Assessment and site-specific manipulation of DNA (hydroxy-)methylation during mouse corticogenesis

Purpose: In this study we investigated the molecular role of cytosine modification in the developing cortex of mice. To this aim we isolated thee linage related cell populations of the developing cortex and mapped both, 5mC as well as 5hmC on a genome-wide scale. Furthermore we established a system to site-specifcally demethylate DNA by using a dCas9-Tet1 fusion protein. Methods: Neuronal stem cells (Btg2-/Tubb3-), neurogenic progenitors (Btg2+/Tubb3-) and neurons (Tubb3+) were isolated from E14.5 Btg2-RFP/Tubb3-GFP double heterozygous mouse embryos (Aprea et al. 2013) and were used for a comparative immunoprecipitation of 5mC and 5hmC (Tan et al. 2013). Results: We were able to identify several genomic regions that change dynamically (hydroxy-)methylation patterns in specific cell type. Specifically for regions losing 5mC and gaining 5hmC we found a consistent enrichment in neurogenesis related GOterms, genomic features, transcription factor binding motifs and different histone marks. Furthermore we found that loss in 5mC and gain in 5hmC correlate with each other and lead to the upregulation of nearby genes. Site-specific demethylation of the identified differentially (hydroxy-)methylated regions in vivo, led to a premature upregulation of the nearby genes which resulted in an altered development of the brain. Genome-wide maps of 5-methylcytosine as well as 5-hydroxymethylcytosine in three lineage-related cell populations of the developing cortex of mice (E14.5).研究目的：本研究旨在探究胞嘧啶修饰在小鼠发育大脑皮层中的分子调控机制。为此，我们分离得到小鼠发育皮层内三种谱系相关的细胞群体，并在全基因组范围内绘制了5-甲基胞嘧啶（5-methylcytosine, 5mC）与5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）的表观基因组图谱。此外，我们建立了一套基于dCas9-Tet1融合蛋白的DNA位点特异性去甲基化实验体系。 实验方法：从胚胎发育第14.5天（E14.5）的Btg2-RFP/Tubb3-GFP双杂合小鼠胚胎（Aprea等，2013）中，分别分离出神经干细胞（Btg2⁻/Tubb3⁻）、神经发生祖细胞（Btg2⁺/Tubb3⁻）以及成熟神经元（Tubb3⁺），并采用比较免疫沉淀法对5mC与5hmC进行富集分析（Tan等，2013）。 研究结果：我们成功鉴定出多个在特定细胞类型中呈现动态（羟）甲基化模式变化的基因组区域。具体而言，在5mC水平下调、5hmC水平上调的基因组区域中，我们发现其显著富集于神经发生相关的基因本体（Gene Ontology, GO）术语、特异性基因组特征、转录因子结合基序以及多种组蛋白修饰标记。此外，我们证实5mC的丢失与5hmC的增加呈显著正相关，且该表观遗传变化会导致邻近基因的表达上调。在体内对鉴定得到的差异（羟）甲基化区域进行位点特异性去甲基化，可促使邻近基因提前激活表达，最终引发大脑发育进程异常。 本数据集包含小鼠发育皮层（E14.5）三种谱系相关细胞群体的全基因组5-甲基胞嘧啶与5-羟甲基胞嘧啶图谱。