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

Rett综合征小鼠模型分子病理的细胞基因组学分析 摘要 甲基-CpG结合蛋白2基因（MeCP 2）的突变导致Rett综合征，一种自闭症谱系障碍。RTT是 假设是由于缺乏神经元成熟和可塑性，可能通过异常的经验依赖性 突触发育和维持。MeCP 2通过以下途径参与染色质和转录调节： 结合甲基胞嘧啶，包括5-甲基胞嘧啶（5 mC）和5-羟甲基胞嘧啶（5 hmC）。而 5 mC通常被认为是一种“沉默标记”，5 hmC可能代表甲基化动力学并在基因组中起着复杂的作用。 基因调控然而，5 mC和5 hmC在脑细胞中的基因组分布的特征很差， 目前还不清楚MeCP 2如何解释5 mC和5 hmC模式，从而影响转录。它仍然存在争议 MeCP 2是否主要是一种转录抑制因子，调节基因特异性靶点或在染色质中全面发挥作用 调控确定转录影响是理解MeCP 2机制的关键一步。 功能表征相关脑区、神经元类型、发育和生理中的转录改变 在小鼠模型中的上下文对于解开RTT的致病机制是必要的。 研究MeCP 2在大脑基因调控中的作用的一个关键挑战是细胞异质性。例如它 已经很难在确定的神经元类型中实现甲基化组的高分辨率映射。此外，神经细胞改变 它们的表观基因组和转录组在发育过程中以及对神经元输入的反应。因此，一个额外的挑战 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 用于基于细胞的基因组分析和用于在良好建立的 皮层可塑性的范例。我们的一般假设是，通过跟踪基因组范围的5 mC和5 hmC分布， 动力学，MeCP 2以细胞类型和细胞状态依赖性方式调节染色质结构和基因转录 经历依赖的神经元成熟过程中。我们将应用基于细胞的DNA甲基化组分析（包括5 mC 和5 hmC）和转录组，以检查皮质多巴胺能和GABA能神经元中这些谱之间的关系。 神经元在出生后的成熟。然后，我们将研究MeCP 2突变对甲基化的影响， 这些神经元中的转录组。我们将进一步研究MeCP 2是否起“活性调节制动器”的作用， 经验驱动的小清蛋白阳性GABA中间神经元的成熟，其时间的开始和进展， 初级视皮层的关键期可塑性。结合细胞特异性、甲基化组的碱基解析分析， 在神经发育的明确背景下，它们对转录组的影响，这些研究将提供深入了解 胞嘧啶甲基化、MeCP 2功能和基因表达之间的调控关系，是 大脑的表观遗传调控我们的目标是揭示MeCP 2的发育轨迹和遗传结构， 突变，并提出干预RTT的策略。

Cell-based Genomic Analysis of molecular pathology in Mouse Models of Rett Syndrome ABSTRACT 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.