Investigating the Epigenetic Regulation of Imprinted Gene Grb10 in Neurodevelopment

项目摘要 这项建议的目的是研究印迹的基因组印记的表观遗传学机制。 基因生长因子受体结合蛋白-10(GRB10)在神经性和非神经性发育中的作用。 印记基因是哺乳动物独有的，以单等位基因、亲本特有的方式表达。 印记基因表达的失调与神经、行为和生长障碍有关。 Grb10与生长障碍Silver-Russell综合征有关，它是从父亲的等位基因中唯一表达的 在神经细胞中和在非神经元细胞中来自母体等位基因。引人注目的是，母方和父方 转录起始于不同的启动子区域，这种新的开关在神经元中的性质尚不清楚。 这项建议旨在研究Grb10基因座的等位基因和异构体开关是如何发生的，以实现唯一的 亲本起源在神经元中的表达。我们和其他人已经确定了关键DNA的结合部位 差异甲基化Grb10印迹控制区(ICR)内的建筑蛋白CTCF。我们的 实验室还描述了CTCF在控制小鼠单等位基因Grb10表达方面的重要性 胚胎干细胞(MESCs)。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。CTCFs催化神经元特异性的能力 将通过在体外神经元分化中突变ICR内的结合位点来评估其表达 模特。目标2将验证组织特异性增强剂调节神经元中Grb10开关的假设。一个 在mESCs中，通过CRISPR编辑将移除可能的增强子，并将组织特异性Grb10表达 在衍生的神经元中进行测量。本方案中的研究以印记基因Grb10为模型进行研究 控制组织特异性基因表达的表观遗传学和分子机制。考虑到这么多基因 在中枢神经系统中也是组织和等位基因特异的，识别协调的表观遗传因素 Grb10在神经元中的表达将阐明其他复杂基因是如何在 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.