Investigating the Epigenetic Regulation of Imprinted Gene Grb10 in Neurodevelopment

项目概要 该提案的目的是研究印迹基因组印记的表观遗传机制 基因生长因子受体结合蛋白 10 (GRB10) 在神经元与非神经元发育中的作用。 印记基因是哺乳动物所独有的，并且以单等位基因、亲本特异性方式表达。 印记基因表达失调与神经、行为和生长障碍有关。 GRB10 与生长障碍 Silver-Russell 综合征有关，由父系等位基因独特表达 在神经元细胞中来自母体等位基因，在非神经元细胞中来自母体等位基因。令人惊讶的是，母亲和父亲 转录本从不同的启动子区域起始，神经元中这种新开关的性质尚不清楚。 该提案旨在研究等位基因和异构体转换如何在 Grb10 位点发生，以实现独特的 神经元中的亲本表达。我们和其他人已经确定了关键 DNA 的结合位点 差异甲基化 Grb10 印记控制区 (ICR) 内的结构蛋白 CTCF。我们的 实验室还描述了 CTCF 在控制小鼠单等位基因 Grb10 表达中的重要性 胚胎干细胞（mESC）。目标 1 将检验以下假设：需要 CTCF 来协调 Grb10 表达中的印迹和分子开关。 CTCF 催化神经元特异性的能力 通过在体外神经元分化中突变 ICR 内的结合位点来评估表达 模型。目标 2 将检验组织特异性增强子调节神经元中 Grb10 开关的假设。一个 mESC 中的 CRISPR 编辑将去除推定的增强子，并且组织特异性 Grb10 表达将被 在派生神经元中测量。本提案中的研究使用印记基因Grb10作为模型来研究 控制组织特异性基因表达的表观遗传和分子机制。鉴于许多基因 在中枢神经系统中也具有组织和等位基因特异性，识别协调的表观遗传因素 神经元中的 Grb10 表达将阐明其他复杂基因如何在神经元中受到差异调节 神经发育。

Project Summary The objective of this proposal is to investigate the epigenetic mechanism of genomic imprinting of the imprinted gene Growth Factor Receptor Bound Protein-10 (GRB10) in neuronal versus non-neuronal development. Imprinted genes are unique to mammals and are expressed in a monoallelic, parent-of-origin-specific manner. Dysregulation of imprinted gene expression is associated with neurological, behavioral and growth disorders. GRB10, implicated in growth disorder Silver-Russell syndrome, is uniquely expressed from the paternal allele in neuronal cells and from the maternal allele in non-neuronal cells. Strikingly, the maternal and paternal transcripts initiate from different promoter regions and the nature of this novel switch in neurons is unclear. This proposal aims to investigate how the allelic and isoform switch occurs at the Grb10 locus to enable unique parent-of-origin expression in neurons. We and others have identified binding sites for the critical DNA architectural protein CTCF within the differentially methylated Grb10 imprinting control region (ICR). Our laboratory also described the importance of CTCF in controlling monoallelic Grb10 expression in mouse embryonic stem cells (mESCs). Aim 1 will test the hypothesis that CTCF is required for coordinating the imprinted and molecular switch in Grb10 expression. The ability of CTCF to catalyze neuronal-specific expression will be assessed by mutating binding sites within the ICR in an in vitro neuronal differentiation model. Aim 2 will test the hypothesis that tissue-specific enhancers regulate the Grb10 switch in neurons. A putative enhancer will be removed by CRISPR editing in mESCs, and tissue-specific Grb10 expression will be measured in derived neurons. The studies in this proposal use the imprinted gene Grb10 as a model to study the epigenetic and molecular mechanisms that govern tissue-specific gene expression. Given that many genes in the central nervous system are also tissue and allele-specific, discerning the epigenetic factors coordinating Grb10 expression in neurons will elucidate how other complex genes are differentially regulated in neurodevelopment.