Chiarella Anna M, Wang Tiffany A, Butler Kyle V, Jin Jian, Hathaway Nathaniel A
Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, University of North Carolina.
College of Arts and Sciences, Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina.
J Vis Exp. 2018 Sep 20(139):58222. doi: 10.3791/58222.
Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.
染色质压缩的调控是高等真核生物中控制基因表达的重要过程。尽管在许多疾病中染色质压缩和基因表达调控通常会受到破坏，但一直缺乏一种位点特异性、内源性且可逆的方法来研究和控制这些作用机制。为了解决这个问题，我们开发并表征了新型的基因调控双功能分子。双功能分子的一个组分与DNA-蛋白质锚定物结合，从而使其被招募到等位基因特异性位点。另一个组分与内源性细胞染色质修饰机制相互作用，将这些蛋白质招募到感兴趣的基因。这些小分子被称为化学表观遗传修饰剂（CEMs），能够以剂量依赖性和可逆的方式控制基因表达和染色质环境。在这里，我们详细介绍一种CEM方法及其在降低小鼠胚胎干细胞（mESCs）中位于Oct4位点的绿色荧光蛋白（GFP）报告基因的基因表达和组蛋白尾巴乙酰化方面的应用。我们使用荧光显微镜、流式细胞术和染色质免疫沉淀（ChIP），随后进行定量聚合酶链反应（qPCR）来表征先导CEM（CEM23）。虽然该系统的能力在Oct4位点得到了证明，但从概念上讲，CEM技术是模块化的，可应用于其他细胞类型和其他基因组位点。
