Yue Hongjun, Fang He, Wei Sijie, Hayes Jeffrey J, Lee Tae-Hee
Department of Chemistry, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
Department of Biochemistry and Biophysics, Rochester University Medical Center , Rochester, New York 14625, United States.
Biochemistry. 2016 Apr 12;55(14):2069-77. doi: 10.1021/acs.biochem.5b01247. Epub 2016 Mar 29.
Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.
连接组蛋白H1调节染色质结构和基因表达。研究H1与DNA及核小体结合的动力学和化学计量对于阐明其功能至关重要。由于H1具有丰富的正电荷和很强的自身亲和力，对其与DNA及核小体结合的定量体外研究产生了差异很大的结果，因此应以系统特异性的方式进行解释。我们试图通过开发一种经过特殊钝化的显微镜载玻片表面来克服这一限制，以便在单分子水平上监测H1与DNA及核小体的结合。根据我们的测量，H1与DNA及核小体结合的化学计量非常不均一，分布范围很广，其平均值与先前发表的值合理一致。我们的研究还表明，在数十分钟的时间尺度上，H1不会从DNA或核小体上解离。我们发现组蛋白伴侣Nap1能使H1从DNA上轻易解离，并使超化学计量结合在核小体上的H1解离，这支持了一种假说，即组蛋白伴侣有助于调节染色质中H1的分布。
