Open AccessDe novo reconstitution of chromatin using wheat germ cell‐free protein synthesis
Author(s) -
Endo Yaeta,
Takemori Nobuaki,
Nagy Szilvia K.,
Okimune Keiichi,
Kamakaka Rohinton,
Onouchi Hitoshi,
Takasuka Taichi E.
Publication year - 2021
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1002/2211-5463.13178
Subject(s) - chromatin , histone , nucleosome , histone modifying enzymes , microbiology and biotechnology , chromatin remodeling , biology , dna , dna supercoil , histone code , dna replication , genetics
DNA is packaged with histones to form chromatin that impinges on all nuclear processes, including transcription, replication and repair, in the eukaryotic nucleus. A complete understanding of these molecular processes requires analysis of chromatin context in vitro . Here, Drosophila four core histones were produced in a native and unmodified form using wheat germ cell‐free protein synthesis. In the assembly reaction, four unpurified core histones and three chromatin assembly factors (dNAP‐1, dAcf1 and dISWI) were incubated with template DNA. We then assessed stoichiometry with the histones, nucleosome arrays, supercoiling and the ability of the chromatin to serve as a substrate for histone‐modifying enzymes. Overall, our method provides a new avenue to produce chromatin that can be useful in a wide range of chromatin research.