Kaczmarczyk Artur, Allahverdi Abdollah, Brouwer Thomas B, Nordenskiöld Lars, Dekker Nynke H, van Noort John
From the Huygens-Kamerlingh Onnes Laboratory, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands.
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands, and.
J Biol Chem. 2017 Oct 20;292(42):17506-17513. doi: 10.1074/jbc.M117.791830. Epub 2017 Aug 30.
The eukaryotic genome is highly compacted into a protein-DNA complex called chromatin. The cell controls access of transcriptional regulators to chromosomal DNA via several mechanisms that act on chromatin-associated proteins and provide a rich spectrum of epigenetic regulation. Elucidating the mechanisms that fold chromatin fibers into higher-order structures is therefore key to understanding the epigenetic regulation of DNA accessibility. Here, using histone H4-V21C and histone H2A-E64C mutations, we employed single-molecule force spectroscopy to measure the unfolding of individual chromatin fibers that are reversibly cross-linked through the histone H4 tail. Fibers with covalently linked nucleosomes featured the same folding characteristics as fibers containing wild-type histones but exhibited increased stability against stretching forces. By stabilizing the secondary structure of chromatin, we confirmed a nucleosome repeat length (NRL)-dependent folding. Consistent with previous crystallographic and cryo-EM studies, the obtained force-extension curves on arrays with 167-bp NRLs best supported an underlying structure consisting of zig-zag, two-start fibers. For arrays with 197-bp NRLs, we previously inferred solenoidal folding, which was further corroborated by force-extension curves of the cross-linked fibers. The different unfolding pathways exhibited by these two types of arrays and reported here extend our understanding of chromatin structure and its potential roles in gene regulation. Importantly, these findings imply that chromatin compaction by nucleosome stacking protects nucleosomal DNA from external forces up to 4 piconewtons.
真核生物基因组高度压缩成一种名为染色质的蛋白质 - DNA 复合物。细胞通过作用于染色质相关蛋白的多种机制来控制转录调节因子对染色体 DNA 的访问，并提供了丰富的表观遗传调控谱。因此，阐明将染色质纤维折叠成高阶结构的机制是理解 DNA 可及性表观遗传调控的关键。在这里，我们使用组蛋白 H4 - V21C 和组蛋白 H2A - E64C 突变，采用单分子力谱法来测量通过组蛋白 H4 尾巴可逆交联的单个染色质纤维的解折叠。具有共价连接核小体的纤维具有与含有野生型组蛋白的纤维相同的折叠特征，但对拉伸力表现出更高的稳定性。通过稳定染色质的二级结构，我们证实了依赖核小体重复长度（NRL）的折叠。与先前的晶体学和冷冻电镜研究一致，在具有 167 - bp NRL 的阵列上获得的力 - 伸长曲线最有力地支持了由之字形双起始纤维组成的基础结构。对于具有 197 - bp NRL 的阵列，我们先前推断为螺线管折叠，交联纤维的力 - 伸长曲线进一步证实了这一点。这里报道的这两种类型阵列所表现出的不同解折叠途径扩展了我们对染色质结构及其在基因调控中潜在作用的理解。重要的是，这些发现意味着通过核小体堆积实现的染色质压缩可保护核小体 DNA 免受高达 4 皮牛顿的外力作用。
