Department of Chemistry, The Pennsylvania State University, State College, Pennsylvania 16801, United States.
N. K. Koltsov Institute of Developmental Biology of Russian Academy of Sciences, Vavilova Street 26, Moscow 119334, Russia.
ACS Chem Biol. 2023 Mar 17;18(3):537-548. doi: 10.1021/acschembio.2c00887. Epub 2023 Mar 1.
Post-translational modifications of histone proteins often mediate gene regulation by altering the global and local stability of the nucleosome, the basic gene-packing unit of eukaryotes. We employed semisynthetic approaches to introduce histone H2B ubiquitylations at K34 (H2BK34ub) and K120 (H2BK120ub) and H3K79 trimethylation (H3K79me3). With these modified histones, we investigated their effects on the kinetics of transcription elongation by RNA polymerase II (Pol II) using single-molecule FRET. Pol II pauses at several locations within the nucleosome for a few seconds to minutes, which governs the overall transcription efficiency. We found that H2B ubiquitylations suppress pauses and shorten the pause durations near the nucleosome entry while H3K79me3 shortens the pause durations and increases the rate of RNA elongation near the center of the nucleosome. We also found that H2BK34ub facilitates partial rewrapping of the nucleosome upon Pol II passage. These observations suggest that H2B ubiquitylations promote transcription elongation and help maintain the chromatin structure by inducing and stabilizing nucleosome intermediates and that H3K79me3 facilitates Pol II progression possibly by destabilizing the local structure of the nucleosome. Our results provide the mechanisms of how these modifications coupled by a network of regulatory proteins facilitate transcription in two different regions of the nucleosome and help maintain the chromatin structure during active transcription.
组蛋白蛋白的翻译后修饰通常通过改变核小体（真核生物的基本基因包装单位）的全局和局部稳定性来介导基因调控。我们采用半合成方法在 K34（H2BK34ub）和 K120（H2BK120ub）以及 H3K79 上引入组蛋白 H2B 的泛素化和 H3K79 的三甲基化。使用这些修饰的组蛋白，我们通过单分子 FRET 研究了它们对 RNA 聚合酶 II（Pol II）转录延伸动力学的影响。Pol II 在核小体内部的几个位置暂停几秒钟到几分钟，这决定了整体转录效率。我们发现 H2B 泛素化抑制暂停并缩短核小体进入处的暂停持续时间，而 H3K79me3 缩短暂停持续时间并增加核小体中心附近的 RNA 延伸速率。我们还发现 H2BK34ub 促进 Pol II 通过时核小体的部分重新缠绕。这些观察结果表明，H2B 泛素化通过诱导和稳定核小体中间体促进转录延伸并有助于维持染色质结构，而 H3K79me3 可能通过破坏核小体的局部结构促进 Pol II 进展。我们的结果提供了这些修饰如何通过调控蛋白网络在核小体的两个不同区域促进转录并在活跃转录过程中帮助维持染色质结构的机制。
