A Robust Method for the Purification and Characterization of Recombinant Human Histone H1 Variants
A Robust Method for the Purification and Characterization of Recombinant Human Histone H1 Variants
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DOI：
10.1021/acs.biochem.8b01060
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发表时间：
2019-01-22
期刊：
影响因子：
2.9
通讯作者：
David, Yael
中科院分区：
文献类型：
--
作者：
Osunsade, Adewola;Prescott, Nicholas A.;David, Yael
真核基因组的高阶压缩是调节所有 DNA 模板过程（包括转录）的关键。这个严格控制的过程涉及单核小体（染色质的基本单位）的形成，在 HI 连接子组蛋白依赖性过程中包装成更高阶的结构。尽管已经做了很多工作来描述这一事件在体外和体内的精确机制，但仍然存在重大差距，这主要是由于缺乏分子工具。具体而言，从未对所有人类 HI 变体进行成功的纯化和生化表征。在这里，我们提出了一种稳健的方法来纯化 HI，并说明其在纯化所有体细胞变异和一种种系变异中的效用。此外，我们进行了首次并排生化比较，揭示了核小体结合亲和力和压缩能力的梯度。这些数据提供了对 H1 冗余的新见解，并为疾病驱动突变的机制研究奠定了基础。
Higher order compaction of the eukaryotic genome is key to the regulation of all DNA-templated processes, including transcription. This tightly controlled process involves the formation of mononucleosomes, the fundamental unit of chromatin, packaged into higher order architectures in an HI linker histone-dependent process. While much work has been done to delineate the precise mechanism of this event in vitro and in vivo, major gaps still exist, primarily due to a lack of molecular tools. Specifically, there has never been a successful purification and biochemical characterization of all human HI variants. Here we present a robust method to purify HI and illustrate its utility in the purification of all somatic variants and one germline variant. In addition, we performed a first ever side-by-side biochemical comparison, which revealed a gradient of nucleosome binding affinities and compaction capabilities. These data provide new insight into H1 redundancy and lay the groundwork for the mechanistic investigation of disease-driving mutations.