半合成
核小体
组蛋白
染色质
组蛋白密码
计算生物学
化学
细胞生物学
生物
生物化学
DNA
作者
Nora Guidotti,Beat Fierz
出处
期刊:Methods in molecular biology
日期:2020-01-01
卷期号:: 263-291
被引量:1
标识
DOI:10.1007/978-1-0716-0434-2_13
摘要
Nucleosomes, the basic unit of chromatin, contain a protein core of histone proteins, which are heavily posttranslationally modified. These modifications form a combinatorial language which defines the functional state of the underlying genome. As each histone type exists in two copies in a nucleosome, the modification patterns can differ between the individual histones, resulting in asymmetry and increasing combinatorial complexity. To systematically explore the regulation of chromatin regulatory enzymes (writers, erasers, or readers), chemically defined nucleosomes are required. We have developed strategies to chemically modify histones and control nucleosome assembly, thereby enabling the reconstitution of asymmetric histone modification patterns. Here, we report a detailed protocol for the modular assembly of such nucleosomes. Employing a three-segment ligation strategy for the semisynthesis of H3, coupled with the use of the protease cleavable “lnc-tag,” we provide an efficient and traceless method for the controlled semisynthesis and reconstitution of asymmetrically modified nucleosomes.
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