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年行为受到早期生活经历的影响。与环境引起的影响的持久性有关的机制仍然存在疑问。有证据表明，怀孕期间和产后早期的条件会在后代中产生稳定的差异，部分原因是通过改变某些细胞类型和特定大脑区域的基因表达。染色质修饰和DNA甲基化是调控基因表达的两种全局机制。反过来，代谢信号可以影响修饰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甲基化标记的个体间差异对于确定早期生活经历以及改变代谢过程的干预措施的影响以及最终神经基因表达对大脑发育和成人行为的影响具有巨大潜力。我们最近证明，使用啮齿动物的行为和生理措施沿着与途径集中的基因表达分析，荧光显微镜，免疫沉淀和亚硫酸氢盐焦磷酸测序-为基础的方法，妊娠应激抑制修饰染色质的酶的表达，这反过来又抑制表达的雷帕霉素（mTOR）网络，调节DNA修复，代谢和细胞生长的机制目标。这一意想不到的发现提供了一个机会，以对比染色质结构-代谢关系，神经细胞的生长和生存的关键，在怀孕期间和产后早期的条件，以及它们如何与认知，社会和情感行为的发展。这是一项具有挑战性的任务，需要细胞水平上的敏感结构探针，可与生理和行为测试技术一起用于分析。为此，我组建了一个由学生和合作者组成的强大团队，他们在以下技术方面经验丰富：用于自动化啮齿动物行为观察的视频跟踪系统;用于询问活细胞中代谢的细胞外通量分析仪;用于染色质成像的共聚焦显微镜;用于分离特定细胞群的激光捕获和FACS分析;用于测量单细胞全基因组和候选基因表达的数字RT-qPCR; ATAC-Seq和亚硫酸氢盐焦磷酸测序用于核小体和DNA甲基化分析，ChIP-Seq用于蛋白质-DNA相互作用分析;原代培养系统和细胞系用于神经元的时间和功能分析。我们将结合联合收割机这些尖端的方法与转基因动物模型和人脑组织，提供一个完整的图片如何经验传播从外部到内部变量，什么样的作用染色质结构和重塑基因发挥锥形和非锥形途径的生物嵌入早期在生命中的影响，对大脑发育和成人行为表型。