Mechanisms of Immediate Early Gene Regulation in Neurons

描述(申请人提供)：获取、存储和回忆记忆的能力是大脑的一个决定性特征，从人类到果蝇都是如此。即刻早期基因(IEG)是由神经元活动刺激其表达的基因，通常是瞬时的，这一特征使它们成为改变神经可塑性的主要候选基因。然而，人们对IEGs在任何生物体中的调节和功能知之甚少。神经可塑性、学习和记忆的异常可能导致严重的神经疾病，如阿尔茨海默氏症、痴呆症、创伤后应激和自闭症，这突显了阐明记忆背后的分子机制的必要性。我们已经使用RNAseq分析在果蝇大脑中鉴定了一套288个IEG，在暴露于不同持续时间(10、20、30、45分钟)的感觉刺激后，其中65%在人类中具有同源性。根据表达调控的动力学，IEGs聚为5个组，表明调控机制不同。使用一种无偏见的生物信息学方法，我们能够在每个IEG簇的上游区域识别出过度代表的DNA序列基序。簇1和簇2的基序非常相似，并与GATA转录因子家族的结合位点相匹配。遗传和分子分析表明，IEGs的表达确实需要一个GATA因子--谷粒。这项提议的目的是检验这样的假设，即颗粒和其他GATA因子在IEG表达的活动依赖的调节中发挥作用，从而在记忆的获得和保持中发挥核心作用。这一提议意义重大，因为这代表了自CREB以来首批主要的IEG调控途径之一，并可能为理解神经元活动、IEG表达和记忆之间的联系提供了一个机会。此外，该提案将使用IEG启动子来提供转基因工具，以定位活的行为苍蝇大脑中激活的神经回路。通过GATA因素分析IEG监管的方法将在这里概述的两个具体目标中实现。(1)第一个目标将验证谷物在IEG调节中的作用，确定其全基因组靶点，绘制其在大脑中定位的变化图，并研究其在记忆中的作用。(2)第二个目标将调查依赖GATA的IEGs的调控DNA序列，以测试直接调控。此外，它还将开发转基因果蝇，其中IEG启动子将用于编码标记激活神经元的遗传荧光报告。成功完成拟议的研究将发现转录因子和神经回路潜在的学习和记忆的基本机制，并建立将广泛用于神经回路的解剖学研究的工具。

DESCRIPTION (provided by applicant): The ability to acquire, store and recall memories is a defining feature of the brain that is conserved from humans to Drosophila. Immediate Early Genes (IEGs) are genes whose expression is stimulated by neuronal activity, and is often transient, a feature that makes them prime candidates for modifying neural plasticity. Yet very little is understood about the regulation and function of IEGs in any organism. Aberrations in neural plasticity, learning and memory can cause severe neurological conditions such as Alzheimer's, dementia, post-traumatic stress and autism, underscoring the need for elucidating molecular mechanisms that underlie memory. We have identified a suite of 288 IEGs in the Drosophila brain using RNASeq analysis after exposure to sensory stimuli of various durations (10, 20, 30, 45 mins), 65% of which have homologs in humans. Based on the kinetics of expression modulation the IEGs cluster into 5 groups suggesting differences in regulatory mechanisms. Using an unbiased bioinformatics approach we were able to identify over-represented DNA sequence motifs in the upstream regions of each cluster of IEGs. The motifs for cluster 1 and 2 are remarkably alike, and match the binding site of the GATA family of transcription factors. Genetic and molecular analysis showed that a GATA factor, grain, was indeed required for expression of IEGs. The goal of this proposal is to test the hypothesis that grain and other GATA factors play a role in activity-dependent modulation of IEG expression and therefore a central role in acquisition and retention of memory. The proposal is highly significant since this represents one of the first major IEG regulatory pathways since CREB and could present an opportunity to understand the link between neuronal activity, IEG expression and memory. In addition, the proposal will use IEG promoters to provide transgenic tools to map activated neural circuits in brains of live behaving flies. The approach for analysis of IEG regulation by GATA factors will be accomplished in two specific aims outlined here. (1) The first aim will validate the role of grain in IEG regulation, identify its genome-wide targets, map changes in its localization in the brain and investigate its role in memory. (2) The second aim will investigate the regulatory DNA sequences of the GATA- dependent IEGs to test for direct regulation. In addition it will develop transgenic flies where the IEG promoter will be used to encode a genetic fluorescent reporter that marks activated neurons. Successful completion of the proposed studies will uncover transcription factors and neuronal circuits underlying fundamental mechanisms of learning and memory and establish tools that will be widely useful for anatomical studies of neural circuits.