Jiang Shuangying, Liu Yan, Xu Caiyue, Wang Yun, Gong Jianhui, Shen Yue, Wu Qingyu, Boeke Jef D, Dai Junbiao
Ministry of Education Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, PR China.
Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, PR China.
G3 (Bethesda). 2017 Dec 4;7(12):3857-3866. doi: 10.1534/g3.117.300252.
Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function of each amino acid residue involved in various stress conditions including exposure to different DNA damage-inducing reagents, different growth temperatures, and other chemicals. H2B N- and H2A C-termini were critical for maintaining nucleosome function and mutations in these regions led to pleiotropic phenotypes. Additionally, two screens were performed using this library, monitoring heterochromatin gene silencing and genome stability, to identify residues that could compromise normal function when mutated. Many distinctive regions within the nucleosome were revealed. Furthermore, we used the barcode sequencing (bar-seq) method to profile the mutant composition of many libraries in one high-throughput sequencing experiment, greatly reducing the labor and increasing the capacity. This study not only demonstrates the applications of the versatile histone library, but also reveals many previously unknown functions of histone H2A and H2B.
我们使用了一个全面的组蛋白H2A和H2B突变体文库，评估了参与各种应激条件的每个氨基酸残基的生物学功能，这些应激条件包括暴露于不同的DNA损伤诱导试剂、不同的生长温度和其他化学物质。H2B的N端和H2A的C端对于维持核小体功能至关重要，这些区域的突变会导致多效性表型。此外，利用该文库进行了两项筛选，监测异染色质基因沉默和基因组稳定性，以鉴定突变时可能损害正常功能的残基。揭示了核小体内许多独特的区域。此外，我们使用条形码测序（bar-seq）方法在一次高通量测序实验中分析许多文库的突变体组成，大大减少了工作量并提高了通量。这项研究不仅展示了通用组蛋白文库的应用，还揭示了组蛋白H2A和H2B许多以前未知的功能。
