Establishing an intersection between epigenetic control of MTOR gene expression with genetic and environmental factors regulating learning and memory

在 MTOR 基因表达的表观遗传控制与调节学习和记忆的遗传和环境因素之间建立交叉点

• 批准号: RGPIN-2022-05208
• 负责人: Weaver, Ian
• 金额: $ 2.33万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=764314
• 关键词: Establishing; intersection; between; epigenetic; control

Mammalian brain development and adult behaviour are influenced by early life experiences. The mechanism associated with the persistence of environmentally induced effects remains in question. Evidence has revealed that conditions during pregnancy and early postpartum generate stable differences in the offspring, in part, via altered gene expression in certain cell types and specific brain regions. Chromatin modification and DNA methylation are two global mechanisms that regulate gene expression. Metabolic signaling, in turn, can affect the expression and activity of the enzymes that modify chromatin and gene expression. Herein, assessing interindividual differences in chromatin modifications and DNA methylation marks holds great potential for determining the impact of both early life experience as well as the effect of interventions that alter metabolic processes, and ultimately neural gene expression, on brain development and adult behaviour. We recently demonstrated, using rodent behavioural and physiological measures along with pathway focused gene expression analysis, fluorescent microscopy, immunoprecipitation and bisulfite pyrosequencing--based approaches, that gestational stress inhibits the expression of enzymes that modify chromatin, which in turn inhibits expression of the mechanistic target of rapamycin (mTOR) network that regulates DNA repair, metabolism and cell growth. This unexpected finding offers the opportunity to contrast the chromatin structural--metabolic relationships, critical for neural cell growth and survival, in response to conditions during pregnancy and early postpartum, and how they relate to the development of cognitive, social and emotional behaviours. This is a challenging task and requires sensitive structural probes at the cellular level that can be used in concert for analysis with physiological and behavioural testing techniques. To this end, I have assembled a strong team of students and collaborators, experienced in the following techniques: video tracking system for automating rodent behavioural observations; extracellular flux analyzer for interrogating metabolism in live cells; confocal microscopy for chromatin imaging; laser capture and FACS analysis to isolate specific cell populations; digital RT--qPCR to measure genome--wide and candidate gene expression from single cells; ATAC--Seq, and bisulfite pyrosequencing for nucleosome and DNA methylation analysis, and ChIP---Seq for protein-DNA interaction analysis; primary culture systems and cell lines for temporal and functional analysis of neurons. We will combine these cutting--edge approaches with transgenic animal models and human brain tissues to provide a complete picture of how experiences propagate from external to internal variables, and what role chromatin structure and remodelling genes play in conical and non--conical pathways underlying the biological embedding of early in life effects on brain development and adult behavioural phenotypes.

哺乳动物的大脑发育和成人行为受早期生活经历的影响。与环境诱发效应持续相关的机制仍然存在。有证据表明，怀孕期间和产后早期的状况会在某些细胞类型和特定大脑区域中的基因表达改变，从而部分地在后代产生稳定的差异。染色质修饰和DNA甲基化是调节基因表达的两种全局机制。代谢信号转导可以影响改变染色质和基因表达的酶的表达和活性。在此，评估染色质修饰和DNA甲基化标记的个体差异具有确定早期生活经验的影响以及改变代谢过程以及最终神经基因表达，对脑发育和成人行为的干预措施的影响。我们最近证明，使用啮齿动物的行为和生理测量以及集中途径的基因表达分析，荧光显微镜，免疫沉淀和基于亚硫酸硫酸硫酸硫酸硫酸硫化物 - 基于硫磺的方法，妊娠应力抑制了酶的表达，从而抑制了染色质的酶的表达，从而抑制了机械性的表达。雷帕霉素（MTOR）网络的靶标，该网络调节DNA修复，代谢和细胞生长。这一出乎意料的发现提供了对比染色质结构的机会 - 对神经细胞生长和生存至关重要，这是对怀孕和产后早期疾病的响应，以及它们与认知，社会和情感行为的发展如何相关。这是一项具有挑战性的任务，需要在细胞水平上进行敏感的结构探针，该探针可以协同使用，以与生理和行为测试技术进行分析。为此，我组建了一个强大的学生和合作者团队，这些团队在以下技术中经验丰富：用于自动化啮齿动物行为观察的视频跟踪系统；细胞外通量分析仪，用于询问活细胞中的代谢；染色质成像的共聚焦显微镜；激光捕获和FACS分析以分离特定的细胞群体；数字RT- QPCR测量基因组 - 范围内的单个细胞和候选基因表达； ATAC-塞克和亚硫酸硫酸硫酸硫酸硫酸含核小体和DNA甲基化分析，以及芯片--- seq，用于蛋白质-DNA相互作用分析；用于神经元时间和功能分析的原发性培养系统和细胞系。我们将将这些切割的方法与转基因动物模型和人脑组织结合在一起，以完整地了解体验如何从外部变量传播到内部变量，以及在圆锥形和非良性途径中起什么作用染色质结构和重塑基因生命早期对脑发育和成人行为表型的影响。