Understanding the role of DNA methylation dynamics at Bdnf during fear conditioning

项目摘要/摘要 -- 创伤后应激障碍(PTSD)在一定程度上是由大脑中异常的恐惧巩固引起的。 神经营养因子和染色质动力学的信号传递对记忆巩固至关重要。信令 脑源性神经营养因子(BDNF)及其主要受体酪氨酸受体激酶B(TrkB)， 在巩固杏仁核中的恐惧方面起着重要作用。事实上，事实证明，两者都 杏仁核中BDNF的特异性敲除和BDNF-TrkB信号的阻断干扰了巩固。因此， 突触前转录和突触后信号对其功能都是至关重要的。此外，研究 已经证明DNA甲基化(5-MC)水平与 提示表观遗传修饰正在驱动BDNF基因的转录调控过程 在巩固恐惧记忆的时候。我们的目标是描述DNA羟甲基化(5-HMC)水平在BDNF和 利用Cas9-Tet1构建研究定点表观遗传之间的因果关系 调制和恐惧整合。 与其他组织相比，神经组织中含有非常高水平的5-羟甲基纤维素。规范地说，5-MC 已经被认为是一个相对稳定的表观遗传标记，描绘了组织特异性启动子的激活。 然而，两个重要的证据已经证明了5-MC监管的重要性 巩固：动态DNA甲基化是恐惧巩固和操纵Tet1所必需的 酶，一种负责5-MC向5-HMC转化的酶，干扰了背景恐惧 记忆。我们假设在恐惧巩固过程中，5-HMC将动态调节BDNF，并且这些 5-HMC的改变对于巩固恐惧记忆是必要的。在目标1中，我们将使用全基因组 以及在恐惧条件反射过程中对BDNF的5-HMC和5-MC进行基因定位的方法。在《目标2》中我们将 在体外和表现良好的小鼠中进行概念验证Cas9-Tet1表观基因组工程实验，以及 我们将阐明BDNF候选基因座的动态5-HMC与恐惧巩固的关系。

Project Summary/Abstract   Post-Traumatic Stress Disorder (PTSD) is driven, in part, by aberrant fear consolidation in the brain. Signaling by neurotrophic factors and chromatin dynamics are crucial for this memory consolidation. Signaling between brain-derived neurotrophic factor (Bdnf) and its primary receptor, tyrosine-receptor kinase B (TrkB), plays an important role in the consolidation of fear in the amygdala. Indeed, it has been shown that both amygdala specific knockout of Bdnf and blockage of Bdnf-TrkB signaling interfere with consolidation. Thus both presynaptic transcription as well as post-synaptic signaling is crucial for its function. Furthermore, studies have demonstrated an associated between DNA methylation (5-mC) levels, and differential transcription of Bdnf, suggesting that epigenetic modifications are driving transcriptional regulatory processes at the Bdnf gene during fear memory consolidation. We aim to profile the DNA hydroxymethylation (5-hmC) levels at Bdnf and employ a Cas9-Tet1 construct to investigate the causal relationship between site-directed epigenetic modulation and fear consolidation. Neural tissues possess very high levels of 5-hmC, as compared to other tissues. Canonically, 5-mC has been considered to be a relatively stable epigenetic mark, delineating tissue-specific promoter activation. However, two important pieces of evidence have demonstrated the importance of 5-mC regulation for fear consolidation: dynamic DNA methylation is necessary for fear consolidation, and manipulation of the Tet1 enzyme, one enzyme responsible for the conversion of 5-mC to 5-hmC, interfered with contextual fear memory. We hypothesize that 5-hmC will dynamically regulate Bdnf during fear consolidation, and that these 5-hmC alterations will be necessary for the consolidation of fear memory. In aim 1 we will use genome-wide and locus-specific approaches to profile 5-hmC and 5-mC at Bdnf during fear conditioning. In aim 2 we will perform proof-of-concept Cas9-Tet1 epigenomic engineering experiments in vitro and in behaving mice, and we will elucidate the relationship between dynamic 5-hmC at candidate loci within Bdnf and fear consolidation.