Researchers reveal the mechanism of nucleosome assembly by chromatin assembly factor-1

Inheritance of chromatin during cell division involves DNA replication and assembly of nucleosomes on the replicated DNA, because passage of the DNA replication fork disrupts nucleosomes on the DNA. Half of the histones for the assembly of transcription-associated nucleosomes come from malfunctioning parental nucleosomes, and the other half are newly synthesized.

Chromatin assembly factor-1 (CAF-1) is an evolutionarily conserved triplomeric protein complex that is responsible for the deposition of newly synthesized histones H3 and H4 on DNA. However, the lack of structural information about CAF-1 has hampered understanding of the molecular mechanism underlying neonucleosome assembly.

In a study published in Sciences On August 25, a research team led by Professor Xu Ruiming of the Institute of Biophysics (IBP) of the Chinese Academy of Sciences, in collaboration with Professor Li Guohong, Professor Zhu Bing, and Professor Liu Chaopei, all from IBP, reported high-resolution structures of CAF- 1 and CAF-1 bound to histones H3-H4.

The authors first determined the crystal structure of the core domain of human CAF-1 in the absence of histones and the electron microscopy (cryo-EM) structure of CAF-1 in complex with histones H3 and H4.

The results show that the CAF-1 complex binds to the H3-H4 heterodimer mainly through the p60 subunit and the ED loop of the p150 subunit. The C-terminal portion of the ED loop is key to the biological function of CAF-1, judged by confirming structural information with in vitro histone binding assays and plasmid supercoiling assays, as well as in nascent nucleosome assembly mapping and transcriptome analyses.

The researchers also found that a 30-bp DNA oligomer reduced the CAF-1-H3-H4 complex, resolving the cryo-EM structure of the 2:2 CAF-1-H3-H4 complex. This structure showed that two adjacent H3-H4 heterodimers prepare to form an H3-H4 tetramer via dimerization of the two H3s, but the position of the heterodimers is not yet in the exact geometric arrangement of the H3-H4 tetramer, indicating that the histone H3-H4 linker remodeling By CAF-1, possibly with the binding of a longer DNA fragment, it is necessary for the assembly of the H3-H4 tetramer which is a basic unit of the nucleosome.

Using a DNA fragment of 147 bp, the authors observed a right-handed tetramer structure bound to CAF-1, which was further confirmed by analysis using the free-rotating single-molecule magnetic tweezers (FOMT) method at physiological salt concentration. . This finding indicated the possible involvement of right nucleosome precursors in nucleosome assembly associated with transcription, and provided a new direction for the mechanistic investigation of the nucleosome assembly process.