Analysis of the distinct regulatory functions of DNA methylation in memory-related gene expression

记忆形成的一个重要步骤是基因转录激活。转录调控是实现记忆相关基因正确表达的关键。DNA的甲基化被认为是这一过程的关键媒介。DNA甲基转移酶蛋白（Dnmts）负责甲基化标记的书写，而甲基结合蛋白（如MeCP2）则负责读取并将这些标记翻译成染色质结构的变化和转录的可及性。DNA甲基化在记忆形成过程中调控转录的确切机制还远未被理解。该提案描述了一个旨在进一步调查这个问题的研究计划。在我之前的工作中，我发现Dnmt3a2和Dnmt3b的水平受到神经元活动的调节- Dnmt3a2上调，而Dnmt3b下调- Dnmt3a2是记忆形成所必需的，并以积极的方式调节记忆相关基因的表达。此外，我发现Dnmt3a2的基础和学习诱导表达在衰老海马中受损，这与衰老依赖性认知衰退有因果关系。我现在提出一个新的概念，通过DNA甲基化调控基因表达。我推测Dnmt3b作为一种记忆抑制基因，在记忆巩固过程中需要抑制其表达，以使记忆相关基因的最佳表达。这一假设将通过评估Dnmt3b水平在记忆形成中的调节效果得到实验验证。此外，我提出记忆相关基因表达的微调是通过一个中间步骤来实现的，即Dnmt3a2调节相关mirna的表达。因此，我提供了一种机制，通过这种机制，单个基因可以调节多个mrna的表达。我的初步工作确定了一系列假定的Dnmt3a2靶mirna （dtm）。我现在提出一组实验（结合分子和行为方法），以评估dtm表达的调节是否在记忆形成中起作用。DNA甲基化标记是由Dnmts催化的，而甲基化模式的解释是由包括MeCP2在内的甲基结合蛋白完成的。因此，MeCP2将甲基化标记转化为功能变化。我认为MeCP2的DNA占用构成了成熟大脑中基因表达调控的另一个关键水平。根据这一假设，我将测试成年小鼠的记忆形成是否需要海马MeCP2，以及MeCP2基因组占用是否随着年龄的增长而变化及其对基因表达的影响。这项研究将为记忆形成中基因表达调控的机制提供新的、基本的见解。它还将导致识别新的候选对象，以开发治疗衰老相关疾病的方法。

An essential step for memory formation is gene transcription activation. The regulation of transcription is critical for the achievement of proper expression of the memory related genes. The methylation of DNA has been proposed as a key mediator in this process. DNA methyltransferase proteins (Dnmts) are responsible for the writing of methylation marks and the methyl-binding proteins (such as MeCP2) for reading and translating this marks into changes in chromatin structure and accessibility for transcription. The precise mechanism by which DNA methylation regulates transcription during memory formation is far from being understood. This proposal describes a research program that aims at further investigating this question.In my previous work I found that the levels of Dnmt3a2 and Dnmt3b are regulated by neuronal activity - Dnmt3a2 is upregulated, whereas Dnmt3b is downregulated - that Dnmt3a2, is required for memory formation and regulates the expression of memory related genes in a positive manner. Moreover, I showed that the basal and learning-induced expression of Dnmt3a2 is impaired in the aging hippocampus and that this is causally linked with aging-dependent cognitive decline.I now propose a novel concept in the regulation of gene expression by DNA methylation. I hypothesize that Dnmt3b functions as a memory suppressor gene, which expression needs to be repressed during memory consolidation for the optimal expression of memory related genes. This hypothesis will be experimentally validated by assessing the effect of the modulation of Dnmt3b levels in memory formation. Moreover, I propose that the fine-tuning of memory related gene expression is achieved via an intermediate step, the regulation of expression of relevant miRNAs by Dnmt3a2. Thus, I provide a mechanism through which a single gene can modulate the expression of several mRNAs. My preliminary work identified a list of putative Dnmt3a2 target miRNAs (DTMs). I now propose a set of experiments (combination of molecular and behavioral approaches) that will allow assessing whether the regulation of expression of DTMs plays a role in memory formation. The DNA methylation marks are catalyzed by Dnmts whereas the interpretation of the methylation pattern is performed by the methyl binding proteins that include MeCP2. Therefore MeCP2 translates methylation marks into functional changes. I propose that the DNA occupancy by MeCP2 constitutes another level critical for the regulation of gene expression in the mature brain. In line with this hypothesis, I will test whether hippocampal MeCP2 is required for memory formation in adult mice and whether MeCP2 genomic occupancy changes with aging and the subsequent impact on gene expression.This study will provide new, fundamental insights into the mechanism of gene expression regulation in memory formation. It will also lead to the identification of novel candidates for the development of therapies for aging-related diseases.