Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States.
Elife. 2017 Sep 12;6:e28836. doi: 10.7554/eLife.28836.
The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.
染色质的重复亚基核小体包含每个核心组蛋白的两个拷贝，最近的几项研究报告称，不对称修饰的核小体存在于体内的调控元件中。为了探究组蛋白修饰被读取的机制，我们设计了一对必需的 H3 异二聚体，称为 H3X 和 H3Y，我们通过遗传和生化方法对其进行了广泛验证。通过比较不对称组蛋白尾部点突变与对称双突变的影响，我们发现每个核小体中仅一个甲基化 H3K36 就足以在体内沉默隐蔽转录。我们还展示了该系统在分析组蛋白修饰串扰方面的实用性，使用质谱法分别鉴定不对称核小体中每个 H3 分子上的修饰。在体内和体外生成不对称核小体的能力为探究效应蛋白在细胞中读取 H3 尾部的机制提供了一种强大且可推广的工具。
