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

哺乳动物的大脑发育和成年行为会受到早期生活经历的影响。与环境引起的影响持续相关的机制仍然存在疑问。有证据表明，怀孕和产后早期的条件会在后代中产生稳定的差异，部分原因是某些细胞类型和特定大脑区域的基因表达发生了变化。染色质修饰和DNA甲基化是调节基因表达的两种全球性机制。代谢信号反过来可以影响改变染色质和基因表达的酶的表达和活性。因此，评估染色质修饰和DNA甲基化标记的个体间差异具有确定早期生活经验以及改变代谢过程的干预措施的影响，最终改变神经基因表达对大脑发育和成人行为的影响的巨大潜力。最近，我们利用啮齿动物的行为和生理指标，以及基于途径的基因表达分析、荧光显微镜、免疫沉淀和亚硫酸盐焦磷酸测序的方法，证明了妊娠应激抑制了修饰染色质的酶的表达，进而抑制了调节DNA修复、代谢和细胞生长的雷帕霉素(MTOR)网络的机械靶的表达。这一意想不到的发现提供了对比染色质结构--对神经细胞生长和存活至关重要的代谢关系--与怀孕和产后早期状况的反应，以及它们与认知、社会和情感行为发展的关系的机会。这是一项具有挑战性的任务，需要在细胞水平上使用敏感的结构探针，这些探针可以与生理和行为测试技术结合使用进行分析。为此，我组建了一支强大的学生团队和合作伙伴，他们在下列技术方面经验丰富：视频跟踪系统用于自动化啮齿动物行为观察；细胞外通量分析仪用于询问活细胞的新陈代谢；共焦显微镜用于染色质成像；激光捕获和FACS分析用于分离特定的细胞群体；数字RT-qPCR用于测量基因组范围和单个细胞中候选基因的表达；ATAC--SEQ和亚硫酸氢盐焦序分析用于核小体和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。我们将把这些尖端方法与转基因动物模型和人脑组织结合起来，提供经验如何从外部变量传播到内部变量的完整图景，以及染色质结构和重塑基因在锥形和非锥形通路中扮演的角色，这些通路支撑着生命早期对大脑发育和成人行为表型的生物嵌入效应。

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.