1] Graduate School of Frontier Biosciences, Osaka University, Osaka, 565-0871, Japan [2] Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA [3] Transcription Imaging Consortium, Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA.
1] Graduate School of Frontier Biosciences, Osaka University, Osaka, 565-0871, Japan [2] Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Kawaguchi, Saitama, 332-0012, Japan [3] Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan.
Nature. 2014 Dec 11;516(7530):272-5. doi: 10.1038/nature13714. Epub 2014 Sep 21.
In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.
在真核细胞中，翻译后组蛋白修饰在基因调控中起着重要作用。从早期的组蛋白乙酰化研究开始，各种残基特异性修饰现已与 RNA 聚合酶 II（RNAP2）活性相关联，但这些标记是否是转录的有效调节剂，还是仅仅是被动的副产物，仍不清楚。这是因为传统的研究依赖于固定的细胞群体，这意味着时间分辨率最多只能达到分钟级，而且相关的因素实际上可能不在同一时间存在于同一细胞中。因此，需要互补的方法来在单个活细胞中以更高的时间分辨率探测组蛋白修饰和 RNAP2 的动态相互作用。在这里，我们通过开发一种荧光显微镜系统来解决这个问题，该系统可以跟踪活细胞中残基特异性组蛋白修饰和 RNAP2 的磷酸化。这将时间分辨率提高到了几十秒的范围。我们的单细胞分析表明，基因座处的组蛋白 H3 赖氨酸 27 乙酰化可以改变下游转录动力学高达 50%，影响两个时间上分离的事件。首先，乙酰化增强了转录激活剂的搜索动力学，随后，乙酰化加速了 RNAP2 从起始到延伸的转变。使用染色质免疫沉淀 followed by sequencing 可以在全基因组范围内找到后者的特征。我们认为这种调节导致了稳健且具有潜在可调节性的转录反应。
