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.

项目总结/摘要 神经元中快速和短暂的基因表达诱导是记忆形成所必需的。基因 大脑中响应刺激而激活的转录因子通过释放稳定的转录因子来调节 状态，其中RNA聚合酶II（RNAP 2）在启动后仅在基因启动子下游暂停 转录。当钙依赖性信号级联被触发时，RNAP 2暂停被释放。这 释放允许RNAP 2在基因的长度上伸长，并且信使RNA（mRNA）转录物被释放。 生成的.当神经元受到刺激时，有一个转录不应期持续数小时。 转录上对随后的刺激没有反应，这可能与重置平衡所需的时间有关。 RNAP 2.这段转录反应减弱的时期可能解释了动物 在训练期之间的时间很少，训练间隔更长的动物学习效果不好 尽管各组之间的总训练时间相等，但仍然没有制定训练时间表。初步实验表明， 六亚甲基二乙酰胺诱导蛋白1（HEXIM 1）作为建立和重置 由于其能够隔离正转录延伸因子B b（P-TEF B）， 蛋白复合物，其负责释放RNAP 2转录暂停。虽然其他监管机构 P-TEFb与人类认知疾病有关，包括智力障碍，阿尔茨海默氏症，情绪 然而，对于大脑中的HEXIM 1知之甚少。 该项目的中心假设是，神经元中HEXIM 1对P-TEFb的抑制是神经元的生长所必需的。 RNAP 2建立一个平衡状态，这样可以诱导基因表达的爆发来响应刺激， 当转录爆发后，平衡状态建立起来时，学习能力受到了损害。我来测试一下 有三个具体目标的假设。目的1将探索P-TEFb/HEXIM 1复合物的调节， 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HEXIM 1之间的分子关联及其对钙相关蛋白的依赖性 磷酸化事件将在原代神经元培养物中使用生物化学测定进行测试。目标2将测试如何 RNAP 2通过一系列步骤循环（平衡，伸长，脱离，然后再次平衡），这些步骤决定了基因水平， 在神经元去极化后诱导。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 在神经元刺激期间和之后，在含有平衡的、伸长的和无活性的RNAP 2的亚隔室中。 目的3将探索HEXIM 1在记忆和确定学习速率中的作用。行为测试将 在海马中敲低或过表达Hexim 1基因后的小鼠中进行。这个职业 发展奖将进一步发展我的翻译技能在研究转录调控 学习和记忆的机制。