Mittal Chitvan, Blacketer Melissa J, Shogren-Knaak Michael A
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.
Anal Biochem. 2014 Jul 15;457:51-8. doi: 10.1016/j.ab.2014.04.024. Epub 2014 Apr 24.
The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.
翻译后染色质修饰的建立是调节基因组DNA利用方式的主要机制。然而，目前的体外染色质检测方法无法监测单个核小体上的组蛋白修饰。在此，我们描述了一种策略——核小体乙酰化测序，它使我们能够读取每个核小体上的修饰量。在这种方法中，一种与珠子结合的三核小体底物被来自酿酒酵母的SAGA复合物用放射性标记的乙酰辅酶A进行酶促乙酰化。产物用在核小体之间独特位点切割的限制性内切酶消化，然后计数以量化每个核小体位点的乙酰化程度。我们发现我们能够灵敏、特异且可重复地追踪酶介导的核小体乙酰化。应用该策略，当乙酰化广泛进行时，其在核小体上的分布相对均匀。然而，当使用在SAGA介导的乙酰化主要位点发生突变的核小体底物，或者在初始速率条件下进行研究时，可以观察到乙酰化分布的变化。核小体乙酰化测序应适用于分析广泛的修饰。此外，由于我们的三核小体合成策略具有高度模块化和高效性，它可用于生成核小体阵列中核小体组成不同的核小体系统。
