MECHANISMS OF EPIGENETIC REGULATION IN NERVOUS SYSTEM DEVELOPMENT

人类遗传学的进步已经鉴定出大量与遗传相关的基因。 自闭症谱系障碍（ASD）、智力残疾（ID）和相关的神经发育疾病。一个关键的新 挑战是了解这些基因在神经元中的分子通路，并剖析它们如何在神经元中发挥作用。 干扰会改变神经系统功能。DNA中胞嘧啶的甲基化通常只发生在CG 二核苷酸，但最近的研究也揭示了神经元中独特的非CpG DNA甲基化的功能 （主要发生在CA二核苷酸，mCA）。我们已经发现甲基DNA结合蛋白MeCP2， 对神经系统正常功能至关重要，它与mCA结合在基因转录区域内， 神经元下调其表达。新的研究发现了DNMT3A的突变， 在ID和ASD患者中，mCA在大脑中沉积所需的甲基转移酶。这些发现 支持mCA介导的基因调控破坏导致以下疾病的假设： DNMT3A突变我们提出的研究将通过追求三个具体目标来测试这一假设：目标1将使用 体外细胞培养系统，以确定DNMT3A中的疾病相关突变如何影响酶功能， 改变mCA在神经元中的沉积。Aim 2将使用新产生的携带杂合子的转基因小鼠， DNMT3A突变，以确定这种破坏对神经元DNA甲基化，染色质结构， 转录和细胞功能。目的3将探讨mCA和MeCP 2直接作用于细胞的机制。 调节神经元中的转录，测试这些成分与新的基因调控相互作用的假设， 控制转录的位点。这些研究将共同确定mCA的分子机制， 调节神经元中的基因表达，并确定ASD和ID中的分子病理学位点。

Abstract Advances in human genetics have led to the identification of a large number of genes associated with autism spectrum disorder (ASD), intellectual disability (ID), and related neurodevelopmental diseases. A critical new challenge is to understand the molecular pathways these genes contribute to in neurons and to dissect how their disruption alters nervous system function. Methylation of cytosines in DNA classically occurs only at CG dinucleotides, but recent studies have also uncovered functions for unique non-CpG DNA methylation in neurons (which occur largely at CA dinucleotides, mCA). We have discovered that the methyl-DNA-binding protein MeCP2, which is critical for proper nervous system function, binds to mCA within the transcribed regions of genes in neurons to downregulate their expression. New studies have identified mutations in DNMT3A, the DNA methyltransferase required for the deposition of mCA in the brain, in individuals with ID and ASD. These findings support the hypothesis that disruption of gene regulation mediated by mCA contributes to disorders caused by mutation of DNMT3A. Our proposed studies will test this hypothesis by pursuing three specific aims: Aim 1 will use in vitro cell culture systems to determine how disease-associated mutations in DNMT3A affect enzyme function and alter deposition of mCA in neurons. Aim 2 will employ newly-generated transgenic mice carrying heterozygous mutations in DNMT3A to determine the effects of this disruption on neuronal DNA methylation, chromatin structure, transcription and cellular functions. Aim 3 will interrogate the mechanisms by which mCA and MeCP2 directly regulate transcription in neurons, testing the hypothesis that these components interact with novel gene-regulatory sites to control transcription. Together these studies will determine the molecular mechanisms by which mCA regulates gene expression in neurons, and define a site of molecular pathology in ASD and ID.