Molecular Regulation of a Transcriptionally Poised State in Neurons and its Role in Learning

神经元转录平衡状态的分子调节及其在学习中的作用

• 批准号: 10704696
• 负责人: Celeste Brittany Greer
• 金额: $ 13.63万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-14 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10704696
• 关键词: Affect; Alzheimer' s Disease; Animal Behavior; Animals; Behavior; Behavioral; Binding; Biochemical; Biological Assay; Brain; Brain Diseases; Calcium; Calcium Channel; Calcium Signaling; Cell Nucleus; Cells; Co-Immunoprecipitations; Cognition Disorders; Complex; Confocal Microscopy; Data; Dependence; Dependovirus; Dissociation; Environment; Enzymes; Event; Faculty; Fluorescent in Situ Hybridization; Gene Activation; Gene Expression; Genes; Genetic Transcription; Grant; Hexamethylene Bisacetamide; Hippocampus; Hour; Human; Immediate-Early Genes; Impairment; In Situ Hybridization; Intellectual functioning disability; K-Series Research Career Programs; Learning; Length; Link; Mammals; Memory; Mentors; Messenger RNA; Molecular; Mood Disorders; Mus; Neurons; Nuclear; Operative Surgical Procedures; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Positive Transcriptional Elongation Factor B; Process; Processed Genes; Protein Dephosphorylation; Proteins; RNA; RNA Polymerase II; Refractory; Regulation; Research Support; Role; Schedule; Signal Pathway; Signal Transduction; Stimulus; Testing; Time; Training; Transcript; Transcription Elongation; Transcription Initiation; Transcriptional Regulation; Translations; Writing; behavior test; brain dysfunction; career; cell type; cognitive process; experimental study; gene induction; immunocytochemistry; inducible gene expression; knock-down; novel; overexpression; pharmacologic; promoter; protein complex; response; skills; therapeutic target; timeline

PROJECT SUMMARY/ABSTRACT Rapid and transient induction of gene expression in neurons is necessary for memory formation. Genes that are activated in the brain in response to stimuli are regulated by the release of a poised transcriptional state, where RNA polymerase II (RNAP2) pauses just downstream of the gene promoter after initiating transcription. When calcium-dependent signaling cascades are triggered, the RNAP2 pause is released. This release allows RNAP2 to elongate across the length of the gene, and messenger RNA (mRNA) transcripts are generated. There is a transcriptional refractory period that lasts for hours after a stimulus when neurons are transcriptionally unresponsive to subsequent stimuli that may be linked to the time it takes to reset poised RNAP2. This period of dampened transcriptional response may explain the phenomenon where animals that have little time between training sessions do not learn as well as animals with more spaced out training session schedules, despite total training time being equal between groups. Preliminary experiments point to Hexamethylene bisacetamide inducible protein 1 (HEXIM1) as a critical factor for setting up and resetting the poised state in neurons due to its ability to sequester the positive transcription elongation factor b (P-TEFb) protein complex, which is responsible for releasing the RNAP2 transcriptional pause. While other regulators of P-TEFb have been linked to human cognitive diseases including intellectual disability, Alzheimer’s, mood disorders, and others, very little is known about HEXIM1 in the brain. The central hypothesis of this project is that suppression of P-TEFb by HEXIM1 in neurons is required for RNAP2 to set up a poised state so a burst of gene expression can be induced in response to a stimulus, and that while the poised state is getting set up following a transcriptional burst, learning is impaired. I will test this hypothesis with three specific aims. Aim 1 will explore the regulation of the P-TEFb/HEXIM1 complex by calcium channels to identify which memory-associated calcium signaling pathways impact P-TEFb activity. Molecular associations between P-TEFb and HEXIM1, and their dependence on calcium-associated phosphorylation events will be tested using biochemical assays in primary neuron cultures. Aim 2 will test how RNAP2 cycles through a set of steps (poise, elongate, disengage, then poise again) that dictate levels of gene inducibility following neuronal depolarization. Confocal microscopy, immunocytochemistry, and florescence in situ hybridization will be combined to ascertain the association of inducible genes with nuclear subcompartments in containing poised, elongating, and inactive RNAP2 during and after neuronal stimulation. Aim 3 will probe the role of HEXIM1 in memory and determining the rate of learning. Behavioral tests will be conducted in mice after knockdown or overexpression of the Hexim1 gene in the hippocampus. This career development award will further develop the translation of my skillsets in studying transcriptional regulation mechanisms to the study of learning and memory.

项目概要/摘要 神经元中基因表达的快速和瞬时诱导对于记忆形成是必要的。基因 大脑中响应刺激而被激活的神经元，是通过释放稳定的转录因子来调节的 状态，其中 RNA 聚合酶 II (RNAP2) 在启动后暂停在基因启动子下游 转录。当钙依赖性信号级联被触发时，RNAP2 暂停被释放。这 释放使 RNAP2 能够延长基因的长度，并且信使 RNA (mRNA) 转录物 生成的。当神经元受到刺激后，有一个转录不应期持续数小时。 对后续刺激的转录反应迟钝，这可能与重置平衡所需的时间有关 RNAP2。这段转录反应减弱的时期可以解释动物 训练之间的时间很短，训练间隔时间较长的动物学习效果不佳 尽管各组之间的总训练时间相同，但训练计划却有所不同。初步实验表明 六亚甲基双乙酰胺诱导蛋白 1 (HEXIM1) 作为建立和重置 由于其能够隔离正转录延伸因子 b (P-TEFb)，神经元处于平衡状态 蛋白质复合物，负责释放 RNAP2 转录暂停。而其他监管机构 P-TEFb 与人类认知疾病有关，包括智力障碍、阿尔茨海默病、情绪障碍 疾病和其他疾病，人们对大脑中的 HEXIM1 知之甚少。 该项目的中心假设是，神经元中的 HEXIM1 抑制 P-TEFb 是 RNAP2 建立平衡状态，以便响应刺激而诱导基因表达爆发，并且 当转录爆发后建立平衡状态时，学习就会受到损害。我会测试这个 具有三个具体目标的假设。目标 1 将探索 P-TEFb/HEXIM1 复合物的调节 钙通道来识别哪些与记忆相关的钙信号通路影响 P-TEFb 活性。 P-TEFb 和 HEXIM1 之间的分子关联及其对钙相关的依赖性 将使用原代神经元培养物中的生化测定来测试磷酸化事件。目标 2 将测试如何 RNAP2 通过一系列决定基因水平的步骤（平衡、伸长、脱离、然后再次平衡）循环 神经元去极化后的诱导性。共聚焦显微镜、免疫细胞化学和荧光 原位杂交将结合以确定诱导基因与核的关联 在神经元刺激期间和之后，亚区室含有平衡、伸长和失活的RNAP2。 目标 3 将探讨 HEXIM1 在记忆和确定学习速率中的作用。行为测试将 在小鼠海马区 Hexim1 基因敲低或过度表达后进行。这个职业 发展奖将进一步发展我研究转录调控的技能 研究学习和记忆的机制。