within neural networks. We intend to uncover the mechanisms of this IEG-specific deactivation by taking advantage of the recent comprehensive identification of neural activity-dependent enhancers. There are conflicting models concerning whether the kinetic transcriptional differences between IEGs and DRGs are encoded in cis by the enhancers, the promoters, or both. Thus, we are well positioned to test each of these predictions. We will accomplish this via three key approaches: In Aim 1, we will use ChIP-seq and eRNA-sequencing to monitor genome-wide enhancer activity over a time course of neural activity to identify subsets of enhancers that are deactivated in the continued presence of activity. In Aim 2, we will isolate each enhancer sequence of interest and study its intrinsic activity in a global and unbiased reporter assay to identify those specific enhancer sequences that inactivate under continued activity and may therefore coordinate IEG negative regulation. In Aim 3, we will evaluate at representative genomic loci whether endogenous enhancer landscapes are sufficient to encode IEG-specific deactivation, independent of the core promoter sequences. We will accomplish Aim 3 by using genomic engineering techniques to exchange core promoters of two IEGs and two DRGs to determine whether IEG enhancers possess intrinsic negative regulatory activity that down-regulates the transcription of target promoters under prolonged depolarization conditions. This work will identify cis regulatory sequences that coordinate IEG negative regulation and will provide fundamental insight into the mechanisms of activity-regulated enhancer functioning and the encoded inducible-regulatory logic that differentiates promoters and enhancers. It will advance our pursuit of the molecular mechanisms governing IEG deactivation and has the potential to identify novel molecular targets that could aid in the development of therapeutics to treat diseases of cognition or memory decline.