Nwaobi Sinifunanya E, Olsen Michelle L
Department of Cell Developmental and Integrative Biology, University of Alabama at Birmingham.
Department of Cell Developmental and Integrative Biology, University of Alabama at Birmingham;
J Vis Exp. 2015 Sep 26(103):52406. doi: 10.3791/52406.
DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.
DNA甲基化通过将甲基共价连接到胞嘧啶-鸟嘌呤二核苷酸中胞嘧啶的C5位置来调节基因表达。虽然DNA甲基化能使基因表达产生持久而稳定的变化，但DNA甲基化的模式和水平也会基于各种信号和刺激而发生改变。因此，DNA甲基化作为一种强大而动态的基因表达调节因子发挥作用。神经表观遗传学研究揭示了多种与DNA甲基化的整体及基因特异性变化相关的生理和病理状态。具体而言，在神经精神疾病、神经退行性疾病、突触可塑性过程以及中枢神经系统损伤后，基因表达变化与DNA甲基化之间存在显著相关性。然而，随着神经表观遗传学领域不断拓展对DNA甲基化在中枢神经系统生理学中作用的理解，明确基因表达变化与DNA甲基化之间的因果关系至关重要。此外，在更广泛的神经科学领域，存在大量区域和细胞特异性差异，这就需要在研究转录组、蛋白质组和表观基因组时采用能够解决这些差异的技术。在此，我们描述了一种用于皮质星形胶质细胞的荧光激活细胞分选（FACS）方法，该方法可用于后续对RNA转录和DNA甲基化的检测。此外，我们详细介绍了一种检测DNA甲基化的技术——甲基化敏感高分辨率熔解分析（MS-HRMA）以及荧光素酶启动子分析。通过使用这些组合技术，不仅能够探究DNA甲基化与基因表达之间的相关变化，还能直接评估给定基因区域的DNA甲基化状态变化是否足以影响转录活性。
