Cell-based Genomic Analysis of molecular pathology in Mouse Models of Rett Syndro

描述(由申请人提供)：甲基CpG结合蛋白2基因(MeCP2)的突变导致Rett综合征，一种自闭症谱系障碍。RTT被认为是神经元成熟和可塑性不足的结果，可能是通过异常的经验依赖型突触发育和维持。MeCP2通过与甲基胞嘧啶(包括5-甲基胞嘧啶(5mC)和5-羟甲基胞嘧啶(5hmC))结合参与染色质和转录调节。虽然5mC通常被视为“沉默标记”，但5hmC可能代表甲基化动态，并在基因调控中发挥复杂的作用。然而，5mC和5hmC在脑细胞中的基因组分布特征很差，目前还不清楚MeCP2如何解释5mC和5hmC模式，从而影响转录。MeCP2主要是一种转录抑制因子，调节基因特异性靶标，还是在染色质调节中起全局性作用，目前仍存在争议。确定转录影响是了解MeCP2功能机制的关键一步。在小鼠模型中确定相关脑区、神经元类型、发育和生理环境中转录变化的特征对于揭示RTT的发病机制是必要的。研究MeCP2在大脑基因调控中的作用的一个关键挑战是细胞异质性。例如，很难在已定义的神经元类型中实现高分辨率的甲基组映射。此外，神经细胞在发育期间和对神经元输入的反应中修改其表观基因组和转录本。因此，另一个挑战是在适当的发育和可塑性背景下检查功能相关的细胞。我们已经开发了基于细胞的基因组分析的方法和实验系统，并在已建立的皮质可塑性范例中研究MeCP2。我们的一般假设是，通过跟踪全基因组5mC和5hmC的分布和动态，MeCP2在经验依赖的神经元成熟过程中以一种依赖于细胞类型和细胞状态的方式调节染色质结构和基因转录。我们将应用基于细胞的DNA甲基组(包括5mC和5hmC)和转录组的分析来研究皮质谷氨酸能和GABA能神经元在出生后成熟过程中这些特征之间的关系。然后，我们将研究MeCP2突变对这些神经元中的甲基组和转录组的影响。我们将进一步研究MeCP2是否作为经验驱动的小白蛋白阳性GABA中间神经元成熟的“活动调节刹车”，这是初级视皮层关键期可塑性的开始和进展的时间。结合细胞特异性、碱基分辨率分析 在明确定义的神经发育背景下，这些研究将提供对胞嘧啶甲基化、MeCP2功能和基因表达之间的调控关系的洞察，这是大脑表观遗传调控的一个基本问题。我们旨在揭示MeCP2突变的发育轨迹和遗传结构，并提出RTT的干预策略。

DESCRIPTION (provided by applicant): Mutations in the Methyl-CpG binding protein 2 gene (MeCP2) cause Rett syndrome, an autism spectrum disorder. RTT is hypothesized to result from deficient neuronal maturation and plasticity, possibly through abnormal experience-dependent synapse development and maintenance. MeCP2 has been implicated in chromatin and transcription regulation through binding to methyl-cytosines, which includes 5-methylcytosine (5mC) as well as 5-hydroxymethylcytosine (5hmC). While 5mC is generally viewed as a "silencing mark", 5hmC might represent methylation dynamics and play complex roles in gene regulation. However, the genomic distributions of 5mC and 5hmC in brain cells are poorly characterized, and it is unclear how MeCP2 might interpret 5mC and 5hmC patterns, thereby influencing transcription. It remains controversial whether MeCP2 is primarily a transcriptional repressor that modulates gene-specific targets or act globally in chromatin regulation. Identifying the transcriptional impact is a critical step toward understanding the mechanism of MeCP2 function. Characterizing altered transcription in relevant brain regions, neuron types, developmental and physiological context in mouse models is necessary for unraveling the pathogenic mechanism of RTT. A key challenge in studying the role of MeCP2 in gene regulation in the brain is cellular heterogeneity. For example, it has been difficult to achieve high-resolution mapping of methylome in defined neuron types. Further, nerve cells modify their epigenomes and transcriptomes during development and in response to neuronal input. Thus an additional challenge is to examine functionally relevant cells in an appropriate developmental and plasticity context. We have developed methods and experimental systems for cell-based genomic analysis and for studying MeCP2 in a well established paradigm of cortical plasticity. Our General Hypothesis is that by tracking genome wide 5mC and 5hmC distributions and dynamics, MeCP2 regulates chromatin structures and gene transcription in a cell type- and cell state-dependent manner during experience-dependent neuronal maturation. We will apply cell-based analysis of DNA methylome (including 5mC and 5hmC) and transcriptome to examine the relationship among these profiles in cortical glutamatergic and GABAergic neurons during postnatal maturation. We will then examine the impact of MeCP2 mutations on methylomes and transcriptome in these neurons. We will further examine whether MeCP2 functions as an "activity-regulated brake" of experience-driven maturation of parvalbumin-positive GABA interneurons, which time the onset and progression of the critical period plasticity in primary visual cortex. Combining cell specificity, base resolution analysis of methylomes and their impact on transcriptomes in a well-defined context of neural development, these studies will provide insight into the regulatory relationships among cytosine methylation, MeCP2 function and gene expression, a fundamental issue in epigenetic regulation of the brain. We aim to reveal the developmental trajectory and genetic architecture of MeCP2 mutation and suggest strategies for intervention for RTT.