Uncovering the function of histone variant H2BE in neurons

项目摘要 本研究的目的是阐明组蛋白变异体H_2BE在神经元中的分子和行为功能。 组蛋白变异体由不同的基因编码，可以替代典型的组蛋白(H_2A， H2B、H3和H4)，并参与许多细胞过程和基因表达的调节。组蛋白 在小鼠的主嗅觉上皮中发现了不同的H2BE，它会影响嗅神经元的功能 和长寿。虽然之前认为H2BE只在嗅觉系统中表达，但我们的实验室 开发了一种针对H2BE的高度特异的抗体，并证明了H2BE存在于整个大脑中。 然而，尽管组蛋白变体在控制神经元功能方面很重要，但到目前为止，H2BE仍然存在 在嗅觉系统之外没有被研究过。在这里，我建议确定H2BE是如何改变染色质结构的， 神经元基因表达和动物行为。我推测H_2BE减少了连接物组蛋白的结合。 H1控制着依赖活动的基因的表达，对认知灵活性、空间学习、 和恐惧记忆。为了验证我的假设，我将结合全基因组测序、小鼠模型和动物 来自染色质生物学领域的分子和生化技术的行为。在目标1中，我将确定 H2BE的表达如何改变染色质结构。我将使用芯片测序来定义基因组定位 在基线和对外部信号的响应中，H_2BE的浓度。我的初步数据显示，H2BE促进 开放的染色质构型，减少异染色质相关蛋白的结合。因此，我会 研究过氧化氢对染色质纤维的组成有何影响。具体地说，我将使用Chip-seq来确定 H_2BE掺入如何影响连接物组蛋白H1的定位。目标2阐述了H2BE在神经元中的作用 基因表达和小鼠行为。首先，我将测试H2BE基因敲除对RNA测序的影响 有外部刺激和没有外部刺激的神经元。其次，我将使用H2BE执行一系列行为测试 WT和KO小鼠旨在确定受H2BE丢失影响最大的特定脑区，并完全 描述H2BE在认知中的作用。这里提出的工作将揭示组蛋白如何变异H2BE 有助于大脑中复杂的染色质环境。这一发现对于理解 神经元使用环境信号来控制转录，并最终控制行为。在开发更多的 完全了解神经元中的染色质情况，我们还将深入了解潜在的 治疗与表观遗传学中断有关的许多神经疾病 大脑中的调节。

Project Summary The goal of this proposal is to elucidate the molecular and behavioral function of histone variant H2BE in neurons. Histone variants, which are encoded by separate genes, can substitute for the canonical histone proteins (H2A, H2B, H3, and H4) and are involved in regulation of many cellular processes and gene expression. The histone variant H2BE was discovered in the mouse main olfactory epithelium, where it affects olfactory neuron function and longevity. While H2BE was previously thought to be exclusively expressed in the olfactory system, our lab developed a highly specific antibody against H2BE and demonstrated that H2BE is present throughout the brain. However, despite the importance of histone variants in controlling neuronal function, to date, H2BE remains unstudied outside of the olfactory system. Here, I propose to determine how H2BE alters chromatin structure, neuronal gene expression, and animal behavior. I hypothesize that H2BE decreases binding of linker histone H1, controls expression of activity-dependent genes, and is necessary for cognitive flexibility, spatial learning, and fear memory. To test my hypothesis, I will combine genome-wide sequencing, mouse models, and animal behavior with molecular and biochemical techniques from the chromatin biology field. In Aim 1, I will determine how H2BE expression alters chromatin structure. I will use ChIP-sequencing to define the genomic localization of H2BE at baseline and in response to external signals. My preliminary data demonstrates that H2BE promotes an open chromatin configuration and decreases binding of heterochromatin-associated proteins. Therefore, I will examine how H2BE affects the composition of the chromatin fiber. Specifically, I will use ChIP-seq to determine how H2BE incorporation affects localization of linker histone H1. Aim 2 addresses the role of H2BE in neuronal gene expression and mouse behavior. First, I will test the effects of H2BE knockout on RNA-sequencing of neurons with and without external stimulation. Second, I will perform a battery of behavioral tests using H2BE WT and KO mice designed to determine the specific brain regions most affected by H2BE loss and to fully characterize the role of H2BE in cognition. The work proposed here will reveal how histone variant H2BE contributes to the complex chromatin environment in the brain. This discovery is critical to understanding how neurons use environmental signals to control transcription and ultimately govern behavior. In developing a more complete understanding of the chromatin landscape in neurons, we will also gain insight into potential therapeutics for the treatment of the many neurological disorders that are linked to disruption of epigenetic regulation in the brain.