Gene expression profiles of wild type and Mecp2-null mice in three different regions of the brain

Background. Rett syndrome (RTT) is a complex neurodevelopmental disorder that is one of the most frequent causes of mental retardation in women. A great landmark in research in this field was the discovery of a relationship between the disease and the presence of mutations in the gene that codes for the methyl-CpG binding protein 2 (MeCP2). Currently, MeCP2 is thought to act as a transcriptional repressor that couples DNA methylation and transcriptional silencing. The present study aimed to identify new target genes directly regulated by Mecp2 in a mouse model of RTT. Methodology. We have compared the gene expression profiles of wild type (WT) and Mecp2-null (KO) mice in three regions of the brain (cortex, midbrain, and cerebellum) by using cDNA microarrays. The results obtained were confirmed by quantitative real-time PCR. Subsequent chromatin immunoprecipitation assays revealed seven direct target genes of Mecp2 bound in vivo (Dlk1, Mobp, Plagl1, Ddc, Mllt2h, Eya2, and S100a9), and two overexpressed genes due to an indirect effect of a lack of Mecp2 (Irak1 and Prodh). Bisulfite sequencing analysis of the methylation patterns of promoters of the described genes showed no differences between WT and KO mice, demonstrating that methylation differences were not the cause of the observed expression changes. Moreover, the regions bound by Mecp2 were always methylated, suggesting the involvement of the methyl-CpG binding domain of the protein in the mechanism of interaction. Conclusions. We identified new genes that are overexpressed in KO mice and are excellent candidate genes for involvement in various features of the neurodevelopmental disease. Our results demonstrate new targets of MeCP2 and provide us with a better understanding of the underlying mechanisms of RTT. Comparative experiment: Mecp2-null (KO) mice vs. their corresponding age-mated wild type (WT) littermates (CONTROLS). Four couples of KO-WT animals are used and three different brain regions are studied from each couple; cortex, midbrain, and cerebellum.背景。雷特综合征（Rett syndrome, RTT）是一类复杂的神经发育障碍，亦是导致女性智力障碍的最常见病因之一。该领域研究的重大里程碑，在于发现该疾病与编码甲基CpG结合蛋白2（methyl-CpG binding protein 2, MeCP2）的基因发生突变存在关联。目前学界普遍认为，MeCP2作为转录抑制因子，可耦联DNA甲基化与转录沉默过程。本研究旨在通过雷特综合征小鼠模型，鉴定受Mecp2直接调控的全新靶基因。方法学。本研究采用cDNA微阵列技术，对比野生型（wild type, WT）与Mecp2敲除（Mecp2-null, KO）小鼠大脑三个区域（皮层、中脑与小脑）的基因表达谱。所得结果通过实时定量聚合酶链反应进行验证。后续染色质免疫沉淀实验共鉴定出7个在体内与Mecp2特异性结合的直接靶基因（Dlk1、Mobp、Plagl1、Ddc、Mllt2h、Eya2及S100a9），以及2个因Mecp2缺失的间接效应而上调表达的基因（Irak1与Prodh）。对上述基因启动子区域甲基化模式的亚硫酸氢盐测序分析显示，WT与KO小鼠之间未检测到甲基化水平差异，证实甲基化差异并非观测到的基因表达变化的诱因。此外，Mecp2结合的基因组区域始终处于甲基化状态，提示该蛋白的甲基-CpG结合结构域参与了其相互作用机制。结论。本研究鉴定出在KO小鼠中过表达的全新基因，这些基因可作为参与该神经发育疾病多种表型的优质候选靶点。本研究结果明确了MeCP2的全新调控靶标，为深入解析雷特综合征的潜在发病机制提供了新的理论依据。对照实验：Mecp2敲除（Mecp2-null, KO）小鼠与其同月龄、同窝野生型（wild type, WT）对照小鼠的对比实验。本研究共纳入4对KO-WT实验动物，每对动物均采集三个不同脑区样本进行分析，分别为皮层、中脑与小脑。