Saxton Matthew N, Morisaki Tatsuya, Krapf Diego, Kimura Hiroshi, Stasevich Timothy J
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA.
Department of Electrical and Computer Engineering, and School of Biomedical Engineering, Colorado State University, Fort Collins, CO, USA.
Sci Adv. 2023 Oct 6;9(40):eadh4819. doi: 10.1126/sciadv.adh4819. Epub 2023 Oct 4.
Histone acetylation and RNA polymerase II phosphorylation are associated with transcriptionally active chromatin, but their spatiotemporal relationship in live cells remains poorly understood. To address this problem, we combine Fab-based labeling of endogenous protein modifications with single-molecule tracking to quantify the dynamics of chromatin enriched with histone H3 lysine-27 acetylation (H3K27ac) and RNA polymerase II serine-5 phosphorylation (RNAP2-Ser5ph). Our analysis reveals that chromatin enriched with these two modifications is generally separate. In these separated sites, we show that the two modifications are inversely correlated with one another on the minutes time scale and that single nucleosomes within each region display distinct and opposing dynamics on the subsecond time scale. While nucleosomes diffuse ~15% faster in chromatin enriched with H3K27ac, they diffuse ~15% slower in chromatin enriched with RNAP2-Ser5ph. These results argue that high levels of H3K27ac and RNAP2-Ser5ph are not often present together at the same place and time, but rather each marks distinct transcriptionally poised or active sites, respectively.
组蛋白乙酰化和RNA聚合酶II磷酸化与转录活跃的染色质相关，但它们在活细胞中的时空关系仍知之甚少。为了解决这个问题，我们将基于荧光抗体片段（Fab）的内源性蛋白质修饰标记与单分子追踪相结合，以量化富含组蛋白H3赖氨酸-27乙酰化（H3K27ac）和RNA聚合酶II丝氨酸-5磷酸化（RNAP2-Ser5ph）的染色质动力学。我们的分析表明，富含这两种修饰的染色质通常是分开的。在这些分开的位点，我们发现这两种修饰在分钟时间尺度上彼此呈负相关，并且每个区域内的单个核小体在亚秒时间尺度上表现出不同且相反的动力学。虽然核小体在富含H3K27ac的染色质中扩散速度快约15%，但在富含RNAP2-Ser5ph的染色质中扩散速度慢约15%。这些结果表明，高水平的H3K27ac和RNAP2-Ser5ph并不经常在同一时间出现在同一位置，而是分别标记不同的转录就绪或活跃位点。
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