W. H. Coulter Department of Biomedical Engineering, Emory University School of Medicine, Atlanta, GA, USA.
Methods Mol Biol. 2023;2599:191-214. doi: 10.1007/978-1-0716-2847-8_14.
Chromatin is the nucleoprotein complex that organizes genomic DNA in the nuclei of eukaryotic cells. Chromatin-modifying enzymes and chromatin-binding regulators generate chromatin states that affect DNA compaction, repair, gene expression, and ultimately cell phenotype. Many natural chromatin mediators contain subdomains that can be isolated and recombined to build synthetic regulators and probes. Engineered chromatin proteins make up a growing collection of new tools for cell engineering and can help deepen our understanding of the mechanism by which chromatin features, such as modifications of histones and DNA, contribute to the epigenetic states that govern DNA-templated processes. To support efficient exploration of the large combinatorial design space of synthetic chromatin proteins, we have developed a Golden Gate assembly method for one-step construction of protein-encoding recombinant DNA. A set of standard 2-amino acid linkers allows facile assembly of any combination of up to four protein modules, obviating the need to design different compatible overhangs to ligate different modules. Beginning with the identification of protein modules of interest, a synthetic chromatin protein can be built and expressed in vitro or in cells in under 2 weeks.
染色质是真核细胞细胞核中组织基因组 DNA 的核蛋白复合物。染色质修饰酶和染色质结合调节剂产生影响 DNA 压缩、修复、基因表达，最终影响细胞表型的染色质状态。许多天然染色质介质包含可以分离和重组的亚结构域，以构建合成调节剂和探针。工程化染色质蛋白构成了用于细胞工程的越来越多的新工具，有助于加深我们对染色质特征（如组蛋白和 DNA 的修饰）如何影响控制 DNA 模板过程的表观遗传状态的理解。为了支持对合成染色质蛋白的大型组合设计空间的有效探索，我们开发了一种 Golden Gate 组装方法，用于一步构建蛋白质编码重组 DNA。一组标准的 2 氨基酸接头允许轻松组装多达四个蛋白质模块的任意组合，避免了为连接不同模块而设计不同兼容突出端的需要。从感兴趣的蛋白质模块的鉴定开始，合成染色质蛋白可以在不到 2 周的时间内在体外或细胞中构建和表达。
