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: 10591307
• 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/10591307
• 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; Cognition Disorders; Complex; Confocal Microscopy; Data; Dependence; Dependovirus; Dissociation; Environment; Enzymes; Event; Faculty; Gene Activation; Gene Expression; Genes; Genetic Transcription; Grant; Hexamethylene Bisacetamide; Hippocampus (Brain); Hour; Human; Immediate-Early Genes; Impairment; In Situ; In Situ Hybridization; Intellectual functioning disability; K-Series Research Career Programs; Lead; Learning; Length; Link; Mammals; Memory; Mentors; Messenger RNA; Molecular; Mood Disorders; Mus; Neurons; Nuclear; Operative Surgical Procedures; Pharmacology; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Positive Transcriptional Elongation Factor B; Process; Processed Genes; Proteins; Quantitative Reverse Transcriptase PCR; RNA; RNA Polymerase II; Refractory; Regulation; Research Support; Role; Schedule; Signal Pathway; Signal Transduction; Stimulus; Testing; Time; TimeLine; Training; Transcript; Transcription Elongation; 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; promoter; protein complex; response; skills; therapeutic target

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.

项目摘要/摘要 快速而短暂地诱导神经元中的基因表达对于记忆的形成是必要的。基因 大脑对刺激的反应被激活，通过释放一种稳定的转录来调节 启动后，RNA聚合酶II(RNAP2)在基因启动子下游暂停 抄写。当钙依赖信号级联被触发时，RNAP2暂停被释放。这 释放允许RNAP2在整个基因长度上延伸，信使RNA(MRNA)转录本 已生成。有一个转录不应期，在刺激后持续几个小时，当神经元 转录上对随后的刺激无反应，这可能与重置平衡所需的时间有关 RNAP2.这段转录反应受抑的时期可能解释了动物 训练课之间的时间很少，没有学习的动物和更分散训练的动物 课程安排，尽管小组之间的总培训时间相等。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 神经元的稳定状态，因为它有能力隔离正转录延伸因子b(P-TEFb) 蛋白质复合体，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，包括智力残疾、阿尔茨海默氏症、情绪 疾病和其他疾病，对大脑中的HEXIM1知之甚少。 该项目的中心假设是，HEXIM1对神经元中P-TEFb的抑制是 RNAP2来建立一种稳定的状态，这样就可以在刺激下诱导基因表达的爆发，以及 虽然在转录爆发后，镇定的状态正在建立，但学习受到了损害。我要测试一下这个 有三个具体目标的假设。目的1探讨P-TEFb/HEXIM1复合体的调节机制 钙通道，以确定哪些记忆相关的钙信号通路影响P-TEFb活性。 P-TEFb和HEXIM1的分子结合及其对钙离子的依赖 磷酸化事件将在原代神经元培养中使用生化分析进行测试。Aim 2将测试如何 RNAP2通过一系列决定基因水平的步骤(保持平衡、伸长、脱离，然后再次保持稳定)进行循环 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 将结合原位杂交来确定诱导基因与核的联系。 在神经元刺激期间和之后，含有稳定的、延长的和非活性的RNAP2的亚室。 目标3将探讨HEXIM1在记忆和决定学习速度中的作用。行为测试将会是 在海马区HEXIM1基因被敲除或过度表达后，在小鼠身上进行。这份职业 发展奖将进一步发展我在研究转录调控方面的翻译技能 学习和记忆研究的机制。